STAPLED ANTIMICROBIAL PEPTIDES (STAMPS) AND USES THEREOF

Abstract

Provided are stapled antimicrobial peptides (i.e., StAMPs) and methods of using the same (e.g., for treating bacterial infections caused by Gram-negative bacteria). In certain embodiments, the stapled peptides are based on the amino acid sequence of the antimicrobial peptide Magainin II, but include certain modifications that have been found to confer advantageous properties (e.g., improved antimicrobial activity, selectivity for killing Gram-negative bacteria, and/or reduced toxicity). Also provided are unstapled peptides which can serve as synthetic precursors to the stapled peptides provided herein

Claims

1. A peptide comprising the amino acid sequence: TABLE-US-00039 (SEQIDNO:1) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4, or a pharmaceutically acceptable salt thereof, wherein: X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and the amino acid sequence includes 1 to 9 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid substitution is at H7, S8, G18, or E19; and provided that the amino acid substitution at G18 is not G18H.

2. A peptide comprising the amino acid sequence: TABLE-US-00040 (SEQIDNO:1) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4, or a pharmaceutically acceptable salt thereof, wherein: X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and the amino acid sequence includes 1 to 9 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid substitution is at H7, S8, G18, or E19; and provided that the amino acid substitution at G18 is not G18H.

3. The peptide of claim 1 or 2, comprising the amino acid sequence: TABLE-US-00041 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive.

4. The peptide of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 to 7 amino acid substitutions, inclusive.

5. The peptide of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 to 5 amino acid substitutions, inclusive.

6. The peptide of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 or 2 amino acid substitutions.

7. The peptide of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises 1 to 5 amino acid substitutions, inclusive, independently at K4, H7, S8, G18, or E19.

8. The peptide of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises 1 or 2 amino acid substitutions independently at K4, H7, S8, G18, or E19.

9. The peptide of any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at H7.

10. The peptide of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at S8.

11. The peptide of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises amino acid substitutions at both H7 and S8.

12. The peptide of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at H7 is selected from H7K, H7W, H7Dab, H7Orn, H7Dap, H7R, and H7hArg.

13. The peptide of claim 12, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at H7 is H7K.

14. The peptide of any one of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at S8 is selected from S8K, S8W, S8N, S8Q, S8T, S8Y, S8Dab, S8Orn, S8Dap, S8R, and S8hArg.

15. The peptide of claim 14, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at S8 is S8K.

16. The peptide of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at G18.

17. The peptide of claim 16, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at G18 is selected from G18W, G18V, G18L, G18Y, G18F, G18T, G18F.sup.1, G18F.sup.2, G18F.sup.4, G18F.sup.5, G18F.sup.3, and G18S.

18. The peptide of claim 17, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at G18 is G18W.

19. The peptide of any one of claims 1-18, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at E19.

20. The peptide of claim 19, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at E19 is selected from E19W, E19D, E19Q, and E19N.

21. The peptide of any one of claims 1-20, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at K4.

22. The peptide of claim 21, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at K4 is selected from K4S, K4Dab, K4Orn, K4Dap, K4R, and K4hArg.

23. The peptide of any one of claims 10-22, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises H7K and S8K amino acid substitutions.

24. The peptide of any one of claims 1-23, or a pharmaceutically acceptable salt thereof, comprising one or more amino acid substitution selected from G13S, A15S, G18S, and A21S.

25. A peptide comprising the amino acid sequence: TABLE-US-00042 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, or a pharmaceutically acceptable salt thereof, wherein: X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid is substituted by W.

26. A peptide of comprising the amino acid sequence: TABLE-US-00043 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, or a pharmaceutically acceptable salt thereof, wherein: X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid is substituted by W.

27. The peptide of claim 25 or 26, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 to 5 amino acid substitutions, inclusive.

28. The peptide of any one of claims 25-27, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 or 2 amino acid substitutions.

29. The peptide of any one of claims 25-28, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 or 2 amino acids substituted by W.

30. A peptide comprising the amino acid sequence: TABLE-US-00044 (SEQIDNO:3) GX.sup.1GKFX.sup.2KKKKKX.sup.3VGEX.sup.4AKK, or a pharmaceutically acceptable salt thereof, wherein: X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4.

31. A peptide comprising the amino acid sequence: TABLE-US-00045 (SEQIDNO:3) GX.sup.1GKFX.sup.2KKKKKX.sup.3VGEX.sup.4AKK, or a pharmaceutically acceptable salt thereof, wherein: X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4.

32. A peptide comprising the amino acid sequence: TABLE-US-00046 (SEQIDNO:101) ZX.sup.1GKFX.sup.2HSAKKFGKAFV, or a pharmaceutically acceptable salt thereof, wherein: X.sup.1 and X.sup.2 are independently amino acids; X.sup.1 and X.sup.2 are connected via a crosslink; and the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1 and X.sup.2.

33. A peptides comprising the amino acid sequence: TABLE-US-00047 (SEQIDNO:101) ZX.sup.1GKFX.sup.2HSAKKFGKAFV, or a pharmaceutically acceptable salt thereof, wherein: X.sup.1 and X.sup.2 are independently amino acids; X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other; and the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1 and X.sup.2.

34. The peptide of any one of claims 30-33, wherein the amino acid sequence optionally includes 1 or 2 amino acid substitutions.

35. The peptide of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, wherein one or more instances of K are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg.

36. The peptide of claim 35, or a pharmaceutically acceptable salt thereof, wherein each instance of K is independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg.

37. The peptide of any one of claims 1-36, wherein one or more instances of F are independently substituted by an amino acid selected from F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3.

38. The peptide of claim 37, or a pharmaceutically acceptable salt thereof, wherein each instance of F is independently substituted by an amino acid selected from F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3.

39. The peptide of any one of claims 1-38, or a pharmaceutically acceptable salt thereof, comprising a small molecule, lipophilic group, or polymer conjugated to the C-terminus of the peptide.

40. The peptide of claim 39, or a pharmaceutically acceptable salt thereof, wherein the lipophilic group is a lipid or fatty acid.

41. The peptide of claim 39, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises PEG conjugated to the C-terminus.

42. The peptide of claim 41, or a pharmaceutically acceptable salt thereof, wherein the C-terminus comprises PEG3 or (PEG3).sub.2.

43. The peptide of any one of claims 1-42, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated.

44. The peptide of claim 43, wherein the C-terminus is amidated with NH.sub.2.

45. The peptide of claim 43, or a pharmaceutically acceptable salt thereof, wherein the peptide is amidated at the C-terminus with a group of the formula: NH(CH.sub.2CH.sub.2O).sub.1-20CH.sub.2CH.sub.2CONH.sub.2.

46. The peptide of claim 45, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with a selected from: NHCH.sub.2CH.sub.2OCH.sub.2CH.sub.2CONH.sub.2, NH(CH.sub.2CH.sub.2O).sub.2CH.sub.2CH.sub.2CONH.sub.2, NH(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2CONH.sub.2, NH(CH.sub.2CH.sub.2O).sub.4CH.sub.2CH.sub.2CONH.sub.2, and NH(CH.sub.2CH.sub.2O).sub.5CH.sub.2CH.sub.2CONH.sub.2.

47. The peptide of any one of claims 1-38, or a pharmaceutically acceptable salt thereof, comprising an amino acid or peptide conjugated to the C-terminus of the peptide.

48. The peptide of claim 47, or a pharmaceutically acceptable salt thereof, comprising one of the following amino acid sequences conjugated to the C-terminus of the peptide: TABLE-US-00048 GE, AG, AA, GG, GGE, GGS, GGG, GGK, GGQ, (SEQIDNO:78) GGGE, (SEQIDNO:79) GGEE, or (SEQIDNO:80) GGSGGS.

49. The peptide of claim 39, or a pharmaceutically acceptable salt thereof, comprising a polymyxin conjugated to the C-terminus of the peptide.

50. The peptide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, wherein the peptide is 100 amino acids or fewer in length.

51. The peptide of any one of claims 1-50, or a pharmaceutically acceptable salt thereof, wherein the peptide is 30 amino acids or fewer in length.

52. The peptide of any one of claims 1-3, wherein the peptide comprises one of the following amino acid sequences: TABLE-US-00049 (SEQIDNO:4) GX.sup.1GKFX.sup.2KSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:5) GX.sup.1GKFX.sup.2HKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:6) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:7) GX.sup.1GSFX.sup.2HKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:8) GX.sup.1GKFX.sup.2HNKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:9) GX.sup.1GKFX.sup.2HQKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:10) GX.sup.1GKFX.sup.2HTKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:11) GX.sup.1GKFX.sup.2HYKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:12) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VWEX.sup.4AKK, (SEQIDNO:13) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:14) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VLEX.sup.4AKK, (SEQIDNO:15) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VYEX.sup.4AKK, (SEQIDNO:16) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VFEX.sup.4AKK, (SEQIDNO:17) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VTEX.sup.4AKK, (SEQIDNO:18) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGDX.sup.4AKK, (SEQIDNO:19) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGQX.sup.4AKK, (SEQIDNO:20) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGNX.sup.4AKK, (SEQIDNO:21) GX.sup.1GKFX.sup.2KSKKKFGKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:22) GX.sup.1GKFX.sup.2KSKKKFGKAX.sup.3VFEX.sup.4AKK, (SEQIDNO:23) GX.sup.1GKFX.sup.2HKKKKFGKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:24) GX.sup.1GKFX.sup.2HKKKKFGKAX.sup.3VFEX.sup.4AKK, (SEQIDNO:25) GX.sup.1GDabFX.sup.2DabDabDabDabDabFGDabAX.sup.3VGEX.sup.4ADabDab, (SEQIDNO:26) GX.sup.1GOrnFX.sup.2OrnOrnOrnOrnOrnFGOrnAX.sup.3VGEX.sup.4AOrnOrn, (SEQIDNO:27) GX.sup.1GDapFX.sup.2DapDapDapDapDapFGDapAX.sup.3VGEX.sup.4ADapDap, (SEQIDNO:28) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:29) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4K, (SEQIDNO:30) GX.sup.1GKF.sup.1X.sup.2HKKKKF.sup.1GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:31) GX.sup.1GKF.sup.4X.sup.2HKKKKF.sup.4GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:32) GX.sup.1GKF5X.sup.2HKKKKF5GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:33) GX.sup.1GKF.sup.2X.sup.2HKKKKP.sup.2GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:34) GX.sup.1GKF.sup.3X.sup.2HKKKKF.sup.3GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:35) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VVEX.sup.4AKKGGE, (SEQIDNO:36) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VFEX.sup.4AKKGGE, (SEQIDNO:37) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VFLEX.sup.4AKKGGE, (SEQIDNO:38) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VF.sup.2EX.sup.4AKKGGE, (SEQIDNO:39) GX.sup.1GKFX.sup.2KKIKKFGKAX.sup.3VF.sup.3EX.sup.4AKKGGE, (SEQIDNO:59) GX.sup.1GDabFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:60) GX.sup.1GKFX.sup.2KDabKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:61) GX.sup.1GKFX.sup.2KKKKKFGDabAX.sup.3VGEX.sup.4AKK, (SEQIDNO:62) GX.sup.1GKFX.sup.2KKDapKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:63) GX.sup.1GKFX.sup.2DabKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:64) GX.sup.1GKFX.sup.2KKDabKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:65) GX.sup.1GKFX.sup.2KKKDabKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:66) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4ADabK, (SEQIDNO:67) GX.sup.1GDapFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:68) GX.sup.1GKFX.sup.2DapKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:69) GX.sup.1GKFX.sup.2KDapKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:70) GX.sup.1GKFX.sup.2KKKDapKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:71) GX.sup.1GKFX.sup.2KKKKDapFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:72) GX.sup.1GKFX.sup.2KKKKKFGDapAX.sup.3VGEX.sup.4AKK, (SEQIDNO:73) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4ADapK, (SEQIDNO:74) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKDap, (SEQIDNO:75) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGE, (SEQIDNO:76) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGGE, (SEQIDNO:77) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGEE, (SEQIDNO:81) GX.sup.1GDapFX.sup.2DapDapDapDapDapFGDapAX.sup.3VGEX.sup.4ADapDapG, (SEQIDNO:82) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGQ, (SEQIDNO:83) GX.sup.1GDapFX.sup.2DapDapDapDapDapFGDapAX.sup.3VGEX.sup.4AKK, (SEQIDNO:84) GX.sup.1GKFX.sup.2DabKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:85) GX.sup.1GKFX.sup.2DapKKKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:86) GX.sup.1GKFX.sup.2KKDapKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:87) GX.sup.1GKFX.sup.2DapKDapKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:88) GX.sup.1GKFX.sup.2DapKDapKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:89) JX.sup.1GDapFX.sup.2DapDapDapDapDapFGDapAX.sup.3VGEX.sup.4ADapDap, (SEQIDNO:90) GX.sup.1GKFX.sup.2DapSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:91) GX.sup.1GKFX.sup.2DapKKKKFSKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:92) GX.sup.1GKFX.sup.2DapKKKKFGKSX.sup.3VGEX.sup.4AKK, (SEQIDNO:93) GX.sup.1GKFX.sup.2DapKKKKFGKAX.sup.3VSEX.sup.4AKK, (SEQIDNO:94) GX.sup.1GKFX.sup.2DapKKKKFGKAX.sup.3VGEX.sup.4SKK, (SEQIDNO:95) GX.sup.1GKFX.sup.2KSKKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:96) GX.sup.1GKFX.sup.2KKKKKFSKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:97) GX.sup.1GKFX.sup.2KKKKKFGKSX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:98) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VSEX.sup.4AKKGGE, (SEQIDNO:99) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4SKKGGE, (SEQIDNO:100) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGEJ, or a pharmaceutically acceptable salt thereof.

53. The peptide of claim 52, or a pharmaceutically acceptable salt thereof, wherein the peptide is of one of SEQ ID NOs: 4-39, 59-77, or 81-100; and wherein the C-terminus is amidated with NH.sub.2.

54. The peptide of claim 25 or 26, wherein the peptide comprises one of the following amino acid sequences: TABLE-US-00050 (SEQIDNO:40) WX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:41) GX.sup.1WKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:42) GX.sup.1GWFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:43) GX.sup.1GKWX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:44) GX.sup.1GKFX.sup.2WSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:45) GX.sup.1GKFX.sup.2HWKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:46) GX.sup.1GKFX.sup.2HSKKKWWKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:47) GX.sup.1GKFX.sup.2HSKKKFGKWX.sup.3VGEX.sup.4AKK, (SEQIDNO:48) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3WGEX.sup.4AKK, (SEQIDNO:49) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VWEX.sup.4AKK, (SEQIDNO:50) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGWX.sup.4AKK, (SEQIDNO:51) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKW, (SEQIDNO:52) GX.sup.1GKFX.sup.2HSKKKWGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:53) GX.sup.1GKFX.sup.2HSKKKFWKAX.sup.3VGEX.sup.4AKK, or a pharmaceutically acceptable salt thereof.

55. The peptide of claim 54, or a pharmaceutically acceptable sale thereof, wherein the peptide is of one of SEQ ID NOs: 40-53; and wherein the C-terminus is amidated with NH.sub.2.

56. The peptide of claim 30 or 31, wherein the peptide comprises the following amino acid sequence: TABLE-US-00051 (SEQIDNO:3) GX.sup.1GKFX.sup.2KKKKKX.sup.3VGEX.sup.4AKK, or a pharmaceutically acceptable salt thereof.

57. The peptide of claim 56, or a pharmaceutically acceptable salt thereof, wherein the peptide is of SEQ ID NO: 3; and wherein the C-terminus is amidated with NH.sub.2.

58. The peptide of claim 32 or 33, wherein the peptide comprises one of the following amino acid sequences: TABLE-US-00052 (SEQIDNO:102) ZX.sup.1GKFX.sup.2HSKKKFGKAFV, (SEQIDNO:103) ZX.sup.1GKFX.sup.2KSKKKFGKAFV, (SEQIDNO:104) ZX.sup.1GKFX.sup.2KKKKKFGKAFV, (SEQIDNO:105) ZX.sup.1GKFX.sup.2DapKKKKFGKAFV, or a pharmaceutically acceptable salt thereof.

59. The peptide of any one of claims 32, 33, or 58, or a pharmaceutically acceptable salt thereof, wherein the peptide is of any one of SEQ ID NOs: 101-105; and wherein the C-terminus is amidated with NH.sub.2.

60. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the crosslinks are attached to the ?-positions of the amino acids X.sup.1, X.sup.2, X.sup.3, and X.sup.4.

61. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein each crosslink is independently optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted acylene, or any combination thereof.

62. The peptide of claim 61, or a pharmaceutically acceptable salt thereof, wherein each crosslink is a hydrocarbon crosslink independently selected from optionally substituted alkylene, optionally substituted alkenylene, and optionally substituted alkynylene.

63. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein each crosslink is independently of the following formula: ##STR00070## wherein each n is independently an integer from 1-10, inclusive.

64. The peptide of claim 63, or a pharmaceutically acceptable salt thereof, wherein the sum of two n on the same crosslink is 6.

65. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently ?,?-disubstituted amino acids.

66. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are each independently connected by a crosslink to form the following formula: ##STR00071## wherein ? denotes the ?-carbons of the amino acids; and wherein each instance of R.sup.1 is independently optionally substituted C.sub.1-6 alkyl.

67. The peptide of claim 66, or a pharmaceutically acceptable salt thereof, wherein at least one instance of R.sup.1 is methyl.

68. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are each connected by a crosslink to form the following formula: ##STR00072## wherein ? denotes the ?-carbons of the amino acids.

69. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein each crosslink is independently about 10 ? to about 16 ? in length, inclusive.

70. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the length of each crosslink is approximately equal to the length of 5 to 13 carbon-carbon bonds, inclusive.

71. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein at least one crosslink spans at least one turn of an ?-helix of the peptide.

72. The peptide of claim 71, or a pharmaceutically acceptable salt thereof, wherein at least one crosslink stabilizes an ?-helix of the peptide.

73. The peptide of claim 71 or 72, or a pharmaceutically acceptable salt thereof, wherein the peptide has increased ?-helicity as compared to a corresponding uncrosslinked peptide.

74. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 independently comprise ?-sidechains comprising the reactive moieties.

75. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the reactive moieties are independently selected from alkenes and alkynes.

76. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 independently comprise ?-sidechains of the following formula: ##STR00073## wherein each n is independently an integer from 1-10, inclusive.

77. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently ?,?-disubstituted amino acids.

78. The peptide of claim 76 or 77, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 each independently comprise the formula: ##STR00074## wherein ? denotes the ?-carbon of the amino acids; and each instance of R.sup.1 is optionally substituted C.sub.1-6 alkyl.

