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ANTIVIRAL STRUCTURALLY-STABILIZED ACE2 HELIX 1 PEPTIDES AND USES THEREOF

20240131124 ยท 2024-04-25

    Inventors

    Cpc classification

    International classification

    Abstract

    Disclosed herein are structurally stabilized peptides of ACE2 helix 1 useful for diagnosing, preventing, and treating coronavirus infection by targeting the receptor binding domain of SARS-CoV-2 and thereby blocking its interaction with the human ACE2 receptor, which is involved in coronavirus infection and pathogenesis.

    Claims

    1. A polypeptide comprising an amino acid sequence that is at least 30% and less than 81% identical to, or that has at least 4 and up to 14 amino acid additions or substitutions in, EQAKTFLDKFNHEAEDLFYQ (SEQ ID NO:77), wherein the polypeptide has one or more of the following properties: (i) binds the peptide of SEQ ID NO: 64 or SEQ ID NO: 65; (ii) inhibits interaction between human angiotensin converting enzyme 2 (ACE2) protein and an S1 protein subunit of SARS-CoV-2; (iii) inhibits interaction between a carboxypeptidase domain of human ACE2 protein and the S1 protein subunit of SARS-CoV-2; (iv) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and a receptor binding domain (RBD) of the S1 protein subunit of SARS-CoV-2; (v) competes for human ACE2-SARS-CoV-2 S1 protein binding; (vi) binds S1 protein of a SARS-CoV-2 variant and/or RBD of the SARS-CoV-2 variant; and (vii) inhibits a SARS virus infection, optionally, wherein the SARS infection is SARS-CoV-2 infection.

    2.-3. (canceled)

    4. The polypeptide of claim 1, comprising the amino acid sequence: TABLE-US-00014 (i) (SEQIDNO:125) QEEQAKDAADHANHEAEYQAYQSA, (ii) (SEQIDNO:113) IEEQAKTAADKANHEAEDAAYQSA, (iii) (SEQIDNO:118) IEEQAKTAADKANHEAEQAAYQSA, iv) (SEQIDNO:117) AEEQAKTAADKAAHEAEQAAYQAA, (v) (SEQIDNO:123) IQEQAKTDADKHNHEAEDYQYQSA, or (vi) (SEQIDNO:127) ETVDFFAEWFDVEAEDKDYL[[;]].

    5. The polypeptide of claim 1, comprising the amino acid sequence of any one of the sequences set forth in SEQ ID NOs.: 90-95, 98-100, 105-108, and 110.

    6. (canceled)

    7. The polypeptide of claim 1, wherein the polypeptide is hydrocarbon stapled or stitched.

    8. The polypeptide of claim 1, which is not stapled or stitched.

    9. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 10-12, 17-20, and 113-133.

    10. The polypeptide of claim 7, comprising the amino acid sequence set forth in: TABLE-US-00015 (i) (SEQIDNO:115) IEEQXKTAXDKANHEXEDAXYQSA, (ii) (SEQIDNO:116) IEEQXKEAXDKANHEXEDAXYQSA, (iii) (SEQIDNO:120) IEEQXKTAXDKANHEXEQAXYQSA, (iv) (SEQIDNO:121) IEEQXKEAXDKANHEXEQAXYQSA, (v) (SEQIDNO:126) QEEQXKDAXDHANHEXEYQXYQSA, (vi) (SEQIDNO:132) ETXDFLXEWFDVXAEDXDYL, (vii) (SEQIDNO:133) ETXDFYXEWFDVXAEDXDYL, (viii) (SEQIDNO:130) ETXDFFXEWFDVXAEDXDYL, [[or]] (ix) (SEQIDNO:131) ETXDFEXEWFDVXAEDXDYL, (x) (SEQIDNO:128) ETXDFFXEWADVXAEDXDYL, (xii) (SEQIDNO:129) ETXDFFXEWADVEXEDKXYL, (xiii) (SEQIDNO:100) IEEQXKTFLDKXNHEAEDLFYQS, (xiv) (SEQIDNO:105) IEEQAKTFLXKFNHEAXDLFYQS, (XV) (SEQIDNO:106) IEEQAKTFLDXFNHEAEXLFYQS, or (xvi) (SEQIDNO:107) IEEQAKTFLDKXNHEAEDXFYQS, wherein each X in the sequences above is an ?, ?-disubstituted non-natural amino acid with olefinic side chains.

    11. The polypeptide of claim 7, further comprising ?, ?-disubstituted non-natural amino acids with olefinic side chains that are internally cross-linked, wherein the ?, ?-disubstituted non-natural amino acids are inserted at positions 3 and 7 and/or positions 14 and 15 of SEQ ID NO:77 (wherein the position numbering is from the N-terminal E (position 1) to the C-terminal Q (position 20), or wherein the ?, ?-disubstituted non-natural amino acids are inserted at positions 3 and 7 and/or positions 13 and 17 of SEQ ID NO:77 (wherein the position numbering is from the N-terminal E (position 1) to the C-terminal Q (position 20).

    12. (canceled)

    13. The polypeptide of claim 7, further comprising ?, ?-disubstituted non-natural amino acids with olefinic side chains that can be internally cross-linked, wherein the ?, ?-disubstituted non-natural amino acids are inserted at positions SEQ ID NO:77 (wherein the position numbering is from the N-terminal E (position 1) to the C-terminal Q (position 20): (i) positions 5 and 12, (ii) positions 11 and 18, (iii) positions 12 and 19, (iv) positions 14 and 18, (v) positions 15 and 19, or (vi) positions 16 and 20.

    14. (canceled)

    15. The polypeptide of claim 1, wherein the polypeptide is not substituted at one or more of positions 1, 6, 10, 14, 15, 16 and 19 of SEQ ID NO:77 (wherein the position numbering is from the N-terminal E (position 1) to the C-terminal Q (position 20).

    16. The polypeptide of claim 1, which is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

    17. (canceled)

    18. A polypeptide comprising an amino acid sequence set forth below: TABLE-US-00016 (i) (SEQIDNO:2) X.sub.1EEQX.sub.2KTFLDKFNHEAEDLFYQSSXaa.sub.1Xaa.sub.2, (ii) (SEQIDNO:3) IEEQX.sub.1KTFX.sub.2DKFNHEAEDLFYQSSXaa.sub.1Xaa.sub.2, (iii) (SEQIDNO:4) IEEQAKTX.sub.1LDKX.sub.2NHEAEDLFYQSSXaa.sub.1Xaa.sub.2, (iv) (SEQIDNO:5) IEEQAKTFLDKX.sub.1NHEX.sub.2EDLFYQSSXaa.sub.1Xaa.sub.2, (v) (SEQIDNO:6) IEEQAKTFLDKFNHEX.sub.1EDLX.sub.2YQSSXaa.sub.1Xaa.sub.2, (vi) (SEQIDNO:7) IEEQAKTF8DKFNHEXEDLFYQSSXaa.sub.1Xaa.sub.2, (vii) (SEQIDNO:8) IEEQ8KTFLDKXNHEAEDLFYQSSXaa.sub.1Xaa.sub.2, (viii) (SEQIDNO:9) IEEQAKTFLDK8NHEAEDXFYQSSXaa.sub.1Xaa.sub.2, (ix) (SEQIDNO:10) X.sub.1EEQX.sub.2KTFLDKFNHEX.sub.3EDLX.sub.4YQSSXaa.sub.1Xaa.sub.2, (x) (SEQIDNO:11) X.sub.1EEQX.sub.2KTFLDKX.sub.3NHEX.sub.4EDLFYQSSXaa.sub.1Xaa.sub.2, (xi) (SEQIDNO:12) IEEQX.sub.1KTFX.sub.2DKFNHEX.sub.3EDLX.sub.4YQSSXaa.sub.1Xaa.sub.2, (xii) Xaa.sub.11EEQXaa.sub.12KXaa.sub.13Xaa.sub.14Xaa.sub.15DKXaa.sub.16Xaa.sub.17HEXaa.sub.18EDXaa.sub.19Xaa.sub.20 YXa.sub.21Xaa.sub.22Xaa.sub.23Xaa.sub.24Xaa.sub.25, wherein Xaa.sub.11=I, A, or a stapling amino acid Xaa.sub.12=A or a stapling amino acid Xaa.sub.13=T, E, or F Xaa.sub.14=F, A, or a stapling amino acid Xaa.sub.15=L, A, or a stapling amino acid Xaa.sub.16=F, A, or a stapling amino acid Xaa.sub.17=N or A Xaa.sub.18=A or a stapling amino acid Xaa.sub.19=L, A, or a stapling amino acid Xaa.sub.20=F, A, or a stapling amino acid Xaa.sub.21=Q or Y Xaa.sub.22=S or A Xaa.sub.23=S or A Xaa.sub.24=L, A, or absent Xaa.sub.25=A or absent (SEQ ID NO:17), (xiii) Xaa.sub.11Xaa.sub.12EQXaa.sub.13Xaa.sub.14TXaa.sub.15Xaa.sub.16DKXaa.sub.17Xaa.sub.18HEXaa.sub.19Xaa.sub.20Xaa.sub.21 Xaa.sub.22Xaa.sub.23YQXaa.sub.24Xaa.sub.25LXaa.sub.26, wherein Xaa.sub.11=I, A, or a stapling amino acid Xaa.sub.12=E or A Xaa.sub.13=A or a stapling amino acid Xaa.sub.14=K, R, or A Xaa.sub.15=F, A, or a stapling amino acid Xaa.sub.16=L, A, or a stapling amino acid Xaa.sub.17=F, A, or a stapling amino acid Xaa.sub.18=N or A Xaa.sub.19=A or a stapling amino acid Xaa.sub.20=E, D, or A Xaa.sub.21=D, E, or A Xaa.sub.22=L, A, or a stapling amino acid Xaa.sub.23=F, A, or a stapling amino acid Xaa.sub.24=S or A Xaa.sub.25=S or A Xaa.sub.26=A or absent (SEQ ID NO: 18), (xiv) IEEQAK.sub.6TFLD.sub.10K.sub.11FNHEAED.sub.18LFYQSSLA, wherein K.sub.6 and D.sub.10 are linked by a lactam bridge; or wherein K.sub.11 and D.sub.18 are linked by a lactam bridge (SEQ ID NO: 19), (xv) Xaa.sub.11EEQXaa.sub.12K.sub.6Xaa.sub.13Xaa.sub.14Xaa.sub.15D.sub.10K.sub.11Xaa.sub.16Xaa.sub.17HEXaa.sub.18ED.sub.18 Xaa.sub.19Xaa.sub.20YXaa.sub.21Xaa.sub.22Xaa.sub.23Xaa.sub.24Xaa.sub.25, wherein Xaa.sub.11=I or A, Xaa.sub.12=A Xaa.sub.13=T, E, or F Xaa.sub.14=F or A Xaa.sub.15=L or A Xaa.sub.16=F or A Xaa.sub.17=N or A Xaa.sub.18=A Xaa.sub.19=L or A Xaa.sub.20=F or A Xaa.sub.21=Q or Y Xaa.sub.22=S or A Xaa.sub.23=S or A Xaa.sub.24=L or A Xaa.sub.25=A or absent, wherein K.sub.6 and D.sub.10 are linked by a lactam bridge; or wherein K.sub.11 and Dis are linked by a lactam bridge (SEQ ID NO: 20), TABLE-US-00017 (xvi) (SEQIDNO:78) XEEQXKTFLDKFNHEAEDLFYQS, (xvii) (SEQIDNO:79) IXEQAXTFLDKFNHEAEDLFYQS, (xviii) (SEQIDNO:80) IEXQAKXFLDKFNHEAEDLFYQS, (xix) (SEQIDNO:81) IEEQXKTFXDKFNHEAEDLFYQS, (xx) (SEQIDNO:82) IEEQAXTFLXKFNHEAEDLFYQS, (xxi) (SEQIDNO:83) IEEQAKXFLDXFNHEAEDLFYQS, (xxii) (SEQIDNO:84) IEEQAKTXLDKXNHEAEDLFYQS, (xxiii) (SEQIDNO:85) IEEQAKTFXDKFXHEAEDLFYQS, (xxiv) (SEQIDNO:86) IEEQAKTFLXKFNXEAEDLFYQS, (xxv) (SEQIDNO:87) IEEQAKTFLDXFNHXAEDLFYQS, (xxvi) (SEQIDNO:88) IEEQAKTFLDKXNHEXEDLFYQS, (xxvii) (SEQIDNO:89) IEEQAKTFLDKFXHEAXDLFYQS, (xxviii) (SEQIDNO:90) IEEQAKTFLDKFNXEAEXLFYQS, (xxix) (SEQIDNO:91) IEEQAKTFLDKFNHXAEDXFYQS, (xxx) (SEQIDNO:92) IEEQAKTFLDKFNHEXEDLXYQS, (xxxi) (SEQIDNO:93) IEEQAKTFLDKFNHEAXDLFXQS, (xxxii) (SEQIDNO:94) IEEQAKTFLDKFNHEAEXLFYXS, (xxxiii) (SEQIDNO:95) IEEQAKTFLDKFNHEAEDXFYQX, (xxxiv) (SEQIDNO:96) 8EEQAKTXLDKFNHEAEDLFYQS, (xxxv) (SEQIDNO:97) I8EQAKTFXDKFNHEAEDLFYQS, (xxxvi) (SEQIDNO:98) IE8QAKTFLXKFNHEAEDLFYQS, (xxxvii) (SEQIDNO:99) IEE8AKTFLDXFNHEAEDLFYQS, (xxxviii) (SEQIDNO:100) IEEQ8KTFLDKXNHEAEDLFYQS, (xxxix) (SEQIDNO:101) IEEQA8TFLDKFXHEAEDLFYQS, (xl) (SEQIDNO:102) IEEQAK8FLDKFNXEAEDLFYQS, (xli) (SEQIDNO:103) IEEQAKT8LDKFNHXAEDLFYQS, (xlii) (SEQIDNO:104) IEEQAKTF8DKFNHEXEDLFYQS, (xliii) (SEQIDNO:105) IEEQAKTFL8KFNHEAXDLFYQS, (xliv) (SEQIDNO:106) IEEQAKTFLD8FNHEAEXLFYQS, (xlv) (SEQIDNO:107) IEEQAKTFLDK8NHEAEDXFYQS, (xlvi) (SEQIDNO:108) IEEQAKTFLDKF8HEAEDLXYQS, (xlvii) (SEQIDNO:109) IEEQAKTFLDKFN8EAEDLFXQS, (xlviii) (SEQIDNO:110) IEEQAKTFLDKFNH8AEDLFYXS, or (xlix) (SEQIDNO:111) IEEQAKTFLDKFNHE8EDLFYQX, wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4, X, and 8 are non-natural amino acids with olefinic side chains; (i) binds the peptide of SEQ ID NO: 64 or SEQ ID NO: 65; (ii) inhibits interaction between human ACE2 protein and an S1 protein subunit of SARS-CoV-2; (iii) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and the S1 protein subunit of SARS-CoV-2; (iv) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and an RBD of the S1 protein subunit of SARS-CoV-2; (v) competes for human ACE2-SARS-CoV-2 S1 protein binding; (vi) binds an S1 protein of a SARS-CoV-2 variant and/or the RBD of the S1 protein subunit of the SARS-CoV-2 variant; and (vii) inhibits SARS virus infection.

    19. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

    20.-21. (canceled)

    22. A nanoparticle composition comprising the polypeptide of claim 1.

    23. A diagnostic reagent for detecting coronavirus whose receptor-binding domain binds to ACE2, the diagnostic reagent comprising a surface comprising the polypeptide of claim 1.

    24. (canceled)

    25. A method for treating or preventing a viral infection caused by (i) a coronavirus infection, or (ii) a virus that infects cells by binding to ACE2 or treating post-acute sequelae of SARS-CoV-2 infection in a subject in need thereof, the method comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 1.

    26.-28. (canceled)

    29. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of claim 1, wherein the subject has been, or is at risk of being, infected with a variant of SARS-CoV-2, or wherein the subject has post-acute sequelae of SARS-CoV-2 infection.

    30. A method of preventing or inhibiting interaction between the receptor binding domain of a virus and ACE2 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of claim 1.

    31.-33. (canceled)

    34. A method of making an internally cross-linked polypeptide, the method comprising: (a) providing the polypeptide of claim 1; and (b) cross-linking the polypeptide by a ruthenium catalyzed metathesis reaction thereby generating an internally cross-linked polypeptide, and (c) formulating the internally cross-linked polypeptide as a sterile pharmaceutical composition.

    35. A method of detecting the presence of a virus whose receptor binding domain binds to ACE2, the method comprising: (I) (a) providing a biological sample of a subject; (b) mixing the biological sample with a plurality of peptides comprising the polypeptide of claim 1 to create a mixture, wherein: at least one stabilized peptide in the plurality comprises a detection moiety; and at least one stabilized peptide in the plurality comprises a capture moiety; (c) providing a diagnostic reagent for detecting the presence of a virus whose receptor-binding domain binds to ACE2; (d) contacting the diagnostic reagent with the mixture; and (e) detecting the presence or absence of the virus; or (II) (a) providing a detection agent, wherein the detection agent is a first polypeptide of claim 1, wherein the first polypeptide binds to the receptor binding domain of the virus, and wherein the first polypeptide is linked to a detection label; (b) providing a capture agent wherein the capture agent is a second polypeptide of claim 1, wherein the second polypeptide binds to the receptor binding domain of the virus, and wherein the second polypeptide is linked to an affinity label; (c) mixing a biological sample from a subject with the detection agent and the capture agent to form a mixture: (d) contacting the mixture with a solid support that binds the capture agent; and (e) detecting presence or absence of the virus.

    36. A compound comprising a stabilized peptide comprising a sequence having the formula: ##STR00011## or a pharmaceutically acceptable salt thereof, wherein: (a) each R.sub.1 and R.sub.2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted; (b) each R.sub.3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; (c) each x is independently 2, 3, or 6; (d) each w and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; (e) z is 1, 2, or 3; and (f) each Xaa is independently an amino acid, wherein the sequence comprises the polypeptide of claim 1, with: (i) 2 to 5 amino acids that are substituted with ?, ?-disubstituted non-natural amino acids with olefinic side chains that form an internal cross-link, (ii) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 additional amino acid substitutions; (iii) 0, 1, 2, 3, 4, or 5 deletions at the N and/or C-terminus of the sequence; and/or (iv) 0, 1, 2, 3, 4, or 5 amino acid additions at the N and/or C-terminus of the sequence, and wherein the stabilized peptide has one or more of the following properties: (i) binds the peptide of SEQ ID NO: 64 or SEQ ID NO: 65; (ii) inhibits interaction between human ACE2 protein and an S1 protein subunit of SARS-CoV-2; (iii) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and the S1 protein subunit of SARS-CoV-2; (iv) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and an RBD of the S1 protein subunit of SARS-CoV-2; (v) competes for human ACE2-SARS-CoV-2 S1 protein binding; (vi) binds S1 protein of a SARS-CoV-2 variant and/or the RBD of the S1 protein subunit of the SARS-CoV-2 variant; and (vii) inhibits a SARS virus infection, optionally, wherein the SARS infection is SARS-CoV-2 infection.

    37.-40. (canceled)

    41. A method of treating or preventing a coronavirus infection or treating post-acute sequelae of a coronavirus infection in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof of claim 36.

    42.-44. (canceled)

    45. A pharmaceutical composition comprising the polypeptide of claim 18, and a pharmaceutically acceptable carrier.

    46. A nanoparticle composition comprising the polypeptide of claim 18.

    47. A diagnostic reagent for detecting coronavirus whose receptor-binding domain binds to ACE2, the diagnostic reagent comprising a surface comprising the polypeptide of claim 18.

    48. A method of detecting the presence of a virus whose receptor binding domain binds to ACE2, the method comprising: (I) (a) providing a biological sample of a subject; (b) mixing the biological sample with a plurality of peptides comprising the polypeptide of claim 18 to create a mixture, wherein: at least one stabilized peptide in the plurality comprises a detection moiety; and at least one stabilized peptide in the plurality comprises a capture moiety; (c) providing a diagnostic reagent for detecting the presence of a virus whose receptor-binding domain binds to ACE2; (d) contacting the diagnostic reagent with the mixture; and (e) detecting the presence or absence of the virus; or (II) (a) providing a detection agent, wherein the detection agent is a first polypeptide of claim 18, wherein the first polypeptide binds to the receptor binding domain of the virus, and wherein the first polypeptide is linked to a detection label; (b) providing a capture agent wherein the capture agent is a second polypeptide of claim 18, wherein the second polypeptide binds to the receptor binding domain of the virus, and wherein the second polypeptide is linked to an affinity label; (c) mixing a biological sample from a subject with the detection agent and the capture agent to form a mixture; (d) contacting the mixture with a solid support that binds the capture agent; and (e) detecting presence or absence of the virus.

    49. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of claim 18, wherein the subject is infected with, or at risk of being infected with, a variant of SARS-CoV-2, or wherein the subject has post-acute sequelae of SARS-CoV-2 infection.

    50. A method of preventing or inhibiting interaction between the receptor binding domain of a virus and ACE2 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of claim 18.

    51. A method for treating or preventing a viral infection caused by (i) a coronavirus infection, or (ii) a virus that infects cells by binding to ACE2 or treating post-acute sequelae of SARS-CoV-2 infection in a subject in need thereof, the method comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide of claim 18.

    Description

    BRIEF DESCRIPTION OF DRAWINGS

    [0178] FIG. 1 shows an enlarged region of the crystal structure of the interaction between the ACE2 receptor (left) and the SARS-CoV-2 receptor binding domain (RBD; right), highlighting the location of the ACE2 helix 1 whose interacting face residues (facing right) are shown to directly engage a surface of the SARS-CoV-2 RBD. The non-interacting face residues of ACE2 helix 1 are facing left.

    [0179] FIG. 2 shows the first 80 amino acids (SEQ ID NO:66) of human ACE2 (SEQ ID NO:22) compared to Mus musculus (NP_081562.2; SEQ ID NO:167); Rattus norvegicus (NP_001012006.1; SEQ ID NO:168); Danio rerio (XP_005169416.1; SEQ ID NO:169); Sus scrofa (NP_001116542.1; SEQ ID NO:170); Bos taurus (XP_005228485.1; SEQ ID NO:71); Pan troglodytes (XP_016798468.1; SEQ ID NO:72); Macaca mulatta (NP_001129168.1; SEQ ID NO:73); and Canis lupus familiaris (NP_001158732.1; SEQ ID NO:74). The boxed sequences is the ACE2 ?1 helix sequence (SEQ ID NO:1).

    [0180] FIG. 3 shows a non-limiting list of exemplary SAH-ACE2h1 peptides whereby single and double staples are inserted into the ACE2h1 peptide sequence and mutant derivatives thereof.

    [0181] FIG. 4 shows a variety of non-natural amino acids containing olefinic tethers that can be used to generate hydrocarbon stapled ACE2h1 peptides spanning i, i+3; i, i+4; and i, i+7 positions and single staple scanning to generate a library of singly stapled ACE2h1 peptides.