79. The peptide of claim 78, or a pharmaceutically acceptable salt thereof, wherein at least one instance of R.sup.1 is methyl.

80. The peptide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein each instance of X.sup.1, X.sup.2, X.sup.3, and X.sup.4 is an amino acid of the formula: ##STR00075##

81. A pharmaceutical composition comprising a peptide of any one of claims 1-73, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

82. A method of treating an infectious disease in a subject comprising administering to the subject a peptide of any one of claims 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

83. The method of claim 82, wherein the infectious disease is a bacterial infection, viral infection, protozoal infection, or fungal infection.

84. A method of treating a bacterial infection in a subject comprising administering to the subject a peptide of any one of claims 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

85. The method of claim 84, wherein the bacterial infection is a Gram-negative bacterial infection.

86. The method of claim 84 or 85, wherein the bacterial infection is an antibiotic-resistant bacterial infection.

87. The method of any one of claims 84-86, wherein the bacterial infection is a Gram-negative, antibiotic-resistant bacterial infection.

88. The method of claim 86 or 87, wherein the antibiotic-resistant bacterial infection is caused by a bacteria resistant to one or more antibiotics selected from the group consisting of polymyxin, carbapenem, aminoglycosides, vancomycin, methicillin, clarithromycin, cephalosporin, penicillin, ampicillin, fluoroquinolones, tetracycline, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, tobramycin, and ?-lactams.

89. The method of any one of claims 84-88, wherein the bacterial infection is caused by E. coli, A. baumannii, P. aeruginosa, or K. pneumoniae.

90. The method of any one of claims 82-89, wherein the peptide has reduced renal toxicity as compared to a reference.

91. The method of claim 90, wherein the peptide has reduced renal toxicity as compared to a corresponding peptide comprising SEQ ID NO: 1 or 2, or as compared to pexiganan.

92. The method of any one of claims 82-91, wherein the peptide has reduced hepatic toxicity as compared to a reference.

93. The method of claim 92, wherein the peptide has reduced hepatic toxicity as compared to a corresponding peptide comprising SEQ ID NO: 1 or 2, or as compared to pexiganan.

94. The method of any one of claims 82-93, wherein the peptide has reduced hemolytic activity as compared to a reference.

95. The method of claim 94, wherein the peptide has reduced hemolytic activity as compared to a corresponding peptide comprising SEQ ID NO: 1 or 2, or as compared to melittin.

96. The method of any one of claims 82-95, wherein the peptide, pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof is administered intravenously.

97. The method of any one of claims 82-96, wherein the subject is a human.

98. A method of killing and/or inhibiting the growth of bacteria comprising contacting the bacteria with a peptide of any one of claims 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

99. The method of claim 98, wherein the bacteria are E. coli, A. baumannii, P. aeruginosa, or K. pneumoniae.

100. A method of selectively killing and/or inhibiting the growth of microbial cells over mammalian cells comprising contacting the microbial and mammalian cells with a peptide of any one of claims 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

101. A method of selectively lysing microbial cells over mammalian cells comprising contacting the microbial and mammalian cells with a peptide of any one of claims 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

102. The method of claim 100 or 101, wherein the microbial cells are bacterial cells.

103. The method of claim 102, wherein the bacterial cells are E. coli cells, A. baumannii cells, P. aeruginosa cells, or K. pneumoniae cells.

104. A peptide of any one of claims 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in a method of any one of the preceding claims.

105. Use of a peptide of any one of claims 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a medicament.

106. A kit comprising a peptide of any one of claims 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof; and optionally instructions for use.

107. A method of preparing a crosslinked peptide of any one of the preceding claims comprising a step of reacting an uncrosslinked peptide of any one of the preceding claims under conditions sufficient to form the crosslinks connecting X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4.

108. The method of claim 107, wherein the step of reacting involves a ring-closing metathesis (RCM) reaction.

109. A formulation comprising a stapled antimicrobial peptide (StAMP) and one or more lipids.

110. The formulation of claim 109, wherein the formulation is a micellar, liposomal, or lipid nanoparticle formulation.

111. The formulation of claim 109 or 110, wherein the StAMP is a peptide of any one of the preceding claims.

112. The formulation of any one of claims 109-111, wherein at least one of the one or more lipids comprises a phospholipid.

113. The formulation of claim 112, wherein at least one of the one or more lipids is a PEGylated phospholipid.

114. The formulation of claim 113, wherein the PEGylated phospholipid is DSPE-PEG.

115. The formulation of claim 114, wherein the PEGylated phospholipid is DSPE-MPEG or DSPE-PEG (Amine).

116. The formulation of claim 115, wherein the PEGylated phospholipid is DSPE-MPEG(2000) or DSPE-PEG(2000) Amine.

117. The formulation of any one of claims 109-116, comprising a peptide:lipid ratio from about 1:1 to about 1:25 (w/w), inclusive.

118. The formulation of any one of claims 109-117, comprising a peptide:lipid ratio from about 1:2.5 to about 1:20 (w/w), inclusive.

119. The formulation of any one of claims 109-118, comprising a peptide:lipid ratio of about 1:2.5, about 1:5, about 1:10, or about 1:20 (w/w).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0115] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, provide non-limiting examples of the invention.

[0116] FIG. 1. Pharmacokinetic profile of a subcutaneous injection of StAMP 4 (Table A) in mice at 20 mg/kg using various StAMP:lipid ratios (w/w). Lipid=DSPE-MPEG(2000).

[0117] FIG. 2. Efficacy of StAMP 26 (Table A) in a neutropenic mouse pneumonia model of P. aeruginosa at four hours post-treatment.

[0118] FIG. 3. Efficacy of StAMP 46 (Table A) in a neutropenic mouse pneumonia model of P. aeruginosa at four hours post-treatment.

[0119] FIG. 4. Overnight efficacy of StAMP 26 and StAMP 46 in a neutropenic mouse pneumonia model of P. aeruginosa.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0120] Provided herein are stapled antimicrobial peptides (StAMPs), pharmaceutical compositions thereof, and kits comprising the same. The peptides provided herein have antimicrobial properties and can therefore be used to treat and prevent infectious diseases (e.g., bacterial infections) in a subject, to kill and/or inhibit the growth of bacteria, and more.

[0121] Also provided herein are unstapled peptides which can serve as synthetic precursors to the stapled peptides provided herein.

Stapled Peptides

[0122] In one aspect, provided herein are stapled (i.e., crosslinked) peptides. In certain embodiments, the stapled peptides provided herein are based on the amino acid sequence of the antimicrobial peptide Magainin II, but they include certain modifications that have been found to confer advantageous properties (e.g., improved antimicrobial activity, selectivity for killing Gram-negative bacteria, and/or reduced toxicity). In certain embodiments, the stapled peptides provided herein comprise two staples (i.e., crosslinks) that connect the amino acid residues X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, as included in the amino acid sequences herein.

Amino Acid Sequences

[0123] Provided herein are stapled (i.e., crosslinked) peptides comprising the amino acid sequence:

TABLE-US-00005 (SEQIDNO:1) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4,
and pharmaceutically acceptable salts thereof, wherein: [0124] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids (i.e., crosslinked amino acids); [0125] X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and [0126] the amino acid sequence includes 1 to 9 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid substitution is at H7, S8, G18, or E19. In certain embodiments, the amino acid substitution at G18 is not G18H.

[0127] In certain embodiments, the stapled peptide or pharmaceutically acceptable salt thereof comprises the amino acid sequence:

TABLE-US-00006 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK,
wherein: [0128] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids (i.e., crosslinked amino acids); [0129] X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and [0130] the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid substitution is at H7, S8, G18, or E19. In certain embodiments, the amino acid substitution at G18 is not G18H.

[0131] In certain embodiments, a stapled peptide provided herein is not of, or does not comprise, one of the following amino acid sequences:

TABLE-US-00007 (SEQIDNO:57) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VHEX.sup.4BES (SEQIDNO:58) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VHEX.sup.4.

[0132] As described herein, an amino acid sequences provided herein (e.g., SEQ ID NO: 1 or 2) can include one or more amino acid substitutions. The amino acids can be independently substituted by any natural or unnatural amino acid, including, but not limited to, those amino acids provided herein. In certain embodiments, the amino acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In certain embodiments, the amino acid sequence includes 1 amino acid substitution. In certain embodiments, the amino acid sequence includes 1 or 2 amino acid substitutions. In certain embodiments, the amino acid sequence includes 1 to 3 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 4 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 5 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 6 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 7 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 8 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 9 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 10 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive.

[0133] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises 1 to 5 amino acid substitutions, inclusive, independently at K4, H7, S8, G18, or E19. In certain embodiments, the amino acid sequence comprises 1 to 4 amino acid substitutions, inclusive, independently at K4, H7, S8, G18, or E19. In certain embodiments, the amino acid sequence comprises 1 to 3 amino acid substitutions, inclusive, independently at K4, H7, S8, G18, or E19. In certain embodiments, the amino acid sequence comprises 1 or 2 amino acid substitutions independently at K4, H7, S8, G18, or E19. In certain embodiments, the amino acid sequence comprises 1 amino acid substitution at H7, S8, G18, or E19.

[0134] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises an amino acid substitution at H7. In certain embodiments, the amino acid substitution at H7 is selected from H7K, H7W, H7Dab, H7Orn, H7Dap, H7R, and H7hArg. In certain embodiments, the amino acid sequence comprises an H7K substitution. In certain embodiments, the amino acid sequence comprises an H7W substitution. In certain embodiments, the amino acid sequence comprises an H7Dab substitution. In certain embodiments, the amino acid sequence comprises an H7Orn substitution. In certain embodiments, the amino acid sequence comprises an H7Dap substitution. In certain embodiments, the amino acid sequence comprises an H7R substitution. In certain embodiments, the amino acid sequence comprises an H7hArg substitution.

[0135] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises an amino acid substitution at S8. In certain embodiments, the amino acid substitution at S8 is selected from S8K, S8W, S8N, S8Q, S8T, S8Y, S8Dab, S8Orn, S8Dap, S8R, and S8hArg. In certain embodiments, the amino acid sequence comprises a S8K substitution. In certain embodiments, the amino acid sequence comprises a S8W substitution. In certain embodiments, the amino acid sequence comprises a S8N substitution. In certain embodiments, the amino acid sequence comprises a S8Q substitution. In certain embodiments, the amino acid sequence comprises a S8T substitution. In certain embodiments, the amino acid sequence comprises a S8Y substitution. In certain embodiments, the amino acid sequence comprises a S8Dab substitution. In certain embodiments, the amino acid sequence comprises a S8Orn substitution. In certain embodiments, the amino acid sequence comprises a S8Dap substitution. In certain embodiments, the amino acid sequence comprises a S8R substitution. In certain embodiments, the amino acid sequence comprises a S8hArg substitution.

[0136] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises amino acid substitutions at both H7 and S8. In certain embodiments, the amino acid sequence comprises an H7K substitution and a S8K substitution.

[0137] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises an amino acid substitution at G18. In certain embodiments, the amino acid substitution at G18 is selected from G18W, G18V, G18L, G18Y, G18F, G18T, G18F.sup.1, G18F.sup.2, G18F.sup.4, G18F.sup.5, G18F.sup.3, and G18S. In certain embodiments, the amino acid sequence comprises a G18W substitution. In certain embodiments, the amino acid sequence comprises a G18V substitution. In certain embodiments, the amino acid sequence comprises a G18L substitution. In certain embodiments, the amino acid sequence comprises a G18Y substitution. In certain embodiments, the amino acid sequence comprises a G18F substitution. In certain embodiments, the amino acid sequence comprises a G18T substitution. In certain embodiments, the amino acid sequence comprises a G18F.sup.1 substitution. In certain embodiments, the amino acid sequence comprises a G18F.sup.2 substitution. In certain embodiments, the amino acid sequence comprises a G18F.sup.4. substitution. In certain embodiments, the amino acid sequence comprises a G18F.sup.5 substitution. In certain embodiments, the amino acid sequence comprises a G18F.sup.3 substitution. In certain embodiments, the amino acid sequence comprises a G18S substitution.

[0138] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises an amino acid substitution at E19. In certain embodiments, the amino acid substitution at E19 is selected from E19W, E19D, E19Q, and E19N. In certain embodiments, the amino acid sequence comprises an E19W substitution. In certain embodiments, the amino acid sequence comprises an E19D substitution. In certain embodiments, the amino acid sequence comprises an E19Q substitution. In certain embodiments, the amino acid sequence comprises an E19N substitution.

[0139] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises an amino acid substitution at K4. In certain embodiments, the amino acid substitution at K4 is selected from K4S, K4Dab, K4Orn, K4Dap, K4R, and K4hArg. In certain embodiments, the amino acid sequence comprises a K4S substitution. In certain embodiments, the amino acid sequence comprises a K4Dab substitution. In certain embodiments, the amino acid sequence comprises a K4Orn substitution. In certain embodiments, the amino acid sequence comprises a K4Dap substitution. In certain embodiments, the amino acid sequence comprises a K4R substitution. In certain embodiments, the amino acid sequence comprises a K4hArg substitution.

[0140] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises an amino acid substitution at G13. In certain embodiments, the amino acid substitution at G13 is G13S.

[0141] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises an amino acid substitution at A15. In certain embodiments, the amino acid substitution at A15 is A15S.

[0142] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises an amino acid substitution at A21. In certain embodiments, the amino acid substitution at A21 is A21S.

[0143] An amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) can comprise any combination of the foregoing amino acid substitutions.

[0144] In certain embodiments, a stapled peptide or pharmaceutically acceptable salt thereof provided herein comprises one of the following amino acid sequences:

TABLE-US-00008 (SEQIDNO:4) GX.sup.1GKFX.sup.2KSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:5) GX.sup.1GKFX.sup.2HKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:6) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:7) GX.sup.1GSFX.sup.2HKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:8) GX.sup.1GKFX.sup.2HNKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:9) GX.sup.1GKFX.sup.2HQKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:10) GX.sup.1GKFX.sup.2HTKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:11) GX.sup.1GKFX.sup.2HYKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:12) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VWEX.sup.4AKK, (SEQIDNO:13) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:14) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VLEX.sup.4AKK, (SEQIDNO:15) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VYEX.sup.4AKK, (SEQIDNO:16) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VFEX.sup.4AKK, (SEQIDNO:17) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VTEX.sup.4AKK, (SEQIDNO:18) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGDX.sup.4AKK, (SEQIDNO:19) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGQX.sup.4AKK, (SEQIDNO:20) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGNX.sup.4AKK, (SEQIDNO:21) GX.sup.1GKFX.sup.2KSKKKFGKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:22) GX.sup.1GKFX.sup.2KSKKKFGKAX.sup.3VFEX.sup.4AKK, (SEQIDNO:23) GX.sup.1GKFX.sup.2HKKKKFGKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:24) GX.sup.1GKFX.sup.2HKKKKFGKAX.sup.3VFEX.sup.4AKK, (SEQIDNO:25) GX.sup.1GDabFX.sup.2DabDabDabDabDabFGDabAX.sup.3VGEX.sup.4ADabDab, (SEQIDNO:26) GX.sup.1GOrnFX.sup.2OrnOrnOrnOrnOrnFGOrnAX.sup.3VGEX.sup.4AOrnOrn, (SEQIDNO:27) GX.sup.1GDapFX.sup.2DapDapDapDapDapFGDapAX.sup.3VGEX.sup.4ADapDap, (SEQIDNO:28) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:29) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4K, (SEQIDNO:30) GX.sup.1GKF.sup.1X.sup.2HKKKKF.sup.1GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:31) GX.sup.1GKF.sup.4X.sup.2HKKKKF.sup.4GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:32) GX.sup.1GKF.sup.5X.sup.2HKKKKF.sup.5GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:33) GX.sup.1GKF.sup.2X.sup.2HKKKKF.sup.2GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:34) GX.sup.1GKF.sup.3X.sup.2HKKKKF.sup.3GKAX.sup.3VVEX.sup.4AKK, (SEQIDNO:35) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VVEX.sup.4AKKGGE, (SEQIDNO:36) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VFEX.sup.4AKKGGE, (SEQIDNO:37) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VFLEX.sup.4AKKGGE, (SEQIDNO:38) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VF.sup.2EX.sup.4AKKGGE, (SEQIDNO:39) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VF.sup.3EX.sup.4AKKGGE, (SEQIDNO:59) GX.sup.1GDabFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:60) GX.sup.1GKFX.sup.2KDabKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:61) GX.sup.1GKFX.sup.2KKKKKFGDabAX.sup.3VGEX.sup.4AKK, (SEQIDNO:62) GX.sup.1GKFX.sup.2KKDapKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:63) GX.sup.1GKFX.sup.2DabKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:64) GX.sup.1GKFX.sup.2KKDabKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:65) GX.sup.1GKFX.sup.2KKKDabKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:66) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4ADabK, (SEQIDNO:67) GX.sup.1GDapFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:68) GX.sup.1GKFX.sup.2DapKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:69) GX.sup.1GKFX.sup.2KDapKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:70) GX.sup.1GKFX.sup.2KKKDapKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:71) GX.sup.1GKFX.sup.2KKKKDapFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:72) GX.sup.1GKFX.sup.2KKKKKFGDapAX.sup.3VGEX.sup.4AKK, (SEQIDNO:73) GX.sup.1GKFX.sup.2KKKKFGKAX.sup.3VGEX.sup.4ADapK, (SEQIDNO:74) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKDap, (SEQIDNO:75) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGE, (SEQIDNO:76) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGGE, (SEQIDNO:77) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGEE, (SEQIDNO:81) GX.sup.1GDapFX.sup.2DapDapDapDapDapFGDapAX.sup.3VGEX.sup.4ADapDapG, (SEQIDNO:82) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGQ, (SEQIDNO:83) GX.sup.1GDapFX.sup.2DapDapDapDapDapFGDapAX.sup.3VGEX.sup.4AKK, (SEQIDNO:84) GX.sup.1GKFX.sup.2DabKKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:85) GX.sup.1GKFX.sup.2DapKKKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:86) GX.sup.1GKFX.sup.2KKDapKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:87) GX.sup.1GKFX.sup.2DapKDapKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:88) GX.sup.1GKFX.sup.2DapKDapKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:89) JX.sup.1GDapFX.sup.2DapDapDapDapDapFGDapAX.sup.3VGEX.sup.4ADapDap, (SEQIDNO:90) GX.sup.1GKFX.sup.2DapSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:91) GX.sup.1GKFX.sup.2DapKKKKFSKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:92) GX.sup.1GKFX.sup.2DapKKKKFGKSX.sup.3VGEX.sup.4AKK, (SEQIDNO:93) GX.sup.1GKFX.sup.2DapKKKKFGKAX.sup.3VSEX.sup.4AKK, (SEQIDNO:94) GX.sup.1GKFX.sup.2DapKKKKFGKAX.sup.3VGEX.sup.4SKK, (SEQIDNO:95) GX.sup.1GKFX.sup.2KSKKKFGKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:96) GX.sup.1GKFX.sup.2KKKKKFSKAX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:97) GX.sup.1GKFX.sup.2KKKKKFGKSX.sup.3VGEX.sup.4AKKGGE, (SEQIDNO:98) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VSEX.sup.4AKKGGE, (SEQIDNO:99) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4SKKGGE, or (SEQIDNO:100) GX.sup.1GKFX.sup.2KKKKKFGKAX.sup.3VGEX.sup.4AKKGGEJ.

[0145] In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 4-39 or 59-77, or a pharmaceutically acceptable salt thereof. In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 4-39 or 59-77, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with NH.sub.2. In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 4-39 or 59-77, or a pharmaceutically acceptable salt thereof, wherein X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are connected to form a crosslink of the formula (alk). In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 4-39 or 59-77, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with NH.sub.2; and X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are connected to form a crosslink of the formula (alk).

[0146] In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 81-100, or a pharmaceutically acceptable salt thereof. In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 81-100, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with NH.sub.2. In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 81-100, or a pharmaceutically acceptable salt thereof, wherein X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are connected to form a crosslink of the formula (alk). In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 81-100, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with NH.sub.2; and X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are connected to form a crosslink of the formula (alk).

[0147] Also provided herein are stapled (i.e., crosslinked) peptides comprising the amino acid sequence:

TABLE-US-00009 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK,
and pharmaceutically acceptable salts thereof, wherein: [0148] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids (i.e., crosslinked amino acids); [0149] X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and [0150] the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid is substituted by W.

[0151] In certain embodiments, the amino acid sequence of SEQ ID NO: 2 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In certain embodiments, the amino acid sequence includes 1 amino acid substitution. In certain embodiments, the amino acid sequence includes 1 or 2 amino acid substitutions. In certain embodiments, the amino acid sequence includes 1 to 3 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 4 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 5 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 6 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 7 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 8 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 9 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 10 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive. The amino acid substitutions can be selected from any of those provided herein.

[0152] In certain embodiments, the amino acid sequence of SEQ ID NO: 2 comprises 1 amino acid substituted by W. In certain embodiments, the amino acid sequence comprises 2 amino acids substituted by W.

[0153] In certain embodiments, a stapled peptide or pharmaceutically acceptable salt thereof provided herein comprises one of the following amino acid sequences:

TABLE-US-00010 (SEQIDNO:40) WX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:41) GX.sup.1WKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:42) GX.sup.1GWFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:43) GX.sup.1GKWX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:44) GX.sup.1GKFX.sup.2WSKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:45) GX.sup.1GKFX.sup.2HWKKKFGKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:46) GX.sup.1GKFX.sup.2HSKKKWWKAX.sup.3VGEX.sup.4AKK, (SEQIDNO:47) GX.sup.1GKFX.sup.2HSKKKFGKWX.sup.3VGEX.sup.4AKK, (SEQIDNO:48) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3WGEX.sup.4AKK, (SEQIDNO:49) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VWEX.sup.4AKK, (SEQIDNO:50) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGWX.sup.4AKK, (SEQIDNO:51) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKW, (SEQIDNO:52) GX.sup.1GKFX.sup.2HSKKKWGKAX.sup.3VGEX.sup.4AKK, or (SEQIDNO:53) GX.sup.1GKFX.sup.2HSKKKFWKAX.sup.3VGEX.sup.4AKK.

[0154] In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 40-53, or a pharmaceutically acceptable salt thereof. In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 40-53, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with NH.sub.2. In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 40-53, or a pharmaceutically acceptable salt thereof, wherein X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are connected to form a crosslink of the formula (alk). In certain embodiments, a stapled peptide provided herein is of one of SEQ ID NOs: 40-53, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with NH.sub.2; and X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are connected to form a crosslink of the formula (alk).

[0155] Also provided herein are stapled (i.e., crosslinked) peptides comprising the amino acid sequence:

TABLE-US-00011 (SEQIDNO:3) GX.sup.1GKFX.sup.2KKKKKX.sup.3VGEX.sup.4AKK,
and pharmaceutically acceptable salts thereof, wherein: [0156] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids (i.e., crosslinked amino acids); [0157] X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and [0158] the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4.

[0159] In certain embodiments, the amino acid sequence of SEQ ID NO: 3 includes 1, 2, 3, 4, or 5 amino acid substitutions. In certain embodiments, the amino acid sequence includes 1 amino acid substitution. In certain embodiments, the amino acid sequence includes 1 or 2 amino acid substitutions. In certain embodiments, the amino acid sequence includes 1 to 3 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 4 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 5 amino acid substitutions, inclusive. The amino acid substitutions can be selected from any of those provided herein.

[0160] In certain embodiments, a stapled peptide or a pharmaceutically acceptable salt thereof provided herein comprises SEQ ID NO: 3. In certain embodiments, a stapled peptide provided herein is of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof. In certain embodiments, a stapled peptide provided herein is of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with NH.sub.2. In certain embodiments, a stapled peptide provided herein is of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof, wherein X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are connected to form a crosslink of the formula (alk). In certain embodiments, a stapled peptide provided herein is of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with NH.sub.2; and X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are connected to form a crosslink of the formula (alk).

[0161] Also provided herein are stapled (i.e., crosslinked) peptides comprising the amino acid sequence:

TABLE-US-00012 (SEQIDNO:101) ZX.sup.1GKFX.sup.2HSAKKFGKAFV;
and pharmaceutically acceptable salts thereof, wherein: [0162] X.sup.1 and X.sup.2 are independently amino acids (i.e., crosslinked amino acids); [0163] X.sup.1 and X.sup.2 are connected via a crosslink; and [0164] the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1 and X.sup.2.

[0165] In certain embodiments, the amino acid sequence of SEQ ID NO: 101 includes 1, 2, 3, 4, or 5 amino acid substitutions. In certain embodiments, the amino acid sequence includes 1 amino acid substitution. In certain embodiments, the amino acid sequence includes 1 or 2 amino acid substitutions. In certain embodiments, the amino acid sequence includes 1 to 3 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 4 amino acid substitutions, inclusive. In certain embodiments, the amino acid sequence includes 1 to 5 amino acid substitutions, inclusive. The amino acid substitutions can be selected from any of those provided herein.

[0166] In certain embodiments, a stapled peptide or pharmaceutically acceptable salt thereof provided herein comprises one of the following amino acid sequences:

TABLE-US-00013 (SEQIDNO:102) ZX.sup.1GKFX.sup.2HSKKKFGKAFV, (SEQIDNO:103) ZX.sup.1GKFX.sup.2KSKKKFGKAFV, (SEQIDNO:104) ZX.sup.1GKFX.sup.2KKKKKFGKAFV, or (SEQIDNO:105) ZX.sup.1GKFX.sup.2DapKKKKFGKAFV.

[0167] In certain embodiments, a stapled peptide or a pharmaceutically acceptable salt thereof provided herein comprises any one of SEQ ID NOs: 101-105. In certain embodiments, a stapled peptide provided herein is of any one of SEQ ID NOs: 101-105, or a pharmaceutically acceptable salt thereof. In certain embodiments, a stapled peptide provided herein is of any one of SEQ ID NOs: 101-105, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with NH.sub.2. In certain embodiments, a stapled peptide provided herein is of any one of SEQ ID NOs: 101-105, or a pharmaceutically acceptable salt thereof, wherein X.sup.1 and X.sup.2 are connected to form a crosslink of the formula (alk). In certain embodiments, a stapled peptide provided herein is of any one of SEQ ID NOs: 101-105, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with NH.sub.2; and X.sup.1 and X.sup.2 are connected to form a crosslink of the formula (alk).

[0168] In addition to pharmaceutically acceptable salts of the stapled peptides provided herein, also provided herein are stereoisomers, tautomers, isotopically labeled derivatives, solvates, hydrates, polymorphs, co-crystals, and prodrugs of the stapled peptides provided herein.

[0169] As described herein, stapling (e.g., crosslinking) a peptide can stabilize a secondary structure (e.g., ?-helical secondary structure) of the peptide. In certain embodiments, one or more crosslinks of a stapled peptide provided herein stabilize an ?-helix of the peptide. In certain embodiments, a peptide has increased ?-helicity as compared to a corresponding unstapled (e.g., uncrosslinked) peptide.

[0170] A stapled peptide provided herein can exhibit ?-helical stability by the maintenance of ?-helical structure as measured by circular dichroism or NMR. For example, in certain embodiments, the stapled peptide exhibits at least a 1.1, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, or 2-fold increase in ?-helicity (e.g., as determined by circular dichroism or NMR) compared to a corresponding unstapled peptide. In certain embodiments, a stapled peptide provided herein can exhibit about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% ?-helicity (e.g., as determined by circular dichroism or NMR) compared to a corresponding unstapled peptide.

Additional Amino Acid Substitutions

[0171] Another aspect of the present disclosure relates to the discovery that replacing lysine (K) and/or phenylalanine (F) residues of amino acid sequences recited herein with modified variants can help confer advantageous properties (e.g., improved antimicrobial activity, reduced toxicity, etc.). For example, it has been found that replacing one or more lysine (K) residues with shorter lysine residues (e.g., amino acids Orn, Dab, and Dap, shown below in Table 2) can help reduce toxicity (e.g., renal toxicity) of the peptides.

TABLE-US-00014 TABLE 2 Shorter Lysine Amino Acids [00019] [00020] [00021] Orn Dab Dap

[0172] Therefore, also provided herein are peptides and pharmaceutically acceptable salts thereof comprising any amino acid sequence provided herein (e.g., SEQ ID NOs: 1-53, 59-77, and 81-105), optionally wherein one or more instances of K are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg. In certain embodiments, one or more instances of K (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 instances of K) are independently substituted by an amino acid selected from Orn, Dab, and Dap. In certain embodiments, one or more instances of K (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 instances of K) are substituted by Orn. In certain embodiments, one or more instances of K (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 instances of K) are substituted by Dab. In certain embodiments, one or more instances of K (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 instances of K) are substituted by Dap. In certain embodiments, one or more instances of K (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 instances of K) are substituted by R. In certain embodiments, one or more instances of K (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 instances of K) are substituted by and hArg.

[0173] In certain embodiments, each instance of K is independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg. In certain embodiments, each instance of K is substituted by Orn. In certain embodiments, each instance of K is substituted by Dab. In certain embodiments, each instance of K is substituted by Dap. In certain embodiments, each instance of K is substituted by R. In certain embodiments, each instance of K is substituted by hArg.

[0174] Also provided herein are peptides and pharmaceutically acceptable salts thereof comprising any amino acid sequence provided herein (e.g., SEQ ID NOs: 1-53, 59-77, and 81-105), optionally wherein one or more instances of F (e.g., each instance of F) are independently substituted by a modified phenylalanine. Modified phenylalanine for the purpose of this disclosure means a stereoisomer of phenylalanine (e.g., D-Phe) or any unnatural phenylalanine analog wherein the phenyl ring is ortho-, meta-, and/or para-substituted with one or more non-hydrogen substituents (e.g., optionally substituted alkyl (e.g., Me, Et, n-Pr, i-Pr, n-Bu, t-Bu), halogen (e.g., F, Cl, Br, I), haloalkyl (e.g., CF.sub.3), optionally substituted hydroxyl (e.g., OH), optionally substituted amino (e.g., NH.sub.2), optionally substituted thio (e.g., SH), optionally substituted acyl (e.g., C(?O)Me, C(?O)OH, C(?O)NH.sub.2), CN, SCN, NO.sub.3, N.sub.3). In certain embodiments, one or more instances of F are independently substituted by a modified phenylalanine. In certain embodiments, each instance of F is independently substituted by a modified phenylalanine.

[0175] In certain embodiments, one or more instances of F (e.g., 1, 2, 3 instances of F) are independently substituted by an amino acid selected from F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3. In certain embodiments, one or more instances of F (e.g., 1, 2, 3 instances of F) are independently substituted by F.sup.1. In certain embodiments, one or more instances of F (e.g., 1, 2, 3 instances of F) are independently substituted by F.sup.5. In certain embodiments, one or more instances of F (e.g., 1, 2, 3 instances of F) are independently substituted by F.sup.4. In certain embodiments, one or more instances of F (e.g., 1, 2, 3 instances of F) are independently substituted by F.sup.2. In certain embodiments, one or more instances of F (e.g., 1, 2, 3 instances of F) are independently substituted by F.sup.3.

[0176] In certain embodiments, each instance of F is substituted by an amino acid selected from F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3. In certain embodiments, each instance of F is substituted by F.sup.1. In certain embodiments, each instance of F is substituted by F.sup.5. In certain embodiments, each instance of F is substituted by F.sup.4. In certain embodiments, each instance of F is substituted by F.sup.2. In certain embodiments, each instance of F is substituted by F.sup.3.

[0177] Also provided herein are peptides and pharmaceutically acceptable salts thereof comprising any amino acid sequence provided herein (e.g., SEQ ID NOs: 1-53, 59-77, and 81-105), optionally wherein one or more instances of K are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg; and optionally wherein one or more instances of F are independently substituted by a modified phenylalanine.

[0178] All combinations of the foregoing amino acid substitutions (i.e., H7 substitutions, S8 substitutions, G18 substitutions, E19 substitutions, K substitutions, F substitutions, and others) are included in the present disclosure.

[0179] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises 1 to 5 (e.g., 1, 2, 4, 3, 4, 5) amino acid substitutions, inclusive, independently at K4 (e.g., K4S, K4Dab, K4Orn, K4Dap, K4R, or K4hArg), H7 (e.g., H7K, H7W, H7Dab, H7Orn, H7Dap, H7R, or H7hArg), S8 (e.g., S8K, S8W, S8N, S8Q, S8T, S8Y, S8Dab, S8Orn, S8Dap, S8R, or S8hArg), G18 (e.g., G18W, G18V, G18L, G18Y, G18F, G18T, G18F.sup.1, G18F.sup.2, G18F.sup.4, G18F.sup.5, G18F.sup.3, or G18S), or E19 (e.g., E19W, E19D, E19Q, or E19N); optionally further wherein one or more instances of K (e.g., each instance of K) are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg; and optionally further wherein one or more instances of F (e.g., each instance of F) are independently substituted by a modified phenylalanine (e.g., F.sup.1, F.sup.5, F.sup.4, F.sup.2, or F.sup.3).

[0180] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) comprises 1 to 5 (e.g., 1, 2, 4, 3, 4, 5) amino acid substitutions, inclusive, independently at K4 (e.g., K4S, K4Dab, K4Orn, K4Dap, K4R, or K4hArg), H7 (e.g., H7K, H7W, H7Dab, H7Orn, H7Dap, H7R, or H7hArg), S8 (e.g., S8K, S8W, S8N, S8Q, S8T, S8Y, S8Dab, S8Orn, S8Dap, S8R, or S8hArg), G13 (e.g., G13S), A15 (e.g., A15S), G18 (e.g., G18W, G18V, G18L, G18Y, G18F, G18T, G18F.sup.1, G18F.sup.2, G18F.sup.4, G18F.sup.5, G18F.sup.3, or G18S), E19 (e.g., E19W, E19D, E19Q, or E19N), or A21 (e.g., A21S); optionally further wherein one or more instances of K (e.g., each instance of K) are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg; and optionally further wherein one or more instances of F (e.g., each instance of F) are independently substituted by a modified phenylalanine (e.g., F.sup.1, F.sup.5, F.sup.4, F.sup.2, or F.sup.3).

[0181] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1, 2, or 3) comprises 1 or 2 amino acids substituted by W; optionally further wherein one or more instances of K (e.g., each instance of K) are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg; and optionally further wherein one or more instances of F (e.g., each instance of F) are independently substituted by a modified phenylalanine (e.g., F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3).

[0182] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) can comprise any combination of the following amino acid substitutions: [0183] (i) 1 to 5 (e.g., 1, 2, 4, 3, 4, 5) amino acid substitutions, inclusive, independently at K4 (e.g., K4S, K4Dab, K4Orn, K4Dap, K4R, or K4hArg), H7 (e.g., H7K, H7W, H7Dab, H7Orn, H7Dap, H7R, or H7hArg), S8 (e.g., S8K, S8W, S8N, S8Q, S8T, S8Y, S8Dab, S8Orn, S8Dap, S8R, or S8hArg), G18 (e.g., G18W, G18V, G18L, G18Y, G18F, G18T, G18F.sup.1, G18F.sup.2, G18F.sup.4, G18F.sup.5, G18F.sup.3, or G18S), or E19 (e.g., E19W, E19D, E19Q, or E19N); [0184] (ii) one or more instances of K (e.g., each instance of K) are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg; and/or [0185] (iii) one or more instances of F (e.g., each instance of F) are independently substituted by a modified phenylalanine (e.g., F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3).

[0186] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) can comprise any combination of the following amino acid substitutions: [0187] (i) 1 to 5 (e.g., 1, 2, 4, 3, 4, 5) amino acid substitutions, inclusive, independently at K4 (e.g., K4S, K4Dab, K4Orn, K4Dap, K4R, or K4hArg), H7 (e.g., H7K, H7W, H7Dab, H7Orn, H7Dap, H7R, or H7hArg), S8 (e.g., S8K, S8W, S8N, S8Q, S8T, S8Y, S8Dab, S8Orn, S8Dap, S8R, or S8hArg), G18 (e.g., G18W, G18V, G18L, G18Y, G18F, G18T, G18F.sup.1, G18F.sup.2, G18F.sup.4, G18F.sup.5, G18F.sup.3, or G18S), or E19 (e.g., E19W, E19D, E19Q, or E19N); [0188] (ii) 1 or 2 amino acids substituted by W; [0189] (iii) one or more instances of K (e.g., each instance of K) are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg; and/or [0190] (iv) one or more instances of F (e.g., each instance of F) are independently substituted by a modified phenylalanine (e.g., F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3).