    [0182] FIG. 5 shows a variety of staple compositions in multiply stapled peptides and staple scanning to generate a library of multiply stapled ACE2h1 peptides.

    [0183] FIG. 6 shows a variety of staple compositions in tandem stitched peptides to generate a library of stitched ACE2h1 peptides.

    [0184] FIG. 7 is an illustration of an exemplary approach to designing, synthesizing, and identifying optimal SAH-ACE2h1 constructs to target the receptor binding domain of SARS-CoV-2 and block its critical interaction with the ACE2 receptor, including the generation of Ala scan, staple scan, and variable N- and C-terminal deletion, addition, and derivatization libraries. Singly and doubly stapled constructs, including alanine and staple scans, are used to identify optimal SAH-ACE2h1 peptides for in vitro and in vivo analyses.

    [0185] FIG. 8 shows a listing of synthesized SAH-ACE2h1 peptides, including i, i+4 and i, i+7 staple scanning libraries, and a series of single and double stapled analogs bearing point mutations of the ACE2h1 template sequence.

    [0186] FIGS. 9A-9B show how native and mutated ACE2h1 sequences, when synthesized as a peptide and evaluated by circular dichroism, do not retain the natural alpha-helical structure found in the context of the ACE2 receptor. In contrast, inserting staples at specific locations can restore alpha-helical shape. For the ACE2h1 mutant sequence shown in FIG. 9A, a single staple at a specific location can restore alpha-helical shape, with ?-helicity improved even further upon double stapling at the indicated locations. For a distinct ACE2h1 mutant sequence shown in FIG. 9B, installing double staples in the corresponding positions shown in FIG. 9A, converts the random coil conformation of the unstapled sequences into an ?-helix.

    [0187] FIG. 10 shows that insertion of double staples at the indicated positions into ACE2h1 sequences bearing mutations confers striking in vitro proteinase K resistance compared to the unstapled wild-type ACE2h1 sequence.

    [0188] FIG. 11 shows the mouse plasma stability of an unstapled ACE2h1 mutant sequence and a double-stapled analog. Whereas the unstapled peptide demonstrates a half-life of 374 min, essentially no degradation was observed upon insertion of double staples at the indicated locations.

    [0189] FIGS. 12A-12B show a bead-binding assay in which His-tagged SARS-CoV-2 receptor binding domain (RBD) protein is bound to Ni-NTA beads and then FITC-ACE2h1 peptides are applied in order to measure binding activity, as monitored by fluorescence imaging of the beads, and thus detection of RBD protein. FIG. 12A shows that an unstapled and mutated ACE2h1 peptide has little to no detectable binding to the RBD-coated beads (FIG. 12A, column A; SEQ ID NO:113), whereas insertion of a single staple at the indicated location (FIG. 12, columns A and B; SEQ ID NOs: 113 and 114, respectively) and then double staples at the indicated locations (FIG. 12A, columns C and E; SEQ ID NOs: 115 and 116, respectively) leads to progressively enhanced binding activity and RBD detection. Importantly, when no RBD protein is added to the beads, the positive fluorescence signal of an exemplary double stapled ACE2h1 peptide seen with RBD-coated beads (FIG. 12A, column C; SEQ ID NO:115) is completely lost (FIG. 12A, column D; SEQ ID NO:115), highlighting the specificity of double stapled peptide binding activity for RBD. FIG. 12B shows that peptide templates bearing a series of mutations can enhance binding to the RBD-coated beads compared to the native sequence (FIG. 12A, columns A-C; SEQ ID NOs: 76, 123, and 125, respectively), including a double stapled analog (FIG. 12A, column D, SEQ ID NO:126).

    [0190] FIGS. 13A-13C show that the bead-binding results in FIGS. 12A-12B, namely the capacity of a FITC-labeled and stapled ACE2h1 peptide sequence to bind and detect SARS-CoV2 RBD protein, can afford simple and rapid virus detection methods. For example, Peptide A (detection tag) and B (affinity tag) are mixed in a solution (FIG. 13A, part A). The patient sample is added to the peptide solution and mixed (FIG. 13A, part B). The beads are added, mixed, and collected by gravity or centrifugation (FIG. 13A, part C). If the result is negative (no virus present) the peptide containing the chromophore remains in solution (FIG. 13A, part D, top). When the result is positive (virus present in sample), the virus is captured by the beads via peptide B, which is collected along with simultaneous virus-bound peptide A, leading to an immediate read-out (FIG. 13A, part D, bottom). An alternative approach is shown in FIG. 13B and is based on a pregnancy-type strip or ELISA set up in which SAH-ACE2h1 peptide is fixed to a solid support (FIG. 13B, parts A and B), a patient sample is added to the strip or plate well (FIG. 13B, part C), and then application of a second SAH-ACE2h1 peptide is applied (FIG. 13B, part D) that allows for a colorimetric read-out, such as biotinylated SAH-ACE2h1 peptide detected by streptavidin HRP (FIG. 13B, part E) and incubation with chromogenic substrate (FIG. 13B, part F).

    [0191] FIG. 13C demonstrates the successful development of a SAH-ACE2h1-based test strip, based on this concept of an enzyme-linked stapled peptide assay (ELIPSA), which dose-responsively detects a serial dilution of inactivated SARS-CoV-2 virus (starting titer of 109).

    [0192] FIGS. 14A-14C show the binding activities of differentially stapled and mutated ACE2h1 peptides for recombinant SARS-CoV-2 proteins containing the RBD. In FIG. 14A, a direct fluorescence polarization binding assay (FPA) is shown in which FITC-labeled double i, i+4 stapled ACE2h1 peptides bearing a series of mutations demonstrate dose-responsive binding activity when incubated with a serial dilution of GST-tagged recombinant SARS-CoV-2 RBD protein. In FIG. 14B, the direct binding interaction between a FITC-labeled double i, i+4 stapled ACE2h1 peptides bearing a series of mutations and the SARS-CoV-2 spike protein is used to screen biotinylated (non-fluorescent) and differentially stapled ACE2h1 peptides, with and without mutations, for competitive spike protein binding activity in solution, revealing constructs that were capable (black bar). In FIG. 14C, a direct fluorescence polarization binding assay (FPA) is shown in which two FITC-labeled double i, i+4 stapled ACE2h1 peptides bearing a series of mutations bound to wild-type SARS-CoV-2 RBD protein and retain at least equivalent, or exhibit more, binding activity to SARS-CoV-2 RBD proteins bearing clinical variants such as the UK (N501Y), or B.1.1.7 lineage, and South African (K.sub.417N, E484K and N501Y), or B.1.351 lineage, with the latter variant protein in particular showing enhanced binding activity to both SAH-ACE2h1 peptides.

    [0193] FIGS. 15A-15B show an alternative binding analysis in which biotinylated stapled ACE2h1 peptides were applied to a streptavidin-coated tip (solid support) and tested for recombinant SARS-CoV-2 RBD binding activity by biolayer interferometry (BLI). In FIG. 15A, single i, i+4 or single i, i+7 stapled ACE2h1 peptides were tested for SARS-CoV-2 RBD binding activity at a screening dose, revealing compounds that do or do not bind to RBD based on peptide sequence, staple type, and/or staple position. In FIG. 15B, double i, i+4 stapled ACE2h1 peptides bearing a series of mutations show dose-responsive RBD binding activity.

    [0194] FIGS. 16A-16B show a SARS-CoV-2 pseudovirus assay in which a virus coated with the SARS-CoV-2 Spike protein containing RNA coding for GFP is used to infect 293T cells that are treated with ACE2h1 peptides, followed by fluorescence microscopy imaging to detect and compare the infection levels between vehicle and peptide treatments to assay for inhibition of viral infection (FIG. 16A), which was quantitated by image analysis (FIG. 16B). Insertion of a single staple conferred antiviral activity compared to the unstapled template peptide that showed no activity, with double stapling enhancing antiviral activity further. Installing a series of mutations into the template sequence or double stapling an N- and C-terminally truncated construct bearing a series of distinct mutations also conferred marked antiviral activity in the pseudovirus assay.

    [0195] FIGS. 17A-17C show the comparative inhibitory effects of differentially stapled and mutated ACE2h1 peptides on Vero E6 cell infection by native SARS-CoV-2 virus, which is detected and quantitated by a high throughput immunofluorescence assay.

    [0196] FIG. 17A shows how peptides are initially screened for inhibitory activity at a 25 ?M test dose, which is followed by evaluating hits by 2-fold serial dilution from a 25 ?M starting dose, as shown in FIG. 17B and FIG. 17C, revealing differentially stapled and mutated ACE2h1 peptides with dose-responsive antiviral activity.

    [0197] FIGS. 18A-18C shows how lead SAH-ACE2h1 peptides are identified based on consistency of activity across a diversity of assays, including binding assays with peptides in solution or on solid support and antiviral assays using SARS-CoV-2 pseudovirus or native virus. FIG. 18A shows an exemplary single i, i+4 stapled peptide of the native sequence with no biological activity across 4 assays and 5 single i, i+4 stapled peptides of the native sequence that demonstrated biological activity in 3 of 4 functional assays. FIG. 18B shows two exemplary i, i+7 single stapled peptides of the native sequence with no biological activity across 4 assays and 8 single i, i+7 single stapled peptides of the native sequence that demonstrated biological activity in at least 3 of 4 functional assays, with 3 compositions demonstrating efficacy across all RBD binding and antiviral assays. FIG. 18C shows how favorable staple types and locations identified by staple scanning and incorporated into templates iterated by amino acid mutagenesis can lead to a series of lead constructs with consistent biological activity across 4 independent assays, spanning soluble binding, solid phase binding, pseudovirus infectivity, and native virus infectivity assays.

    DETAILED DESCRIPTION

    [0198] Coronavirus disease 2019 is an infectious disease that has spread across the world. It is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein of SARS-CoV-2, which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2, while the S2 subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain. This disclosure relates, in part, to antiviral peptides targeting the S protein.

    [0199] The present disclosure features structurally-stabilized (e.g., stapled, stitched) peptides of the ACE2 ?1 helix that can block or inhibit binding by one or more coronaviruses (e.g., betacoronaviruses such as SARS-CoV-2) to a cell (e.g., a human respiratory epithelial cell). In the case of SARS-CoV-1 and SARS-CoV-2, the RBD in the spike glycoprotein (i.e., S1 subunit) of the coronavirus interacts with the al helix of ACE2. The stabilized peptides of this disclosure inhibit or block the interaction between ACE2 on a cell with the receptor binding domain (RBD) of a virus that binds ACE2, such as a coronavirus (e.g., SARS-CoV-2). Accordingly, the present disclosure provides novel methods and compositions (e.g., combinations of compositions) for treating, for developing treatments for, for preventing infection with, and for diagnosing infection by one or more coronaviruses (e.g., betacoronaviruses such as SARS-CoV-2).

    Angiotensin I Converting Enzyme 2 (ACE2)

    [0200] The mechanism for SARS-CoV-2 infection is the requisite binding of the virus to the membrane-bound form of angiotensin-converting enzyme 2 (ACE2) and internalization of the complex by the host cell. Functionally, there is an interaction between ACE2 ?1 helix and the S1 protein of the SARS-CoV-2 virus. S1 contains the receptor binding domain (RBD), which directly binds to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2). Li et al., Science 309, 1864-1868 (2005).

    [0201] The ACE2 domain involved in the interaction with RBD is called the carboxypeptidase domain and encompasses residues 1-612 of human ACE 2 protein. The ACE2 ?1 helix sequence spans from approximately position 20 to position 54 of the human ACE2 protein. An exemplary sequence for ACE2 ?1 helix sequence that engages the RBD of SARS-CoV-2 (e.g. amino acids 21 to 46) is provided as IEEQAKTFLDKFNHEAEDLFYQSSLA (SEQ ID NO:1). The amino acid sequence of an exemplary human angiotensin I converting enzyme 2 (ACE2) helix 1 sequence that interacts with the RBD is provided as SEQ ID NO:22, shown below.

    TABLE-US-00005 (SEQIDNO:22) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKENHEAEDLE YQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLA QMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTIL NTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNE RLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYG DYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHL HAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGREWTNLYS LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGL PNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDELTAHHEMGHIQYDMAYAAQPFLLRNGANEGE HEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINELL KQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEM KREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTL YQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLENMLRL GKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNK NSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEM YLERSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRIS ENFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDN SLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVIL IFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDD VQTSF

    [0202] Underlined in SEQ ID NO:22 is the ACE2 ?1 helix sequence that spans from position 21 to position 46 (i.e., SEQ ID NO:1). In some instances, the ACE2 ?1 helix sequence targets by the Receptor Binding Domain (RBD) and Receptor Binding Motif (RBM) of SARS-CoV-2 and other ACE2 interacting viruses (e.g., SARS-CoV-1, HCoV-NL63). In some instances, the ACE2 ?1 helix sequence targets a variant of SARS-CoV-2. Variants are disclosed in Peacock et al., Journal of General Virology, (2021); 102:001584, which is incorporated by reference in its entirety. In some instances, the RBD amino acid sequence has the amino acid sequence of SEQ ID NO:64.

    TABLE-US-00006 (SEQIDNO:64) 1 rvqptesivrfpnitnlcpfgevfnatrfasvyawnrkrisncvadysvlynsasfstfk 61 cygvsptklndlcftnvyadsfvirgdevrqiapgqtgkiadynyklpddftgcviawns 121 nnldskvggnynylyrlfrksnlkpferdisteiyqagstpcngvegfncyfplqsygfq 181 ptngvgyqpyrvvvlsfellhapatvcgpkkstnlvknkcvnf
    In some instances, the RBD is an ACE2 binding fragment of the amino acid sequence of SEQ ID NO:64. In one embodiment the ACE2 binding fragment of RBD is

    TABLE-US-00007 (SEQIDNO:65) fpnitnlcpfgevfnatrfasvyawnrkrisncvadysvlynsasfstfk 61 cygvsptklndlcftnvyadsfvirgdevrqiapgqtgkiadynyklpddftgcviawns 121 nnldskvggnynylyrlfrksnlkpferdisteiyqagstpcngvegfncyfplqsygfq 181 ptngvgyqpyrvvvlsfell.

    [0203] In certain instances, each of the peptides and stabilized peptides described herein can bind SEQ ID NO:64 or 65.

    [0204] Exemplary amino acid sequence of the ACE2 ?1 helix sequence and variants thereof are provided in Table 1. Note that each of these amino acid sequences may further include an N-terminal threonine (T) amino acid residue. All such variants and their use in the methods described herein are also encompassed by this disclosure.

    TABLE-US-00008 TABLE1 ACE2Helix1PeptideAnalogs. Name Sequence SEQIDNO: ACE2h1 IEEQAKTFLDKFNHEAEDLFYQSSLA 1 (native)-26 ACE2h1 IEEQAKTFLDKFNHEAEDLFYQSS 21 (native)-24 ACE2h1 IEEQAKTFLDKFNHEAEDLFYQS 76 (native)-23 (21-43) ACE2h1 EQAKTFLDKFNHEAEDLFYQ 77 (native)-20 (23-42) SAH- X.sub.aa1EEQAKTX.sub.aa2X.sub.aa3DKX.sub.aa4X.sub.aa5HEAEDX.sub.aa6 13 ACE2h1- X.sub.aa7YQX.sub.aa8X.sub.aa9X.sub.aa10X.sub.aa11,wherein 12 X.sub.aa1=IorA X.sub.aa2=ForA X.sub.aa3=LorA X.sub.aa4=ForA X.sub.aa5=NorA X.sub.aa6=LorA X.sub.aa7=ForA X.sub.aa8=SorA X.sub.aa9=SorA X.sub.aa10=L,A,orabsent X.sub.aa11=Aorabsent SAH- IEEQAKTFLDKFNHEAEDLFYX.sub.aa1SSX.sub.aa2X.sub.aa3, 14 ACE2h1- wherein 13 X.sub.aa1=QorY X.sub.aa2=L,A,orabsent X.sub.aa3=Aorabsent SAH- IEEQAKX.sub.aa1FLDKFNHEAEDLFYQSSX.sub.aa2X.sub.aa3, 15 ACE2h1- wherein 14 X.sub.aa1=TorE X.sub.aa2=L,A,orabsent X.sub.aa3=Aorabsent SAH- IEEQAKX.sub.aa1FLDKFNHEAEDLFYQSSX.sub.aa2X.sub.aa3, 16 ACE2h1- wherein 15 X.sub.aa1=TorF X.sub.aa2=L,A,orabsent X.sub.aa3=Aorabsent SAH- X.sub.aa1EEQAKTX.sub.aa2X.sub.aa3DKX.sub.aa4X.sub.aa5HEAE(D/Q) 145 ACE2h1- X.sub.aa6X.sub.aa7YQX.sub.aa8X.sub.aa9,wherein 12-alt X.sub.aa1=IorA X.sub.aa2=ForA X.sub.aa3=LorA X.sub.aa4=ForA X.sub.aa5=NorA X.sub.aa6=LorA X.sub.aa7=ForA X.sub.aa8=SorA X.sub.aa9=SorA SAH- Xaa1EEQAKTXaa2Xaa3DKXaa4Xaa5HEAE(D/Q) 145 ACE2h1- Xaa6Xaa7YQXaa8Xaa9,wherein 12-alt SAH- IEEQAKTFLDKFNHEAE(D/Q)LFYX.sub.aa1SS, 146 ACE2h1- whereinX.sub.aa1=QorY 13-alt SAH- IEEQAKX.sub.aa1FLDKFNHEAE(D/Q)LFYQSS, 147 ACE2h1- whereinX.sub.aa1=TorE 14-alt SAH- IEEQAKX.sub.aa1FLDKFNHEAE(D/Q)LFYQSS, 148 ACE2h1- whereinX.sub.aa1=TorF 15-alt Native IEEQAKTFLDKFNHEAEDLFYQSXaa.sub.1Xaa.sub.2Xaa.sub.3, 49 ACE2h1 whereinXaa.sub.1isAorabsent,Xaa.sub.2isL,A,orabsent, peptide Xaa.sub.2isAorabsent,andXaa.sub.3isAorabsent SAH- IEEQAKTAADKANHEAEDAAYQSAXaa.sub.1Xaa.sub.2, 50 ACE2h1- whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2isAorabsent mut-1 SAH- IQEQAKTDADKHNHEAEDYQYQSAXaa.sub.1Xaa.sub.2, 53 ACE2h1- whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2isAorabsent mut-4 SAH- QEEQAKDAADHANHEAEYQAYQSAXaa.sub.1X.sub.aa.sub.2, 54 ACE2h1- whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2isAorabsent mut-5 SAH- IEEQAKTAADKANHEAEQAAYQSAXaa.sub.1Xaa.sub.2, 56 ACE2h1- whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2isAorabsent mut-7

    [0205] In certain instances, the ACE2 ?1 helix sequence peptides described herein (e.g., SEQ ID NOs: 1, 13-16, 21, 49, 50, 53, 54, 56, 76, 77, 112, 113, 117, 118, 123, 125, 127, or 145-148 may also contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) amino acid substitutions. These substitutions may be conservative and/or non-conservative amino acid substitutions. In addition, in some instances at least two (e.g., 2, 3, 4, 5, or 6) amino acids of SEQ ID NOs: 1, 13-16, 21, 49, 50, 53, 54, 56, 76, 77, 112, 113, 117, 118, 123, 125, or 127, or 145-148 may be substituted by ?, ?-disubstituted non-natural amino acids with olefinic side chains.

    [0206] In some instances, any of the peptides in Table 1 (SEQ ID NO: 1, 13-16, 21, 49, 50, 53, 54, 56, or 145-148) may include Xaa.sub.1 (wherein Xaa.sub.1 is L or absent), or Xaa.sub.2 (wherein Xaa1 is A, or absent). In some instances, Xaa1, and Xaa.sub.2 are absent, in any of SEQ ID NO: 1, 13-16, 21, 49-56, or 145-148 and these peptides include an N-terminal threonine (T). In some instances, when both Xaa1, and Xaa.sub.2 are both present in any of SEQ ID NOs: 1, 13-16, 21, 49-56, 76, 77, or 145-148 these peptides can further include an N-terminal threonine (T).

    [0207] In some instances, the disclosed polypeptides comprises EQAKTFLDKFNHEAEDLFYQ (SEQ ID NO:77). In some instances, the polypeptides comprises an amino acid sequence that is at least 30%, and less than 81% identical to, or that has at least 4 and up to 14 amino acid substitutions in, (SEQ ID NO:77). In some instances, the polypeptide comprises an amino acid sequence that is 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 70%, at least 71%, at least 75% or at least 81% identical to SEQ ID NO:77. In some instances, the polypeptides comprises an amino acid sequence that is at less than 61%, 71%, or 81% identical to SEQ ID NO:77. In some instances, the polypeptide comprises an amino acid sequence that has at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 amino acid substitutions compared to SEQ ID NO:77. As disclosed herein, substitutions include changes from an amino acid of SEQ ID NO:77 to a naturally-occurring amino acid or to a stabilized amino acid (e.g., comprising a stitch).

    [0208] In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, and 20 of SEQ ID NO:77 (wherein the position numbering is from the N-terminal E (position 1) to the C-terminal Q (position 20). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 2 (Q). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 3 (A). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 4 (K). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 5 (T). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 6 (F). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 7 (L). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 8 (D). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 9 (K). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 10 (F). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 11 (N). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 12 (H). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 13 (E). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 17 (L). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 18 (F). In some instances, the polypeptide comprises SEQ ID NO:77 and is substituted at position 20 (Q). The polypeptide has one or more of the following properties: (i) binds the peptide of SEQ ID NO: 64 or SEQ ID NO: 65; (ii) inhibits interaction between human ACE2 protein and the S1 protein subunit of SARS-CoV-2; (iii) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and the S1 protein subunit of SARS-CoV-2; (iv) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and the RBD of the S1 protein subunit of SARS-CoV-2; (v) competes for human ACE2-SARS-CoV-2 S1 protein subunit binding; (vi) binds the S1 protein subunit (e.g., the RBD) of a SARS-CoV-2 variant; and (vii) inhibits SARS virus infection, optionally, wherein the SARS infection is SARS-CoV-2 infection.

    [0209] A conservative amino acid substitution means that the substitution replaces one amino acid with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), and acidic side chains and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine).

    [0210] In some instances, the ACE2 ?1 helix sequence peptides described herein (e.g., SEQ ID NO: 1, 13-16, 21, 49, 50, 53, 54, 56, 76, 77, 112, 113, 117, 118, 123, 125, 127, or 145-148) may also contain at least one, at least 2, at least 3, at least 4, or at least 5 amino acids added to or deleted from the N-terminus of the peptide. In one instance any one of the peptides of SEQ ID NO: 1, 13-16, 21, 49, 50, 53, 54, 56, 76, 77, 112, 113, 117, 118, 123, 125, 127, or 145-148 has an added threonine (T) at the N-terminus of the sequence. In some instances, the ACE2 ?1 helix sequence peptides described herein (e.g., SEQ ID NO: 1, 13-16, or 21, 49, 50, 53, 54, 56, 76, 77, 112, 113, 117, 118, 123, 125, 127, or 145-148) may also contain at least one, at least 2, at least 3, at least 4, or at least 5 amino acids added to or deleted from the C-terminus of the peptide.

    [0211] In some instances, the amino acids of the interacting face of SEQ ID NO:1 are at positions 3 (corresponding to E23 of SEQ ID NO:22), 4 (Q24), 7 (T27), 10 (D30), 11 (K31), 14 (H34), 15 (E35), 21 (Y41), 22 (Q42), and 25 (L45). In some cases, the amino acids of the interacting face are not substituted. In some cases 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acids of the interacting face are substituted (e.g., conservative amino acid substitutions). In some cases 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acids of the interacting face are substituted to alanine. In some instances, the amino acids of the non-interacting face of SEQ ID NO:1 are at positions 1 (corresponding to 121 of SEQ ID NO:22), 2 (E22), 5 (A25), 6 (K26), 8 (F28), 9 (L29), 12 (F32), 13 (N33), 16 (A36), 17 (E37), 18 (D38), 19 (L39), 20 (F40), 23 (S43), 24 (S44), and 26 (A46). In some cases, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of the non-interacting face are substituted (e.g., non-conservative and/or conservative amino acid substitutions). In some cases, the amino acids of the non-interacting face at one or more of positions 2, 5, 8, 9, 12, and 13 of SEQ ID NO:1 are substituted by non-conservative and/or conservative amino acid substitutions. In some cases, the amino acids of the non-interacting face at one or more of positions 1, 2, 8, 9, 12, 13, 19, 20, 23, or 24 of SEQ ID NO:1 are substituted by non-conservative and/or conservative amino acid substitutions. In some cases, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 of the amino acids of the non-interacting face are substituted. In some cases the substitutions are non-conservative amino acid substitutions. In other cases, the substitutions are conservative amino acid substitutions. In some cases, the substitutions include both conservative and non-conservative amino acid substitutions.

    [0212] In some instances, substitutions can be made at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) of positions 1, 2, 4-20, 22-24, 25, and 26, wherein the position numbering is provided with respect to SEQ ID NO:1. In some instances, ?,?-disubstituted non natural amino acids can be introduced in to the polypeptide (to enable internal cross-linking) at one or more of the following groups of positions: 5 and 9; 15 and 19; 16 and 20; 14 and 18; 18 and 22; 19 and 23; 9 and 16; 10 and 17; 11 and 18; 12 and 19; 13 and 70; or 15 and 22, wherein the position numbering is provided with respect to SEQ ID NO:1.

    [0213] In some instances, each peptide in Table 1 can include beta alanine at the N-terminus. In some embodiments, a conjugate can be coupled to the N-terminus of the peptides in Table 1. In some instances, the conjugate is a detection moiety disclosed herein (e.g., FITC (fluorescein isothiocyanate)). In some instances, the conjugate is a capture moiety disclosed herein (e.g., a biotin moiety).

    [0214] Further exemplary amino acid sequences of the ACE2 in Mus musculus (NP_081562.2); Rattus norvegicus (NP_001012006.1); Danio rerio (XP_005169416.1); Sus scrofa (NP_001116542.1); Bos taurus (XP_005228485.1); Pan troglodytes (XP_016798468.1); Macaca mulatta (NP_001129168.1); and Canis lupusfamiliaris (NP_001158732.1) are aligned to the first 80 amino acids of SEQ ID NO:22, as shown in FIG. 2, with the ACE2 ?1 helix sequence boxed. In some instances, IEEQAKTFLDKFNHEAEDLFYQSSLA (SEQ ID NO:1) varies at different amino acid positions based on differences in amino acid sequences provided in FIG. 2 and listed in Table 2.

    TABLE-US-00009 TABLE2 ACE2Helix1PeptideAnalogs. Name Sequence SEQIDNO: ACE2h1 IEEQAKTFLDKFNHEAEDLFYQSSLA 1 (native) ACE2h1 Xaa.sub.1EXaa.sub.2Xaa.sub.3Xaa.sub.4Xaa.sub.5Xaa.sub.6FLXaa.sub.7Xaa.sub.8FXaa.sub.9 151 homologous Xaa.sub.10EAXaa.sub.11Xaa.sub.12Xaa.sub.13Xaa.sub.14YQXaa.sub.15Xaa.sub.16SS variant LA,wherein Xaa.sub.1=I,T,V,orisabsent Xaa.sub.2=EorD Xaa.sub.3=Q,N,K,R,orL Xaa.sub.4=AorV Xaa.sub.5=K,E,orR Xaa.sub.6=T,S,orE Xaa.sub.7=D,N,orE Xaa.sub.8=KorN Xaa.sub.9=NorD Xaa.sub.10=H,Q,E,L,orY Xaa.sub.11=EorS Xaa.sub.12=DorE Xaa.sub.13=LorI Xaa.sub.14=F,S,M,Nle(B),orA Xaa.sub.15=SorY Xaa.sub.16=SorT

    [0215] In some instances, each peptide in Table 2 can include beta alanine at the N-terminus. In some embodiments, a conjugate can be coupled to the N-terminus of the peptides listed in Table 2. In some instances, the conjugate is a detection moiety disclosed herein (e.g., FITC (fluorescein isothiocyanate)). In some instances, the conjugate is a capture moiety disclosed herein (e.g., a biotin moiety).

    [0216] The above described polypeptides have one or more (e.g., 1, 2, 3, 4, 5, 6, 7) of the following properties: (i) binds the peptide of SEQ ID NO: 64 or SEQ ID NO: 65; (ii) inhibits interaction between human ACE2 protein and the S1 protein subunit of SARS-CoV-2; (iii) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and the S1 protein subunit of SARS-CoV-2; (iv) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and the RBD of the S1 protein subunit of SARS-CoV-2; (v) competes for human ACE2-SARS-CoV-2 S1 protein subunit binding; (vi) binds the S1 protein subunit (e.g., the RBD) of a SARS-CoV-2 variant (e.g., one or more of B.1.1.7, B.1.351, P.1, B.1.427, B.1.429, B.1.167); and (vii) inhibits SARS virus infection, optionally, wherein the SARS infection is SARS-CoV-2 infection.

    Structurally-Stabilized Peptides of ACE2 ?1 Helix

    [0217] Disclosed herein are structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides. In some instances, the structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides are derived from ACE2 ?1 helix peptides (IEEQAKTFLDKFNHEAEDLFYQSSLA (SEQ ID NO:1)). In some instances, the structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides are derived from ACE2 ?1 helix peptides (IEEQAKTFLDKFNHEAEDLFYQSS (SEQ ID NO:21)). In some instances, the structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides are derived from SEQ ID NO:1. In some instances, the structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides are derived from SEQ ID NO:76. In some instances, the structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides are derived from SEQ ID NO:77. In some instances, the structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides are derived from any one of SEQ ID NOs.: 112, 113, 117, 118, 123, 125, or 127. In some instances, the structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides are variants of any one of SEQ ID NOs.: 1, 21, 76, 77, 112, 113, 117, 118, 123, 125, or 127. Such variants differ from the recited sequence at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids and have one or more (e.g., 1, 2, 3, 4, 5, 6, 7) of the following properties: (i) binds the peptide of SEQ ID NO: 64 or SEQ ID NO: 65; (ii) inhibits interaction between human ACE2 protein and the S1 protein subunit of SARS-CoV-2; (iii) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and the S1 protein subunit of SARS-CoV-2; (iv) inhibits interaction between the carboxypeptidase domain of human ACE2 protein and the RBD of the S1 protein subunit of SARS-CoV-2; (v) competes for human ACE2-SARS-CoV-2 S1 protein subunit binding; (vi) binds the S1 protein subunit (e.g., the RBD) of a SARS-CoV-2 variant (e.g., one or more of B.1.1.7, B.1.351, P.1, B.1.427, B.1.429, B.1.167); and (vii) inhibits SARS virus infection, optionally, wherein the SARS infection is SARS-CoV-2 infection.

    [0218] In some instances, the structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides has an interacting face (i.e., the face of the helix that interacts with the RBD of the virus) comprising positions 3 (corresponding to E23 of SEQ ID NO:22), 4 (Q24), 7 (T27), 10 (D30), 11 (K31), 14 (H34), 15 (E35), 21 (Y41), 22 (Q42), and 25 (L45) of SEQ ID NO:1. In some cases, the amino acids of the interacting face are not substituted. In some cases 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acids of the interacting face are substituted (e.g., conservative amino acid substitutions or to alanine). In some instances, the structurally-stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides has a non-interacting face (i.e., the face of the helix that does not interact with the RBD of the virus) comprising positions 1 (corresponding to 121 of SEQ ID NO:22), 2 (E22), 5 (A25), 6 (K26), 8 (F28), 9 (L29), 12 (F32), 13 (N33), 16 (A36), 17 (E37), 18 (D38), 19 (L39), 20 (F40), 23 (S43), 24 (S44), and 26 (A46) of SEQ ID NO:1. In some cases 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acids of the non-interacting face are substituted (e.g., non-conservative or conservative amino acid substitutions). In some cases, the amino acids of the non-interacting face at one or more of positions 2, 5, 8, 9, 12, 13, and 14 are substituted. In some cases, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 of the amino acids of the non-interacting face are substituted. In some cases, the substitutions are non-conservative amino acid substitutions. In other cases, the substitutions are conservative amino acid substitutions. In some cases, the substitutions include both conservative and non-conservative amino acid substitutions.

    [0219] In one aspect, this disclosure features an internally cross-linked ACE2h1 peptide that binds to both the S1 protein and/or RBD of SARS-CoV-2 and the S1 protein and/or RBD of one or more SARS-CoV-2 variants, optionally wherein the SARS-CoV-2 variant B.1.1.7, B.1.351, P.1, B.1.427, B.1.429, or B.1.167. In some instances, the internally cross-linked peptide has an amino acid sequence that differs from any one of SEQ ID NOs.: 77, 90-95, 98-100, 105-108, 110, 112, 113, 117, 118, 123, 125, or 127 at 2, 3, 4, 5, 6, 7, or 8 amino acid positions. In some instances, the internally cross-linked peptide comprises ?, ?-disubstituted non-natural amino acids with olefinic side chains that are internally cross-linked, wherein the ?, ?-disubstituted non-natural amino acids are inserted at one or more of (i)-(vi): (i) positions 5 and 12, (ii) positions 11 and 18, (iii) positions 12 and 19, (iv) positions 14 and 18, (v) positions 15 and 19, or (vi) positions 16 and 20, wherein the position numbering is provided based on the N-terminal E (position 1) to the C-terminal Q (position 20) of SEQ ID NO:77. In some cases, the peptides is 20 to 25, 20 to 30, 20 to 35, 20 to 40, 20 to 45, 20 to 50, 20 to 60, to 70, 20 to 80, 20 to 90, or 20 to 100 amino acids in length. Also featured are pharmaceutical compositions comprising the above internally cross-linked peptide and a pharmaceutically acceptable carrier. These internally cross-linked peptides can be used to treat or prevent a coronavirus infection (e.g., SARS-CoV-1, SARS-CoV-2, HcoV-NL63). These internally cross-linked peptides can be used to treat or prevent post-acute sequelae of SARS-CoV-2. These internally cross-linked peptides are especially useful to treat or prevent infection by variants of SARS-CoV-2.

    [0220] In some instances, the structurally stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides derived from SEQ ID NO:1 or 21 include SAH-ACE2h1-1-SAH-ACE2h-land SAH-ACE2h1-21-SAH-ACE2h1-24 (e.g., SEQ ID NOs: 2-12, 17-20, 51, 52, 55, 57-60, 134-143, and 172), as shown in Table 3 below. In some instances, the structurally stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides derived from SEQ ID NO:76 include SEQ ID NOs: 78-111, as shown in Table 3 below. In some instances, the structurally stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides derived from SEQ ID NO:21 include SEQ TD NOs: 112-126, as shown in Table 3 below. In some instances, the structurally stabilized (e.g., stapled or stitched) ACE2 ?1 helix peptides derived from SEQ TD NO:77 include SEQ ID NOs: 127-133, as shown in Table 3 below.

    TABLE-US-00010 TABLE3 ACE2Helix1PeptideAnalogs. Name Sequence SEQIDNO: SAH- X.sub.1EEQX.sub.2KTFLDKFNHEAEDLFYQSSXaa.sub.1Xaa.sub.2, 2 ACE2h1- whereinX.sub.1andX.sub.2arenon-naturalaminoacidswith 1 olefinicsidechains;Xaa.sub.1=Lorabsent;Xaa.sub.2=Aor absent SAH- IEEQX.sub.1KTFX.sub.2DKFNHEAEDLFYQSSXaa.sub.1Xaa.sub.2, 3 ACE2h1- whereinX.sub.1andX.sub.2arenon-naturalaminoacidswith 2 olefinicsidechains;Xaa.sub.l=Lorabsent;Xaa.sub.2=Aor absent SAH- IEEQAKTX.sub.1LDKX.sub.2NHEAEDLFYQSSXaa.sub.1Xaa.sub.2, 4 ACE2h1- whereinX.sub.1andX.sub.2arenon-naturalaminoacidswith 3 olefinicsidechains;Xaa.sub.1=Lorabsent;Xaa.sub.2=Aor absent SAH- IEEQAKTFLDKX.sub.1NHEX.sub.2EDLFYQSSXaa.sub.1Xaa.sub.2, 5 ACE2h1- whereinX.sub.1andX.sub.2arenon-naturalaminoacidswith 4 olefinicsidechains;Xaa.sub.1=Lorabsent;Xaa.sub.2=Aor absent SAH- IEEQAKTFLDKFNHEX.sub.1EDLX.sub.2YQSSXaa.sub.1Xaa.sub.2, 6 ACE2h1- whereinX.sub.1andX.sub.2arenon-naturalaminoacidswith 5 olefinicsidechains;Xaa.sub.1=Lorabsent;Xaa.sub.2=Aor absent SAH- IEEQAKTF8DKFNHEXEDLFYQSSXaa.sub.1Xaa.sub.2, 7 ACE2h1- wherein8andXarenon-naturalaminoacidswith 6 olefinicsidechains;Xaa.sub.1=Lorabsent;Xaa.sub.2=Aor absent SAH- IEEQ8KTFLDKXNHEAEDLFYQSSXaa.sub.1Xaa.sub.2, 8 ACE2h1- wherein8andXarenon-naturalaminoacidswith 7 olefinicsidechains;Xaa.sub.1=Lorabsent;Xaa.sub.2=Aor absent SAH- IEEQAKTFLDK8NHEAEDXFYQSSXaa.sub.1Xaa.sub.2, 9 ACE2h1- wherein8andXarenon-naturalaminoacidswith 8 olefinicsidechains;Xaa.sub.1=Lorabsent;Xaa.sub.2=Aor absent SAH- X.sub.1EEQX.sub.2KTFLDKFNHEX.sub.3EDLX.sub.4YQSSXaa.sub.1Xaa.sub.2, 10 ACE2h1- whereinX.sub.1,X.sub.2,X.sub.3,andX.sub.4arenon-naturalamino 9 acidswitholefinicsidechains;Xaa.sub.1=Lorabsent; Xaa.sub.2=Aorabsent SAH- X.sub.1EEQX.sub.2KTFLDKX.sub.3NHEX.sub.4EDLFYQSSXaa.sub.1Xaa.sub.2, 11 ACE2h1- whereinX.sub.1,X.sub.2,X.sub.3,andX.sub.4arenon-naturalamino 10 acidswitholefinicsidechains;Xaa.sub.1=Lorabsent; Xaa.sub.2=Aorabsent SAH- IEEQX.sub.1KTFX.sub.2DKFNHEX.sub.3EDLX4YQSSXaa.sub.1Xaa.sub.2, 12 ACE2h1- whereinX.sub.1,X.sub.2,X.sub.3,andX.sub.4arenon-naturalamino 11 acidswitholefinicsidechains;Xaa.sub.1=Lorabsent; Xaa.sub.2=Aorabsent SAH- X.sub.aa1EEQX.sub.aa2KX.sub.aa3X.sub.aa4X.sub.aa5DKX.sub.aa6X.sub.aa7HEX.sub.aa8ED 17 ACE2h1- X.sub.aa9X.sub.aa10YX.sub.aa11X.sub.aa12X.sub.aa13X.sub.aa14X.sub.aa15,wherein 21 X.sub.aa1=I,A,orastaplingaminoacid X.sub.aa2=Aorastaplingaminoacid X.sub.aa3=T,E,orF X.sub.aa4=F,A,orastaplingaminoacid X.sub.aa5=L,A,orastaplingaminoacid X.sub.aa6=F,A,orastaplingaminoacid X.sub.aa7=NorA X.sub.aa8=Aorastaplingaminoacid X.sub.aa9=L,A,orastaplingaminoacid X.sub.aa10=F,A,orastaplingaminoacid X.sub.aa11=QorY X.sub.aa12=SorA X.sub.aa13=SorA X.sub.aa14=L,A,orabsent X.sub.aa15=Aorabsent SAH- X.sub.1EEQX.sub.2KTFLDKFNHEAEDLFYQSS 172 ACE2h1- 1-alt SAH- IEEQX.sub.1KTFX.sub.2DKFNHEAEDLFYQSS 134 ACE2h1- 2-alt SAH- IEEQAKTX.sub.1LDKX.sub.2NHEAEDLFYQSS 135 ACE2h1- 3-alt SAH- IEEQAKTFLDKX.sub.1NHEX.sub.2EDLFYQSS 136 ACE2h1- 4-alt SAH- IEEQAKTFLDKFNHEX.sub.1EDLX.sub.2YQSS 137 ACE2h1- 5-alt SAH- IEEQAKTF8DKFNHEXEDLFYQSS 138 ACE2h1- 6-alt SAH- IEEQ8KTFLDKXNHEAEDLFYQSS 139 ACE2h1- 7-alt SAH- IEEQAKTFLDK8NHEAEDXFYQSS 140 ACE2h1- 8-alt SAH- X.sub.1EEQX.sub.2KTFLDKFNHEX.sub.3EDLX.sub.4YQSS 141 ACE2h1- 9-alt SAH- X.sub.1EEQX.sub.2KTFLDKX.sub.3NHEX.sub.4EDLFYQSS 142 ACE2h1- 10-alt SAH- IEEQX.sub.1KTFX.sub.2DKFNHEX.sub.3EDLX.sub.4YQSS 143 ACE2h1- 11-alt SAH- X.sub.aa1X.sub.aa2EQX.sub.aa3X.sub.aa4TX.sub.aa5X.sub.aa6DKX.sub.aa7X.sub.aa8HEX.sub.aa9 18 ACE2h1- X.sub.aa10X.sub.aa11X.sub.aa12X.sub.aa13YQX.sub.aa14X.sub.aa15LX.sub.aa16,wherein 22 X.sub.aa1=I,A,orastaplingaminoacid X.sub.aa2=EorA X.sub.aa3=Aorastaplingaminoacid X.sub.aa4=K,R,orA X.sub.aa5=F,A,orastaplingaminoacid X.sub.aa6=L,A,orastaplingaminoacid X.sub.aa7=F,A,orastaplingaminoacid X.sub.aa8=NorA X.sub.aa9=Aorastaplingaminoacid X.sub.aa10=E,D,orA X.sub.aa11=D,E,orA X.sub.aa12=L,A,orastaplingaminoacid X.sub.aa13=F,A,orastaplingaminoacid X.sub.aa14=SorA X.sub.aa15=SorA X.sub.aal6=Aorabsent SAH- IEEQAK.sub.6TFLD.sub.10K.sub.11FNHEAED18LFYQSSLA, 19 ACE2h1- whereinK.sub.6andD.sub.10arelinkedbyalactambridge;or 23 whereinK.sub.11andD.sub.18arelinkedbyalactambridge SAH- X.sub.aa1EEQX.sub.aa2K.sub.6X.sub.aa3X.sub.aa4X.sub.aa5D.sub.10K.sub.11X.sub.aa6X.sub.aa7HE 20 ACE2h1- X.sub.aa8ED.sub.18X.sub.aa9X.sub.aa10YX.sub.a11X.sub.aa12X.sub.aa13X.sub.aa14X.sub.aa15, 24 wherein X.sub.aa1=IorA, X.sub.aa2=A X.sub.aa3=T,E,orF X.sub.aa4=ForA X.sub.aa5=LorA X.sub.aa6=ForA X.sub.aa7=NorA X.sub.aa8=A= X.sub.aa9=LorA X.sub.aa10=ForA X.sub.aa11=QorY X.sub.aa12=SorA X.sub.aa13=SorA X.sub.aa14=LorA X.sub.aa15=Aorabsent, whereinK.sub.6andD.sub.10arelinkedbyalactambridge;or whereinK.sub.11andD.sub.18arelinkedbyalactambridge SAH- XEEQXKTFLDKFNHEAEDLFYQS 78 ACE2h1- (21-43)- A SAH- IXEQAXTFLDKFNHEAEDLFYQS 79 ACE2h1- (21-43)- B SAH- IEXQAKXFLDKFNHEAEDLFYQS 80 ACE2h1- (21-43)- C SAH- IEEQXKTFXDKFNHEAEDLFYQS 81 ACE2h1- (21-43)- D SAH- IEEQAXTFLXKFNHEAEDLFYQS 82 ACE2h1- (21-43)- E SAH- IEEQAKXFLDXFNHEAEDLFYQS 83 ACE2h1- (21-43)- F SAH- IEEQAKTXLDKXNHEAEDLFYQS 84 ACE2h1- (21-43)- G SAH- IEEQAKTFXDKFXHEAEDLFYQS 85 ACE2h1- (21-43)- H SAH- IEEQAKTFLXKFNXEAEDLFYQS 86 ACE2h1- (21-43)- I SAH- IEEQAKTFLDXFNHXAEDLFYQS 87 ACE2h1- (21-43)- J SAH- IEEQAKTFLDKXNHEXEDLFYQS 88 ACE2h1- (21-43)- K SAH- IEEQAKTFLDKFXHEAXDLFYQS 89 ACE2h1- (21-43)- L SAH- IEEQAKTFLDKFNXEAEXLFYQS 90 ACE2h1- (21-43)- M SAH- IEEQAKTFLDKFNHXAEDXFYQS 91 ACE2h1- (21-43)- N SAH- IEEQAKTFLDKFNHEXEDLXYQS 92 ACE2h1- (21-43)- O SAH- IEEQAKTFLDKFNHEAXDLFXQS 93 ACE2h1- (21-43)- P SAH- IEEQAKTFLDKFNHEAEXLFYXS 94 ACE2h1- (21-43)- Q SAH- IEEQAKTFLDKFNHEAEDXFYQX 95 ACE2h1- (21-43)- R SAH- 8EEQAKTXLDKFNHEAEDLFYQS 96 ACE2h1- (21-43)- S SAH- I8EQAKTFXDKFNHEAEDLFYQS 97 ACE2h1- (21-43)- T SAH- IE8QAKTFLXKFNHEAEDLFYQS 98 ACE2h1- (21-43)- U SAH- IEE8AKTFLDXFNHEAEDLFYQS 99 ACE2h1- (21-43)- V SAH- IEEQ8KTFLDKXNHEAEDLFYQS 100 ACE2h1- (21-43)- W SAH- IEEQA8TFLDKFXHEAEDLFYQS 101 ACE2h1- (21-43)- X SAH- IEEQAK8FLDKFNXEAEDLFYQS 102 ACE2h1- (21-43)- Y SAH- IEEQAKT8LDKFNHXAEDLFYQS 103 ACE2h1- (21-43)- Z SAH- IEEQAKTF8DKFNHEXEDLFYQS 104 ACE2h1- (21-43)-a SAH- IEEQAKTFL8KFNHEAXDLFYQS 105 ACE2h1- (21-43)-b SAH- IEEQAKTFLD8FNHEAEXLFYQS 106 ACE2h1- (21-43)-c SAH- IEEQAKTFLDK8NHEAEDXFYQS 107 ACE2h1- (21-43)-d SAH- IEEQAKTFLDKF8HEAEDLXYQS 108 ACE2h1- (21-43)-e SAH- IEEQAKTFLDKFN8EAEDLFXQS 109 ACE2h1- (21-43)-f SAH- IEEQAKTFLDKFNH8AEDLFYXS 110 ACE2h1- (21-43)-g SAH- IEEQAKTFLDKFNHE8EDLFYQX 111 ACE2h1- (21-43)-h SAH- IEEQXKTAXDKANHEAEDAAYQSAXaa.sub.1Xaa.sub.2, 51 ACE2h1- whereinXisanon-naturalaminoacidwitholefinic mut-2 sidechains,Xaa.sub.1isL,A,orabsent,andXaa.sub.2isAor absent SAH- IEEQXKTAXDKANHEXEDAXYQSAXaa.sub.1Xaa.sub.2, 52 ACE2h1- whereinXisanon-naturalaminoacidwitholefinic mut-3 sidechains,whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2 isAorabsent SAH- QEEQXKDAXDHANHEXEYQXYQSAXaa.sub.1Xaa.sub.2, 55 ACE2h1- whereinXisanon-naturalaminoacidwitholefinic mut-6 sidechains,Xaa.sub.1isL,A,orabsent,andXaa.sub.2isAor absent SAH- IEEQAKTXADKXNHEAEQAAYQSAXaa.sub.1Xaa.sub.2, 57 ACE2h1- whereinXisanon-naturalaminoacidwitholefinic mut-8 sidechains,Xaa.sub.1isL,A,orabsent,andXaa.sub.2isAor absent SAH- IEEQXKTAXDKANHEXEQAXYQSAXaa.sub.1Xaa.sub.2, 58 ACE2h1- whereinXisanon-naturalaminoacidwitholefinic mut-9 sidechains,Xaa.sub.1isL,A,orabsent,andXaa.sub.2isAor absent SAH- IEEQXKEAXDKANHEXEQAXYQSAXaa.sub.1Xaa.sub.2, 59 ACE2h1- whereinXisanon-naturalaminoacidwitholefinic mut-10 sidechains,Xaa.sub.1isL,A,orabsent,andXaazisAor absent SAH- IEEQAKTA8DKANHEXEQAAYQSAXaa.sub.1Xaa.sub.2, 60 ACE2h1- wherein8andXarenon-naturalaminoacidswith mut-11 olefinicsidechains;Xaa.sub.1=L,A,orabsent;Xaa.sub.2= Aorabsent SAH- IEEQAKTFLDKFNHEAEDLFYQSA 112 ACE2h1- (21-44)- mut-12 SAH- IEEQAKTAADKANHEAEDAAYQSA 113 ACE2h1- (21-44)- mut-13 SAH- IEEQXKTAXDKANHEAEDAAYQSA 114 ACE2h1- (21-44)- mut- 13(D) SAH- IEEQXKTAXDKANHEXEDAXYQSA 115 ACE2h1- (21-44)- mut- 13(D,O) SAH- IEEQXKEAXDKANHEXEDAXYQSA 116 ACE2h1- (21-44)- mut- 14(D,O) SAH- AEEQAKTAADKAAHEAEQAAYQAA 117 ACE2h1- (21-44)- mut-15 SAH- IEEQAKTAADKANHEAEQAAYQSA 118 ACE2h1- (21-44)- mut-16 SAH- IEEQAKTXADKXNHEAEQAAYQSA 119 ACE2h1- (21-44)- mut- 16(G) SAH- IEEQXKTAXDKANHEXEQAXYQSA 120 ACE2h1- (21-44)- mut- 16(D,O) SAH- IEEQXKEAXDKANHEXEQAXYQSA 121 ACE2h1- (21-44)- mut- 17(D,O) SAH- IEEQAKTA8DKANHEXEQAAYQSA 122 ACE2h1- (21-44)- mut- 18(D,O) SAH- IQEQAKTDADKHNHEAEDYQYQSA 123 ACE2h1- (21-44)- mut-19 SAH- IQEQXKTDXDKHNHEAEDYQYQSA 124 ACE2h1- (21-44)- mut-20 SAH- QEEQAKDAADHANHEAEYQAYQSA 125 ACE2h1- (21-43)- mut-21 SAH- QEEQXKDAXDHANHEXEYQXYQSA 126 ACE2h1- (21-43)- mut- 21(D,O) SAH- ETVDFFAEWFDVEAEDKDYL 127 ACE2h1- (23-42)- mut-22 SAH- ETXDFFXEWADVXAEDXDYL 128 ACE2h1- (23-42)- mut- 23(D,N) SAH- ETXDFFXEWADVEXEDKXYL 129 ACE2h1- (23-42)- mut- 23(D,O) SAH- ETXDFFXEWFDVXAEDXDYL 130 ACE2h1- (23-42)- mut- 22(D,N) SAH- ETXDFEXEWFDVXAEDXDYL 131 ACE2h1- (23-42)- mut- 24(D,N) SAH- ETXDFLXEWFDVXAEDXDYL 132 ACE2h1- (23-42)- mut- 25(D,N) SAH- ETXDFYXEWFDVXAEDXDYL 133 ACE2h1- (23-42)- mut- 26(D,N)