[0191] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) can comprise any combination of the following amino acid substitutions: [0192] (i) 1 to 5 (e.g., 1, 2, 4, 3, 4, 5) amino acid substitutions, inclusive, independently at K4 (e.g., K4S, K4Dab, K4Orn, K4Dap, K4R, or K4hArg), H7 (e.g., H7K, H7W, H7Dab, H7Orn, H7Dap, H7R, or H7hArg), S8 (e.g., S8K, S8W, S8N, S8Q, S8T, S8Y, S8Dab, S8Orn, S8Dap, S8R, or S8hArg), G13 (e.g., G13S), A15 (e.g., A15S), G18 (e.g., G18W, G18V, G18L, G18Y, G18F, G18T, G18F.sup.1, G18F.sup.2, G18F.sup.4, G18F.sup.5, G18F.sup.3, or G18S), E19 (e.g., E19W, E19D, E19Q, or E19N), or A21 (e.g., A21S); [0193] (ii) one or more instances of K (e.g., each instance of K) are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg; and/or [0194] (iii) one or more instances of F (e.g., each instance of F) are independently substituted by a modified phenylalanine (e.g., F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3).

[0195] In certain embodiments, an amino acid sequence provided herein (e.g., SEQ ID NO: 1 or 2) can comprise any combination of the following amino acid substitutions: [0196] (i) 1 to 5 (e.g., 1, 2, 4, 3, 4, 5) amino acid substitutions, inclusive, independently at K4 (e.g., K4S, K4Dab, K4Orn, K4Dap, K4R, or K4hArg), H7 (e.g., H7K, H7W, H7Dab, H7Orn, H7Dap, H7R, or H7hArg), S8 (e.g., S8K, S8W, S8N, S8Q, S8T, S8Y, S8Dab, S8Orn, S8Dap, S8R, or S8hArg), G13 (e.g., G13S), A15 (e.g., A15S), G18 (e.g., G18W, G18V, G18L, G18Y, G18F, G18T, G18F.sup.1, G18F.sup.2, G18F.sup.4, G18F.sup.5, G18F.sup.3, or G18S), E19 (e.g., E19W, E19D, E19Q, or E19N), or A21 (e.g., A21S); [0197] (ii) 1 or 2 amino acids substituted by W; [0198] (iii) one or more instances of K (e.g., each instance of K) are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg; and/or [0199] (iv) one or more instances of F (e.g., each instance of F) are independently substituted by a modified phenylalanine (e.g., F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3).

Stapled Peptide Crosslinks

[0200] The stapled peptides provided herein comprise crosslinks (e.g., staples), wherein each crosslink connects two amino acids (i.e., crosslinked amino acids) to form a macrocycle. In certain embodiments, when an amino acid sequence comprises X.sup.1 and X.sup.2, X.sup.1 and X.sup.2 are crosslinked amino acids connected via a crosslink. Likewise, in certain embodiments, when an amino acid sequence comprises X.sup.3 and X.sup.4, X.sup.3 and X.sup.4 are crosslinked amino acids connected via a crosslink.

[0201] In certain embodiments, the crosslinks are independently attached to the ?-positions of the crosslinked amino acids (e.g., ?-positions of X.sup.1, X.sup.2, X.sup.3, and X.sup.4). In certain embodiments, the crosslinks are independently attached to the ?-positions of the crosslinked amino acids (e.g., X.sup.1, X.sup.2, X.sup.3, and X.sup.4), and the crosslinked amino acids are independently ?,?-disubstituted amino acids.

[0202] In certain embodiments, each crosslink is independently from about 5 ? to about 25 ? in length, inclusive. In certain embodiments, each crosslink is independently from about 6 ? to about 22 ? in length, inclusive. In certain embodiments, each crosslink is independently from about 7 ? to about 20 ? in length, inclusive. In certain embodiments, each crosslink is independently from about 8 ? to about 18 ? in length, inclusive. In certain embodiments, each crosslink is independently from about 9 ? to about 17 ? in length, inclusive. each crosslink is independently about 10 ? to about 16 ? in length, inclusive. In certain embodiments, each crosslink is independently from about 11 ? to about 15 ? in length, inclusive. In certain embodiments, each crosslink is independently from about 12 ? to about 14 ? in length, inclusive. In certain embodiments, each crosslink is independently about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 ? in length.

[0203] In certain embodiments, the length of each crosslink is approximately equal to the length of 5 to 25 carbon-carbon bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 5 to 20 carbon-carbon bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 5 to 15 carbon-carbon bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 5 to 13 carbon-carbon bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 6 to 12 carbon-carbon bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 7 to 11 carbon-carbon bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 8 to 10 carbon-carbon bonds, inclusive. In certain embodiments, the length of each crosslink is approximately equal to the length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon-carbon bonds, inclusive.

[0204] In certain embodiments, at least one crosslink spans at least one turn of an ?-helix of the peptide. In certain embodiments, each crosslink spans at least one turn of an ?-helix of the peptide. In certain embodiments, at least one crosslink spans one turn of an ?-helix of the peptide. In certain embodiments, each crosslink spans one turn of an ?-helix of the peptide.

[0205] In certain embodiments, each pair of crosslinked amino acids (e.g., X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4) are independently connected by a crosslink to form the following formula:

##STR00022##

wherein ? denotes the ?-carbons of the crosslinked amino acids; L.sup.1 is a crosslink; and each instance of R.sup.1 is independently hydrogen or optionally substituted C.sub.1-6 alkyl.

[0206] In certain embodiments, each crosslink (e.g., L.sup.1) is independently optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted acylene, or any combination thereof.

[0207] In certain embodiments, each crosslink (e.g., L.sup.1) is independently a hydrocarbon crosslink. Hydrocarbon crosslink for the purposes of this disclosure is a crosslink consisting of optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, and combinations thereof.

[0208] In certain embodiments, each crosslink (e.g., L.sup.1) is independently optionally substituted alkenylene (e.g., unsubstituted alkenylene). In certain embodiments, each crosslink is independently of the following formula:

##STR00023##

wherein each n is independently an integer from 1-10, inclusive. In certain embodiments, the sum of two n on the same crosslink is 6.

[0209] In certain embodiments, the crosslinked amino acids (e.g., X.sup.1, X.sup.2, X.sup.3, and X.sup.4) are independently ?,?-disubstituted amino acids. For instance, in certain embodiments, each pair of crosslinked amino acids (e.g., X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4) are independently connected by a crosslink to form the following formula:

##STR00024##

wherein ? denotes the ?-carbons of the crosslinked amino acids; and wherein each instance of R.sup.1 is independently optionally substituted C.sub.1-6 alkyl. In certain embodiments, the sum of two n on the same crosslink is 6.

[0210] For example, in certain embodiments, a crosslink (e.g., L.sup.1) is independently of the formula:

##STR00025##

[0211] For example, in certain embodiments, a pair of crosslinked amino acids (e.g., X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4) are independently connected via a crosslink to form the following formula:

##STR00026##

wherein ? denotes the ?-carbons of the crosslinked amino acids.

[0212] In certain embodiments, X.sup.1 and X.sup.2 are connected to form the formula (alk).

[0213] In certain embodiments, X.sup.3 and X.sup.4 are connected to form the formula (alk).

[0214] In certain embodiments, a crosslink (e.g., L.sup.1) is independently optionally substituted alkylene (e.g., unsubstituted alkylene). In certain embodiments, each crosslink is independently of the following formula:

##STR00027##

wherein m is an integer from 1-20, inclusive. In certain embodiments, m is 6.

[0215] In certain embodiments, a pair of crosslinked amino acids (e.g., X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4) are independently joined by a crosslink to form the following formula:

##STR00028##

wherein ? denotes the ?-carbons of the crosslinked amino acids; and wherein each instance of R.sup.1 is independently optionally substituted C.sub.1-6 alkyl. In certain embodiments, m is 6.

[0216] For example, in certain embodiments, a crosslink (e.g., L.sup.1) is independently of the formula:

##STR00029##

[0217] For example, in certain embodiments, a pair of crosslinked amino acids (e.g., X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4) are connected via a crosslink to form the following formula:

##STR00030##

wherein ? denotes the ?-carbons of the crosslinked amino acids.

[0218] In certain embodiments, a crosslink (e.g., L.sup.1) is independently optionally substituted alkynylene (e.g., unsubstituted alkynylene).

[0219] In certain embodiments, a crosslink (e.g., L.sup.1) is independently a dithio crosslink. For the purposes of this disclosure, a dithio crosslink is a crosslink comprising two thioethers (i.e., two S groups). In certain embodiments, a crosslink is independently a dithio crosslink of the following formula:

##STR00031##

wherein each n is independently an integer from 1-10, inclusive; [0220] L.sup.2 is optionally substituted alkylene,

##STR00032##

optionally substituted arylene, optionally substituted heteroarylene, or -A.sup.1-A.sup.1-; wherein each instance of A.sup.1 is independently optionally substituted arylene or optionally substituted heteroarylene. In certain embodiments, each instance of n is 1.

[0221] In certain embodiments, a crosslink is independently a dithio crosslink of one of the following formulae:

##STR00033##

In certain embodiments, each instance of n is 1.

[0222] For example, in certain embodiments, a crosslink is independently a dithio crosslink of one of the following formulae:

##STR00034##

[0223] In other embodiments, a crosslink is independently a dithio crosslink of the following formula:

##STR00035##

wherein each n is independently an integer from 1-10, inclusive; [0224] L.sup.2 is an optionally substituted aromatic ring (e.g., a polyhalogenated aryl or heteroaryl ring) or -A.sup.1-A.sup.1-; wherein each instance of A.sup.1 is independently an optionally substituted aromatic ring (e.g., a polyhalogenated aryl or heteroaryl ring). In certain embodiments, each instance of n is 1.

[0225] In certain embodiments, a crosslink is independently a dithio crosslink of the following formula:

##STR00036##

wherein each n is independently an integer from 1-10, inclusive. In certain embodiments, each instance of n is 1.

[0226] For example, in certain embodiments, a crosslink is independently of the following formula:

##STR00037##

[0227] In certain embodiments, a crosslink (e.g., L.sup.1) is independently a triazolylene crosslink. For the purpose of this disclosure, a triazolylene crosslink is a crosslink interrupted by at least one triazolylene moiety

##STR00038##

[0228] In certain embodiments, a crosslink is independently a triazolylene crosslink of the following formula:

##STR00039##

wherein each n is independently an integer from 1-10, inclusive. In certain embodiments, the sum of two n on the same crosslink is 5.

[0229] For example, in certain embodiments, a crosslink is independently a triazolylene crosslink of one of the following formulae:

##STR00040##

[0230] The following embodiments for n and R.sup.1 apply to all generic formulae and subgenera provided herein, as well as all stapled and unstapled peptides provided herein.

[0231] In certain embodiments, the sum of two n on the same crosslink is an integer from 3-9, inclusive. In certain embodiments, the sum of two n on the same crosslink is an integer from 4-8, inclusive. In certain embodiments, the sum of two n on the same crosslink is an integer from 5-7, inclusive. In certain embodiments, the sum of two n on the same crosslink is 5. In certain embodiments, the sum of two n on the same crosslink is 6. In certain embodiments, the sum of two n on the same crosslink is 7.

[0232] In certain embodiments, at least one instance of n is 1. In certain embodiments, at least one instance of n is 2. In certain embodiments, at least one instance of n is 3. In certain embodiments, at least one instance of n is 4. In certain embodiments, at least one instance of n is 5. In certain embodiments, at least one instance of n is 6. In certain embodiments, at least one instance of n is 7. In certain embodiments, at least one instance of n is 8. In certain embodiments, at least one instance of n is 9. In certain embodiments, at least one instance of n is 10.

[0233] In certain embodiments, m is an integer from 3-9, inclusive. In certain embodiments, m is an integer from 4-8, inclusive. In certain embodiments, m is an integer from 5-7, inclusive. In certain embodiments, m is 5. In certain embodiments, m is 6. In certain embodiments, m is 7.

[0234] In certain embodiments, at least one instance of R.sup.1 is hydrogen. In certain embodiments, each instance of R.sup.1 is hydrogen. In certain embodiments, at least one instance of R.sup.1 is unsubstituted C.sub.1-6 alkyl. In certain embodiments, at least one instance of R.sup.1 is unsubstituted C.sub.1-3 alkyl. In certain embodiments, at least one instance of R.sup.1 is methyl. In certain embodiments, each instance of R.sup.1 is methyl.

[0235] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Peptide C-Terminus Modifications

[0236] Peptides provided herein can comprise one or more additional modifications anywhere on the peptide (e.g., on an amino acid sidechain, on an ?-carbon of an amino acid, on a peptidic nitrogen, at the C-terminus, N-terminus, etc.). Peptides provided herein can comprise modifications to the C-terminus and/or N-terminus of the polypeptide. In certain embodiments, a polypeptide comprises a modified C-terminus. Examples of C-terminus modifications are described herein.

[0237] In certain embodiments, a peptide provided herein comprises an amidated C-terminus. Traditionally, peptides comprise a carboxyl group (C(?O)OH) at the C-terminus. Peptides provided herein may comprise an amide at the C-terminus (e.g., C(?O)NR.sub.2, wherein the group NR.sub.2 is NH.sub.2, monosubstituted amino, disubstituted amino, or trisubstituted amino), referred to as amidated C-terminus. For example, a peptide with a C-terminus amidated with NH.sub.2 comprises the group C(?O)NH.sub.2 at the C-terminus instead of carboxyl (C(?O)OH). An amidated C-terminus can also be represented by including NR.sub.2 (e.g., NH.sub.2) at the C-terminus end of an amino acid sequence.

[0238] Peptides provided herein may also be amidated at the C-terminus with an amino acid, peptide, or protein. The amino acid, peptide, or protein can be natural or unnatural. In certain embodiments, the peptide comprises a peptide conjugated to the C-terminus. In certain embodiments, the peptide is from 2 to 6 amino acids in length, inclusive, and comprises amino acids selected from G, E, and S. In certain embodiments, the peptide is from 2 to 6 amino acids in length, inclusive, and comprises amino acids selected from G and E. In certain embodiments, the peptide is 2 amino acids in length and comprises amino acids selected from G and E. In certain embodiments, the peptide is 3 amino acids in length and comprises amino acids selected from G and E. In certain embodiments, the peptide is 4 amino acids in length and comprises amino acids selected from G and E.

[0239] Non-limited examples of peptides which can be conjugated to the C-terminus are the following:

TABLE-US-00015 GE, AG, AA, GG, GGE, GGS, GGG, GGK, GGQ, (SEQIDNO:78) GGGE (SEQIDNO:79) GGEE or (SEQIDNO:80) GGSGGS.

[0240] Peptides provided herein may also comprise a small molecule, lipophilic group, or polymer conjugated to the C-terminus of the peptide. In certain embodiments, the peptide comprises a lipophilic group conjugated to the C-terminus of the peptide. In certain embodiments, the lipophilic group is a lipid or fatty acid. In certain embodiments, the lipophilic group is a hydrocarbon chain.

[0241] In certain embodiments, the peptide comprises a polymer conjugated to the C-terminus of the peptide. In certain embodiments, the polymer is a polyether, e.g., polyethylene glycol (PEG). In certain embodiments, the polymer is PEG. In certain embodiments, the C-terminus comprises PEG3. In certain embodiments, the C-terminus comprises (PEG3).sub.2. In certain embodiments, the C-terminus comprises NH(PEG). In certain embodiments, the C-terminus comprises NH(PEG3). In certain embodiments, the C-terminus comprises NH(PEG3).sub.2.

[0242] In certain embodiments, the peptide is amidated at the C-terminus with a group of the following formula: NH-(PEG)-CONH.sub.2, wherein PEG is polyethylene glycol. In certain embodiments, the peptide is amidated at the C-terminus with a group of the following formula: NH(CH.sub.2CH.sub.2O).sub.1-20CH.sub.2CH.sub.2CONH.sub.2. In certain embodiments, the polymer is amidated at the C-terminus with a group of one of the following formulae: NHCH.sub.2CH.sub.2OCH.sub.2CH.sub.2CONH.sub.2, NH(CH.sub.2CH.sub.2O).sub.2CH.sub.2CH.sub.2CONH.sub.2, NH(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2CONH.sub.2, NH(CH.sub.2CH.sub.2O).sub.4CH.sub.2CH.sub.2CONH.sub.2, or NH(CH.sub.2CH.sub.2O).sub.5CH.sub.2CH.sub.2CONH.sub.2.

[0243] In certain embodiments, the peptide comprises a small molecule conjugated to the C-terminus of the peptide. In certain embodiments, the small molecule is an antimicrobial agent such as a small-molecule antibiotic. In certain embodiments, the small molecule is cyclic antimicrobial peptide conjugated to the C-terminus. Polymyxins are a non-limiting example of cyclic antimicrobial peptides.

[0244] In certain embodiments, a stapled peptide or pharmaceutically acceptable salt thereof comprises, or is one of, the following amino acid sequences with corresponding C-terminus modifications:

TABLE-US-00016 SEQ ID NO: C-terminus Modification 6 NH(CH.sub.2CH.sub.2O)CH.sub.2CH.sub.2CONH.sub.2 6 NH(CH.sub.2CH.sub.2O).sub.2CH.sub.2CH.sub.2CONH.sub.2 6 NH(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2CONH.sub.2 6 NH(CH.sub.2CH.sub.2O).sub.4CH.sub.2CH.sub.2CONH2 6 NH(CH.sub.2CH.sub.2O).sub.5CH.sub.2CH.sub.2CONH.sub.2 6 (PEG3).sub.2 68 PEG3 62 PEG3 87 PEG3

Unstapled Peptides

[0245] Also provided herein are unstapled (i.e., uncrosslinked) peptides which can serve as synthetic precursors to the stapled (i.e., crosslinked) peptides provided herein. Such unstapled peptides may be referred to an unstapled precursor peptides, unstapled precursors, and the like. Unstapled precursor peptides comprise pairs of amino acids which comprise reactive moieties capable of reacting to form crosslinks.

Amino Acid Sequences and Substitutions

[0246] Provided herein are unstapled (i.e., uncrosslinked) peptides comprising the amino acid sequence:

TABLE-US-00017 (SEQIDNO:1) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4,
and pharmaceutically acceptable salts thereof, wherein: [0247] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids (i.e., amino acid comprising reactive moieties); [0248] X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and [0249] the amino acid sequence includes 1 to 9 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid substitution is at H7, S8, G18, or E19; and provided that the amino acid substitution at G18 is not G18H.

[0250] In certain embodiments, the unstapled peptide or pharmaceutically acceptable salt thereof comprises the amino acid sequence:

TABLE-US-00018 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK,
wherein: [0251] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids (i.e., amino acid comprising reactive moieties); [0252] X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and [0253] the amino acid sequence includes 1 to 9 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid substitution is at H7, S8, G18, or E19; and provided that the amino acid substitution at G18 is not G18H.

[0254] In certain embodiments, an unstapled peptide provided herein is not of, or does not comprise, one of the following amino acid sequences:

TABLE-US-00019 (SEQIDNO:57) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VHEX.sup.4BES (SEQIDNO:58) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VHEX.sup.4.