    [0221] In Table 3, X.sub.1, Xa, X.sub.3, X.sub.4, 8, and X are all ?,?-disubstituted non-natural amino acids with olefinic side chains (which can be cross-linked by e.g., a RCM reaction). R.sub.8 and S5 are ?-methylated and the final stabilized (e.g., stapled) peptide is produced by ring-closing metathesis (and loss of ethylene). In some instances, X.sub.1, X.sub.2, X.sub.3, X.sub.4=(4-pentenyl)alanine or (S)-pentenyl alanine (S5); 8=2-(7-octenyl)alanine or (R)-octenyl alanine residues (R8); and X=(4-pentenyl)alanine or S5.

    [0222] In some instances (e.g., SEQ ID NOs: 2-9, 51, 57, and 60), the structurally-stabilized peptide is single-stapled peptide. In some instances (e.g., SEQ ID NOs: 10-12, 52, 55, 58, and 59) the structurally-stabilized peptide is a double-stapled peptide. In some instances (e.g., SEQ TD NO: 17 or 18), the structurally-stabilized peptide is stapled peptide and includes 1 or 2 staples. In some instances (e.g., SEQ TD NO: 19 or 20), the structurally-stabilized peptide includes lactam bridge links between a lysine (K) and an aspartic acid (D) of the ACE2 ?1 helix or a variant thereof.

    [0223] In some instances, each peptide in Table 3 can include beta alanine at the N-terminus. In some embodiments, a conjugate can be coupled to the N-terminus of the peptides in Table 3. In some instances, the conjugate is a detection moiety disclosed herein (e.g., FITC (fluorescein isothiocyanate)). In some instances, the conjugate is a capture moiety disclosed herein (e.g., a biotin moiety).

    [0224] In some instances, the stapled peptide is an internally cross-linked peptide of any one of the sequences set forth in any one of SEQ ID NOs.:78-111, 114-116, 119-122, 124, 126, or 128-133.

    [0225] In some instances, disclosed herein is a stapled or stitched peptide comprising or consisting of any one of the amino acids sequences of SEQ ID NO: 1, 21, 76, 77, 112, 113, 117, 118, 123, 125, or 127, except that at least two (e.g., 2, 3, 4, 5, 6) amino acids of SEQ ID NO: 1 or 21 are replaced with a non-natural amino acid capable of forming a staple or stitch. In some instances, the non-natural amino acid is an ?, ?-disubstituted non-natural amino acids with olefinic side chains. In some instances, the stapled peptide of SEQ ID NO: 1, 21, 76, 77, 112, 113, 117, 118, 123, 125, or 127 further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions and can bind the S1 protein and/or RBD of SARS-CoV-2 or a SARS-CoV-2 variant.

    [0226] In some instances, the non-natural amino acids that may be used as stapling amino acids or stitching amino acids are: (R)-2-(7-octenyl)alanine; (R)-2-(4-pentenyl)alanine; (R)-?-(7-octenyl)alanine; (R)-?-(4-pentenyl)alanine; (S)-?-(7-octenyl)alanine; (S)-2-(7-octenyl)alanine; (S)-?-(4-pentenyl)alanine; (S)-2-(4-pentenyl)alanine; ?,?-Bis(4-pentenyl)glycine; and ?,?-Bis(7-octeny)glycine.

    [0227] In some embodiments, an internal staple replaces the side chains of 2 amino acids, i.e., each staple is between two amino acids separated by, for example, 2, 3, or 6 amino acids. In some embodiments, an internal stitch replaces the side chains of 3 amino acids, i.e., the stitch is a pair of crosslinks between three amino acids separated by, for example, 2, 3, or 6 amino acids. In some embodiments, the amino acids forming the staple or stitch are at each of positions i and i+3 of the staple. In some embodiments, the amino acids forming the staple or stitch are at each of positions i and i+4 of the staple. In some embodiments, the amino acids forming the staple or stitch are at each of positions i and i+7 of the staple. For example, where a peptide has the sequence . . . X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7, X.sub.8, X.sub.9 . . . , cross-links between X.sub.1 and X.sub.4 (i and i+3), or between X.sub.1 and X.sub.5 (i and i+4), or between X.sub.1 and X.sub.8 (i and i+7) are useful hydrocarbon stapled forms of that peptide. The use of multiple cross-links (e.g., 2, 3, 4, or more) is also contemplated. Additional description regarding making and use of hydrocarbon-stapled peptides can be found, e.g., in U.S. Patent Publication Nos. 2012/0172285, 2010/0286057, and 2005/0250680, the contents of which are incorporated by reference herein in their entireties.

    [0228] In some embodiments, the amino acids forming the staple or stitch are at each of positions i and i+4 of the staple. In some instances, to achieve the various staple lengths, ?-methyl, ?-alkenyl amino acids may be installed at i, i+4 positions using two (S)-pentenyl alanine residues (S5). In some instances, ?-methyl, ?-alkenyl amino acids may be installed at i, i+4 positions using (R)-octenyl alanine residues (R.sub.8). In some embodiments, the amino acids forming the staple or stitch are at each of positions i and i+7 of the staple. In some instances, to achieve the various staple lengths, ?-methyl, ?-alkenyl amino acids may be installed at i, i+7 positions using one (S)-pentenyl alanine residues (S5) at a first position i and one (R)-octenyl alanine residues (R8) at a second position i+7. In one instance, to achieve the various staple lengths, ?-methyl, ?-alkenyl amino acids may be installed at i, i+7 positions using one (R)-octenyl alanine residues (R8) at a first position i and one (S)-pentenyl alanine residues (S5) at a second position i+7.

    [0229] Peptide stapling is a term coined from a synthetic methodology wherein two olefin-containing side-chains (e.g., cross-linkable side chains) present in a peptide chain are covalently joined (e.g., stapled together) using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (see, e.g., Blackwell et al., J. Org. Chem., 66: 5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). The structural-stabilization may be by, e.g., stapling the peptide (see, e.g., Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are incorporated by reference herein in its entirety). In some cases, the staple is a hydrocarbon staple.

    [0230] In some instances, the structural-stabilization is a stitch. The term peptide stitching, as used herein, refers to multiple and tandem stapling events in a single peptide chain to provide a stitched (e.g., tandem or multiply stapled) peptide, in which two staples, for example, are linked to a common residue. Peptide stitching is disclosed, e.g., in WO 2008/121767 and WO 2010/068684, which are both hereby incorporated by reference in their entirety.

    [0231] In some instances, a staple or stitch used herein is a lactam staple or stitch. In some instances, the lactam staple or stitch couples a lysine residue side chain and to an aspartic acid or glutamic acid residues side-chain.

    [0232] In some instances, a staple or stitch used herein is a UV-cycloaddition staple or stitch; an oxime staple or stitch; a thioether staple or stitch; a double-click staple or stitch; a bis-lactam staple or stitch; a bis-arylation staple or stitch; or a combination of any two or more thereof. Stabilized peptides as described herein include stapled peptides and stitched peptides as well as peptides containing multiple stitches, multiple staples or a mix of staples and stitches, or any other chemical strategies for structural reinforcement (see, e.g., Balaram P. Cur. Opin. Struct. Biol. 1992; 2:845; Kemp D S, et al., J. Am. Chem. Soc. 1996; 118:4240; Orner B P, et al., J. Am. Chem. Soc. 2001; 123: 5382; Chin J W, et al., Int. Ed. 2001; 40:3806; Chapman R N, et al., J. Am. Chem. Soc. 2004; 126: 12252; Horne W S, et al., Chem., Int. Ed. 2008; 47:2853; Madden et al., Chem Commun (Camb). 2009 Oct. 7; (37): 5588-5590; Lau et al., Chem. Soc. Rev., 2015, 44:91-102; and Gunnoo et al., Org. Biomol. Chem., 2016, 14:8002-8013; each of which is incorporated by reference herein in its entirety).

    [0233] A peptide is structurally-stabilized in that it maintains its native secondary structure. For example, stapling allows a peptide, predisposed to have an ?-helical secondary structure, to maintain its native ?-helical conformation. This secondary structure increases resistance of the peptide to proteolytic cleavage and heat, and may increase target binding affinity, hydrophobicity, and cell permeability. Accordingly, the stapled (cross-linked) peptides described herein have improved biological activity relative to a corresponding non-stapled (un-cross-linked) peptide.

    [0234] In certain instances, the modification(s) to introduce structural stabilization (e.g., internal cross-linking, e.g., stapling, stitching) into the ACE2 ?1 helix peptides described herein may be positioned on the face of the ACE2 ?1 helix that does not interact with SARS-CoV-2 S1. In certain instances, the modification(s) to introduce structural stabilization (e.g., internal cross-linking, e.g., stapling, stitching) into the ACE2 ?1 helix peptides described herein may be positioned on the face of the ACE2 ?1 helix that interacts with SARS-CoV-2 S1.

    [0235] In some instances, the modifications to introduce structural stabilization (e.g., internal cross-linking, e.g., stapling or stitching) into the ACE2 ?1 helix peptides described herein are positioned at the amino acid positions in the ACE2 ?1 helix peptide corresponding to residues: [0236] (i) 1 and 5 of SEQ ID NO: 1 or 21; [0237] (ii) 5 and 9 of SEQ ID NO: 1 or 21; [0238] (iii) 8 and 12 of SEQ ID NO: 1 or 21; [0239] (iv) 12 and 16 of SEQ ID NO: 1 or 21; [0240] (v) 9 and 16 of SEQ ID NO: 1 or 21; [0241] (vi) 5 and 12 of SEQ ID NO: 1 or 21; [0242] (vii) 12 and 19 of SEQ ID NO: 1 or 21; [0243] (viii) 1, 5, 16, and 20 of SEQ ID NO: 1 or 21; [0244] (ix) 1, 5, 12, and 16 of SEQ ID NO: 1 or 21; or [0245] (x) 5, 9, 16, and 20 of SEQ ID NO: 1 or 21.

    [0246] In some instances, the modifications to introduce structural stabilization (e.g., internal cross-linking, e.g., stapling or stitching) into the ACE2 ?1 helix peptides described herein are positioned at the amino acid positions in the ACE2 ?1 helix peptide corresponding to residues 1, 5, 8, 9, 12, 16, 19, 20, or a combination thereof, of SEQ ID NO: 1 or 21.

    [0247] In some instances, the modifications to introduce structural stabilization (e.g., internal cross-linking, e.g., stapling or stitching) into the ACE2 ?1 helix peptides described herein are positioned at the amino acid positions in the ACE2 ?1 helix peptide corresponding to residues 1, 5, 8, 9, 12, 16, 19, 20, or a combination thereof, of SEQ ID NO: 1 or 21.

    [0248] In certain instances, the ACE2 ?1 helix peptides described herein (e.g., SEQ ID NOs: 11-52) may also contain one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions (relative to an amino acid sequence set forth in any one of SEQ ID NOs: 2-12 17-20, 134-143, and 172), e.g., one or more (e.g., 1, 2, 3, 4, or 5) conservative and/or non-conservative amino acid substitutions. In some instances, the ACE2 ?1 helix peptides described herein (e.g., SEQ ID NOs: 2-12 17-20, 134-143, and 172) may also contain at least one, at least 2, at least 3, at least 4, or at least 5 amino acids added to the N-terminus of the peptide. In some instances, the ACE2 ?1 helix peptides described herein (e.g., SEQ ID NOs: 2-12 17-20, 134-143, and 172) may also contain at least one, at least 2, at least 3, at least 4, or at least 5 amino acids added to the C-terminus of the peptide.

    [0249] In one aspect, the structurally-stabilized ACE2 ?1 helix peptide comprises Formula (I),

    ##STR00004##

    or a pharmaceutically acceptable salt thereof. In some instances, each R.sub.1 and R.sub.2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted. In some instances, each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; [0250] each x is independently 2, 3, or 6. In some instances, each w and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some instances, z is 1, 2, or 3. In some instances, each Xaa is independently an amino acid. In some instances, the stabilized peptide prevents or inhibits the interaction between Angiotensin converting enzyme 2 (ACE2) and a virus whose receptor binding domain binds ACE2. In some instances, the sequence of the peptide of pharmaceutically acceptable salt thereof has SEQ ID NO: 1 with: [0251] (i) at least 2 amino acids that are substituted with ?, ?-disubstituted non-natural amino acids with olefinic side chains that form an internal cross-link, [0252] (ii) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 additional amino acid substitutions; and [0253] (iii) 0, 1, 2, 3, 4, or 5 deletions at the N and/or C-terminus of the sequence.

    [0254] In some embodiments, each of the [Xaa].sub.w of Formula (I), the [Xaa].sub.x of Formula (I), and the [Xaa].sub.y of Formula (I) is as described for any one of constructs 1-8 or 12-14 in Table 4. For example, for a stabilized peptide comprising the [Xaa].sub.w, the [Xaa].sub.x, and the [Xaa].sub.y of construct 2 of Table 4, the [Xaa].sub.w, the [Xaa].sub.x, and the [Xaa].sub.y is IEEQ (SEQ ID NO: 24), KTF, and DKFNHiEAEDLFYQSSXaa1Xaa2 (SEQ ID NO: 153), respectively. As another example, for a stabilized peptide comprising the [Xaa].sub.w, the [Xaa].sub.x, and the [Xaa].sub.y of construct 3 of Table 4, the [Xaa].sub.w, the [Xaa].sub.x, and the [Xaa].sub.y is IEEQAKT (SEQ ID NO: 26), LDK, and NHEAEDLFYQSSXaa1Xaa2 (SEQ ID NO: 156), respectively.

    TABLE-US-00011 TABLE4 [Xaa].sub.w,[Xaa].sub.x,and[Xaa].sub.ysequencesforFormula(I)constructs1-8 and12-14. Construct [Xaa].sub.w [Xaa].sub.x [Xaa].sub.y 1(SAH- EEQ KTFLDKFNHEAE(D/Q) ACE2h1-1) LFYQSSXaa.sub.1Xaa.sub.2 (SEQIDNO:23), wherein Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 2(SAH- IEEQ(SEQIDNO:24) KTF DKFNHEAE(D/Q)LFY ACE2h1-2) QSSXaa.sub.1Xaa.sub.2(SEQID NO:153),wherein Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 3(SAH- IEEQAKT(SEQIDNO: LDK NHEAE(D/Q)LFYQSS ACE2h1-3) 26) Xaa.sub.1Xaa.sub.2(SEQIDNO: 156),wherein Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 4(SAH- IEEQAKTFLDK(SEQ NHE E(D/Q)LFYQSSXaa.sub.1Xaa.sub.2 ACE2h1-4) IDNO:29) (SEQIDNO:31), wherein Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 5(SAH- IEEQAKTFLDKFNHE EDL YQSSXaa.sub.1Xaa.sub.2(SEQ ACE2h1-5) (SEQIDNO:32) IDNO:33),wherein Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 6(SAH- IEEQAKTF(SEQID DKFNHE(SEQID E(D/Q)LFYQSS ACE2h1-6) NO:34) NO:35) Xaa.sub.1Xaa.sub.2(SEQIDNO: 36),wherein Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 7(SAH- IEEQ(SEQIDNO:24) KTFLDK(SEQID NHEAE(D/Q)LFYQSS ACE2h1-7) NO:38) Xaa.sub.1Xaa.sub.2(SEQID NO:39),wherein Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 8(SAH- IEEQAKTFLDK(SEQ NHEAE(D/Q) FYQSSXaa.sub.1Xaa.sub.2(SEQ ACE2h1-8) IDNO:29) (SEQIDNO:41) IDNO:42),wherein Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 12 IEEQ(SEQIDNO:24) KTA DKANHEAE(D/Q)AAYQ SAXaa.sub.1Xaa.sub.2(SEQID NO:67),whereinXaa.sub.1is L,A,orabsent,andXaa.sub.2 isAorabsent 13 IEEQAKT(SEQIDNO: ADK NHEAEQAAYQSAXaa.sub.1 26) Xaa.sub.2(SEQIDNO:68), whereinXaa.sub.1isL,A,or absent,andXaa.sub.2isAor absent 14 IEEQAKTA(SEQID DKANHE(SEQID EQAAYQSAXaa.sub.1Xaa.sub.2 NO:61) NO:62) (SEQIDNO:69),wherein Xaa.sub.1isL,A,orabsent, andXaa2isAorabsent

    [0255] In certain instances, the sequences set forth above in Table 4 can have at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid substitution or deletion. All of these peptides and their variants bind the RBD of the virus (e.g., SARS-CoV-2) and inhibit its interaction with ACE2 on the cell (e.g., human respiratory cell) surface. The ACE2 ?1 helix peptides can include any amino acid sequence described herein.

    [0256] In addition to the sequences provided in Table 4, peptides comprising Formula (I) also include SEQ ID NOs: 78-111, 114, 119, and 122, as shown in FIG. 8.

    [0257] In some instances, a peptide comprising Formula (I) comprising the sequences set forth above in Table 4 can have one or more of the properties listed below: (i) the peptide prevents or inhibits the interaction between Angiotensin converting enzyme 2 (ACE2) and a virus whose receptor binding domain binds ACE2; (ii) the peptide is alpha helical; (iii) the peptide is protease resistant; and/or (iv) the peptide blocks or inhibits infection of human ACE2 expressing epithelial cells. In some instances, the epithelial cells are located in the respiratory system.

    [0258] In some instances, the peptide of Formula (I) has a sequence of any one of SEQ ID NOs.: 78-111, wherein [Xaa].sub.w refers to the amino acids corresponding to those before the first (N-terminal most) stapling amino acid; [Xaa].sub.x refers to the amino acids corresponding to those between the first (N-terminal most) and second (N-terminal most) stapling amino acid; and Xaa].sub.y refers to the amino acids corresponding to those after the last (C-terminal most) stapling amino acid in these sequences.

    [0259] The tether of Formula (I) can include an alkyl, alkenyl, or alkynyl moiety (e.g., C.sub.5, C.sub.8, C.sub.11, or C.sub.12 alkyl, a C.sub.5, C.sub.8, or C.sub.11 alkenyl, or C.sub.5, C.sub.8, C.sub.11, or C.sub.12 alkynyl). The tethered amino acid can be alpha disubstituted (e.g., C.sub.1-C.sub.3 or methyl).

    [0260] In some instances of Formula (I), x is 2, 3, or 6. In some instances of Formula (I), each y is independently an integer between 0 and 15, or 3 and 15. In some instances of Formula (I), R.sub.1 and R.sub.2 are each independently H or C.sub.1-C.sub.3 alkyl. In some instances of Formula (I), R.sub.1 and R.sub.2 are each independently C.sub.1-C.sub.3 alkyl. In some instances of Formula (I), at least one of R.sub.1 and R.sub.2 are methyl. For example, R.sub.1 and R.sub.2 can both be methyl. In some instances of Formula (I), R.sub.3 is alkyl (e.g., C.sub.8 alkyl) and x is 3. In some instances of Formula (I), R.sub.3 is C.sub.11 alkyl and x is 6. In some instances of Formula (I), R.sub.3 is alkenyl (e.g., C.sub.8 alkenyl) and x is 3. In some instances of Formula (I), x is 6 and R.sub.3 is C.sub.11 alkenyl. In some instances, R.sub.3 is a straight chain alkyl, alkenyl, or alkynyl. In some instances, R.sub.3 is CH.sub.2CH.sub.2CH.sub.2CH?CHCH.sub.2CH.sub.2CH.sub.2.

    [0261] In another aspect of Formula (I), the two alpha, alpha disubstituted stereocenters are both in the R configuration or S configuration (e.g., i, i+4 cross-link), or one stereocenter is R and the other is S (e.g., i, i+7 cross-link). Thus, where Formula (I) is depicted as:

    ##STR00005##

    [0262] The C and C disubstituted stereocenters can both be in the R configuration or they can both be in the S configuration, e.g., when x is 3. When x is 6 in Formula (I), the C disubstituted stereocenter is in the R configuration and the C disubstituted stereocenter is in the S configuration. The R.sub.3 double bond of Formula (I) can be in the E or Z stereochemical configuration.

    [0263] In some instances of Formula (I), R.sub.3 is [R.sub.4KR.sub.4].sub.n; and R.sub.4 is a straight chain alkyl, alkenyl, or alkynyl.

    [0264] In some instances, z of Formula (I) is greater than one. In some instances, z=2, as shown in Formula (II). In this instance, the peptide includes more than one staple.

    [0265] In one aspect, disclosed herein is a compound comprising a stabilized peptide comprising a sequence having the formula:

    ##STR00006##

    or a pharmaceutically acceptable salt thereof. In some instances, each R.sub.1, R.sub.3, R.sub.4, and R.sub.6 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted. In some instances, each R.sub.2 and R.sub.5 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; optionally wherein R.sub.2 and R.sub.5 are either C.sub.8 alkylene, C.sub.8 alkenylene, or C.sub.8 alkylene; or C.sub.11 alkylene, C.sub.11 alkenylene, or C.sub.11 alkylene. In some instances, each u and x is independently 2, 3, or 6. In some instances, each t, v, and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some instances, each Xaa is independently an amino acid. In some instances, the stabilized peptide prevents or inhibits the interaction between Angiotensin converting enzyme 2 (ACE2) and a virus whose receptor binding domain binds ACE2. In some instances, the sequence of the peptide or the pharmaceutically acceptable salt thereof has SEQ ID NO: 1 with: [0266] (i) at least 4 amino acids that are substituted with ?, ?-disubstituted non-natural amino acids with olefinic side chains that form an internal cross-link; [0267] (ii) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 additional amino acid substitutions, and/or [0268] (iii) 0, 1, 2, 3, 4, or 5 amino acid deletions at the N and/or C-terminus of the sequence.

    [0269] In some embodiments, each of the [Xaa].sub.t of Formula (II), the [Xaa].sub.u of Formula (II), the [Xaa].sub.v of Formula (II), the [Xaa].sub.x of Formula (II), and the [Xaa].sub.y of Formula (II) is as described for any one of constructs 9-11 of Table 5. For example, for a stabilized peptide comprising the [Xaa].sub.t, the [Xaa].sub.u, the [Xaa].sub.v, the [Xaa].sub.x, and the [Xaa].sub.y of construct 9 of Table 5, the [Xaa].sub.t, the [Xaa].sub.u, the [Xaa].sub.v, the [Xaa].sub.x, and the [Xaa].sub.y is absent; EEQ; KTFLDKFNHE (SEQ ID NO:43); EDL; and YQSS (SEQ ID NO: 161), respectively. As another example, for a stabilized peptide comprising the [Xaa].sub.t, the [Xaa].sub.u, the [Xaa].sub.v, the [Xaa].sub.x, and the [Xaa].sub.y of construct 9 of Table 5, the [Xaa].sub.t, the [Xaa].sub.u, the [Xaa].sub.v, the [Xaa].sub.x, and the [Xaa].sub.y is absent; EEQ; KTFLDK (SEQ ID NO:38); NHE; and EDLFYQSS (SEQ ID NO: 159), respectively.

    TABLE-US-00012 TABLE5 [Xaa].sub.t,[Xaa].sub.u,[Xaa].sub.v,[Xaa].sub.x,and[Xaa].sub.ysequencesforFormula(II) constructs9-11and15-18. Construct [Xaa].sub.t [Xaa].sub.u [Xaa].sub.v [Xaa].sub.x [Xaa].sub.y 9(SAH- absent EEQ KTFLDKFNHE EDL YQSSXaa.sub.1Xaa.sub.2(SEQID ACE2h1- (SEQIDNO: NO:161),wherein 8) 43) Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 10(SAH- absent EEQ KTFLDK(SEQ NHE EDLFYQSSXaa.sub.1Xaa.sub.2 ACE2h1- IDNO:38) (SEQIDNO:159), 9) wherein Xaa.sub.1=Lorabsent Xaa.sub.2=Aorabsent 11(SAH- IEEQ KTF DKFNHE(SEQ EDL YQSSXaa.sub.1Xaa.sub.2(SEQID ACE2h1- (SEQ IDNO:48) NO:161),wherein 10) IDNO: Xaa.sub.1=Lorabsent 24) Xaa.sub.2=Aorabsent 15 IEEQ KTA DKANHE EDA YQSAXaa.sub.1Xaa.sub.2(SEQID (SEQ (SEQIDNO: NO:63),whereinXaa.sub.1isL, IDNO: 62) A,orabsent,andXaa.sub.2isA 24) orabsent 16 QEEQ KDA DHANHE EYQ YQSAXaa.sub.1Xaa.sub.2(SEQID (SEQ (SEQIDNO: NO:63),whereinXaa.sub.1isL, ID 165) A,orabsent,andXaa.sub.2isA NO:70) orabsent 17 IEEQ KTA DKANHE EQA YQSAXaa.sub.1Xaa.sub.2(SEQID (SEQ (SEQIDNO: NO:63),whereinXaa.sub.1isL, IDNO: 62) A,orabsent,andXaa.sub.2isA 24) orabsent 18 IEEQ KEA DKANHE EQA YQSAXaa.sub.1Xaa.sub.2(SEQID (SEQ (SEQIDNO: NO:63),whereinXaa.sub.1isL, IDNO: 62) A,orabsent,andXaa.sub.2isA 24) orabsent

    [0270] In certain instances, the sequences set forth above in Table 5 can have at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid substitution or deletion. The ACE2 ?11 helix peptides can include any amino acid sequence described herein.

    [0271] In addition to the sequences provided in Table 5, peptides comprising Formula (II) also include SEQ TD NOs: 115, 116, 120, 121, 126, and 128-133, as shown in FIG. 8.

    [0272] In some instances, a peptide comprising Formula (II) comprising the sequences set forth above in Table 5 can have one or more of the properties listed below: (i) the peptide prevents or inhibits the interaction between Angiotensin converting enzyme 2 (ACE2) and a virus whose receptor binding domain binds ACE2; (ii) the peptide is alpha helical; (iii) the peptide is protease resistant; and/or (iv) the peptide blocks or inhibits infection of human ACE2 expressing epithelial cells. In some instances, the epithelial cells are located in the respiratory system.

    [0273] The tether of Formula (II) can include an alkyl, alkenyl, or alkynyl moiety (e.g., C.sub.5, C.sub.8, C.sub.11, or C.sub.12 alkyl, a C.sub.5, C.sub.8, or C.sub.11 alkenyl, or C.sub.5, C.sub.8, C.sub.11, or C.sub.12 alkynyl). The tethered amino acid can be alpha disubstituted (e.g., C.sub.1-C.sub.3 or methyl).

    [0274] In some instances of Formula (II), x is 2, 3, or 6. In some instances of Formula (II), each y is independently an integer between 0 and 15, or 3 and 15. In some instances of Formula (II), R.sub.1 and R.sub.2 are each independently H or C.sub.1-C.sub.6 alkyl. In some instances of Formula (II), R.sub.1 and R.sub.2 are each independently C.sub.1-C.sub.3 alkyl. In some instances of Formula (II), at least one of R.sub.1 and R.sub.2 are methyl. For example, R.sub.1 and R.sub.2 can both be methyl. In some instances of Formula (II), R.sub.3 is alkyl (e.g., C.sub.8 alkyl) and x is 3. In some instances of Formula (II), R.sub.3 is C.sub.11 alkyl and x is 6. In some instances of Formula (II), R.sub.3 is alkenyl (e.g., C.sub.8 alkenyl) and x is 3. In some instances of Formula (II), x is 6 and R.sub.3 is C.sub.11 alkenyl. In some instances, R.sub.3 is a straight chain alkyl, alkenyl, or alkynyl. In some instances, R.sub.3 is CH.sub.2CH.sub.2CH.sub.2CH?CHCH.sub.2CH.sub.2CH.sub.2.

    [0275] In another aspect, disclosed herein is a compound comprising a stabilized peptide comprising a sequence having the formula:

    ##STR00007##

    or a pharmaceutically acceptable salt thereof. In some instances, R.sub.1 and R.sub.4 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted. In some instances, each R.sub.2 and R.sub.3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted. In some instances, each u and x is independently 2, 3, or 6. In some instances, each t, v, and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some instances, each Xaa is independently an amino acid. In some instances, the stabilized peptide prevents or inhibits the interaction between Angiotensin converting enzyme 2 (ACE2) and a virus whose receptor binding domain binds ACE2. In some instances, the sequence has SEQ ID NO: 1 with: [0276] (i) at least 3 amino acids that are substituted with ?, ?-disubstituted non-natural amino acids with olefinic side chains that form an internal cross-link; [0277] (ii) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 additional amino acid substitutions, and [0278] (iii) 0, 1, 2, 3, 4, or 5 amino acid deletions at the N and/or C-terminus of the sequence.

    [0279] In some instances, each R.sub.1 and R.sub.4 is independently H or a C.sub.1-10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted. In some instances, each of R.sub.2 and R.sub.3 is independently a C.sub.5-20 alkyl, alkenyl, alkynyl; [R.sub.4KR.sub.4].sub.n; each of which is substituted with 0-6 R.sub.5. In some instances, R.sub.5 is halo, alkyl, OR.sub.6, N(R.sub.6).sub.2, SR.sub.6, SOR.sub.6, SO.sub.2R.sub.6, CO.sub.2R.sub.6, R.sub.6, a fluorescent moiety, or a radioisotope; K is O, S, SO, SO.sub.2, CO, CO.sub.2, CONR.sub.6, or

    ##STR00008##

    In some instances, R.sub.6 is H, alkyl, or a therapeutic agent. In some instances, n is an integer from 1-4. In some instances, [Xaa].sub.w; [Xaa].sub.x; [Xaa].sub.y; and [Xaa]z are provided in Table 5.

    [0280] In some instances, a peptide comprising Formula (III) can have one or more of the properties listed below: (i) the peptide prevents or inhibits the interaction between Angiotensin converting enzyme 2 (ACE2) and a virus whose receptor binding domain binds ACE2; (ii) the peptide is alpha helical; (iii) the peptide is protease resistant; and/or (iv) the peptide blocks or inhibits infection of human ACE2 expressing epithelial cells. In some instances, the epithelial cells are located in the respiratory system.

    [0281] The tether of Formula (III) can include an alkyl, alkenyl, or alkynyl moiety (e.g., C.sub.5, C.sub.8, C.sub.11, or C.sub.12 alkyl, a C.sub.5, C.sub.8, or C.sub.11 alkenyl, or C.sub.5, C.sub.8, C.sub.11, or C.sub.12 alkynyl). The tethered amino acid can be alpha disubstituted (e.g., C.sub.1-C.sub.3 or methyl).

    [0282] In some instances of Formula (III), x is 2, 3, or 6. In some instances of Formula (III), each y is independently an integer between 0 and 15, or 3 and 15. In some instances of Formula (III), R.sub.1 and R.sub.2 are each independently H or C.sub.1-C.sub.6 alkyl. In some instances of Formula (III), R.sub.1 and R.sub.2 are each independently C.sub.1-C.sub.3 alkyl. In some instances of Formula (III), at least one of R.sub.1 and R.sub.2 are methyl. For example, R.sub.1 and R.sub.2 can both be methyl. In some instances of Formula (III), R.sub.3 is alkyl (e.g., C.sub.8 alkyl) and x is 3. In some instances of Formula (III), R.sub.3 is C.sub.11 alkyl and x is 6. In some instances of Formula (III), R.sub.3 is alkenyl (e.g., C.sub.8 alkenyl) and x is 3. In some instances of Formula (III), x is 6 and R.sub.3 is C.sub.11 alkenyl. In some instances, R.sub.3 is a straight chain alkyl, alkenyl, or alkynyl. In some instances, R.sub.3 is CH.sub.2CH.sub.2CH.sub.2CH?CHCH.sub.2CH.sub.2CH.sub.2.

    [0283] In another aspect of Formula (III), of the three alpha, alpha disubstituted stereocenters: (i) two stereocenters are in the R configuration and one stereocenter is in the S configuration; or (ii) two stereocenters are in the S configuration and one stereocenter is in the R configuration. Thus, where Formula (III) is depicted as:

    ##STR00009##

    The C and C disubstituted stereocenters can both be in the R configuration or they can both be in the S configuration. When both C and C are in the R configuration, C is in the S configuration. When both C and C are in the S configuration, C is in the R configuration. The double bond in each of R.sub.2 and R.sub.3 of Formula (III) can be in the E or Z stereochemical configuration.

    [0284] In some instances of Formula (III), R.sub.3 is [R.sub.4KR.sub.4].sub.n; and R.sub.4 is a straight chain alkyl, alkenyl, or alkynyl.

    [0285] As used herein, the term alkyl, employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched. In some embodiments, the alkyl group contains 1 to 7, 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, n-heptyl, and the like. In some embodiments, the alkyl group is methyl, ethyl, or propyl. The term alkylene refers to a linking alkyl group.

    [0286] As used herein, alkenyl, employed alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon double bonds. In some embodiments, the alkenyl moiety contains 2 to 6 or 2 to 4 carbon atoms. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

    [0287] As used herein, alkynyl, employed alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.

    [0288] As used herein, alkynyl, employed alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.

    [0289] As used herein, the term cycloalkylalkyl, employed alone or in combination with other terms, refers to a group of formula cycloalkyl-alkyl-. In some embodiments, the alkyl portion has 1 to 4, 1 to 3, 1 to 2, or 1 carbon atom(s). In some embodiments, the alkyl portion is methylene. In some embodiments, the cycloalkyl portion has 3 to ring members or 3 to 7 ring members. In some embodiments, the cycloalkyl group is monocyclic or bicyclic. In some embodiments, the cycloalkyl portion is monocyclic. In some embodiments, the cycloalkyl portion is a C.sub.3-7 monocyclic cycloalkyl group.

    [0290] As used herein, the term heteroarylalkyl, employed alone or in combination with other terms, refers to a group of formula heteroaryl-alkyl-. In some embodiments, the alkyl portion has 1 to 4, 1 to 3, 1 to 2, or 1 carbon atom(s). In some embodiments, the alkyl portion is methylene. In some embodiments, the heteroaryl portion is a monocyclic or bicyclic group having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl portion has 5 to carbon atoms.

    [0291] As used herein, the term substituted means that a hydrogen atom is replaced by a non-hydrogen group. It is to be understood that substitution at a given atom is limited by valency.

    [0292] As used herein, halo or halogen, employed alone or in combination with other terms, includes fluoro, chloro, bromo, and iodo. In some embodiments, halo is F or Cl.

    [0293] In some embodiments, the disclosure features structurally-stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NO: 1 or 21 (or a modified version thereof), wherein: the side chains of two amino acids separated by two, three, or six amino acids are replaced by an internal staple, the side chains of three amino acids are replaced by an internal stitch, the side chains of four amino acids are replaced by two internal staples, or the side chains of five amino acids are replaced by the combination of an internal staple and an internal stitch. In some embodiments, the disclosure features structurally-stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NO: 1 or 21 (or a modified version thereof), wherein the side chains of two amino acids separated by two, three, or six amino acids are replaced by an internal staple. In some embodiments, the disclosure features structurally-stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NO: 1 or 21 (or a modified version thereof), wherein the side chains of two amino acids separated by three amino acids are replaced by an internal staple. In some embodiments, the disclosure features structurally-stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NO: 1 or 21 (or a modified version thereof), wherein the side chains of two amino acids separated by six amino acids are replaced by an internal staple. In some embodiments, the disclosure features structurally-stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NO: 1 or 21 (or a modified version thereof), wherein the side chains of three amino acids are replaced by an internal stitch.

    [0294] The stapled or stitched peptide can be 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. In one instance, the stapled or stitched peptide is 20-25, 20-30, 20-35, 20-40, 20-45, 20-45, 20-50, 20-60, 20-70, 20-80, 20-85, 20-90, 20-95, or 20-100 amino acids in length. In a specific embodiment, the stapled or stitched peptide is 24-45 amino acids (i.e., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) in length. In a specific embodiment, the stapled or stitched peptide is 24-42 amino acids (i.e., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) amino acids in length. In a specific embodiment, the stapled or stitched peptide is 24-35 amino acids (i.e., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) amino acids in length. In a specific embodiment, the stapled or stitched peptide is 24 amino acids in length. In a specific embodiment, the stapled or stitched peptide is 26 amino acids in length. In another specific embodiment, the stapled or stitched peptide is 35 amino acids in length. In another specific embodiment, the stapled or stitched peptide is 40 amino acids in length. In another specific embodiment, the stapled or stitched peptide is 45 amino acids in length. In another specific embodiment, the stapled or stitched peptide is 50 amino acids in length. Exemplary ACE2 ?1 helix stapled or stitched peptides are shown in Tables 3-5 and described in Formulae (I)-(III). In one embodiment, the ACE2 ?1 helix stapled or stitched peptide comprises or consists of a stapled or stitched version of the amino acid sequence of any one of SEQ ID NOs: 1, 13-16, 21, or 145-148 (e.g., the product of a ring-closing metathesis reaction performed on a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 13-16, 21, or 145-148, respectively). In one embodiment, the ACE2 ?1 helix stapled or stitched peptide comprises or consists of a stapled or stitched version of the amino acid sequence of SEQ ID NO: 1 (e.g., the product of a ring-closing metathesis reaction performed on a peptide comprising the amino acid sequence of SEQ ID NO:1). In one embodiment, the ACE2 ?1 helix stapled or stitched peptide comprises or consists of a stapled or stitched version of the amino acid sequence of SEQ ID NO: 21 (e.g., the product of a ring-closing metathesis reaction performed on a peptide comprising the amino acid sequence of SEQ ID NO: 21).

    [0295] In certain embodiments, the stapled peptide comprises or consists of a variant of the amino acid sequence set forth in any one of SEQ ID NO: 1 or 21, wherein two amino acids each separated by 3 amino acids (i.e., positions i and i+4) are modified to structurally stabilize the peptide (e.g., by substituting them with non-natural amino acids to permit hydrocarbon stitching, i.e., stapling amino acids). In certain embodiments, the stapled peptide comprises or consists of a variant of the amino acid sequence set forth in any one of SEQ ID NO: 1 or 21, wherein two amino acids each separated by 6 amino acids (i.e., positions i and i+7) are modified to structurally stabilize the peptide (e.g., by substituting them with non-natural amino acids to permit hydrocarbon stapling, i.e., with stapling amino acids).

    [0296] While hydrocarbon tethers are common, other tethers can also be employed in the structurally-stabilized ACE2 ?1 helix peptides described herein. For example, the tether can include one or more of an ether, thioether, ester, amine, or amide, or triazole moiety. In some cases, a naturally occurring amino acid side chain can be incorporated into the tether. For example, a tether can be coupled with a functional group such as the hydroxyl in serine, the thiol in cysteine, the primary amine in lysine, the acid in aspartate or glutamate, or the amide in asparagine or glutamine. Accordingly, it is possible to create a tether using naturally occurring amino acids rather than using a tether that is made by coupling two non-naturally occurring amino acids. It is also possible to use a single non-naturally occurring amino acid together with a naturally occurring amino acid. Triazole-containing (e.g., 1, 4 triazole or 1, 5 triazole) crosslinks can be used (see, e.g., Kawamoto et al. 2012 Journal of Medicinal Chemistry 55:1137; WO 2010/060112). In addition, other methods of performing different types of stapling are well known in the art and can be employed with the ACE2 ?1 helix peptides described herein (see, e.g., Lactam stapling: Shepherd et al., J. Am. Chem. Soc., 127:2974-2983 (2005); UV-cycloaddition stapling: Madden et al., Bioorg. Med. Chem. Lett., 21:1472-1475 (2011); Disulfide stapling: Jackson et al., Am. Chem. Soc.,113:9391-9392 (1991); Oxime stapling: Haney et al., Chem. Commun., 47:10915-10917 (2011); Thioether stapling: Brunel and Dawson, Chem. Commun., 552-2554 (2005); Photoswitchable stapling: J. R. Kumita et al., Proc. Natl. Acad. Sci. U.S.A, 97:3803-3808 (2000); Double-click stapling: Lau et al., Chem. Sci., 5:1804-1809 (2014); Bis-lactam stapling: J. C. Phelan et al., J. Am. Chem. Soc., 119:455-460 (1997); and Bis-arylation stapling: A. M. Spokoyny et al., J. Am. Chem. Soc., 135:5946-5949 (2013)).