[0255] As the unstapled peptides provided herein can serve as synthetic precursors to the stapled peptides provided herein, the various numbers, positions, and types of amino acid substitutions described herein with respect to the stapled peptides (e.g., supra) also apply to the unstapled peptides provided herein. Additionally, the possible C-terminus modifications described herein with respect to the stapled peptides (e.g., supra) apply to the unstapled peptides provided herein.

[0256] In certain embodiments, an unstapled peptide or pharmaceutically acceptable salt thereof provided herein comprises one of SEQ ID NOs: 4-39 or 59-77. In certain embodiments, an unstapled peptide provided herein is of one of SEQ ID NOs: 4-39 or 59-77, or a pharmaceutically acceptable salt thereof. In certain embodiments, an unstapled peptide provided herein is of one of SEQ ID NOs: 4-39 or 59-77, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with NH.sub.2.

[0257] In certain embodiments, an unstapled peptide or pharmaceutically acceptable salt thereof provided herein comprises one of SEQ ID NOs: 81-100. In certain embodiments, an unstapled peptide provided herein is of one of SEQ ID NOs: 81-100, or a pharmaceutically acceptable salt thereof. In certain embodiments, an unstapled peptide provided herein is of one of SEQ ID NOs: 81-100, or a pharmaceutically acceptable salt thereof, wherein the C-terminus of the peptide is amidated with NH.sub.2.

[0258] Also provided herein are unstapled (i.e., uncrosslinked) peptides comprising the amino acid sequence:

TABLE-US-00020 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK,
and pharmaceutically acceptable salts thereof, wherein: [0259] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids (i.e., amino acid comprising reactive moieties); [0260] X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and [0261] the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid is substituted by W.

[0262] The various numbers, positions, and types of amino acid substitutions described herein with respect to the stapled peptides (e.g., supra) also apply to the unstapled peptides provided herein. The possible C-terminus modifications described herein with respect to the stapled peptides (e.g., supra) also apply to the unstapled peptides provided herein.

[0263] In certain embodiments, an unstapled peptide or pharmaceutically acceptable salt thereof provided herein comprises one of SEQ ID NOs: 40-53. In certain embodiments, an unstapled peptide provided herein is of one of SEQ ID NOs: 40-53, or a pharmaceutically acceptable salt thereof. In certain embodiments, an unstapled peptide provided herein is of one of SEQ ID NOs: 40-53, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with NH.sub.2.

[0264] Also provided herein are unstapled (i.e., uncrosslinked) peptides comprising the amino acid sequence:

TABLE-US-00021 (SEQIDNO:3) GX.sup.1GKFX.sup.2KKKKKX.sup.3VGEX.sup.4AKK,
or a pharmaceutically acceptable salt thereof, wherein: [0265] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; [0266] X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and [0267] the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4.

[0268] As described herein, the various numbers, positions, and types of amino acid substitutions described herein with respect to the stapled peptides (e.g., supra) also apply to the unstapled peptides provided herein. The possible C-terminus modifications described herein with respect to the stapled peptides (e.g., supra) also apply to the unstapled peptides provided herein.

[0269] In certain embodiments, an unstapled peptide or pharmaceutically acceptable salt thereof provided herein comprises SEQ ID NO: 3. In certain embodiments, an unstapled peptide provided herein is of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof. In certain embodiments, an unstapled peptide provided herein is of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with NH.sub.2.

[0270] Also provided herein are unstapled (i.e., uncrosslinked) peptides comprising the amino acid sequence:

TABLE-US-00022 (SEQIDNO:101) ZX.sup.1GKFX.sup.2HSAKKFGKAFV,
or a pharmaceutically acceptable salt thereof, wherein: [0271] X.sup.1 and X.sup.2 are independently amino acids; [0272] X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other; and [0273] the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1 and X.sup.2.

[0274] As described herein, the various numbers, positions, and types of amino acid substitutions described herein with respect to the stapled peptides (e.g., supra) also apply to the unstapled peptides provided herein. The possible C-terminus modifications described herein with respect to the stapled peptides (e.g., supra) also apply to the unstapled peptides provided herein.

[0275] In certain embodiments, an unstapled peptide or pharmaceutically acceptable salt thereof provided herein comprises any one of SEQ ID NOs: 101-105. In certain embodiments, an unstapled peptide provided herein is of any one of SEQ ID NOs: 101-105, or a pharmaceutically acceptable salt thereof. In certain embodiments, an unstapled peptide provided herein is of any one of SEQ ID NOs: 101-105, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with NH.sub.2.

[0276] In addition to pharmaceutically acceptable salts of the stapled peptides provided herein, also provided herein are stereoisomers, tautomers, isotopically labeled derivatives, solvates, hydrates, polymorphs, co-crystals, and prodrugs of the stapled peptides provided herein.

Reactive Moieties

[0277] As described herein, the unstapled (i.e., uncrosslinked) peptides comprise pairs of amino acids (e.g., X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4) which comprise reactive moieties capable of forming crosslinks. In preferred embodiments, the pairs of amino acids (e.g., X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4) comprise ?-sidechains comprising the reactive moieties.

[0278] For the purposes of this disclosure, reactive moieties are any chemical moieties capable of reacting with another chemical moiety to form a covalent bond or covalent bonds. Non-limiting examples of reactive moieties include alkenes, alkynes, alcohols, amines, thiols, azides, esters, amides, halogens, and the like. In certain embodiments, two reactive moieties are capable of reacting directly with each other to form a crosslink (e.g., alkenes undergoing ring-closing metathesis (RCM), or an alkyne and an azide undergoing 1,3-dipolar cycloaddition to form a triazole). In other embodiments, two reactive moieties react with an intervening crosslinking reagent to form a crosslink (e.g., two cysteine residues reacting with a dihalide (e.g., dibromo xylene)). In certain embodiments, the reactive moieties are click chemistry moieties. Click chemistry moieties are any moieties that can be used in click chemistry reactions (infra).

[0279] In certain embodiments, the reactive moieties are independently selected from alkenes and alkynes (e.g., capable of undergoing RCM reactions to form a hydrocarbon crosslink). In certain embodiments, the reactive moieties are terminal alkenes. In certain embodiments, the reactive moieties are terminal alkynes.

[0280] In certain embodiments, X.sup.1, X.sup.2, X.sup.3, and X.sup.4 independently comprise ?-sidechains of the following formula:

##STR00041##

wherein each n is independently an integer from 1-10, inclusive. In certain embodiments, at least one instance of n is 3. In certain embodiments, the sum of two n between crosslinking amino acids is 6.

[0281] In certain embodiments, X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently ?,?-disubstituted amino acids. In certain embodiments, X.sup.1, X.sup.2, X.sup.3, and X.sup.4 each independently comprise the formula:

##STR00042##

wherein ? denotes the ?-carbon of the amino acids; and each instance of R.sup.1 is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.1 is methyl.

[0282] In certain embodiments, at least one instance of X.sup.1, X.sup.2, X.sup.3, and X.sup.4 is an amino acid of the following formula:

##STR00043##

wherein each n is independently an integer from 1-10, inclusive.

[0283] In certain embodiments, at least one instance of X.sup.1, X.sup.2, X.sup.3, and X.sup.4 is the amino acid S.sup.5:

##STR00044##

[0284] In certain embodiments, X.sup.1 is S.sup.5. In certain embodiments, X.sup.2 is S.sup.5. In certain embodiments, X.sup.3 is S.sup.5. In certain embodiments, X.sup.4 is S.sup.5. In certain embodiments, X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are S5.

[0285] In certain embodiments, X.sup.1 is R.sup.8. In certain embodiments, X.sup.2 is R.sup.8. In certain embodiments, X.sup.3 is R.sup.8. In certain embodiments, X.sup.4 is R.sup.8. In certain embodiments, X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are R.sup.8.

[0286] In certain embodiments, the reactive moieties are thiols (e.g., capable of reacting with a crosslinking reagent such as a dihalide (e.g., dibromo xylene) to form a dithio crosslink). In certain embodiments, X.sup.1, X.sup.2, X.sup.3, and X.sup.4 independently comprise ?-sidechains of the following formula:

##STR00045##

wherein each n is independently an integer from 1-10, inclusive. In certain embodiments, at least one instance of n is 1. In certain embodiments, each instance of n is 1.

[0287] In certain embodiments, each of X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are cysteine (C). In certain embodiments, X.sup.1 and X.sup.2 are C. In certain embodiments, X.sup.3 and X.sup.4 are C.

[0288] In certain embodiments, the reactive moieties are azides and alkynes (e.g., capable of reacting via 1,3-cycloaddition to form a triazolylene crosslink). In certain embodiments, one of X.sup.1 and X.sup.2 comprises an alkyne reactive moiety, and the other comprises an azide reactive moiety. In certain embodiments, one of X.sup.3 and X.sup.4 comprises an alkyne reactive moiety, and the other comprises an azide reactive moiety.

[0289] In certain embodiments, one of X.sup.1 and X.sup.2 comprises an ?-sidechain of the following formula:

##STR00046##

and the other comprises an ?-sidechain of the following formula:

##STR00047##

In certain embodiments, one of X.sup.3 and X.sup.4 comprises an ?-sidechain of the following formula:

##STR00048##

and the other comprises an ?-sidechain of the following formula:

##STR00049##

wherein each n is independently an integer from 1-10, inclusive. In certain embodiments, one of X.sup.1 and X.sup.2 comprises an ?-sidechain of the following formula:

##STR00050##

N.sub.3, and the other comprises an ?-sidechain of the following formula:

##STR00051##

In certain embodiments, one of X.sup.3 and X.sup.4 comprises an ?-sidechain of the following formula:

##STR00052##

and the other comprises an ?-sidechain of the following formula:

##STR00053##

[0290] In certain embodiments, one of X.sup.1 and X.sup.2 is J, and the other is Azi. In certain embodiments, one of X.sup.3 and X.sup.4 is J, and the other is Azi.

Methods of Preparing Stapled Peptides

[0291] Also provided herein are methods of preparing crosslinked (i.e., stapled) peptides described herein comprising reacting uncrosslinked (i.e., unstapled) peptides under conditions sufficient to form the crosslinks.

[0292] For example, in the case of reactions between alkene or alkyne reactive moieties to form hydrocarbon crosslinks, ring-closing metathesis (RCM) reactions may be used. In certain embodiments, the RCM reaction involves reacting the peptide in the presence of a ruthenium complex. In certain embodiments, the ruthenium complex is, e.g., a Grubbs, Grubbs II, Hoveyda-Grubbs I, or Hoveyda-Grubbs II catalyst.

##STR00054##

[0293] Other examples of metathesis (e.g., RCM) catalysts, reagents, and reaction conditions useful in the present methods can be found in, e.g., Schrodi, Y.; Pederson, R. L. Aldrichimica Acta 2007, 40, 45; Adv. Synth. Catal. 2007, 349, 1-268; Grubbs, R. H. Tetrahedron 2004, 60, 7117; Handbook of Metathesis; Grubbs, R. H., Ed.; Wiley-VCH: Weinheim, 2003; Vols. 1-3; Trnka, T. M.; Grubbs, R. H. Acc. Chem. Res. 2001, 34, 18; Furstner, A. Angew. Chem., Int. Ed. 2000, 39, 3012; Schuster, M.; Blechert, S. Angew. Chem., Int. Ed. 1997, 36, 2036; Ritter, T. et al. Organometallics 2006, 25, 5740; Chatterjee, A. K. et al. J. Am. Chem. Soc. 2000, 122, 3783; Chatterjee, A. K.; Grubbs, R. H. Org. Lett. 1999, 1, 1751; Murelli, R. P.; Snapper, M. L. Org. Lett. 2007, 9, 1749; Stewart, I. C. et al. Org. Lett. 2007, 9, 1589; Ung, T. et al. Organometallics 2004, 23, 5399; Benitez, D.; Goddard, W. A., III. J. Am. Chem. Soc. 2005, 127, 12218; Love, J. A. et al. Angew. Chem., Int. Ed. 2002, 41, 4035; Sanford, M. S. et al. Organometallics 2001, 20, 5314; Choi, T.-L.; Grubbs, R. H. Angew. Chem. 2003, 115, 1785; Ritter, T. et al. Organometallics 2006, 25, 5740, the entire contents of each of which is incorporated herein by reference.

[0294] As another example, in the case of reactions between thiol reactive moieties to form dithio crosslinks, thiol-stapling reactions may be used. Thiol-stapling involves reacting two thiol moieties in the presence of a crosslinking reagent (e.g., a dihalide) to form the crosslink. Examples of thiol-stapling reactions can be found in, e.g., US Patent Application Publication No. US 2019/0382443 A1, published Dec. 19, 2019; U.S. Pat. No. 9,670,484 B2, issued Jun. 6, 2017; U.S. Pat. No. 9,644,201 B2, issued May 9, 2017; US Patent Application Publication No. 2017/0067045 A1, published Mar. 9, 2017; Peraro, L. et al. Methods in Enzymology vol. 580 (2016): 303-32; and Kale, S. S. et al. Nature Chem 10, 715-723 (2018), the entire contents of each of which is incorporated herein by reference.

[0295] For example, provided below in Table 3 are examples of crosslinking reagents and the dithio crosslinks they are capable of forming via thiol-stapling reactions.

TABLE-US-00023 TABLE 3 Thiol-Stapling Crosslinking Reagents Crosslinking Reagent Dithio Crosslink [00055] (mxy) [00056] (pxy) [00057] (but) [00058] (bbn) [00059] (bbf) [00060] (bbp) [00061] (pfb)

[0296] As another example, in the case of reactions between an azide and alkyne reactive moieties to form triazolylene crosslinks, alkyne-azide 1,3-cycloadditions may be used (e.g., the Huisgen alkyne-azide cycloaddition). In certain embodiments, the alkyne-azide cycloaddition is copper-catalyzed. In certain embodiments, the alkyne-azide cycloaddition is strain-promoted. Examples of alkyne-azide reactions can be found in, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Kolb and Sharpless, Drug Discov Today (2003) 24: 1128-1137; and Evans, Australian Journal of Chemistry (2007) 60: 384-395.

[0297] Other click chemistry reactions may be used to form the crosslinks of the peptides described herein. Click chemistry is a chemical approach introduced by Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining small units together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395. Exemplary coupling reactions (some of which may be classified as click chemistry) include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-yne addition; imine formation; Michael additions (e.g., maleimide addition); and Diels-Alder reactions (e.g., tetrazine [4+2] cycloaddition).

Pharmaceutical Compositions, Kits, and Administration

[0298] The present disclosure provides pharmaceutical compositions comprising a peptide disclosed herein, or a pharmaceutically acceptable salt thereof. The pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers/excipients. In certain embodiments, a peptide described herein is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount (e.g., for treating a bacterial infection in a subject). In certain embodiments, the effective amount is a prophylactically effective amount (e.g., for preventing a bacterial infection in a subject). In certain embodiments, the effective amount is an amount effective for killing and/or inhibiting the growth of bacteria in a subject or biological sample.

[0299] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the peptide described herein (i.e., the active ingredient) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

[0300] Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A unit dose is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0301] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

[0302] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

[0303] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

[0304] Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0305] Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween? 20), polyoxyethylene sorbitan (Tween? 60), polyoxyethylene sorbitan monooleate (Tween? 80), sorbitan monopalmitate (Span? 40), sorbitan monostearate (Span? 60), sorbitan tristearate (Span? 65), glyceryl monooleate, sorbitan monooleate (Span? 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj? 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol?), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor?), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij? 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic? F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.

[0306] Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum?), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

[0307] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0308] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0309] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0310] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0311] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0312] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0313] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant? Plus, Phenonip?, methylparaben, Germall? 115, Germaben? II, Neolone?, Kathon?, and Euxyl?.

[0314] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0315] Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0316] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof. In certain embodiments, the formulation comprises a polymer excipient. In certain embodiments, the formulation comprises a polyether. In certain embodiments, the formulation comprises polyethylene glycol (PEG) (e.g., PEG200, PEG300, PEG400, and the like).

[0317] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor?, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0318] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0319] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0320] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

[0321] Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.

[0322] Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

[0323] The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.

[0324] Dosage forms for topical and/or transdermal administration of a peptide described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

[0325] Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder/particle delivery devices which use compressed gas to accelerate the peptide in powder form through the outer layers of the skin to the dermis are suitable.

[0326] Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

[0327] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

[0328] A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient. Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device.

[0329] Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein.

[0330] A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient.

[0331] A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.

[0332] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

[0333] Peptides provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

[0334] The peptides and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, topical administration, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).

[0335] The exact amount of a peptide required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular peptide, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a peptide described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell.

[0336] In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 ?g and 1 ?g, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a peptide described herein.

[0337] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0338] A peptide or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents). The peptides or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in reducing the risk to develop a disease in a subject in need thereof), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a peptide described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the peptide and the additional pharmaceutical agent, but not both. In some embodiments, the additional pharmaceutical agent achieves a desired effect for the same disorder. In some embodiments, the additional pharmaceutical agent achieves different effects.

[0339] The peptide or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic peptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.

[0340] The additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-angiogenesis agents, steroidal or non-steroidal anti-inflammatory agents (NSAIDs), immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, anesthetics, anti-coagulants, inhibitors of an enzyme, steroidal agents, steroidal or antihistamine, antigens, vaccines, antibodies, decongestant, sedatives, opioids, analgesics, anti-pyretics, hormones, and prostaglandins.

[0341] In certain embodiments, the additional pharmaceutical agent is an antimicrobial agent. In certain embodiments, the additional pharmaceutical agent is an antibiotic. In certain embodiments, the additional pharmaceutical agent is selected from the group consisting of polymyxins, rifampicin, ofloxacin, vancomycin, and meropenem.

[0342] Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the peptide or composition described herein in a single dose or composition or administered separately in different doses or compositions. The particular combination to employ in a regimen will take into account compatibility of the peptide described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

[0343] Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or peptide described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or peptide described herein. In some embodiments, the pharmaceutical composition or peptide described herein provided in the first container and the second container are combined to form one unit dosage form. Thus, in one aspect, provided are kits including a first container comprising a peptide or pharmaceutical composition described herein. In certain embodiments, the kits are useful for treating a disease (e.g., bacterial infection) in a subject in need thereof. In certain embodiments, the kits are useful for preventing a disease (e.g., bacterial infection) in a subject in need thereof.

[0344] In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits provide instructions for treating a disease (e.g., bacterial infection) in a subject in need thereof. In certain embodiments, the kits provide instructions for preventing a disease (e.g., bacterial infection) in a subject in need thereof. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

Lipid Formulations

[0345] Also provided herein are formulations (i.e., pharmaceutical compositions) comprising one or more stapled antimicrobial peptides (StAMPs) and one or more lipids (lipid formulations). The lipid formulation may be a liposomal, micellar, lipid nanoparticle (LNP) formulation, or the like. In certain embodiments, the lipid formulation is a micellar formulation. In certain embodiments, the lipid formulation is a liposomal formulation. In certain embodiments, the lipid formulation is an LNP formulation. In certain embodiments, a StAMP is encapsulated in a micelle, liposome, LNP, or the like.

[0346] In certain embodiments, the lipid formulation comprises a peptide (e.g., StAMP) provided herein. In certain embodiments, the peptide (e.g., StAMP) is a peptide described in, e.g., International PCT Application Publication Nos. WO 2017/004591, published Jan. 5, 2017; and WO 2019/018499, published Jan. 24, 2019, the entire contents of each of which are incorporated herein by reference.

[0347] Lipid formulations provided herein comprise one or more lipids. In certain embodiments, the lipid comprises a phospholipid (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)). In certain embodiments, the lipid comprises polyethylene glycol (PEG). In certain embodiments, the lipid is a PEGylated phospholipid. A PEGylated phospholipid is a lipid comprising a phospholipid conjugated to PEG. For example, in certain embodiments, the PEGylated phospholipid comprises DSPE and PEG (i.e., DSPE-PEG). In certain embodiments, the PEGylated phospholipid is DSPE-MPEG (e.g., DSPE-MPEG(2000)). In certain embodiments, the PEGylated phospholipid is DSPE-PEG Amine (e.g., DSPE-PEG(2000) Amine). Lipids (e.g., phospholipids) include pharmaceutically acceptable salts of the lipids. For example, the structure of DSPE-MPEG is shown below:

##STR00062##

[0348] For example, the structure of DSPE-PEG Amine is shown below:

##STR00063##

[0349] The lipid formulation can comprise any ratio of peptide:lipid. In certain embodiments, the lipid formulation comprises a peptide:lipid ratio from about 1:1 to about 1:25 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide:lipid ratio from about 1:2 to about 1:25 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide:lipid ratio from about 1:2.5 to about 1:20 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide:lipid ratio from about 1:2 to about 1:3 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide:lipid ratio from about 1:4 to about 1:6 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide:lipid ratio from about 1:8 to about 1:12 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide:lipid ratio from about 1:15 to about 1:25 (w/w), inclusive.

[0350] In certain embodiments, the lipid formulation comprises a peptide:lipid ratio of about 1:2.5 (w/w). In certain embodiments, the lipid formulation comprises a peptide:lipid ratio of about 1:5 (w/w). In certain embodiments, the lipid formulation comprises a peptide:lipid ratio of about 1:10 (w/w). In certain embodiments, the lipid formulation comprises a peptide:lipid ratio of about 1:20 (w/w).

[0351] For example, in certain embodiments, the lipid formulation comprises a peptide:DSPE-MPEG ratio from about 1:1 to about 1:25 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide:DSPE-MPEG ratio from about 1:2 to about 1:25 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-MPEG ratio from about 1:2.5 to about 1:20 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-MPEG ratio from about 1:2 to about 1:3 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-MPEG ratio from about 1:4 to about 1:6 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-MPEG ratio from about 1:8 to about 1:12 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-MPEG ratio from about 1:15 to about 1:25 (w/w), inclusive.

[0352] In certain embodiments, the lipid formulation comprises a peptide: DSPE-MPEG ratio of about 1:2.5 (w/w). In certain embodiments, the lipid formulation comprises a peptide: DSPE-MPEG ratio of about 1:5 (w/w). In certain embodiments, the lipid formulation comprises a peptide: DSPE-MPEG ratio of about 1:10 (w/w). In certain embodiments, the lipid formulation comprises a peptide: DSPE-MPEG ratio of about 1:20 (w/w).

[0353] For example, in certain embodiments, the lipid formulation comprises a peptide:DSPE-PEG Amine ratio from about 1:1 to about 1:25 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-PEG Amine ratio from about 1:2 to about 1:25 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-PEG Amine ratio from about 1:2.5 to about 1:20 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-PEG Amine ratio from about 1:2 to about 1:3 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-PEG Amine ratio from about 1:4 to about 1:6 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide: DSPE-PEG Amine ratio from about 1:8 to about 1:12 (w/w), inclusive. In certain embodiments, the lipid formulation comprises a peptide:DSPE-PEG Amine ratio from about 1:15 to about 1:25 (w/w), inclusive.

[0354] In certain embodiments, the lipid formulation comprises a peptide: DSPE-PEG Amine ratio of about 1:2.5 (w/w). In certain embodiments, the lipid formulation comprises a peptide: DSPE-PEG Amine ratio of about 1:5 (w/w). In certain embodiments, the lipid formulation comprises a peptide: DSPE-PEG Amine ratio of about 1:10 (w/w). In certain embodiments, the lipid formulation comprises a peptide:DSPE-PEG Amine ratio of about 1:20 (w/w).

Methods of Treatment and Uses

[0355] Peptides (e.g., stapled peptides, e.g., StAMPs) of the present disclosure have antimicrobial activity and are therefore useful in various methods such as treating infectious diseases (e.g., bacterial infections) in a subject, and killing and/or inhibiting the growth of microbes (e.g., bacteria).

[0356] Provided herein are methods of treating and/or preventing an infectious disease in a subject comprising administering to the subject a peptide (e.g., StAMP) described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the infectious disease is a bacterial infection. In certain embodiments, the infectious disease is a viral infection. In certain embodiments, the infectious disease is a protozoal infection. In certain embodiments, the infectious disease is a fungal infection. In certain embodiments, the infectious disease is a parasitic infection.

[0357] In certain embodiments, the method is for treating an infectious disease. In certain embodiments, the method is for preventing an infectious disease.

[0358] Provided herein are methods of treating and/or preventing a bacterial infection in a subject comprising administering to the subject a peptide (e.g., StAMP) described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the bacterial infection is a Gram-positive bacterial infection, i.e., is caused by Gram-positive bacteria. In certain embodiments, the bacterial infection is a Gram-negative bacterial infection, i.e., is caused by Gram-negative bacteria.

[0359] In certain embodiments, the method is for treating a bacterial infection. In certain embodiments, the method is for preventing a bacterial infection.

[0360] The Gram-negative bacterial infection may be caused by any Gram-negative bacteria described herein. In certain embodiments, the bacterial infectious is caused by Acineobacter, Escherichia, Pseudomonas, Neisseria, Chlamydia, Yersinia, Proteus, Enterobacter, Serratia, Helicobacter, Salmonella, Shigella, Moraxella, Stenotrophomonas, Bdellovibrio, Klebsiella, Legionella, or acetic acid bacteria. In certain embodiments, the bacterial infection is caused by Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Yersinia pestis, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, Shigella, Klebsiella pneumoniae, or Legionella pneumophila bacteria, or a Gram-negative bacteria that contains a MCR-1 gene.

[0361] In certain embodiments, the bacterial infection is caused by an Escherichia, Acinetobacter, Pseudomonas, or Klebsiella species. In certain embodiments, the bacterial infection is caused by Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), or Klebsiella pneumoniae (K. pneumoniae). In certain embodiments, the bacterial infection is caused by a Stenotrophomonas, Burkholderia, or Klebsiella species. In certain embodiments, the bacterial infection is caused by Stenotrophomonas maltophilia (S. maltophilia), Burkholderia cepacia (B. cepacia), or Klebsiella oxytoca (K. oxytoca). In certain embodiments, the bacterial infection is caused by E. coli. In certain embodiments, the bacterial infection is caused by A. baumannii. In certain embodiments, the bacterial infection is caused by P. aeruginosa. In certain embodiments, the bacterial infection is caused by K. pneumoniae. In certain embodiments, the bacterial infection is caused by S. maltophilia. In certain embodiments, the bacterial infection is caused by B. cepacia. In certain embodiments, the bacterial infection is caused by K. oxytoca.

[0362] In certain embodiments, the bacterial infection is caused by an Enterobacter, Pasteurella, Proteus, or Citrobacter species. Examples of species are provided herein.

[0363] In certain embodiments, the bacterial infection is an antibiotic-resistant bacterial infection, i.e., a bacterial infection caused by antibiotic-resistant bacteria. In certain embodiments, the bacterial infection is a Gram-negative, antibiotic-resistant bacterial infection. Therefore, also provided herein are methods for treating an antibiotic-resistant, Gram-negative bacterial infection in a subject comprising administering to the subject a peptide (e.g., StAMP) described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the antibiotic-resistant bacterial infection is caused by a bacteria resistant to one or more antibiotics selected from the group consisting of polymyxin, carbapenem, aminoglycosides, vancomycin, methicillin, clarithromycin, cephalosporins, penicillins, ampicillin, fluoroquinolones, tetracycline, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, tobramycin, and ?-lactams.

[0364] As discussed herein, the present disclosure relates in part to antimicrobial peptides that have reduced toxicity as compared to known or existing antimicrobial agents (e.g., AMPs, e.g., StAMPs). The reduced toxicity can be, but is not limited to, reduced renal toxicity, reduced hepatic toxicity, and/or reduced hemolytic activity.

[0365] In certain embodiments, a peptide (e.g., StAMP) provided herein has reduced renal (i.e., kidney) toxicity as compared to a reference. Renal toxicity is reduced if the peptide kills and/or damages fewer kidney cells as compared to the reference. In certain embodiments, the peptide has reduced renal toxicity as compared to a corresponding peptide comprising SEQ ID NO: 1 or 2. In certain embodiments, the peptide has reduced renal toxicity as compared to pexiganan. In certain embodiments, the peptide has reduced renal toxicity as compared to Magainin. In certain embodiments, the peptide has reduced renal toxicity as compared to Magainin II.

[0366] In some embodiments, a peptide provided herein has renal toxicity that is at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) lower than the renal toxicity of the reference. The renal toxicity of a peptide can be measured in vitro using a renal proximal tubule epithelial cell (RPTEC) renal toxicity assay.

[0367] In certain embodiments a peptide (e.g., StAMP) provided herein has reduced hepatic (i.e., liver) toxicity as compared to a reference. Liver toxicity is reduced if the peptide kills and/or damages fewer liver cells as compared to the reference. In certain embodiments, the peptide has reduced hepatic toxicity as compared to a corresponding peptide comprising SEQ ID NO: 1 or 2. In certain embodiments, the peptide has reduced hepatic toxicity as compared to pexiganan. In certain embodiments, the peptide has reduced hepatic toxicity as compared to Magainin. In certain embodiments, the peptide has reduced hepatic toxicity as compared to Magainin II.

[0368] In some embodiments, a peptide provided herein has hepatic toxicity that is at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) lower than the hepatic toxicity of the reference. The hepatic toxicity of a peptide can be measured in vitro using a human liver cells (e.g., HepG2) toxicity assay.

[0369] In certain embodiments a peptide (e.g., StAMP) provided herein has reduced hemolytic activity as compared to a reference. Hemolytic activity for the purposes of this disclosure refers to the breakdown or lysis of red blood cells. In certain embodiments, the peptide has reduced hemolytic activity as compared to a corresponding peptide comprising SEQ ID NO: 1 or 2. In certain embodiments, the peptide has reduced hemolytic activity as compared to melittin. In certain embodiments, the peptide has reduced hemolytic activity as compared to Magainin. In certain embodiments, the peptide has reduced hemolytic activity as compared to Magainin II.

[0370] In some embodiments, a peptide provided herein has hemolytic activity that is at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) lower than the hemolytic activity of the reference.

[0371] Also provided herein are method of killing and/or inhibiting the growth of bacteria (e.g., Gram-negative and/or Gram-positive bacteria) comprising contacting the bacteria (e.g., in vitro or in vivo) with a peptide (e.g., StAMP) provided herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0372] In certain embodiments, the bacteria are Gram-positive bacteria. In certain embodiments, the bacteria are Gram-negative bacteria. In certain embodiments, the Gram-negative bacteria are Acineobacter, Escherichia, Pseudomonas, Neisseria, Chlamydia, Yersinia, Proteus, Enterobacter, Serratia, Helicobacter, Salmonella, Shigella, Moraxella, Stenotrophomonas, Bdellovibrio, Klebsiella, Legionella, or acetic acid bacteria. In certain embodiments, the bacteria are Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Yersinia pestis, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, Shigella, Klebsiella pneumoniae, or Legionella pneumophila bacteria, or a Gram-negative bacterium that contains a MCR-1 plasmid.

[0373] In certain embodiments, the bacteria are an Escherichia, Acinetobacter, Pseudomonas aeruginosa, or Klebsiella species. In certain embodiments, the bacteria are Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), or Klebsiella pneumoniae (K. pneumoniae). In certain embodiments, the bacteria are a Stenotrophomonas, Burkholderia, or Klebsiella species. In certain embodiments, the bacteria are Stenotrophomonas maltophilia (S. maltophilia), Burkholderia cepacia (B. cepacia), or Klebsiella oxytoca (K. oxytoca). In certain embodiments, the bacteria are E. coli. In certain embodiments, the bacteria are A. baumannii. In certain embodiments, the bacteria are P. aeruginosa. In certain embodiments, the bacteria are K. pneumoniae. In certain embodiments, the bacteria are S. maltophilia. In certain embodiments, the bacteria are B. cepacia. In certain embodiments, the bacteria are K. oxytoca.

[0374] In certain embodiments, the bacteria are an Enterobacter, Pasteurella, Proteus, or Citrobacter species. Examples of species are provided herein.

[0375] In certain embodiments, the bacteria are antibiotic-resistant bacteria. In certain embodiments, the bacteria are Gram-negative, antibiotic-resistant bacteria. Therefore, also provided herein are methods of killing and/or inhibiting the growth (e.g., in vitro or in vivo) of antibiotic-resistant, Gram-negative bacteria with a peptide (e.g., StAMP) described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the bacteria resistant to one or more antibiotics selected from the group consisting of polymyxin, carbapenem, aminoglycosides, vancomycin, methicillin, clarithromycin, cephalosporin, penicillin, ampicillin, fluoroquinolones, tetracycline, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, tobramycin, and ?-lactams.

[0376] In certain embodiments, a peptide (e.g., StAMP) has a minimum inhibitory concentration (MIC) less than about 64 ?g/mL. In certain embodiments, a peptide (e.g., StAMP) has a minimum inhibitory concentration (MIC) less than or equal to about 32 ?g/mL, less than or equal to about 16 ?g/mL, less than or equal to about 14 ?g/mL, less than or equal to about 12 ?g/mL, or less than or equal to about 10 ?g/mL. In certain embodiments, a peptide (e.g., StAMP) has a minimum inhibitory concentration (MIC) less than or equal to about 10 ?g/mL. In certain embodiments, a peptide (e.g., StAMP) has a MIC less than or equal to about 10 ?g/mL, less than or equal to about 9 ?g/mL, less than or equal to about 8 ?g/mL, less than or equal to about 7 ?g/mL, less than or equal to about 6 ?g/mL, less than or equal to about 5 ?g/mL, less than or equal to about 4 ?g/mL, less than or equal to about 3 ?g/mL, less than or equal to about 2 ?g/mL, less than or equal to about 1 ?g/mL. In certain embodiments, a peptide (e.g., StAMP) has a MIC of about 2 ?g/mL. In certain embodiments, the MIC is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 ?g/mL.

[0377] In certain embodiments, a peptide (e.g., StAMP) has a MIC of from about 0.1 to about 64 ?g/mL, from about 0.1 to about 32 ?g/mL, from about 0.1 to about 16 ?g/mL, from about 0.1 to about 14 ?g/mL, from about 0.1 to about 12 ?g/mL, or from about 0.1 to about 10 ?g/mL. In certain embodiments, a peptide (e.g., StAMP) has a MIC of from about 0.1 to about 10 ?g/mL. In certain embodiments, a peptide (e.g., StAMP) has a MIC of from about 0.1 to about 9 ?g/mL, from about 0.1 to about 8 ?g/mL, from about 0.1 to about 7 ?g/mL, from about 0.1 to about 6 ?g/mL, from about 0.1 to about 5 ?g/mL, from about 0.1 to about 4 ?g/mL, from about 0.1 to about 3 ?g/mL, from about 0.1 to about 2 ?g/mL. In certain embodiments, a peptide (e.g., StAMP) has a MIC of from about 0.1 to about 4 ?g/mL. In certain embodiments, the MIC is at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 ?g/mL.

[0378] In certain embodiments, a peptide (e.g., StAMP) described herein selectively kills and/or inhibits the growth of Gram-negative bacteria over Gram-positive bacteria. In certain embodiments, the peptide selectively kills and/or inhibits the growth of Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), and/or Klebsiella pneumoniae (K. pneumoniae), over Staphylococcus aureus (S. aureus).

[0379] In certain embodiments, peptides (e.g., StAMPs) provided herein have the ability to kill and/or inhibit the growth of microbial cells but not mammalian cells. In certain embodiments, peptides (e.g., StAMPs) provided herein have the ability to selectively lyse microbial cells (e.g., bacterial cells) over mammalian cells. In certain embodiments, the peptides (e.g., StAMPs) are selectively cytotoxic to microbial cells (e.g., bacterial cells) over mammalian cells. This selectively can lead to reduced toxicity (e.g., reduced renal toxicity, hepatic toxicity, hemolytic activity) when the peptide is administered to a subject.

[0380] Provided herein are methods of selectively killing and/or inhibiting the growth of microbial cells (e.g., bacterial cells) over mammalian cells comprising contacting the microbial (e.g., bacterial) and mammalian cells with a peptide (e.g., StAMP) provided herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, peptides (e.g., StAMPs) provided herein lyse microbial cells (e.g., bacterial cells) to a greater extent than mammalian cells. Therefore, provided herein are methods of selectively lysing microbial cells (e.g., bacterial cells) over mammalian cells comprising contacting the microbial (e.g., bacterial) and mammalian cells with a peptide (e.g., StAMP) provided herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0381] In certain embodiments, the microbial cells are bacterial cells. In certain embodiments, the microbial cells are Gram-negative bacterial cells. The cells can be of any bacteria (e.g., Gram-negative bacteria) are provided herein. In certain embodiments, the bacterial cells are Escherichia, Acinetobacter, Pseudomonas aeruginosa, or Klebsiella cells. In certain embodiments, the bacterial cells are Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), or Klebsiella pneumoniae (K. pneumoniae) cells. In certain embodiments, the bacterial cells are Stenotrophomonas, Burkholderia, or Klebsiella cells. In certain embodiments, the bacterial cells are Stenotrophomonas maltophilia (S. maltophilia), Burkholderia cepacia (B. cepacia), or Klebsiella oxytoca (K. oxytoca) cells. In certain embodiments, the bacterial cells are E. coli cells. In certain embodiments, the bacterial cells are A. baumannii cells. In certain embodiments, the bacterial cells are P. aeruginosa cells. In certain embodiments, the bacterial cells are K. pneumoniae cells. In certain embodiments, the bacterial cells are S. maltophilia cells. In certain embodiments, the bacterial cells are B. cepacia cells. In certain embodiments, the bacterial cells are K. oxytoca cells.

[0382] In certain embodiments, the bacterial cells are Enterobacter, Pasteurella, Proteus, or Citrobacter cells. Examples of species are provided herein.

[0383] Also provided here are peptides (e.g., StAMPs) described herein, and pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof, for use in any of the methods described herein (e.g., for treating an infectious disease in a subject, treating a bacterial infection in a subject, killing and/or inhibiting the growth of bacteria in a subject, selectively killing and/or inhibiting the growth of bacterial cells over mammalian cells in a subject, etc.)

[0384] In another aspect, also provided herein are uses of peptides (e.g., StAMPs) described herein, and pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof, for the manufacture of medicament (e.g., for treating an infectious disease in a subject, treating a bacterial infection in a subject, killing and/or inhibiting the growth of bacteria in a subject, selectively killing and/or inhibiting the growth of bacterial cells over mammalian cells in a subject, etc.)

[0385] In certain embodiments, in vivo (i.e., in a subject) methods and uses provided herein comprise administering to a subject an effective amount of a peptide (e.g., StAMP) provided herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount.

[0386] In certain embodiments, in vitro methods and uses provided herein can be carried out, e.g., in a cellular assay or biological sample. In certain embodiments, in vitro methods and uses comprise contacting a microbial cell (e.g., bacterial cell) with a peptide provided herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

EXAMPLES

General Methods

[0387] Solid phase peptide synthesis: Fmoc-based solid-phase peptide synthesis was used to synthesize the antimicrobial peptides and their stapled derivatives. To achieve the i+4 staple lengths, ?-methyl, ?-alkenyl amino acids were used flanking three residues. For the stapling reaction, Grubbs 1st generation ruthenium catalyst dissolved in dichloroethane was added to the peptides while still on resin. To ensure maximal conversion, three to five rounds of stapling were performed. Once stapled, the peptides were cleaved off the resin using trifluoroacetic acid, then precipitated using a hexane:ether (1:1) mixture, and afterwards purified using a prep HPLC. Final peptide characterization for purity was assessed using a UHPLC/MS system.

[0388] Antimicrobial Activity Assay: The following microbroth dilution protocol was adapted from CLSI to determine the minimum inhibitory concentration (MIC) of StAMPs. Briefly, Mueller-Hinton broth (MHB) or cation-adjusted Mueller-Hinton broth (MHB II) was prepared and autoclaved. Then, a colony of bacterial cells was picked and grown overnight in broth at 37? C. and then diluted and allowed to grow again for 3-4 hours. Serial dilutions of peptide stocks in water (10 ?l) were prepared using clear round-bottom polypropylene 96-well plates. Then 90 ?l of bacteria in broth was added to give a final inoculum of 5?10.sup.5 CFU/ml. The plates were then covered with porous tape to reduce evaporation and incubated for 20-24 hours at 37? C. The MIC was the minimum peptide concentration at which no visible bacterial growth was observed.

[0389] Cell culture: RPTEC were maintained in MEM media supplemented with growth factors. HEPG2 cell line was maintained in Eagle's Minimum Essential Medium (EMEM) supplemented with fetal bovine serum to a final concentration of 10%.

[0390] 90 minute cytotoxicity assay: Cells were plated in a 96-well format, and after overnight incubation, media was replaced with fresh media. Serial dilutions of StAMPs from a 5 mg/mL water stock, or vehicle, were added to the cells in a final volume of 100 ?l. After incubating at 37? C. for 90 min, 50 ?l of cell culture media was transferred to a clear 96-well plate, incubated with 50 ?l of lactate dehydrogenase (LDH) assay reagent for 10 min, and absorbance measured at 490 nm on a microplate reader.

[0391] 48 hours cytotoxicity assay: Cells were plated in a 96-well format, and after 4 hour incubation to allow attachment, serial dilutions of StAMPs from a 5 mg/mL water stock, or vehicle, were then added to the cells in a final volume of 100 ?l. After incubating at 37? C. for 48 hours, 100 ?l of CellTiter-Glo? reagent was added to the cells, and the plates were incubated 10 minutes at room temperature. Luminescence was then measured on a microplate reader.

Antimicrobial Activity and Toxicity

[0392] As shown in Tables A-D, antimicrobial peptides (e.g., StAMPs) provided herein exhibit broad-spectrum Gram-negative antimicrobial activity and are selective for bacterial cells over renal and hepatic cells. Renal toxicity was measured after 90 minutes of incubation, while liver toxicity was measured after 48 hours of incubation with StAMPs. N/A=not tested. The following apply to all peptides (i.e., StAMPs) listed in Tables A-C: Crosslinks (X.sup.1X.sup.2 and X.sup.3X.sup.4): (alk); N-terminus: NH.sub.2; C-terminus: amidated with NH.sub.2; counterion: Cl.sup.?. The following apply to all peptides (i.e., StAMPs) listed in Table D: Crosslinks (X.sup.1X.sup.2 and X.sup.3X.sup.4): (alk); N-terminus: NH.sub.2; C-terminus: as shown in table; counterion: Cl.sup.?.

TABLE-US-00024 TABLE A Antimicrobial Activity and Renal/Hepatic Toxicity Inhibitory Concentration at 50% Minimum Inhibitory Concentration Cytotoxicity (IC50) [?g/mL] (MIC) [?g/mL] Human Renal Proximal Human Liver SEQ ID E. A. P. K. S. Tubule Epithelial Cells StAMP # NO: coli baumannii aeruginosa pneumoniae aureus Cells (RPTEC) (HepG2) 1 2 2 2 8 32 >64 294 391 2 4 2 2 4 4 64 116 143 3 5 4 4 4 16 >64 >200 >200 4 6 2 3 3 6 >64 105 127 5 7 4 4 4 >64 >64 >200 >200 6 8 4 4 8 >64 >64 >200 >200 7 9 6 4 8 >64 >64 >200 >200 8 10 8 4 8 >64 >64 >200 >200 9 11 8 4 16 >64 >64 >200 >200 10 12 4 2 4 4 32 110 150 11 13 2 2 6 7 >64 203 227 12 14 2 2 4 4 >64 ~200 ~200 13 15 3 2 4 8 >64 >200 >200 14 16 2 2 4 4 >64 214 222 15 17 6 2 8 16 >64 >200 >200 16 18 12 4 16 >64 >64 >200 >200 17 19 3 2 4 32 >64 ~180 >200 18 20 4 2 4 64 >64 ~200 >200 19 21 2 2 4 4 >64 92 95 20 22 2 2 2 2 64 70 105 21 23 2 2 4 4 >64 251 208 22 24 2 3 4 4 >64 202 >200 23 25 8 8 2 32 64 84 39 24 26 4 2 2 32 64 100 44 25 27 4 32 4 16 >64 >800 >200 26 28 4 4 4 32 >64 366 354 27 29 4 4 4 4 >64 100 117 28 30 64 16 64 >64 >64 N/A 201 29 31 2 2 4 4 >64 86 N/A 30 33 2 2 4 4 32 17 N/A 31 34 4 4 8 8 16 9 101 32 35 4 4 4 8 >64 ~200 >200 33 36 2 2 4 4 >64 ~200 >200 34 37 2 2 4 2 >64 96 109 35 38 2 2 2 2 64 ~200 >200 36 39 2 2 4 2 64 96 >200 37 59 2 2 2 4 >64 113 N/A 38 60 2 2 2 4 >64 117 N/A 39 61 2 2 4 8 >64 115 140 40 62 2 2 4 8 >64 200 203 41 63 2 2 4 4 >64 N/A 115 42 64 4 2 8 16 32 N/A 194 43 65 4 2 4 8 32 N/A 127 44 66 2 2 4 6 32 N/A 87 45 67 2 2 4 4 >64 N/A 104 46 68 2 2 4 8 64 N/A 299 47 69 2 2 4 6 >64 N/A N/A 48 70 4 2 8 16 >64 N/A N/A 49 71 4 2 8 8 >64 N/A N/A 50 72 4 2 8 8 >64 N/A N/A 51 73 2 2 4 4 >64 N/A N/A 52 74 2 2 4 8 >64 N/A N/A 53 75 8 4 16 32 >64 N/A 211 54 76 8 4 8 32 >64 N/A 262 55 77 16 8 16 64 >64 N/A 207 76 81 1 16 16 16 64 >200 >200 77 82 4 3 4 16 >64 >200 >200 78 83 16 8 2 16 >64 >200 >200 79 84 1 0.5 1 8 >64 N/A 113 80 85 8 8 4 >64 >64 N/A >200 81 86 8 4 16 64 >64 N/A >200 82 87 4 1 2 64 >64 N/A 190 83 88 64 64 32 >64 >64 N/A >200 84 89 2 8 16 64 >64 N/A >200 85 90 2 0.5 2 16 64 N/A >200 86 91 2 1 1 32 64 N/A >200 87 92 4 1 2 64 64 N/A >200 88 93 1 1 1 32 >64 N/A 168 89 94 2 1 1 32 64 N/A 199 90 95 4 2 2 32 64 N/A >200 91 96 64 4 64 >64 >64 N/A >200 92 97 16 4 16 >64 >64 N/A >200 93 98 2 1 1 32 >64 N/A >200 94 99 8 4 2 >64 64 N/A >200 95 100 4 2 4 64 >64 N/A >200 96 101 8 8 64 64 >64 N/A N/A 97 102 >64 >64 >64 >64 >64 N/A N/A 98 103 N/A N/A N/A N/A N/A N/A >200 99 104 32 16 16 >64 >64 N/A >200 100 105 64 >64 32 >64 >64 N/A >200

TABLE-US-00025 TABLE B Antimicrobial Activity and Renal/Hepatic Toxicity Inhibitory Concentration at 50% Minimum Inhibitory Concentration Cytotoxicity (IC50) [?g/mL] (MIC) [?g/mL] Human Renal Proximal Human Liver SEQ ID E. A. P. K. S. Tubule Epithelial Cells StAMP # NO: coli baumannii aeruginosa pneumoniae aureus Cells (RPTEC) (HepG2) 56 40 8 4 16 >64 >64 78 200 57 41 4 4 8 16 32 35 70 58 42 16 8 32 >64 >64 >200 >200 59 43 4 4 8 64 >64 >200 >200 60 44 4 4 8 32 >64 108 56 61 45 8 4 8 64 >64 150 >200 62 46 16 4 16 8 >64 30 35 63 47 4 4 6 8 16 32 60 64 48 4 4 8 32 16 >200 >200 65 49 4 2 4 4 32 110 150 66 50 4 4 6 4 32 16 38 67 51 4 4 16 8 16 27 52 68 52 4 4 8 16 >64 117 129 69 53 4 4 8 8 32 24 23

TABLE-US-00026 TABLE C Antimicrobial Activity and Renal/Hepatic Toxicity Inhibitory Concentration at 50% Minimum Inhibitory Concentration Cytotoxicity (IC50) [?g/mL] (MIC) [?g/mL] Human Renal Proximal Human Liver SEQ ID E. A. P. K. S. Tubule Epithelial Cells StAMP # NO: coli baumannii aeruginosa pneumoniae aureus Cells (RPTEC) (HepG2) 70 SEQ ID 4 16 4 4 >64 125 102 NO: 3

TABLE-US-00027 TABLE D Antimicrobial Activity and Renal/Hepatic Toxicity Inhibitory Concentration at 50% Cytotoxicity (IC50) [?g/mL] Human Renal Minimum Inhibitory Concentration Proximal Human (MIC) [?g/mL] Tubule Liver SEQ ID E. A. P. K. S. Epithelial Cells StAMP # NO: C-terminus coli baumannii aeruginosa pneumoniae aureus Cells (RPTEC) (HepG2) 71 6 NH(CH.sub.2CH.sub.2O)CH.sub.2CH.sub.2CONH.sub.2 2 2 4 4 >64 200 247 72 6 NH(CH.sub.2CH.sub.2O).sub.2CH.sub.2CH.sub.2CONH.sub.2 2 2 4 4 >64 200 275 73 6 NH(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2CONH.sub.2 2 2 4 4 >64 671 407 74 6 NH(CH.sub.2CH.sub.2O).sub.4CH.sub.2CH.sub.2CONH.sub.2 2 2 4 4 >64 >200 430 75 6 NH(CH.sub.2CH.sub.2O).sub.5CH.sub.2CH.sub.2CONH.sub.2 4 2 4 8 >64 >200 357 96 6 (PEG3).sub.2 8 2 4 16 >64 N/A >200 97 68 PEG3 4 2 2 8 >64 N/A >200 98 62 PEG3 4 2 8 16 >64 N/A >200 99 87 PEG3 8 4 16 64 >64 N/A >200

Lipid Formulations

[0393] StAMP Micelle Formulation: DSPE-MPEG(2000) was suspended in buffered saline at 50-100 mg/mL overnight. StAMP stock solutions at 5-20 mg/mL in buffered saline were prepared and equal volumes of micelle and StAMP stock solution were mixed at the desired StAMP:Lipid ratio. Suspensions were allowed to sit for 5-10 minutes at room temperature before being used in in vitro and in vivo assays.

[0394] Pharmacokinetic profile in mice: StAMP solution or micellar suspension was injected subcutaneously into 8-week-old C57BL/6 mice. At each time point, mice were euthanized, and blood was collected via cardiac puncture. The plasma was then isolated, followed by compound extraction via protein precipitation and the concentration determined using LC/MS/MS. FIG. 1 shows the pharmacokinetic profile of a subcutaneous injection of StAMP 4 (Table A) in mice at 20 mg/kg using various StAMP:lipid ratios. Lipid=DSPE-MPEG(2000).

[0395] Antimicrobial activity and renal toxicity: Table E below shows results of antimicrobial activity and/or renal toxicity assays with formulations comprising various StAMP:lipid (w/w) ratios. In Table E, the StAMP is StAMP 4 (Table A) and Lipid=DSPE-MPEG(2000).

TABLE-US-00028 TABLE E Antimicrobial Activity and/or Renal Toxicity of StAMP-Lipid Formulations Inhibitory Concentration at 50% Cytotoxicity Minimum Inhibitory Concentration (IC50) [?g/mL] (MIC) [?g/mL] Human Renal Proximal SEQ ID E. A. P. K. S. Tubule Epithelial StAMP # NO: coli baumannii aeruginosa pneumoniae aureus Formulation Cells (RPTEC) 4 6 2 3 3 6 >64 No DSPE-MPEG 105 4 6 N/A N/A 4 N/A N/A DSPE-MPEG Micelle 190 (1:2.5 Peptide:Lipid) 4 6 N/A N/A 8 N/A N/A DSPE-MPEG Micelle N/A (1:5 Peptide:Lipid) 4 6 N/A N/A N/A N/A N/A DSPE-MPEG Micelle 827 (1:10 Peptide:Lipid) 4 6 N/A N/A N/A N/A N/A DSPE-MPEG Micelle >2000 (1:20 Peptide:Lipid)

In Vivo Studies

[0396] FIG. 2 shows the efficacy of StAMP 26 (Table A) in a neutropenic mouse pneumonia model of P. aeruginosa at four hours post-treatment. Neutropenia was established via intraperitoneal administration of cyclophosphamide on Day ?4 and Day ?1 at a dose of 150 mg/kg and 100 mg/kg, respectively. On Day 0, animals were inoculated intranasally with P. aeruginosa (PA01) suspended in saline at a concentration of 10.sup.5-6 CFUs/mL. At two hours, post-inoculation, a dose of StAMP 26 (formulated in phosphate-buffered saline containing DSPE-PEG(2000) Amine) or vehicle (phosphate-buffered saline) was administered intraperitoneally and a control group of animals was euthanized and lungs were harvested for colony counting. At six hours post-inoculation, animals were euthanized and lungs were harvested for colony counting.

[0397] FIG. 3 shows the efficacy of StAMP 46 (Table A) in a neutropenic pneumonia mouse model of P. aeruginosa. Neutropenia was established via intraperitoneal administration of cyclophosphamide on Day ?4 and Day ?1 at a dose of 150 mg/kg and 100 mg/kg, respectively. On Day 0, animals were inoculated intranasally with P. aeruginosa (PA01) suspended in saline at a concentration of 10.sup.5-6 CFUs/mL. At two hours, post-inoculation, a dose of StAMP 46 (formulated in phosphate-buffered saline containing DSPE-PEG(2000) Amine) or vehicle (phosphate-buffered saline containing DSPE-PEG(2000) Amine) was administered intraperitoneally and a control group of animals was euthanized and lungs were harvested for colony counting. At six hours post-inoculation, animals were euthanized and lungs were harvested for colony counting.

[0398] FIG. 4 shows the overnight efficacy of StAMP 26 and StAMP 46 in a neutropenic pneumonia mouse model of P. aeruginosa. Neutropenia was established via intraperitoneal administration of cyclophosphamide on Day ?4 and Day ?1 at a dose of 150 mg/kg and 100 mg/kg, respectively. On Day 0, animals were inoculated intranasally with P. aeruginosa (PA01) suspended in saline at a concentration of 10.sup.5-6 CFUs/mL. At two hours, post-inoculation, a dose of StAMP 26 or StAMP 46 (formulated in phosphate-buffered saline containing DSPE-PEG(2000) Amine) or vehicle (phosphate-buffered saline containing DSPE-PEG(2000) Amine) was administered intraperitoneally and a control group of animals was euthanized and lungs were harvested for colony counting. At six hours, post-inoculation, a second dose of StAMP 26 or StAMP 46 (formulated in phosphate-buffered saline containing DSPE-PEG(2000) Amine) or vehicle (phosphate-buffered saline containing DSPE-PEG(2000) Amine) was administered intraperitoneally. At eighteen hours post-inoculation, animals were euthanized and lungs were harvested for colony counting.