    [0297] It is further envisioned that the length of the tether can be varied. For instance, a shorter length of tether can be used where it is desirable to provide a relatively high degree of constraint on the secondary alpha-helical structure, whereas, in some instances, it is desirable to provide less constraint on the secondary alpha-helical structure, and thus a longer tether may be desired.

    [0298] Additionally, while tethers spanning from amino acids i to i+3; i to i+4; and i to i+7 are common in order to provide a tether that is primarily on a single face of the alpha helix, the tethers can be synthesized to span any combinations of numbers of amino acids and also used in combination to install multiple tethers.

    [0299] In some instances, the hydrocarbon tethers (i.e., cross links) described herein can be further manipulated. In one instance, a double bond of a hydrocarbon alkenyl tether, (e.g., as synthesized using a ruthenium-catalyzed ring closing metathesis (RCM)) can be oxidized (e.g., via epoxidation, aminohydroxylation or dihydroxylation) to provide one of compounds below.

    ##STR00010##

    [0300] Either the epoxide moiety or one of the free hydroxyl moieties can be further functionalized. For example, the epoxide can be treated with a nucleophile, which provides additional functionality that can be used, for example, to attach a therapeutic agent. Such derivatization can alternatively be achieved by synthetic manipulation of the amino or carboxy-terminus of the peptide or via the amino acid side chain. Other agents can be attached to the functionalized tether, e.g., an agent that facilitates entry of the peptide into cells.

    [0301] In some instances, alpha disubstituted amino acids are used in the peptide to improve the stability of the alpha helical secondary structure. However, alpha disubstituted amino acids are not required, and instances using mono-alpha substituents (e.g., in the tethered amino acids) are also envisioned.

    [0302] The structurally-stabilized (e.g., stapled or stitched) peptides can include a drug, a toxin, a derivative of polyethylene glycol; a second peptide; a carbohydrate, etc. Where a polymer or other agent is linked to the structurally-stabilized (e.g., stapled or stitched) peptide, it can be desirable for the composition to be substantially homogeneous.

    [0303] The addition of polyethelene glycol (PEG) molecules can improve the pharmacokinetic and pharmacodynamic properties of the peptide. For example, PEGylation can reduce renal clearance and can result in a more stable plasma concentration. PEG is a water soluble polymer and can be represented as linked to the peptide as formula: [0304] XO(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2Y where n is 2 to 10,000 and X is H or a terminal modification, e.g., a C.sub.1-4 alkyl; and Y is an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the N-terminus) of the peptide. Y may also be a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine). Other methods for linking PEG to a peptide, directly or indirectly, are known to those of ordinary skill in the art. The PEG can be linear or branched. Various forms of PEG including various functionalized derivatives are commercially available.

    [0305] PEG having degradable linkages in the backbone can be used. For example, PEG can be prepared with ester linkages that are subject to hydrolysis. Conjugates having degradable PEG linkages are described in WO 99/34833; WO 99/14259, and U.S. Pat. No. 6,348,558.

    [0306] In certain embodiments, macromolecular polymer (e.g., PEG) is attached to a structurally-stabilized (e.g., stapled or stitched) peptide described herein through an intermediate linker. In certain embodiments, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In other embodiments, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Non-peptide linkers are also possible. For example, alkyl linkers such as NH(CH.sub.2).sub.nC(O), wherein n=2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C.sub.1-C.sub.6) lower acyl, halogen (e.g., Cl, Br), CN, NH.sub.2, phenyl, etc. U.S. Pat. No. 5,446,090 describes a bifunctional PEG linker and its use in forming conjugates having a peptide at each of the PEG linker termini.

    [0307] The structurally-stabilized (e.g., stapled or stitched) peptides can also be modified, e.g., to further facilitate cellular uptake or increase in vivo stability, in some embodiments. For example, acylating or PEGylating a structurally-stabilized peptide facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.

    [0308] In some embodiments, the structurally-stabilized (e.g., stapled or stitched) peptides disclosed herein have an enhanced ability to penetrate cell membranes (e.g., relative to non-stabilized peptides). See, e.g., International Publication No. WO 2017/147283, which is incorporated by reference herein in its entirety.

    Conjugates to the Polypeptides or Stabilized Peptides

    [0309] Disclosed herein are conjugates that associate with (e.g., are conjugated to) a peptide or stabilized peptide disclosed herein. Conjugates are moieties that are used to isolate and/or detect a peptide disclosed herein. In some instances, the moiety increased the half-life of the peptide to which it is conjugated. In some instances, the moiety is a detection moiety. In some instances, the moiety is a capture moiety such as biotin. In some instances, the peptide is conjugated to a bead. In some instances, the bead is magnetic.

    [0310] In some instances, the conjugate functions to extend the half-life of the peptide. In some instances, the peptide is conjugated to a moiety that increases the overall size of the peptide molecule. In some instances, the moiety is a polyethylene glycol (PEG). In some instances, other moieties known in the art are conjugated to a peptide disclosed herein. For example, in one aspect, human serum albumin (HSA) is conjugated to a peptide disclosed herein.

    [0311] In some instances, a detection moiety is conjugated to a peptide disclosed herein. In some instances, the detection moiety is a fluorescent, radioactive, chemiluminescent, or colorimetric detectable markers. Any suitable detectable label can be used. In some embodiments, the detectable label is a fluorophore (e.g., GFP, FITC, Cy5, etc.). In some embodiments, a detectable label is or includes a luminescent or chemiluminescent moiety. Common luminescent/chemiluminescent moieties include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. These protein moieties can catalyze chemiluminescent reactions given the appropriate chemical substrates (e.g., an oxidizing reagent plus a chemiluminescent compound). A number of compound families are known to provide chemiluminescence under a variety of conditions. Non-limiting examples of chemiluminescent compound families include 2,3-dihydro-1,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog. These compounds can luminesce in the presence of alkaline hydrogen peroxide or calcium hypochlorite and base. Other examples of chemiluminescent compound families include, e.g., 2,4,5-triphenylimidazoles, para-dimethylamino and -methoxy substituents, oxalates such as oxalyl active esters, p-nitrophenyl, N-alkyl acridinum esters, luciferins, lucigenins, or acridinium esters.

    [0312] In some instances, the peptide is conjugated to a capture moiety. A capture moiety can be used for purification or capture of the peptide of interest. For example, in some instances, the capture moiety is a biotin molecule. In this setting, an avidin or streptavidin molecule (e.g., on a support) can be used to capture the biotin-conjugated peptide. In some instances, the capture moiety is a fusion protein tag. In some instances, the fusion protein tag includes His, HA, Flu, or FLAG tag.

    Pharmaceutical Compositions

    [0313] One or more of any of the structurally-stabilized (e.g., stapled or stitched) peptides described herein can be formulated for use as or in pharmaceutical compositions. The pharmaceutical compositions may be used in the methods of treatment or prevention described herein (see above). In certain embodiments, the pharmaceutical composition comprises a structurally-stabilized (e.g., stapled or stitched) peptide comprising or consisting of an amino acid sequence that is identical to an amino acid sequence set forth in Table 1, except for 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution, insertion, or deletion. These changes to the amino acid sequences can be made on the non-interacting alpha-helical face of these peptides (i.e., to the amino acids that do not interact with the coronavirus 5 helix bundle) and/or on the interacting alpha-helical face (i.e., to the amino acids that interact with the coronavirus 5 helix bundle). Such compositions can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA's CDER Data Standards Manual, version number 004 (which is available at fda.give/cder/dsm/DRG/drg00301.htm). For example, compositions can be formulated or adapted for administration by inhalation (e.g., oral and/or nasal inhalation (e.g., via nebulizer or spray)), injection (e.g., intravenously, intra-arterial, subdermally, intraperitoneally, intramuscularly, and/or subcutaneously); and/or for oral administration, transmucosal administration, and/or topical administration (including topical (e.g., nasal) sprays and/or solutions).

    [0314] In some instances, pharmaceutical compositions can include an effective amount of one or more structurally-stabilized (e.g., stapled or stitched) peptides. The terms effective amount and effective to treat, as used herein, refer to an amount or a concentration of one or more structurally-stabilized (e.g., stapled or stitched) peptides or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment of infection).

    [0315] Pharmaceutical compositions of this invention can include one or more structurally-stabilized (e.g., stapled or stitched) peptides described herein and any pharmaceutically acceptable carrier and/or vehicle. In some instances, pharmaceuticals can further include one or more additional therapeutic agents in amounts effective for achieving a modulation of disease or disease symptoms.

    [0316] The term pharmaceutically acceptable carrier or adjuvant refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

    [0317] The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intra-cutaneous, intra-venous, intra-muscular, intra-articular, intra-arterial, intra-synovial, intra-sternal, intra-thecal, intra-lesional and intra-cranial injection or infusion techniques.

    [0318] In some instances, one or more structurally-stabilized (e.g., stapled or stitched) peptides disclosed herein can be conjugated, for example, to a carrier protein. Such conjugated compositions can be monovalent or multivalent. For example, conjugated compositions can include one structurally-stabilized (e.g., stapled or stitched) peptide disclosed herein conjugated to a carrier protein. Alternatively, conjugated compositions can include two or more structurally-stabilized (e.g., stapled or stitched) peptides disclosed herein conjugated to a carrier.

    [0319] As used herein, when two entities are conjugated to one another they are linked by a direct or indirect covalent or non-covalent interaction. In certain embodiments, the association is covalent. In other embodiments, the association is non-covalent. Non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. An indirect covalent interaction occurs when two entities are covalently connected, optionally through a linker group.

    [0320] Carrier proteins can include any protein that increases or enhances immunogenicity in a subject. Exemplary carrier proteins are described in the art (see, e.g., Fattom et al., Infect. Immun., 58:2309-2312, 1990; Devi et al., Proc. Natl. Acad. Sci. USA 88:7175-7179, 1991; Li et al., Infect. Immun. 57:3823-3827, 1989; Szu et al., Infect. Immun. 59:4555-4561, 1991; Szu et al., J. Exp. Med. 166:1510-1524, 1987; and Szu et al., Infect. Immun. 62:4440-4444, 1994). Polymeric carriers can be a natural or a synthetic material containing one or more primary and/or secondary amino groups, azido groups, or carboxyl groups. Carriers can be water soluble.

    Methods of Treatment

    [0321] The disclosure features methods of using any of the structurally-stabilized (e.g., stapled or stitched) peptides (or pharmaceutical compositions comprising said structurally-stabilized peptides) described herein for the prevention and/or treatment of a coronavirus (e.g., betacoronavirus) infection or coronavirus disease. The terms treat or treating, as used herein, refers to alleviating, inhibiting, or ameliorating the disease or infection from which the subject (e.g., human) is suffering. In some instances, the subject is an animal. In some embodiments, the subject is a mammal such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human). In some instances, the subject is a domesticated animal (e.g., a dog or cat). In some instances, the subject is a bat. In some instances, the subject is a human. In certain embodiments, such terms refer to a non-human animal (e.g., a non-human animal such as a pig, horse, cow, cat or dog). In some embodiments, such terms refer to a pet or farm animal. In some embodiments, such terms refer to a human.

    [0322] The structurally-stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein can be useful for treating a subject (e.g., human subject) having a coronavirus (e.g., betacoronavirus) infection. The structurally-stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein can also be useful for treating a human subject having a coronavirus disease. In certain embodiments, the coronavirus infection is an infection of one of 229E (alphacoronavirus); NL63 (alphacoronavirus); OC43 (betacoronavirus); HKU1 (betacoronavirus); Middle East respiratory syndrome (MERS); SARS-CoV-1; or SARS-CoV-2. In certain embodiments, the coronavirus disease is caused by a SARS-CoV-2 infection.

    [0323] The structurally-stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein can be useful for preventing (i.e., prophylaxis treatment of) a coronavirus (e.g., betacoronavirus) infection in a subject. The peptides (or compositions comprising the peptides) described herein can also be useful for preventing a coronavirus disease in a subject (e.g., human subject). In certain embodiments, the coronavirus infection is an infection of one of 229E (alphacoronavirus); NL63 (alphacoronavirus); OC43 (betacoronavirus); HKU1 (betacoronavirus); Middle East respiratory syndrome (MERS); SARS-CoV-1; or SARS-CoV-2. In certain embodiments, the coronavirus disease is caused by a SARS-CoV-2 infection.

    [0324] The structurally-stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein can also be useful for treating a subject with post-acute sequelae of SARS-CoV-2 infection.

    [0325] In addition, the structurally-stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein can also be useful for treating or preventing infection by a SARS-CoV-2 variant in a subject.

    [0326] Also provided are methods of preventing or inhibiting interaction between the receptor binding domain of a virus and ACE2 in a subject in need thereof using the structurally-stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein. In some cases, the virus can be a coronavirus (e.g., SARS-CoV-1 or SARS-CoV-2).

    [0327] In certain embodiments, the subject in need thereof is administered a peptide described in Tables 1-5, or a variant thereof. In certain embodiments, the human subject in need thereof is administered a stapled ACE2 ?1 helix peptide comprising or consisting of SEQ ID NO: 1 or a modified version (variant) thereof. In certain embodiments, the human subject in need thereof is administered a stapled ACE2 ?1 helix peptide comprising or consisting of SEQ ID NO:21 or a modified version (variant) thereof.

    [0328] In certain embodiments, the subject in need thereof is administered any one of the peptides having SEQ ID NOs: 1, 13-16, 21, 76, 77, or 145-148 described in Table 1, or a peptide of SEQ ID NOs. 112, 113, 117, 118, 123, 125, or 127, or a variant of any of these peptides that can still bind the S1 protein and/or RBD of SARS-CoV-2 or of a SARS-CoV-2 variant. In certain embodiments, the human subject in need thereof is administered any one of the peptides described in Table 3 or FIG. 8, or a variant thereof that can still bind the S1 protein and/or RBD of SARS-CoV-2 or of a SARS-CoV-2 variant. In certain embodiments, the human subject in need thereof is administered any one of the peptides described in FIG. 8. In one instance, the human subject in need thereof is administered any one of the peptides set forth in SEQ ID NOs.: 90-95, 98-100, 105-108, 110, 115, 116, 120, 121, 126, 130, 131, 132, or 133. In some embodiments, the human subject is infected with a coronavirus (e.g., betacoronavirus). In some embodiments, the human subject is at risk of being infected with a coronavirus (e.g., betacoronavirus). In some embodiments, the human subject is at risk of developing a coronavirus disease (e.g., betacoronavirus). In some instances, a human subject is at risk of being infected with a coronavirus or at risk of developing a coronavirus disease if he or she lives in an area (e.g., city, state, country) subject to an active coronavirus outbreak (e.g., an area where at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or more people have been diagnosed as infected with a coronavirus). In some instances, a human subject is at risk of being infected with a coronavirus or developing a coronavirus disease if he or she lives in an area near (e.g., a bordering city, state, country) a second area (e.g., city, state, country) subject to an active coronavirus outbreak (e.g., an area near (e.g., bordering) a second area where at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or more people have been diagnosed as infected with a coronavirus). In certain embodiments, the coronavirus disease is caused by a SARS-CoV-2 infection.

    [0329] In some instances, also disclosed are methods of treatment or prevention that include a combination therapy. In some instances, the combination therapy treats or prevents a SARS virus infection (e.g., SARS-CoV-2 or a SARS-CoV-2 variant). In some instances, the combination therapy comprises any one of the polypeptides in FIG. 8 or Table 3. In some instances, the combination therapy includes a pharmaceutical composition that comprises any one of the polypeptides in FIG. 8 or Table 3. In some instances, the combination therapy further includes one or more of: dexamethasone, remdesivir, baricitinib in combination with remdesivir, favipiravir, merimepodib, an anticoagulation drug selected from low-dose heparin or enoxaparin, bamlanivimab, a combination of bamlanivimab and etesevimab, a combination of casirivimab and imdevimab, convalescent plasma, an mRNA SARS-CoV-2 vaccine (such as those produced by Moderna or Pfizer), an attenuated SARS-CoV-2 virus vaccine, or a dead SARS-CoV-2 virus vaccine. In some instances, the combination therapy comprises a viral vaccine against SARS-CoV-2 (e.g., an adenovirus vaccine such as those produced by Astra Zeneca and Johnson & Johnson. In some instances, the combination therapy comprises a monoclonal antibody that binds the coronavirus (e.g., SARS-CoV-2) and inhibits infection of a human subject. In some instances, the combination therapy comprises orthogonal entry inhibitors, such as antibodies, peptides, and small molecules; and furin inhibitors such as decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein. See e.g., the agents described in Huang et al., Acta Pharmacologica Sinica (2020) 41:1141-1149; https://doi.org/10,1038/s41401-020-0485-4, which is incorporated by reference herein. In some instances, the combination therapy is with any one or more of a stabilized peptide(s) described in PCT/US2021/020940 (which is incorporated by reference herein).

    [0330] In general, methods include selecting a subject and administering to the subject an effective amount of one or more of the structurally-stabilized (e.g., stapled or stitched) peptides herein, e.g., in or as a pharmaceutical composition, and optionally repeating administration as required for the prevention or treatment of a coronavirus infection or a coronavirus disease and can be administered intranasally (e.g. nose spray), as an inhalant (e.g. nebulization to access the respiratory system), orally, intravenously or topically. A subject can be selected for treatment based on, e.g., determining that the subject has a coronavirus (e.g., betacoronavirus) infection. The peptides of this disclosure can be used to determine if a subject's is infected with a coronavirus.

    [0331] Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.

    [0332] An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. For example, effective amounts can be administered at least once.

    Methods of Diagnosis

    [0333] Provided herein is a method of diagnosing a subject as having an infection caused by SARS-CoV-2 using a structurally-stabilized (e.g., stapled) peptide described herein.

    [0334] In some instances, the peptides used for methods of diagnosis include SEQ ID NOs: 1, 13-16, 21, or 145-148 as shown in Table 1. In some embodiments, the peptides used for methods of diagnosis include SEQ ID NOs: 2-12 17-20, 134-143, and 172 as shown in Table 3. In some instances, the peptides used for methods of diagnosis include SEQ ID NOs: 49-53, as shown in Table 6.

    [0335] The disclosure features methods of using a structurally-stabilized peptide (e.g., any of the structurally-stabilized (e.g., stapled) peptides (or pharmaceutical compositions comprising said structurally-stabilized peptides) described herein) to determine whether a subject would be receptive to therapy using structurally-stabilized (e.g., stapled) peptide described herein.

    [0336] Moreover, the disclosure additionally provides a method for predicting the efficacy of treatment using a structurally-stabilized (e.g., stapled) peptide described herein for a subject having an infection caused by SARS-CoV-2. In some instances, the methods include testing a cell of a subject having an infection caused by SARS-CoV-2 for the presence of SARS-CoV-2, and predicting that a structurally-stabilized (e.g., stapled) peptide would likely inhibit SARS-CoV-2 infection.

    [0337] In some instances, the methods include isolating secretions (e.g., mucous, sputum, saliva) or cells (e.g., biopsy; e.g., liquid biopsy) from a subject having an infection caused by SARS-CoV-2. In some instances, the isolated cells are cultured and treated with one or more of the structurally-stabilized (e.g., stapled) peptides described herein.

    [0338] In some embodiments, the methods can include developing a personalized treatment regimen for a subject having an infection caused by SARS-CoV-2. Such methods can include, e.g., identifying a subject with secretions or cells containing the virus that are sensitive to one or more structurally-stabilized (e.g., stapled) peptides as disclosed herein and treating the subject with one or more structurally-stabilized (e.g., stapled) peptides as disclosed herein. In some embodiments, the methods can include determining the most appropriate treatment for a subject having an infection caused by SARS-CoV-2.

    [0339] In some instances, the method of detecting the presence of a virus (e.g., SARS-CoV-2) whose receptor binding domain causes infection by binding to ACE2 includes isolating a sample from a subject and providing the sample to a plurality of stabilized peptides as disclosed herein. In some instances, the stabilized peptides in this method include stabilized peptides that are conjugated to a detection moiety and stabilized peptides that are conjugated to a capture moiety. After mixing the sample with the two different types of conjugated peptides, the sample is provided to a diagnostic such as a test strip. In some instances, the detection moiety (e.g., any of the detection moieties disclosed herein) can be identified.

    [0340] In some instances, the method of diagnosis is a method described in FIG. 13A or FIG. 13B or a modification thereof.

    [0341] In some instances, the subject can be an animal. In some embodiments, the subject is a mammal such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human). In some instances, the subject is a domesticated animal (e.g., a dog or cat). In some instances, the subject is a bat. In some instances, the subject is a human. In some instances, the subject is a human. In certain embodiments, such terms refer to a non-human animal (e.g., a non-human animal such as a pig, horse, cow, cat or dog). In some embodiments, such terms refer to a pet or farm animal. In some embodiments, such terms refer to a human.

    Kits

    [0342] Provided herein is a kit that is used to detect SARS-CoV-2 infection. In some instances, the kit includes a test strip. In some instances, the kit includes one or more stapled proteins disclosed herein that is conjugated to a detection moiety (e.g., a fluorophore, chromophore, HRP, etc.), In some instances, the kit includes one or more stapled peptide disclosed herein that is conjugated to an affinity moiety (e.g, biotin) that can be captured by a solid support using streptavidin beads. In some instances, the kit further includes a capture resin (e.g. streptavidin beads for a biotin affinity label, Nickel NTA beads for a His-tag affinity label, anti-FLAG beads for a FLAG tag affinity label, etc.). Once captured using the affinity moiety, the peptide can be imaged for the presence of the detection moiety. In some instances, the kit further includes instructions for identification of the presence of the detection moiety. A positive test is determined by the presence of the detection label, such as fluorescence, and a negative test is determined by the absence of the detection label, as measured by eye, spectrometer, or other detection instrument. Like a pregnancy strip test, this diagnostic method can be used as a simple, rapid, and point-of-care method that does not require complex infrastructure or expertise to diagnose the infection. Thus, home use is also envisioned.

    [0343] Also provided herein are kits comprising one or more structurally-stabilized ACE2 ?1 antiviral peptides described herein In some embodiments, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more structurally-stabilized peptides provided herein. In some embodiments, the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Also provided herein are kits that can be used in the above methods

    Methods of Making Stapled or Stitched Peptides

    [0344] Stapled peptide synthesis: Fmoc-based solid-phase peptide synthesis was used to synthesize stapled peptide fusion inhibitors in accordance with our reported methods for generating all-hydrocarbon stapled peptides. To achieve the various staple lengths, ?-methyl, ?-alkenyl amino acids were installed at i, i+4 positions using two S-pentenyl alanine residues (S5). For the stapling reaction, Grubbs 1st generation ruthenium catalyst dissolved in dichloroethane was added to the resin-bound peptides. To ensure maximal conversion, three to five rounds of stapling were performed. The peptides were then cleaved off of the resin using trifluoroacetic acid, precipitated using a hexane:ether (1:1) mixture, air dried, and purified by LC-MS. All peptides were quantified by amino acid analysis.

    [0345] Stitched peptide synthesis: Methods of synthesizing the stitched peptides described herein are known in the art. Nevertheless, the following exemplary method may be used. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3d. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

    [0346] The peptides of this invention can be made by chemical synthesis methods, which are well known to the ordinarily skilled artisan. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Hence, peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with the ?-NH2 protected by either t-Boc or Fmoc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431.

    [0347] One manner of making of the peptides described herein is using solid phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups.

    [0348] Longer peptides could be made by conjoining individual synthetic peptides using native chemical ligation. Insertion of a stitching amino acid may be performed as described in, e.g., Young and Schultz, J Biol Chem. 2010 Apr. 9; 285(15): 11039-11044. Alternatively, the longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of this invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed. Next, a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

    [0349] The peptides can be made in a high-throughput, combinatorial fashion, e.g., using a high-throughput multiple channel combinatorial synthesizer available from, e.g., Advanced Chemtech or Symphony X. Peptide bonds can be replaced, e.g., to increase physiological stability of the peptide, by: a retro-inverso bonds (C(O)NH); a reduced amide bond (NHCH.sub.2); a thiomethylene bond (SCH.sub.2 or CH.sub.2S); an oxomethylene bond (OCH.sub.2 or CH.sub.2-0); an ethylene bond (CH.sub.2CH.sub.2); a thioamide bond (C(S)NH); a trans-olefin bond (CH?CH); a fluoro substituted trans-olefin bond (CF?CH); a ketomethylene bond (C(O)CHR) or CHRC(O) wherein R is H or CH.sub.3; and a fluoro-ketomethylene bond (C(O)CFR or CFRC(O) wherein R is H or F or CH.sub.3.

    [0350] The peptides can be further modified by: acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceination, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and sulfurylation. As indicated above, peptides can be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (e.g., C.sub.1-C.sub.20 straight or branched alkyl groups); fatty acid radicals; and combinations thereof. ?, ?-Disubstituted non-natural amino acids containing olefinic side chains of varying length can be synthesized by known methods (Williams et al. J. Am. Chem. Soc., 113:9276, 1991; Schafmeister et al., J. Am. Chem Soc., 122:5891, 2000; and Bird et al., Methods Enzymol., 446:369, 2008; Bird et al., Current Protocols in Chemical Biology, 2011). In some instances for peptides where an i linked to i+7, i+7 linked to i+14 stitch is used (four turns of the helix stabilized): one R-octenyl alanine (e.g., (R)-?-(7-octenyl)alanine), one one bis-pentenyl glycine (e.g., ?,?-Bis(4-pentenyl)glycine), and one R-octenyl alanine (e.g., (R)-?-(7-octenyl)alanine) is used. In some instances for peptides where an i linked to i+7, i+7 linked to i+14 stitch is used (four turns of the helix stabilized): one S-octenyl alanine (e.g., (S)-?-(7-octenyl)alanine), one one bis-pentenyl glycine (e.g., ?,?-Bis(4-pentenyl)glycine), and one R-octenyl alanine (e.g., (R)-?-(7-octenyl)alanine) is used. In some instances for peptides where an i linked to i+7, i+7 linked to i+14 stitch is used (four turns of the helix stabilized): one S-octenyl alanine (e.g., (S)-?-(7-octenyl)alanine), one bis-pentenyl glycine (e.g., ?,?-Bis(4-pentenyl)glycine), and one S-octenyl alanine (e.g., (S)-?-(7-octenyl)alanine) is used. In some instances for peptides where an i linked to i+7, i+7 linked to i+14 stitch is used (four turns of the helix stabilized): one R-pentenyl alanine (e.g., (R)-?-(4-pentenyl)alanine), one bis-octenyl glycine (e.g., ?,?-Bis(7-octenyl)glycine), and one S-pentenyl alanine (e.g., (S)-?-(4-pentenyl)alanine) is used. In some instances for peptides where an i linked to i+7, i+7 linked to i+14 stitch is used (four turns of the helix stabilized): one R-pentenyl alanine (e.g., (R)-?-(4-pentenyl)alanine), one bis-octenyl glycine (e.g., ?,?-Bis(7-octenyl)glycine), and one R-pentenyl alanine (e.g., (R)-?-(4-pentenyl)alanine) is used. In some instances for peptides where an i linked to i+7, i+7 linked to i+14 stitch is used (four turns of the helix stabilized): one S-pentenyl alanine (e.g., (S)-?-(4-pentenyl)alanine), one bis-octenyl glycine (e.g., ?,?-Bis(7-octenyl)glycine), and one R-pentenyl alanine (e.g., (R)-?-(4-pentenyl)alanine) is used. In some instances for peptides where an i linked to i+7, i+7 linked to i+14 stitch is used (four turns of the helix stabilized): one S-pentenyl alanine (e.g., (S)-?-(4-pentenyl)alanine), one bis-octenyl glycine (e.g., ?,?-Bis(7-octenyl)glycine), and one S-pentenyl alanine (e.g., (S)-?-(4-pentenyl)alanine) is used. R-octenyl alanine is synthesized using the same route, except that the starting chiral auxiliary confers the R-alkyl-stereoisomer. Also, 8-iodooctene is used in place of 5-iodopentene. Inhibitors are synthesized on a solid support using solid-phase peptide synthesis (SPPS) on MBHA resin (see, e.g., WO 2010/148335).

    [0351] Fmoc-protected ?-amino acids (other than the olefinic amino acids N-Fmoc-?,?-Bis(4-pentenyl)glycine, (S)N-Fmoc-?-(4-pentenyl)alanine, (R)N-Fmoc-?-(7-octenyl)alanine, (R)N-Fmoc-?-(7-octenyl)alanine, and (R)N-Fmoc-?-(4-pentenyl)alanine), 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially available from, e.g., Novabiochem (San Diego, CA). Dimethylformamide (DMF), N-methyl-2-pyrrolidinone (NMP), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), fluorescein isothiocyanate (FITC), and piperidine are commercially available from, e.g., Sigma-Aldrich. Olefinic amino acid synthesis is reported in the art (Williams et al., Org. Synth., 80:31, 2003).

    [0352] Again, methods suitable for obtaining (e.g., synthesizing), stitching, and purifying the peptides disclosed herein are also known in the art (see, e.g., Bird et. al., Methods in Enzymol., 446:369-386 (2008); Bird et al., Current Protocols in Chemical Biology, 2011; Walensky et al., Science, 305:1466-1470 (2004); Schafmeister et al., J. Am. Chem. Soc., 122:5891-5892 (2000); U.S. patent application Ser. No. 12/525,123, filed Mar. 18, 2010; and U.S. Pat. No. 7,723,468, issued May 25, 2010, each of which are hereby incorporated by reference in their entirety).

    [0353] In some instances, the peptides are substantially free of non-stitched peptide contaminants or are isolated. Methods for purifying peptides include, for example, synthesizing the peptide on a solid-phase support. Following cyclization, the solid-phase support may be isolated and suspended in a solution of a solvent such as DMSO, DMSO/dichloromethane mixture, or DMSO/NMP mixture. The DMSO/dichloromethane or DMSO/NMP mixture may comprise about 30%, 40%, 50% or 60% DMSO. In a specific instance, a 50%/50% DMSO/NMP solution is used. The solution may be incubated for a period of 1, 6, 12 or 24 hours, following which the resin may be washed, for example with dichloromethane or NMP. In one instance, the resin is washed with NMP. Shaking and bubbling an inert gas into the solution may be performed.

    [0354] Properties of the stitched or stapled peptides of the disclosure can be assayed, for example, using the methods described below and in the Examples.

    Assays to Determine Characteristics and Effectiveness of Stabilized Peptides

    [0355] Assays to Determine ?-Helicity: Compounds are dissolved in an aqueous solution (e.g. 5 ?M potassium phosphate solution at pH 7, or distilled H.sub.2O, to concentrations of 25-50 ?M). Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard measurement parameters (e.g. temperature, 20? C.; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The ?-helical content of each peptide is calculated by dividing the mean residue ellipticity by the reported value for a model helical decapeptide (Yang et al., Methods Enzymol., 1986).

    [0356] Assays to Determine Melting Temperature (Tm): Cross-linked or the unmodified template peptides are dissolved in distilled H.sub.2O or other buffer or solvent (e.g. at a final concentration of 50 ?M) and Tm is determined by measuring the change in ellipticity over a temperature range (e.g. 4 to 95? C.) on a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: 1? C./min; path length, 0.1 cm).

    [0357] In Vitro Protease Resistance Assays: The amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thereby rendering peptidic compounds vulnerable to rapid degradation in vivo. Peptide helix formation, however, typically buries and/or twists and/or shields the amide backbone and therefore may prevent or substantially retard proteolytic cleavage. The peptidomimetic macrocycles of the present invention may be subjected to in vitro enzymatic proteolysis (e.g. trypsin, chymotrypsin, pepsin) to assess for any change in degradation rate compared to a corresponding uncrosslinked or alternatively stapled polypeptide. For example, the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycle and peptidomimetic precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S/E ?125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction displays first order kinetics and the rate constant, k, is determined from a plot of ln[S] versus time.

    [0358] Peptidomimetic macrocycles and/or a corresponding uncrosslinked polypeptide can be each incubated with fresh mouse, rat and/or human serum (e.g. 1-2 mL) at 37? C. for, e.g., 0, 1, 2, 4, 8, and 24 hours. Samples of differing macrocycle concentration may be prepared by serial dilution with serum. To determine the level of intact compound, the following procedure may be used: The samples are extracted, for example, by transferring 100 ?L of sera to 2 ml centrifuge tubes followed by the addition of 10 ?L of 50% formic acid and 500 ?L acetonitrile and centrifugation at 14,000 RPM for 10 min at 4+/?2? C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N2<10 psi, 37? C. The samples are reconstituted in 100 ?L of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis. Equivalent or similar procedures for testing ex vivo stability are known and may be used to determine stability of macrocycles in serum.

    [0359] Plasma Stability Assay: Stapled peptide stability can be tested in freshly drawn mouse plasma collected in lithium heparin tubes. Triplicate incubations are set up with 500 ?l of plasma spiked with 10 ?M of the individual peptides. Samples are gently shaken in an orbital shaker at 37? C. and 25 ?l aliquots are removed at 0, 5, 15, 30, 60, 240, 360 and 480 min and added to 100 ?l of a mixture containing 10% methanol: 10% water: 80% acetonitrile to stop further degradation of the peptides. The samples are allowed to sit on ice for the duration of the assay and then transferred to a MultiScreen? Solvinert 0.45 ?m low-binding hydrophilic PTFE plate (Millipore). The filtrate is directly analyzed by LC-MS/MS. The peptides are detected as double or triple charged ions using a Sciex 5500 mass spectrometer. The percentage of remaining peptide is determined by the decrease in chromatographic peak area and log transformed to calculate the half-life.

    [0360] In Vivo Protease Resistance Assays: A key benefit of peptide stapling is the translation of in vitro protease resistance into markedly improved pharmacokinetics in vivo.

    [0361] Liquid chromatography/mass spectrometry-based analytical assays are used to detect and quantitate SARS-CoV-2 levels in plasma. For pharmacokinetic analysis, peptides are dissolved in sterile aqueous 5% dextrose (1 mg/mL) and administered to C57BL/6 mice (Jackson Laboratory) by bolus tail vein or intraperitoneal injection (e.g. 5, 10, 25, 50 mg/kg). Blood is collected by retro-orbital puncture at 5, 30, 60, 120, and 240 minutes after dosing 5 animals at each time point. Plasma is harvested after centrifugation (2,500?g, 5 minutes, 4? C.) and stored at ?70? C. until assayed. Peptide concentrations in plasma are determined by reversed-phase high performance liquid chromatography with electrospray ionization mass spectrometric detection (Aristoteli et al., Journal of Proteome Res., 2007; Walden et al., Analytical and Bioanalytical Chem., 2004). Study samples are assayed together with a series of 7 calibration standards of peptide in plasma at concentrations ranging from 1.0 to 50.0 ?g/mL, drug-free plasma assayed with and without addition of an internal standard, and 3 quality control samples (e.g. 3.75, 15.0, and 45.0 ?g/mL). Standard curves are constructed by plotting the analyte/internal standard chromatographic peak area ratio against the known drug concentration in each calibration standard. Linear least squares regression is performed with weighting in proportion to the reciprocal of the analyte concentration normalized to the number of calibration standards. Values of the slope and y-intercept of the best-fit line are used to calculate the drug concentration in study samples. Plasma concentration-time curves are analyzed by standard noncompartmental methods using WinNonlin Professional 5.0 software (Pharsight Corp., Cary, NC), yielding pharmacokinetic parameters such as initial and terminal phase plasma half-life, peak plasma levels, total plasma clearance, and apparent volume of distribution.

    [0362] Persistence of stabilized ACE2 ?1 helix peptides in the nasal mucosa after topical administration (i.e. nose drops) and in the respiratory mucosa after nebulization is examined in the context of pre- and post-infection blockade of viral fusion and dissemination. Mice are exposed to single ACE2 ?1 helix peptide treatment by nose drop or nebulizer at a series of intervals preceding intranasal infection with SARS-CoV-2, and the duration of protection from mucosal infection (assessed histologically as described above) used to measure the relative mucosal stability and prophylactic efficacy of ACE2 ?1 helix constructs.

    [0363] In vitro SARS CoV-2 RBD Protein Binding Assays: To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins, a fluorescence polarization assay (FPA) can be used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein, such as in this case, recombinant SARS-CoV-2 RBD protein or recombinant SARS-CoV-2 spike protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution). A positive control binding interaction between a FITC-labeled ACE2h1 peptide and SARS-CoV-2 protein can also be used to conduct competitive FPAs, in which non-fluorescently labeled peptides are incubated with the FITC-peptide/RBD complex to assess the differential capacity of alternate SAH-ACE2h1 peptides to compete with the FITC-peptide for protein binding. Another method for evaluating the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins involves anchoring the test peptides on a solid support, such as the use of biotinylated SAH-ACE2h1 peptides bound to streptavidin-coated tips in a biolayer interferometry assay, whereby the association and dissociation of RBD protein in solution to the peptide-coated tip is monitored and quantitated. Binding assays are performed with FITC-labeled ACE2h1 peptides for direct FPAs, non-fluorescent ACE2h1 peptides for competitive FPAs, and affinity tagged ACE2h1 peptides (such as biotinylated peptides) for peptide capture on a compatible BLI tip (e.g. streptavidin coated tip for capture of biotinylated peptides). Target proteins for analysis include recombinant SARS-CoV-2 RBD or spike proteins expressed in E coli or HEK293 cells with GST or His tags that are either cleaved after purification or retained for affinity capture (e.g. glutathione plates, Ni-NTA beads or plates, etc.).

    [0364] In vitro SARS-CoV-2 RBD Bead Binding Assay: To monitor the association of FITC-labeled ACE2h1 peptides to RBD-coated beads, which can be used for qualitative and quantitative assessment of binding activity, His-tagged RBD protein (25 ?g) or vehicle is incubated with Ni-NTA agarose beads (100 ?L) in PBS for 30 min. The beads are then incubated with FITC-labeled peptide (10 ?M) for 30 min, isolated by benchtop centrifugation (2000?g), resuspended in PBS for plating in 386-well plate format (10 L/well), and imaged using an Olympus wide-field epifluorescence microscope, a 63? LCPlanFL NA 0.7 objective and a CoolSNAP DYNO camera.

    [0365] Antiviral Efficacy Assays: The efficiency of ACE2 ?1 helix peptides in preventing and treating COVID-19 infection are evaluated in monolayer cell cultures. A viral detection platform has been developed for SARS-CoV-2 based on previous screens against Ebolaviruses (see, Anantpadma M. et al., Antimicrob Agents Chemother. 2016; 60(8):4471-81. Epub 2016/05/11. doi: 10.1128/AAC.00543-16. PubMed PMID: 27161622; PMCID: PMC4958205). Vero E6 cells plated in 384-well format are treated for 1 hour with a serial dilution of stapled peptides (e.g. 10-25 ?M starting dose), performed in triplicate, followed by 4 hour challenge with SARS-CoV-2 to achieve control infection of 10-20% cells (the pre-determined optimal infectivity to assess the dynamic range of test compounds in the assay). Infected cells are then washed, fixed with 4% paraformaldehyde, rewashed in PBS, immune-stained with anti-SARS-CoV-2 nucleocapsid monoclonal antibody followed by anti-Ig secondary antibody (Alexa Fluor 488; Life Technologies), and cell bodies counterstained with HCS CellMask blue. Cells are imaged across the z-plane on a Nikon Ti Eclipse automated microscope, analyzed by CellProfiler software, and infection efficiency calculated by dividing infected by total cells. Control cytotoxicity assays are performed using Cell-Titer Glo (Promega) and LDH release (Roche) assays.

    [0366] In an alternative approach, qPCR based viral detection is used in natively-susceptible human-derived Huh770 and Calu-371 cells that express ACE2, and also MatTek Life Sciences primary lung epithelial and alveolar cell models, infected with SARS-CoV-2 virus (e.g. USA-WA1/2020; Hongkon VM20001061). Cultured cells are treated for 1 hour with a serial dilution of stapled peptides followed by challenge with SARS-CoV-2. Culture supernatants are sampled, virus lysed in the presence of RNAse inhibitor, and RT and qPCR performed as described. See Suzuki et al. J Vis Exp. 2018(141). Epub 2018/11/20. doi: 10.3791/58407. CDC-validated BHQ quenched dye pair primers are purchased from IDT and genome equivalents calculated from Ct values.

    [0367] In yet another approach, antiviral activity of ACE2 ?1 helix stapled peptides are assessed using pseudotyped virus. The 293T-hsACE2 stable cell line (Cat #C-HA101) and the pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain) viral particles coated with the SARS-CoV-2 spike protein and carrying RNA coding for GFP (Cat #RVP-701G, Lot #CG-113A) reporters are used (Integral Molecular). The neutralization assay is carried out according to the manufacturers' protocols. In brief, 5 ?L of a single dose of peptide (5 ?M final dose) is incubated with 5 ?L pseudotyped SARS-CoV-2-GFP for 1 hr at 37? C. in a 384 well black clear bottom plate followed by addition of 30 ?L of 1,000 293T-hsACE2 cells in 10% FBS DMEM, phenol red free media and placed in a humidified incubator for 48 or 72 hrs. Hoechst 33342 and DRAQ7 dyes are added and the plate imaged on a Molecular Devices ImageXpress Micro Confocal Laser at 10? magnification. GFP (+) cells are counted and plotted using Prism software (Graphpad). To evaluate the capacity of lead stapled peptides to prevent SARS-CoV-2 infection, K18-hACE2 (Jackson Laboratory) mice (n=10 per arm; 5 male, 5 female) are administered intranasally or by the oropharyngeal route with stapled peptide or vehicle and 24 hours later a viral dosage of 10.sup.4 PFU is inoculated intranasally. Mice are euthanized 4 days later (peak of viremia) for evaluation by necropsy and viral load as quantitated by qPCR from supernatant samples of lung homogenates, prepared as described using a tissuelyzer (Qiagen). See Bao L et al. Nature. 2020. Epub 2020/05/08; doi: 10.1038/s41586-020-2312-y. To evaluate the capacity of lead stapled peptides to treat or mitigate established SARS-CoV-2 infection, K18-hACE2 mice (n=10 per arm; 5 male, 5 female) are inoculated intranasally at a viral dosage of 10.sup.4 PFU on day 1, followed by daily oropharyngeal or intraperitoneal treatment with stapled peptide or vehicle for 10 days (days 2-12). In an alternate design, dosing is delayed until 3-5 days post-inoculation to simulate symptom- or positive test-driven initiation of therapy. Mice are continuously monitored to record body weights and clinical signs, with disease progression scored as >10% body weight loss, labored breathing, and/or failure to thrive. Doses for the most effective compound and route are then be refined in both prevention and treatment studies to determine the minimum dose to protect mice. The same experimental design is used except that the 4 treatment groups (n=10; 5 male, 5 female) receive the original dose and then 3 progressively lowered doses in 4-fold increments.

    [0368] Clinical Trials: To determine the suitability of the cross-linked polypeptides of the invention for treatment of humans, clinical trials can be performed. For example, patients exposed to SARS-CoV-2 infection or diagnosed with SARS-CoV-2 infection are selected and separated in treatment and one or more control groups, wherein the treatment group is administered a crosslinked polypeptide of the invention, while the control groups receive a placebo or a known antiviral drug. The treatment safety and efficacy of the cross-linked polypeptides of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors such as prevention of symptoms, time to resolution of symptoms, and/or overall infection severity. In another example, uninfected patients are identified and are given either a cross-linked polypeptide or a placebo. After receiving treatment, patients are followed. In both examples, the SARS-CoV-2-exposed patient group treated with a cross-linked polypeptide would avoid the development of infection, or a patient group with SARS-CoV-2 infection would show resolution of or relief from symptoms compared to a patient control group treated with a placebo.

    EXAMPLES

    [0369] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

    Example 1: Design and Synthesis of SAH-ACE2h1 Stapled Peptides

    [0370] Hydrocarbon-stapled peptides were synthesized, purified, and quantitated using previously reported methods (Bird et al., Methods Enzymol., 446:369-86 (2008); Bird et al., Curr. Protoc. Chem. Biol., 3(3):99-117 (2011). To design peptides that could block the interaction between the SARS-CoV-2 spike protein and the human ACE2 receptor, a series of stapled peptides bearing differentially localized chemical staples and ACE2h1 sequences, including a series of mutants, were designed (FIG. 3, FIG. 8). The differentially localized chemical staples were located within the ACE2 receptor helix 1 peptide that engages the receptor binding domain of SARS-CoV-2 (see FIGS. 1 and 2) by replacing native residues with ?, ?-disubstituted non-natural olefinic residues (X) at select (i, i+4) or (i, i+7) positions and combinations thereof in the form of double staples or stitches, followed by ruthenium-catalyzed olefin metathesis (FIGS. 4-6). Some designs incorporate staples on the non-interacting amphiphilic face of the helix, on the interacting face of the helix, or at positions at the border of the interacting and non-interacting faces of the helix (FIG. 1). Template sequences used were based on the human ACE2 helix 1 peptide, N- and C-terminal truncations thereof, and/or a series of mutations of the native sequence (FIGS. 2, 3, 7, and 8).