Additional Embodiments

[0399] Additional embodiments of the disclosure are provided according to the following numbered paragraphs:

1. A peptide comprising the amino acid sequence:

TABLE-US-00029 (SEQIDNO:1) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4,
or a pharmaceutically acceptable salt thereof, wherein: [0400] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; [0401] X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and [0402] the amino acid sequence includes 1 to 9 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid substitution is at H7, S8, G18, or E19; and provided that the amino acid substitution at G18 is not G18H.
2. A peptide comprising the amino acid sequence:

TABLE-US-00030 (SEQIDNO:1) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4,
or a pharmaceutically acceptable salt thereof, wherein: [0403] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; [0404] X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and [0405] the amino acid sequence includes 1 to 9 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid substitution is at H7, S8, G18, or E19; and provided that the amino acid substitution at G18 is not G18H.
3. The peptide of paragraph 1 or 2, comprising the amino acid sequence:

TABLE-US-00031 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK,
or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive.
4. The peptide of any one of paragraphs 1-3, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 to 7 amino acid substitutions, inclusive.
5. The peptide of any one of paragraphs 1-4, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 to 5 amino acid substitutions, inclusive.
6. The peptide of any one of paragraphs 1-5, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 or 2 amino acid substitutions.
7. The peptide of any one of paragraphs 1-5, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises 1 to 5 amino acid substitutions, inclusive, independently at K4, H7, S8, G18, or E19.
8. The peptide of any one of paragraphs 1-7, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises 1 or 2 amino acid substitutions independently at K4, H7, S8, G18, or E19.
9. The peptide of any one of paragraphs 1-8, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at H7.
10. The peptide of any one of paragraphs 1-9, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at S8.
11. The peptide of any one of paragraphs 1-10, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises amino acid substitutions at both H7 and S8.
12. The peptide of any one of paragraphs 1-11, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at H7 is selected from H7K, H7W, H7Dab, H7Orn, H7Dap, H7R, and H7hArg.
13. The peptide of paragraph 12, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at H7 is H7K.
14. The peptide of any one of paragraphs 1-13, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at S8 is selected from S8K, S8W, S8N, S8Q, S8T, S8Y, S8Dab, S8Orn, S8Dap, S8R, and S8hArg.
15. The peptide of paragraph 14, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at S8 is S8K.
16. The peptide of any one of paragraphs 1-15, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at G18.
17. The peptide of paragraph 16, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at G18 is selected from G18W, G18V, G18L, G18Y, G18F, G18T, G18F.sup.1, G18F.sup.2, G18F.sup.4, G18F.sup.5, G18F.sup.3, and G18S.
18. The peptide of paragraph 17, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at G18 is G18W.
19. The peptide of any one of paragraphs 1-18, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at E19.
20. The peptide of paragraph 19, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at E19 is selected from E19W, E19D, E19Q, and E19N.
21. The peptide of any one of paragraphs 1-20, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises an amino acid substitution at K4.
22. The peptide of paragraph 21, or a pharmaceutically acceptable salt thereof, wherein the amino acid substitution at K4 is selected from K4S, K4Dab, K4Orn, K4Dap, K4R, and K4hArg.
23. The peptide of any one of paragraphs 10-22, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence comprises H7K and S8K amino acid substitutions.
24. The peptide of any one of paragraphs 1-23, or a pharmaceutically acceptable salt thereof, comprising one or more amino acid substitution selected from G13S, A15S, G18S, and A21S.
25. A peptide comprising the amino acid sequence:

TABLE-US-00032 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK,
or a pharmaceutically acceptable salt thereof, wherein: [0406] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; [0407] X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and [0408] the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid is substituted by W.
26. A peptide of comprising the amino acid sequence:

TABLE-US-00033 (SEQIDNO:2) GX.sup.1GKFX.sup.2HSKKKFGKAX.sup.3VGEX.sup.4AKK,
or a pharmaceutically acceptable salt thereof, wherein: [0409] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; [0410] X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and [0411] the amino acid sequence includes 1 to 11 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4; provided that at least one amino acid is substituted by W.
27. The peptide of paragraph 25 or 26, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 to 5 amino acid substitutions, inclusive.
28. The peptide of any one of paragraphs 25-27, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 or 2 amino acid substitutions.
29. The peptide of any one of paragraphs 25-28, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence includes 1 or 2 amino acids substituted by W.
30. A peptide comprising the amino acid sequence:

TABLE-US-00034 (SEQIDNO:3) GX.sup.1GKFX.sup.2KKKKKX.sup.3VGEX.sup.4AKK,
or a pharmaceutically acceptable salt thereof, wherein: [0412] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; [0413] X.sup.1 and X.sup.2 are connected via a crosslink, and X.sup.3 and X.sup.4 are connected via a crosslink; and [0414] the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4.
31. A peptide comprising the amino acid sequence:

TABLE-US-00035 (SEQIDNO:3) GX.sup.1GKFX.sup.2KKKKKX.sup.3VGEX.sup.4AKK,
or a pharmaceutically acceptable salt thereof, wherein: [0415] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently amino acids; [0416] X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other, and X.sup.3 and X.sup.4 each independently comprise a reactive moiety capable of forming a crosslink with the other; and [0417] the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1, X.sup.2, X.sup.3, and X.sup.4.
32. A peptide comprising the amino acid sequence:

TABLE-US-00036 (SEQIDNO:101) ZX.sup.1GKFX.sup.2HSAKKFGKAFV,
or a pharmaceutically acceptable salt thereof, wherein: [0418] X.sup.1 and X.sup.2 are independently amino acids; [0419] X.sup.1 and X.sup.2 are connected via a crosslink; and [0420] the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1 and X.sup.2.
33. A peptides comprising the amino acid sequence:

TABLE-US-00037 (SEQIDNO:101) ZX.sup.1GKFX.sup.2HSAKKFGKAFV,
or a pharmaceutically acceptable salt thereof, wherein: [0421] X.sup.1 and X.sup.2 are independently amino acids; [0422] X.sup.1 and X.sup.2 each independently comprise a reactive moiety capable of forming a crosslink with the other; and [0423] the amino acid sequence optionally includes 1 to 5 amino acid substitutions, inclusive, at positions other than X.sup.1 and X.sup.2.
34. The peptide of any one of paragraphs 30-33, wherein the amino acid sequence optionally includes 1 or 2 amino acid substitutions.
35. The peptide of any one of paragraphs 1-34, or a pharmaceutically acceptable salt thereof, wherein one or more instances of K are independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg.
36. The peptide of paragraph 35, or a pharmaceutically acceptable salt thereof, wherein each instance of K is independently substituted by an amino acid selected from Orn, Dab, Dap, R, and hArg.
37. The peptide of any one of paragraphs 1-36, wherein one or more instances of F are independently substituted by an amino acid selected from F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3.
38. The peptide of paragraph 37, or a pharmaceutically acceptable salt thereof, wherein each instance of F is independently substituted by an amino acid selected from F.sup.1, F.sup.5, F.sup.4, F.sup.2, and F.sup.3.
39. The peptide of any one of paragraphs 1-38, or a pharmaceutically acceptable salt thereof, comprising a small molecule, lipophilic group, or polymer conjugated to the C-terminus of the peptide.
40. The peptide of paragraph 39, or a pharmaceutically acceptable salt thereof, wherein the lipophilic group is a lipid or fatty acid.
41. The peptide of paragraph 39, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises PEG conjugated to the C-terminus.
42. The peptide of paragraph 41, or a pharmaceutically acceptable salt thereof, wherein the C-terminus comprises PEG3 or (PEG3).sub.2.
43. The peptide of any one of paragraphs 1-42, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated.
44. The peptide of paragraph 43, wherein the C-terminus is amidated with NH.sub.2.
45. The peptide of paragraph 43, or a pharmaceutically acceptable salt thereof, wherein the peptide is amidated at the C-terminus with a group of the formula: [0424] NH(CH.sub.2CH.sub.2O).sub.1-20CH.sub.2CH.sub.2CONH.sub.2.
46. The peptide of paragraph 45, or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated with a selected from: NHCH.sub.2CH.sub.2OCH.sub.2CH.sub.2CONH.sub.2, NH(CH.sub.2CH.sub.2O).sub.2CH.sub.2CH.sub.2CONH.sub.2, NH(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.2CONH.sub.2, NH(CH.sub.2CH.sub.2O).sub.4 CH.sub.2CH.sub.2CONH.sub.2, and NH(CH.sub.2CH.sub.2O).sub.5CH.sub.2CH.sub.2CONH.sub.2.
47. The peptide of any one of paragraphs 1-38, or a pharmaceutically acceptable salt thereof, comprising an amino acid or peptide conjugated to the C-terminus of the peptide.
48. The peptide of paragraph 47, or a pharmaceutically acceptable salt thereof, comprising one of the following amino acid sequences conjugated to the C-terminus of the peptide:

TABLE-US-00038 GE, AG, AA, GG, GGE, GGS, GGG, GGK, GGQ, (SEQIDNO:78) GGGE, (SEQIDNO:79) GGEE, or (SEQIDNO:80) GGSGGS.
49. The peptide of paragraph 39, or a pharmaceutically acceptable salt thereof, comprising a polymyxin conjugated to the C-terminus of the peptide.
50. The peptide of any one of paragraphs 1-49, or a pharmaceutically acceptable salt thereof, wherein the peptide is 100 amino acids or fewer in length.
51. The peptide of any one of paragraphs 1-50, or a pharmaceutically acceptable salt thereof, wherein the peptide is 30 amino acids or fewer in length.
52. The peptide of any one of paragraphs 1-3, wherein the peptide comprises one of the following amino acid sequences: SEQ ID NOs: 4-39, 59-77, and 81-100; or a pharmaceutically acceptable salt thereof.
53. The peptide of paragraph 52, or a pharmaceutically acceptable salt thereof, wherein the peptide is of one of SEQ ID NOs: 4-39, 59-77, or 81-100; and wherein the C-terminus is amidated with NH.sub.2.
54. The peptide of paragraph 25 or 26, wherein the peptide comprises one of the following amino acid sequences: SEQ ID NOs: 40-53; or a pharmaceutically acceptable salt thereof.
55. The peptide of paragraph 54, or a pharmaceutically acceptable sale thereof, wherein the peptide is of one of SEQ ID NOs: 40-53; and wherein the C-terminus is amidated with NH.sub.2.
56. The peptide of paragraph 30 or 31, wherein the peptide comprises the following amino acid sequence: [0425] G X.sup.1 G K F X.sup.2 K K K K K X.sup.3 V G E X.sup.4 A K K (SEQ ID NO: 3),
or a pharmaceutically acceptable salt thereof.
57. The peptide of paragraph 56, or a pharmaceutically acceptable salt thereof, wherein the peptide is of SEQ ID NO: 3; and wherein the C-terminus is amidated with NH.sub.2.
58. The peptide of paragraph 32 or 33, wherein the peptide comprises one of the following amino acid sequences: SEQ ID NOs: 102-105; or a pharmaceutically acceptable salt thereof.
59. The peptide of any one of paragraphs 32, 33, or 58, or a pharmaceutically acceptable salt thereof, wherein the peptide is of any one of SEQ ID NOs: 101-105; and wherein the C-terminus is amidated with NH.sub.2.
60. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein the crosslinks are attached to the ?-positions of the amino acids X.sup.1, X.sup.2, X.sup.3, and X.sup.4.
61. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein each crosslink is independently optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted acylene, or any combination thereof.
62. The peptide of paragraph 61, or a pharmaceutically acceptable salt thereof, wherein each crosslink is a hydrocarbon crosslink independently selected from optionally substituted alkylene, optionally substituted alkenylene, and optionally substituted alkynylene.
63. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein each crosslink is independently of the following formula:

##STR00064##

wherein each n is independently an integer from 1-10, inclusive.
64. The peptide of paragraph 63, or a pharmaceutically acceptable salt thereof, wherein the sum of two n on the same crosslink is 6.
65. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently ?,?-disubstituted amino acids.
66. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are each independently connected by a crosslink to form the following formula:

##STR00065##

wherein ? denotes the ?-carbons of the amino acids; and wherein each instance of R.sup.1 is independently optionally substituted C.sub.1-6 alkyl.
67. The peptide of paragraph 66, or a pharmaceutically acceptable salt thereof, wherein at least one instance of R.sup.1 is methyl.
68. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4, are each connected by a crosslink to form the following formula:

##STR00066##

wherein ? denotes the ?-carbons of the amino acids.
69. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein each crosslink is independently about 10 ? to about 16 ? in length, inclusive.
70. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein the length of each crosslink is approximately equal to the length of 5 to 13 carbon-carbon bonds, inclusive.
71. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein at least one crosslink spans at least one turn of an ?-helix of the peptide.
72. The peptide of paragraph 71, or a pharmaceutically acceptable salt thereof, wherein at least one crosslink stabilizes an ?-helix of the peptide.
73. The peptide of paragraph 71 or 72, or a pharmaceutically acceptable salt thereof, wherein the peptide has increased ?-helicity as compared to a corresponding uncrosslinked peptide.
74. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 independently comprise ?-sidechains comprising the reactive moieties.
75. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein the reactive moieties are independently selected from alkenes and alkynes.
76. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 independently comprise ?-sidechains of the following formula:

##STR00067##

wherein each n is independently an integer from 1-10, inclusive.
77. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently ?,?-disubstituted amino acids.
78. The peptide of paragraph 76 or 77, or a pharmaceutically acceptable salt thereof, wherein X.sup.1, X.sup.2, X.sup.3, and X.sup.4 each independently comprise the formula:

##STR00068##

wherein ? denotes the ?-carbon of the amino acids; and each instance of R.sup.1 is optionally substituted C.sub.1-6 alkyl.
79. The peptide of paragraph 78, or a pharmaceutically acceptable salt thereof, wherein at least one instance of R.sup.1 is methyl.
80. The peptide of any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein each instance of X.sup.1, X.sup.2, X.sup.3, and X.sup.4 is an amino acid of the formula:

##STR00069##

81. A pharmaceutical composition comprising a peptide of any one of paragraphs 1-73, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
82. A method of treating an infectious disease in a subject comprising administering to the subject a peptide of any one of paragraphs 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
83. The method of paragraph 82, wherein the infectious disease is a bacterial infection, viral infection, protozoal infection, or fungal infection.
84. A method of treating a bacterial infection in a subject comprising administering to the subject a peptide of any one of paragraphs 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
85. The method of paragraph 84, wherein the bacterial infection is a Gram-negative bacterial infection.
86. The method of paragraph 84 or 85, wherein the bacterial infection is an antibiotic-resistant bacterial infection.
87. The method of any one of paragraphs 84-86, wherein the bacterial infection is a Gram-negative, antibiotic-resistant bacterial infection.
88. The method of paragraph 86 or 87, wherein the antibiotic-resistant bacterial infection is caused by a bacteria resistant to one or more antibiotics selected from the group consisting of polymyxin, carbapenem, aminoglycosides, vancomycin, methicillin, clarithromycin, cephalosporin, penicillin, ampicillin, fluoroquinolones, tetracycline, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, tobramycin, and ?-lactams.
89. The method of any one of paragraphs 84-88, wherein the bacterial infection is caused by E. coli, A. baumannii, P. aeruginosa, or K. pneumoniae.
90. The method of any one of paragraphs 82-89, wherein the peptide has reduced renal toxicity as compared to a reference.
91. The method of paragraph 90, wherein the peptide has reduced renal toxicity as compared to a corresponding peptide comprising SEQ ID NO: 1 or 2, or as compared to pexiganan.
92. The method of any one of paragraphs 82-91, wherein the peptide has reduced hepatic toxicity as compared to a reference.
93. The method of paragraph 92, wherein the peptide has reduced hepatic toxicity as compared to a corresponding peptide comprising SEQ ID NO: 1 or 2, or as compared to pexiganan.
94. The method of any one of paragraphs 82-93, wherein the peptide has reduced hemolytic activity as compared to a reference.
95. The method of paragraph 94, wherein the peptide has reduced hemolytic activity as compared to a corresponding peptide comprising SEQ ID NO: 1 or 2, or as compared to melittin.
96. The method of any one of paragraphs 82-95, wherein the peptide, pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof is administered intravenously.
97. The method of any one of paragraphs 82-96, wherein the subject is a human.
98. A method of killing and/or inhibiting the growth of bacteria comprising contacting the bacteria with a peptide of any one of paragraphs 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
99. The method of paragraph 98, wherein the bacteria are E. coli, A. baumannii, P. aeruginosa, or K. pneumoniae.
100. A method of selectively killing and/or inhibiting the growth of microbial cells over mammalian cells comprising contacting the microbial and mammalian cells with a peptide of any one of paragraphs 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
101. A method of selectively lysing microbial cells over mammalian cells comprising contacting the microbial and mammalian cells with a peptide of any one of paragraphs 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
102. The method of paragraph 100 or 101, wherein the microbial cells are bacterial cells.
103. The method of paragraph 102, wherein the bacterial cells are E. coli cells, A. baumannii cells, P. aeruginosa cells, or K. pneumoniae cells.
104. A peptide of any one of paragraphs 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in a method of any one of the preceding paragraphs.
105. Use of a peptide of any one of paragraphs 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a medicament.
106. A kit comprising a peptide of any one of paragraphs 1-73, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof; and optionally instructions for use.
107. A method of preparing a crosslinked peptide of any one of the preceding paragraphs comprising a step of reacting an uncrosslinked peptide of any one of the preceding paragraphs under conditions sufficient to form the crosslinks connecting X.sup.1 and X.sup.2, and X.sup.3 and X.sup.4.
108. The method of paragraph 107, wherein the step of reacting involves a ring-closing metathesis (RCM) reaction.
109. A formulation comprising a stapled antimicrobial peptide (StAMP) and one or more lipids.
110. The formulation of paragraph 109, wherein the formulation is a micellar, liposomal, or lipid nanoparticle formulation.
111. The formulation of paragraph 109 or 110, wherein the StAMP is a peptide of any one of the preceding paragraphs.
112. The formulation of any one of paragraphs 109-111, wherein at least one of the one or more lipids comprises a phospholipid.
113. The formulation of paragraph 112, wherein at least one of the one or more lipids is a PEGylated phospholipid.
114. The formulation of paragraph 113, wherein the PEGylated phospholipid is DSPE-PEG.
115. The formulation of paragraph 114, wherein the PEGylated phospholipid is DSPE-MPEG or DSPE-PEG Amine.
116. The formulation of paragraph 115, wherein the PEGylated phospholipid is DSPE-MPEG(2000) or DSPE-PEG(2000) Amine.
117. The formulation of any one of paragraphs 109-116, comprising a peptide:lipid ratio from about 1:1 to about 1:25 (w/w), inclusive.
118. The formulation of any one of paragraphs 109-117, comprising a peptide:lipid ratio from about 1:2.5 to about 1:20 (w/w), inclusive.
119. The formulation of any one of paragraphs 109-118, comprising a peptide:lipid ratio of about 1:2.5, about 1:5, about 1:10, or about 1:20 (w/w).

EQUIVALENTS AND SCOPE

[0426] In the claims, articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0427] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

[0428] It is also noted that the terms comprising and containing are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0429] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0430] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.