    [0371] Stabilized Alpha-Helix of ACE2h1 (SAH-ACE2h1) constructs were designed by replacing two naturally occurring amino acids with the non-natural S-2-(4-pentenyl) alanine (S5) amino acids at i, i+4 positions (i.e. flanking 3 amino acids) to generate a staple spanning one ?-helical turn, or a combination of (R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-dec-9-enoic acid (R.sub.8) and S5 at i, i+7 positions, respectively, to generate a staple spanning two ?-helical turns. Asymmetric syntheses of ?, ?-disubstituted amino acids were performed as previously described in detail (Schafmeister et al., J. Am. Chem. Soc., 2000; Walensky et al., Science, 2004; Bird et al. Current Protocols in Chemical Biology, 2011, each of which is incorporated by reference in its entirety).

    [0372] Staple scanning was performed to respectively identify residues and binding surfaces critical for interaction, which dictates the design of optimized constructs and negative control mutants (FIG. 4). The N-termini of SAHs were capped with acetyl, a fluorophore (e.g. FITC, rhodamine), or affinity tag (e.g. biotin) depending upon the experimental application.

    [0373] Doubly stapled peptides were generated by installing two-S5-S5, two R.sub.8S5, or other combinations of crosslinking non-natural amino acids. Multiply stapled or stitched peptides are generated using similar principles (FIGS. 5 and 6).

    [0374] Synthesis of the SAH-ACE2h peptides shown in FIGS. 3 and 8 were performed using solid phase Fmoc chemistry and ruthenium-catalyzed olefin metathesis, followed by peptide deprotection and cleavage, purification by reverse phase high performance liquid chromatography/mass spectrometry (LC/MS), and quantification by amino acid analysis (AAA) (Bird et al., Methods Enzymol., 2008).

    [0375] The peptides shown in Table 3 were synthesized based on the template sequences in Table 1. SEQ ID NOs: 2-12, 145-148, 18-20, 51-60, 134-143, and 172 in Table 1 and FIG. 3 were generated by mutating SEQ ID NO:21 (IEEQAKTFLDKFNHEAEDLFYQSS) with substitutions of naturally-occurring amino acids and/or by substituting amino acids therein with other naturally-occurring amino acids. SEQ ID NOs: 78-111 in FIG. 8 were generated by mutating SEQ ID NO:76 (IEEQAKTFLDKFNHEAEDLFYQS) with substitutions of naturally-occurring amino acids and by substituting amino acids therein with other naturally-occurring amino acids. SEQ ID NOs: 112-126, 134-143, 145-149, and 172 in FIGS. 3 and 8 were generated by mutating SEQ ID NO:21 (IEEQAKTFLDKFNHEAEDLFYQSS) with substitutions of naturally-occurring amino acids and by substituting amino acids therein with other naturally-occurring amino acids. SEQ ID NOs: 127-133 in FIG. 8 were generated by mutating SEQ ID NO:77 (EQAKTFLDKFNHEAEDLFYQ) with substitutions of naturally-occurring amino acids and by substituting amino acids therein with other naturally-occurring amino acids. SEQ ID NOs: 50, 53, 54, and 56 were generated by mutating SEQ ID NO:49 by substituting amino acids therein with other naturally-occurring amino acids (not shown in Table below). SEQ ID NOs: 51, 52, 55, and 57-60 were generated by mutating peptides in SEQ ID NO:49 by substitution with naturally-occurring amino acids and with non-natural all-hydrocarbon cross-link or staple (indicated by 8 or X in Table 6).

    TABLE-US-00013 TABLE6 GeneratedConjugatedACE2Peptides Name Sequence SEQIDNO SAH- IEEQAKTAADKANHEAEQAAYQSAXaa.sub.1Xaa.sub.2, 56 ACE2h1- whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2isAor mut-7 absent (P7) SAH- IEEQAKTXADKXNHEAEQAAYQSAXaa.sub.1Xaa.sub.2, 57 ACE2h1- whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2isAor mut-8 absent (P8) SAH- IEEQXKTAXDKANHEXEQAXYQSAXaa.sub.1Xaa.sub.2, 58 ACE2h1- whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2isAor mut-9 absent (P9) SAH- IEEQXKEAXDKANHEXEQAXYQSAXaa.sub.1Xaa.sub.2, 59 ACE2h1- whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2isAor mut-10 absent (P10) SAH- IEEQAKTA8DKANHEXEQAAYQSAXaa.sub.1Xaa.sub.2, 60 ACE2h1- whereinXaa.sub.1isL,A,orabsent,andXaa.sub.2isAor mut-11 absent (P11)

    [0376] Tables 3 and 6 provides a list of synthesized peptide analogs, where X and 8 (bolded e.g., in Table 6) indicate non-natural amino acids used to install the all-hydrocarbon cross-link or staple. In some instances, additional amino acids (e.g., a leucine (L) and/or an alanine (A)) is added to the C-terminus of each peptide. In some instances, non-natural amino acids as shown in Tables 3 and 6 are inserted into one of the peptides of Table 1 to generate the peptide analogs.

    [0377] In some instances, each peptide in Tables 3 and 6 can include beta alanine at the N-terminus. For example, SEQ ID NO:56 can include BA-IEEQAKTAADKANHEAEQAAYQSAXaa.sub.1Xaa.sub.2, wherein Xaa1 is L, A, or absent, and Xaa.sub.2 is A or absent (SEQ ID NO:171). In some embodiments, a conjugate can be coupled to the N-terminus of any one of the peptides of Tables 3 or 6. In some instances, the conjugate is a detection moiety (e.g., FITC (fluorescein isothiocyanate)). In some instances, the conjugate is a capture moiety (e.g., a biotin moiety).

    Example 2: Assessing Alpha-Helical Stabilization of ACE2h1 Stapled Peptides

    [0378] Generally, short peptides do not exhibit significant ?-helical structure in solution. This is because the entropic cost of maintaining a conformationally-restricted structure is not overcome by the enthalpic gain from hydrogen bonding of the peptide backbone. To document secondary structure improvements of hydrocarbon-stapled peptides, circular dichroism (CD) spectra was recorded and analyzed on a Model 410 Aviv Biomedical spectrometer. Five scans from 190-260 nm in 0.5 nm increments with 0.5 sec averaging time were collectively averaged to obtain each spectrum using a 1 mm path length cell. The target peptide concentration for CD studies was 25-50 ?M in 50 mM potassium phosphate (pH 7.5) or Milli-Q deionized water, and exact concentrations were confirmed by quantitative AAA of two CD sample dilutions. The CD spectra were initially plotted as wavelength versus millidegree. Once the precise peptide concentration was confirmed, the mean residue ellipticity [0], in units of degree.Math.cm.sup.2.Math.dmol.sup.?1.Math.residue.sup.?1, was derived from the equation, [?]=millidegree/molar concentration/number of amino acid residues. After conversion to mean residue ellipticity, percent ?-helicity was calculated using the equation, % helicity=100?[?]222/max[0]222, where max[?]222=?40,000?[1?(2.5/number of amino acid residues)]. Stapled constructs that reinforce ?-helical structure were advanced to protease-resistance testing, binding analyses, and antiviral activity assays. FIGS. 9A-9B shows how native and mutated ACE2h1 sequences, when synthesized as a peptide and evaluated by circular dichroism, do not retain the natural alpha-helical structure found in the context of the ACE2 receptor. In contrast, inserting staples at specific locations can restore alpha-helical shape. For the ACE2h1 mutant sequence shown in FIG. 9A, a single staple at a specific location (SEQ ID NO:114) can restore alpha-helical shape, with ?-helicity improved even further upon double stapling at the indicated locations (SEQ ID NO:115). For a distinct ACE2h1 mutant sequence shown in FIG. 9B, installing double staples in the corresponding positions (SEQ ID NO:126) shown in FIG. 9A, converts the random coil conformation of the unstapled sequences into an ?-helix.

    Example 3: Determining Protease Resistance of SARS-CoV-2 HR2 Stapled Peptides

    [0379] Linear peptides are susceptible to rapid proteolysis in vitro and in vivo, limiting the application of natural peptides for mechanistic analyses and therapeutic use. In contrast, amide bonds engaged in the hydrogen-bonding network of a structured peptide helix are poor enzymatic substrates, as are residues shielded by the hydrocarbon staple itself (Bird et al., PNAS, 2010). To evaluate the relative protease resistance conferred by hydrocarbon stapling, in vitro proteolytic degradation was measured by LC/MS (Agilent 1200) using the following parameters: 20 ?L injection, 0.6 mL flow rate, 15 min run time consisting of a gradient of water (0.1% formic acid) to 20-80% acetonitrile (0.075% formic acid) over 10 min, 4 min wash to revert to starting gradient conditions, and 0.5 min post-time. The DAD signal was set to 280 nm with an 8 nm bandwidth and MSD set to scan mode with one channel at (M+2H)/2, +/?1 mass units and the other at (M+3H)/3, +/?1 mass units. Integration of each MSD signal yielded areas under the curve of >10.sup.8 counts. Reaction samples were composed of 5 ?L peptide in DMSO (1 mM stock) and 195 ?L of buffer consisting of 50 mM Tris HCl at pH 7.4. Upon injection of the 0 hr time point sample, 2 ?L of 100 ng/?L proteinase K (New England Biolabs) was added and the amount of intact peptide quantitated by serial injection over time. An internal control of acetylated tryptophan carboxamide at a concentration of 100 ?M is used to normalize each MSD data point. A plot of MSD area versus time yielded an exponential decay curve and half-lives were determined by nonlinear regression analysis using Prism software (GraphPad). FIGS. 13A and 13B show how insertion of double staples or stitches into the core template sequence (aa 1169-1197) conferred striking protease stability compare to the unstapled sequence, depending on the sequence, staple type, and staple location. FIG. 10 shows that insertion of double staples at the indicated positions into two ACE2h1 sequences (SEQ ID NOs: 120 and 126) bearing mutations confers striking in vitro proteinase K resistance to both constructs compared to the unstapled wild-type ACE2h1 sequence.

    [0380] The protease resistance and stability of stapled peptides were also measured by use of a mouse plasma stability assay. Stapled peptide stability was tested in freshly drawn mouse plasma collected in lithium heparin tubes. Triplicate incubations were set up with 500 ?l of plasma spiked with 10 ?M of the individual peptides. Samples were gently shaken in an orbital shaker at 37? C. and 25 ?l aliquots were removed at 0, 5, 15, 30, 60, 240, 360 and 480 min and added to 100 ?l of a mixture containing 10% methanol: 10% water: 80% acetonitrile to stop further degradation of the peptides. The samples were allowed to sit on ice for the duration of the assay and then transferred to a MultiScreen Solvinert 0.45 ?m low-binding hydrophilic PTFE plate (Millipore). The filtrate was directly analyzed by LC-MS/MS. The peptides were detected as double or triple charged ions using a Sciex 5500 mass spectrometer. The percentage of remaining peptide was determined by the decrease in chromatographic peak area and log transformed to calculate the half-life. FIG. 11 shows the mouse plasma stability of an unstapled ACE2h1 mutant sequence (SEQ ID NO: 113, top chart) and a double-stapled analog (SEQ ID NO: 115, bottom chart). Whereas the unstapled peptide demonstrates a half-life of 374 min, essentially no degradation was observed upon insertion of double staples at the indicated locations.

    Example 4: Visualization of ACE2h Stapled Peptide Binding Activity to SARS-CoV-2 RBD Protein by Bead Assay

    [0381] To asses and visualize the binding activity of SAH-ACE2h1 peptides for the SARS-CoV-2 receptor binding domain (RBD), 25 ?g of recombinant His-tagged RBD containing amino acids Val 16-Arg 685 of S1 (SEQ ID NO:75) was captured using 100 ?l of Ni-NTA agarose beads (Invitrogen? catalog #R90110) followed by incubation with the FITC-labeled peptides (10 ?M) for 30 min. FIG. 12A shows that an unstapled and mutated ACE2h1 peptide has little to no detectable binding to the RBD-coated beads (FIG. 12A, column A, SEQ ID NO: 113), whereas insertion of a single staple at the indicated location (FIG. 12A, column B, SEQ ID NO: 114) and then double staples at the indicated locations (FIG. 12A, columns C and E, SEQ ID NOs: 115 and 116, respectively) leads to progressively enhanced binding activity and RBD detection. Importantly, when no RBD protein is added to the beads, the positive fluorescence signal of an exemplary double stapled ACE2h1 peptide seen with RBD-coated beads (FIG. 12, columns A-C, SEQ ID NOs: 113-115, respectively) is completely lost (FIG. 12A, column D, SEQ ID NO: 115), highlighting the specificity of double stapled peptide binding activity for RBD. FIG. 12B shows that peptide templates bearing a series of mutations can enhance binding to the RBD-coated beads compared to the native sequence (FIG. 12A, columns A-C, SEQ ID NOs: 76, 123, and 125, respectively), including a double stapled analog (FIG. 12A, column D, SEQ ID NO:126).

    Example 5: Utilization of SAH-ACE2h1 Peptides as a Detection Agent for the Diagnosis of SARS-CoV-2

    [0382] The peptides of the invention can be derivatized with fluorophores, affinity tags, and/or enzymes (e.g. HRP) and incorporated into an assay for the detection of SARS-CoV-2 in human fluid (nasal, oral, respiratory fluid or mucous), as a rapid, capture-and-detect (non-serologic/non-genetic) point-of-care diagnostic test. FIG. 13 shows that the bead-binding results in FIG. 12, namely the capacity of a FITC-labeled and stapled ACE2h1 peptide sequence to bind and detect SARS-CoV-2 RBD protein, can afford simple and rapid virus detection methods. For example, Peptide A (detection tag; e.g. fluorophore) and B (affinity tag; e.g. biotin) are mixed in a solution (FIG. 13A, part A). The patient sample is added to the peptide solution and mixed (FIG. 13A, part B). The virus contains multiple sites to which the differentially-labeled (e.g., comprising a moiety for detection or a moiety for affinity capture) peptides can bind, and therefore, each viral particle can bind to Peptides A and B. The capture beads (e.g. streptavidin beads to capture biotinylated peptide) are added, mixed, and collected by gravity or centrifugation (FIG. 13A, part C). If the result is negative (no virus present) the peptide containing the chromophore/fluorophore remains in solution (FIG. 13A, part D, top). When the result is positive (virus present in sample), the virus is captured by the beads via peptide B, which is collected along with simultaneous virus-bound peptide A, leading to an immediate read-out using a detection instrument (FIG. 13A, part D, bottom). An alternative approach is shown in FIG. 13B and is based on a pregnancy-type strip or ELISA set up in which SAH-ACE2h1 peptide is fixed to a solid support (FIG. 13B, parts A and B; e.g. biotin-peptide saturates a streptavidin coated plate), a patient sample is added to the strip or plate well (FIG. 13B, part C), and then application of a second SAH-ACE2h1 peptide is applied (FIG. 13B, part D) that allows for a colorimetric read-out, such as a second biotinylated SAH-ACE2h1 peptide detected by streptavidin HRP (FIG. 13B, part E) and incubation with chromogenic substrate (FIG. 13B, part F). FIG. 13C demonstrates the successful development of a SAH-ACE2h1-based test strip, based on this concept of an enzyme-linked stapled peptide assay (ELIPSA), which dose-responsively detects a serial dilution of inactivated native SARS-CoV-2 virus (starting titer of 10). Like a pregnancy strip test, this diagnostic ELIPSA method can be used as a simple, rapid, and point-of-care method that does not require complex infrastructure or expertise to diagnose the infection, and thus can also be for home use.

    Example 6: Solution-Phase Binding Analysis of SAH-ACE2h1 Interaction with SARS-CoV-2 RBD and Spike Proteins

    [0383] Direct and competitive fluorescence polarization binding assays were used to evaluate and compare the binding activity of SAH-ACE2h1 peptides for SARS-CoV-2 proteins, including recombinant SARS-CoV-2 RBD and Spike proteins. FIGS. 14A-14C shows the binding activities of differentially stapled and mutated ACE2h1 peptides for recombinant SARS-CoV-2 proteins containing the RBD. In FIG. 14A, a direct fluorescence polarization binding assay (FPA) is shown in which FITC-labeled double i, i+4 stapled ACE2h1 peptides bearing a series of mutations demonstrate dose-responsive binding activity when incubated with a serial dilution of purified, recombinant, GST-tagged SARS-CoV-2 RBD protein expressed in E. coli. In FIG. 14B, the direct binding interaction between a FITC-labeled double i, i+4 stapled ACE2h1 peptides bearing a series of mutations and the recombinant SARS-CoV-2 spike protein is used to screen biotinylated (non-fluorescent) and differentially stapled ACE2h1 peptides, with and without mutations, for competitive spike protein binding activity in solution, revealing constructs that were capable (black bar). In FIG. 14C, a direct fluorescence polarization binding assay (FPA) is shown in which two FITC-labeled double i, i+4 stapled ACE2h1 peptides bearing a series of mutations bound to wild-type SARS-CoV-2 RBD protein expressed in mammalian HEK293 cells and retained at least equivalent, or exhibit more, binding activity to SARS-CoV-2 RBD proteins bearing clinical variants such as the UK (N501Y) and South African (K417N, E484K and N501Y), with the latter variant protein in particular showing enhanced binding activity to both SAH-ACE2h1 peptides.

    Example 7: Solid-Phase Binding Analysis of SAH-ACE2h1 Interaction with SARS-CoV-2 RBD Protein

    [0384] Applying SAH-ACE2h1 peptide to a solid support, such as via biotin-peptide capture by a streptavidin coated tip or chip, can be used to evaluate and compare the binding activity of SAH-ACE2h1 peptides for SARS-CoV-2 RBD protein using methods such as biolayer interferometry (BLI) or surface plasmon resonance. FIGS. 15A-15B show a binding analysis in which biotinylated stapled ACE2h1 peptides were applied to a streptavidin-coated tip (solid support) and tested for recombinant SARS-CoV-2 RBD binding activity by BLL. In FIG. 15A, single i, i+4 or single i, i+7 stapled ACE2h1 peptides were tested for SARS-CoV-2 RBD binding activity at a screening dose, revealing compounds that do or do not bind to RBD based on peptide sequence, staple type, and/or staple position. In FIG. 15B, double i, i+4 stapled ACE2h1 peptides bearing a series of mutations showed dose-responsive RBD binding activity.

    Example 8: Antiviral Activity of SAH-ACE2h1 Peptides as Assessed by Pseudovirus Infection Assay

    [0385] Antiviral activity of ACE2 ?1 helix stapled peptides were assessed using pseudotyped virus. The 293T-hsACE2 stable cell line (Cat #C-HA101) and the pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain) particles with GFP (Cat #RVP-701G, Lot #CG-113A) reporters were used (Integral Molecular). The neutralization assay was carried out according to the manufacturers' protocols. In brief, 5 ?L of a single dose of peptide (5 ?M final dose) was incubated with 5 ?L pseudotyped SARS-CoV-2-GFP for 1 hr at 37? C. in a 384 well black clear bottom plate followed by addition of 30 ?L of 1,000 293T-hsACE2 cells in 10% FBS DMEM, phenol red free media and placed in a humidified incubator for 48 or 72 hrs. Hoechst 33342 and DRAQ7 dyes were added and the plate imaged on a Molecular Devices ImageXpress Micro Confocal Laser at 10? magnification. GFP (+) cells were counted and plotted using Prism software (Graphpad). FIGS. 16A-16B show a SARS-CoV-2 pseudovirus assay in which GFP-coding virus was used to infect 293T cells that were treated with ACE2h1 peptides, followed by fluorescence microscopy imaging to detect and compare the level of virus between vehicle and peptide treatments to assay for inhibition of viral infection (FIG. 16A), which was quantitated by image analysis (FIG. 16B). Insertion of a single staple conferred antiviral activity compared to the unstapled template peptide that showed no activity, with double stapling enhancing antiviral activity further. Installing a series of mutations into the template sequence or double stapling an N- and C-terminally truncated construct bearing a series of distinct mutations also conferred marked antiviral activity in the pseudovirus assay.

    Example 9: Antiviral Activity of SAH-ACE2h1 Peptides as Assessed by a Native SARS-CoV-2 Infectivity Assay

    [0386] Vero E6 cells plated in 384-well format were treated for 1 hour with either a screening dose of stapled peptide (e.g. 25 ?M) or a serial dilution of stapled peptide (e.g. 25 ?M starting dose), performed in triplicate, followed by 4 hour challenge with SARS-CoV-2 to achieve control infection of 10-20% cells (the pre-determined optimal infectivity to assess the dynamic range of test compounds in the assay). Infected cells were then washed, fixed with 4% paraformaldehyde, rewashed in PBS, immune-stained with anti-SARS-CoV-2 nucleocapsid monoclonal antibody followed by anti-Ig secondary antibody (Alexa Fluor 488; Life Technologies), and cell bodies counterstained with HCS CellMask blue. Cells were imaged across the z-plane on a Nikon Ti Eclipse automated microscope, analyzed by CellProfiler software, and infection efficiency calculated by dividing infected by total cells. Control cytotoxicity assays were performed using Cell-Titer Glo (Promega) and LDH release (Roche) assays. FIG. 17A shows that initial screening for inhibitory activity at a 25 ?M test dose demonstrated that single and double stapled constructs of a template ACE2h1 sequence exhibited improved antiviral activity. Positive hits were advanced to serial dilution testing. FIG. 17B shows the differential antiviral activity of a series of SAH-ACE2h1 peptides bearing single i, i+4 or i, i+7 staples, or double i, i+4 staples, in the context of various ACE2h1 template sequences bearing a serious of distinct mutations. FIG. 17C shows additional biological replicates of i, i+4 double stapled peptides of ACE2h1 template sequences bearing a series of mutations and exhibiting dose-responsive antiviral activity.

    Example 10: Identification of Lead SAH-ACE2h1 Peptides by Correlation of Biochemical and Antiviral Activities

    [0387] Synthesis and multidisciplinary testing of libraries of stapled peptides based on diverse sequence templates, which can incorporate individual or a series of mutations, can enable the identification of lead constructs that demonstrate correlations between optimized biophysical properties, target binding affinities, and functional antiviral activity. FIGS. 18A-18C show how lead SAH-ACE2h1 peptides were identified based on consistency of activity across a diversity of assays, including binding assays with peptides in solution or on solid support and antiviral assays using SARS-CoV-2 pseudovirus or native virus. FIG. 18A shows an exemplary single i, i+4 stapled peptide of the native sequence with no biological activity across 4 assays and 5 single i, i+4 stapled peptides of the native sequence that demonstrated biological activity in 3 of 4 functional assays. FIG. 18B shows two exemplary i, i+7 single stapled peptides of the native sequence with no biological activity across 4 assays and 8 single i, i+7 single stapled peptides of the native sequence that demonstrated biological activity in at least 3 of 4 functional assays, with 3 compositions demonstrating efficacy across all RBD binding and antiviral assays. FIG. 18C shows how incorporation of favorable staple types and locations based on staple scanning into templates iterated by amino acid mutagenesis led to identification of a series of lead constructs with consistent biological activity across 4 independent assays, spanning soluble binding, solid phase binding, pseudovirus infectivity, and native virus infectivity assays.

    Other Embodiments

    [0388] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims