METHODS FOR THE IDENTIFICATION OF BIFUNCTIONAL COMPOUNDS
20190144503 ยท 2019-05-16
Assignee
Inventors
Cpc classification
C12Y402/01001
CHEMISTRY; METALLURGY
C07K14/78
CHEMISTRY; METALLURGY
G01N33/74
PHYSICS
C07K16/2866
CHEMISTRY; METALLURGY
C07K7/54
CHEMISTRY; METALLURGY
G01N33/53
PHYSICS
C07K2318/20
CHEMISTRY; METALLURGY
C12Q1/25
CHEMISTRY; METALLURGY
International classification
C07K7/54
CHEMISTRY; METALLURGY
G01N33/53
PHYSICS
C12Q1/25
CHEMISTRY; METALLURGY
G01N33/74
PHYSICS
Abstract
The present disclosure provides bifunctional compounds including a polypeptide targeting moiety conjugated to a small molecule in a site-specific manner both of which bind to the same target protein resulting in potent and specific binding characteristics and methods of identifying such compounds.
Claims
1. A synthetic bifunctional compound, or a pharmaceutically acceptable salt thereof, that modulates the activity of an extracellular target protein, the compound comprising a polypeptide targeting moiety that binds to the extracellular target protein covalently conjugated to a small molecule moiety that binds to the extracellular target protein, wherein the bifunctional compound binds to the target protein with at least 5-fold greater affinity and/or 5-fold greater selectivity than the affinity of each of the polypeptide targeting moiety and the small molecule moiety alone.
2. The synthetic bifunctional compound of claim 1, wherein the polypeptide targeting moiety is an antibody or an antigen binding fragment thereof or a fibronectin type III domain.
3. The synthetic bifunctional compound of claim 2, wherein the antibody or an antigen binding fragment thereof is an antibody or a fibronectin type III domain.
4-19. (canceled)
20. The synthetic bifunctional compound of claim 1, wherein the extracellular target protein is a carbonic anhydrase.
21. (canceled)
22. The synthetic bifunctional compound of claim 20, wherein the small molecule moiety comprises the structure of Formula II: ##STR00023## wherein R.sup.1 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, or optionally substituted C.sub.2-C.sub.9 heteroaryl.
23. The synthetic bifunctional compound of claim 1, wherein the extracellular target protein is a metalloprotease.
24. (canceled)
25. The synthetic bifunctional compound of claim 23, wherein the small molecule moiety comprises the structure of Formula III: ##STR00024## wherein R.sup.2 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, or optionally substituted C.sub.2-C.sub.9 heteroaryl.
26. The synthetic bifunctional compound of claim 1, wherein the extracellular target protein is PSMA.
27. (canceled)
28. The bifunctional compound of claim 26, wherein the small molecule moiety comprises the structure of Formula IV or Formula V: ##STR00025## wherein R.sup.3 is optionally substituted C.sub.1-C.sub.6 alkyl or optionally substituted C.sub.1-C.sub.6 heteroalkyl (e.g., NHC(O)); and R.sup.4 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl.
29. (canceled)
30. The bifunctional compound of claim 1, wherein the extracellular target protein is CXCR4.
31. (canceled)
32. The bifunctional compound of claim 30, wherein the small molecule moiety comprises the structure of Formula VI: ##STR00026## wherein R.sup.5 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl.
33. (canceled)
34. A method of identifying a compound that modulates the activity of a target protein, the method comprising: (a) providing two or more synthetic bifunctional compounds comprising a polypeptide targeting moiety covalently conjugated to a small molecule moiety; and (b) contacting a target protein with the two or more synthetic bifunctional compounds; (c) determining the binding of the two or more synthetic bifunctional compounds to the target protein, wherein a compound is identified as modulating the activity of the target protein if the synthetic bifunctional compound binds to the target protein with at least 5-fold greater affinity and/or 5-fold greater selectivity than the affinity of each of the polypeptide targeting moiety and the small molecule alone.
35-54. (canceled)
55. The method of claim 34, wherein the extracellular target protein is a carbonic anhydrase.
56. (canceled)
57. The method of claim 55, wherein the small molecule moiety comprises the structure of Formula II: ##STR00027## wherein R.sup.1 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, or optionally substituted C.sub.2-C.sub.9 heteroaryl.
58. The method of claim 34, wherein the extracellular target protein is a metalloprotease.
59. (canceled)
60. The method of claim 58, wherein the small molecule moiety comprises the structure of Formula III: ##STR00028## wherein R.sup.2 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, or optionally substituted C.sub.2-C.sub.9 heteroaryl.
61. The method of claim 34, wherein the extracellular target protein is PSMA.
62. (canceled)
63. The method of claim 61, wherein the small molecule moiety comprises the structure of Formula IV or Formula V: ##STR00029## wherein R.sup.3 is optionally substituted C.sub.1-C.sub.6 alkyl or optionally substituted C.sub.1-C.sub.6 heteroalkyl (e.g., NHC(O)); and R.sup.4 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl.
64. (canceled)
65. The method of claim 34, wherein the extracellular target protein is CXCR4.
66. (canceled)
67. The method of claim 65, wherein the small molecule moiety comprises the structure of Formula VI: ##STR00030## wherein R.sup.5 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl.
68-73. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
Small Molecule Moieties
[0163] The small molecule moieties of the invention include low molecular weight organic and/or inorganic compounds which have affinity (e.g., low affinity) for the target protein when not covalently conjugated to the polypeptide targeting moiety. In some embodiments, the small molecule moiety binds at the active site of the target protein. In some embodiments, the small molecule moiety does not specifically bind to the target protein (e.g., the small molecule binds to more than one member of the family of proteins to which the target protein belongs.)
[0164] In some embodiments, a small molecule is an antagonist (e.g., a direct inhibitor) of the target protein. In some embodiments, the small molecule is an agonist or a partial agonist (e.g., a direct agonist of partial agonist) of the target protein.
[0165] In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g., is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulating compound. In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic.
[0166] In some embodiments, the small molecule moiety is a sulfonamide. For example, the small molecule moiety includes the structure of Formula II:
##STR00017##
[0167] wherein R.sup.1 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, or optionally substituted C.sub.2-C.sub.9 heteroaryl.
[0168] In some embodiments, the small molecule moiety is a hydroxamic acid. For example, the small molecule moiety includes the structure of Formula III:
##STR00018##
[0169] wherein R.sup.2 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, or optionally substituted C.sub.2-C.sub.9 heteroaryl.
[0170] In some embodiments, the small molecule moiety is a thiadiazole sulfonamide or includes a glutamate-urea-lysine moiety. For example, the small molecule moiety includes the structure of Formula IV or Formula V:
##STR00019##
[0171] wherein R.sup.3 is optionally substituted C.sub.1-C.sub.6 alkyl or optionally substituted C.sub.1-C.sub.6 heteroalkyl (e.g., NHC(O)); and
[0172] R.sup.4 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl.
[0173] In some embodiments, the small molecule moiety includes the structure:
##STR00020##
[0174] In some embodiments, the small molecule moiety is a cyclic peptide. For example, the small molecule moiety includes the structure of Formula VI:
##STR00021##
[0175] wherein R.sup.5 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.6 heteroaryl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.2-C.sub.6 heteroaryl C.sub.1-C.sub.6 alkyl.
[0176] In some embodiments, the small molecule moiety includes the structure:
##STR00022##
Linkers
[0177] The compounds of the invention include a linker (e.g., moiety linker joining a small molecule moiety to a polypeptide targeting moiety). The linker component of the invention is, at its simplest, a bond, but may also provide a linear, cyclic, or branched molecular skeleton having pendant groups covalently linking two moieties.
[0178] Thus, a linker, when included in a compound and/or conjugate as described herein, achieves linking of two (or more) moieties by covalent means, involving bond formation with one or more functional groups located on either moiety. Examples of chemically reactive functional groups which may be employed for this purpose include, without limitation, amino, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenolic groups.
[0179] In some embodiments, such covalent linking of two (or more) moieties may be effected using a linker that contains reactive moieties capable of reaction with such functional groups present in either moiety. For example, an amine group of a moiety may react with a carboxyl group of the linker, or an activated derivative thereof, resulting in the formation of an amide linking the two.
[0180] Examples of moieties capable of reaction with sulfhydryl groups include -haloacetyl compounds of the type XCH.sub.2CO (where XBr, Cl, or I), which show particular reactivity for sulfhydryl groups, but which can also be used to modify imidazolyl, thioether, phenol, and amino groups as described by Gurd, Methods Enzymol. 11:532 (1967). N-Maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionally be useful in coupling to amino groups under certain conditions. Reagents such as 2-iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), which introduce a thiol group through conversion of an amino group, may be considered as sulfhydryl reagents if linking occurs through the formation of disulfide bridges.
[0181] Examples of reactive moieties capable of reaction with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include: [0182] (i) -haloacetyl compounds, which show specificity towards amino groups in the absence of reactive thiol groups and are of the type XCH.sub.2CO (where XBr, Cl, or I), for example, as described by Wong Biochemistry 24:5337 (1979); [0183] (ii) N-maleimide derivatives, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group, for example, as described by Smyth et al., J. Am. Chem. Soc. 82:4600 (1960) and Biochem. J. 91:589 (1964); [0184] (iii) aryl halides such as reactive nitrohaloaromatic compounds; [0185] (iv) alkyl halides, as described, for example, by McKenzie et al., J. Protein Chem. 7:581 (1988); [0186] (v) aldehydes and ketones capable of Schiff's base formation with amino groups, the adducts formed usually being stabilized through reduction to give a stable amine; [0187] (vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups; [0188] (vii) chlorine-containing derivatives of s-triazines, which are very reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups; [0189] (vii) aziridines based on s-triazine compounds detailed above, e.g., as described by Ross, J. Adv. Cancer Res. 2:1 (1954), which react with nucleophiles such as amino groups by ring opening; [0190] (ix) squaric acid diethyl esters as described by Tietze, Chem. Ber. 124:1215 (1991); and [0191] (x) -haloalkyl ethers, which are more reactive alkylating agents than normal alkyl halides because of the activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem. 28:463 (1993).
[0192] Representative amino-reactive acylating agents include: [0193] (i) isocyanates and isothiocyanates, particularly aromatic derivatives, which form stable urea and thiourea derivatives respectively; [0194] (ii) sulfonyl chlorides, which have been described by Herzig et al., Biopolymers 2:349 (1964); [0195] (iii) acid halides; [0196] (iv) active esters such as nitrophenylesters or N-hydroxysuccinimidyl esters; [0197] (v) acid anhydrides such as mixed, symmetrical, or N-carboxyanhydrides; [0198] (vi) other useful reagents for amide bond formation, for example, as described by M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, 1984; [0199] (vii) acylazides, e.g., wherein the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58:347 (1974); [0200] (vii) imidoesters, which form stable amidines on reaction with amino groups, for example, as described by Hunter and Ludwig, J. Am. Chem. Soc. 84:3491 (1962); and [0201] (ix) haloheteroaryl groups such as halopyridine or halopyrimidine.
[0202] Aldehydes and ketones may be reacted with amines to form Schiff's bases, which may advantageously be stabilized through reductive amination. Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, for example, as described by Webb et al., in Bioconjugate Chem. 1:96 (1990).
[0203] Examples of reactive moieties capable of reaction with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high specificity to generate ester groups, for example, as described by Herriot, Adv. Protein Chem. 3:169 (1947). Carboxyl modifying reagents such as carbodiimides, which react through O-acylurea formation followed by amide bond formation, may also be employed.
[0204] It will be appreciated that functional groups in either moiety may, if desired, be converted to other functional groups prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives; conversion of thiols to carboxyls using reagents such as -haloacetates; conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to the thiol with sodium acetate.
[0205] So-called zero-length linkers, involving direct covalent joining of a reactive chemical group of one moiety with a reactive chemical group of the other without introducing additional linking material may, if desired, be used in accordance with the invention.
[0206] More commonly, however, the linker will include two or more reactive moieties, as described above, connected by a spacer element. The presence of such a spacer permits bifunctional linkers to react with specific functional groups within either moiety, resulting in a covalent linkage between the two. The reactive moieties in a linker may be the same (homobifunctional linker) or different (heterobifunctional linker, or, where several dissimilar reactive moieties are present, heteromultifunctional linker), providing a diversity of potential reagents that may bring about covalent attachment between the two moieties.
[0207] Spacer elements in the linker typically consist of linear or branched chains and may include a C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, C.sub.2-C.sub.100 polyethylene glycol, or C.sub.1-10 heteroalkyl.
[0208] In some embodiments, the spacer element of a linker consists of a polyethylene glycol. Polyethylene glycols of the invention are considered to include an alkoxy chain comprised of one or more momomer units, each monomer unit consisting of OCH2CH2-. Polyethyelene glycol (PEG) is also sometimes referred to as polyethylene oxide (PEO) or polyoxyethylene (POE), and these terms may be considered interchangeable for the purpose of this invention. For example, a polyethylene glycol may have the structure, (CH2)s2(OCH2CH2)s1(CH2)s3O, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), and each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10). In some embodiments, the PEG is PEG.sub.2, PEG.sub.3, PEG.sub.5, or PEG.sub.7, wherein the subscript refers to the number of OCH2CH2-monomer units of the PEG. Polyethylene glycol may also be considered to include an amino-polyethylene glycol of NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1-, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl.
[0209] In some embodiments, the linker includes a polypeptide having two or more amino acid residues. In some embodiments, the linker is a polypeptide having the formula (G.sub.4S).sub.n, where n is 1 or greater (e.g., n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some embodiments, the linker is a polypeptide having the formula (G.sub.4S).sub.5. In some embodiments, the linker is a polypeptide having the formula (G.sub.4S).sub.10. In some embodiments, the linker is a polypeptide having the formula (G.sub.4S).sub.15. In some embodiments, the linker is a polypeptide having the formula (G.sub.4S).sub.n(EAK).sub.m, where n and m each independently have a value or 1 or greater (e.g., n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some embodiments, the linker is a polypeptide having the formula (G.sub.4S).sub.5(EAK).sub.5. In some embodiments, the linker is a polypeptide having the formula (G.sub.4S).sub.10(EAK).sub.5. In some embodiments, the linker is a polypeptide having the formula (G.sub.4S).sub.n(EAK).sub.m(G.sub.4S).sub.k, where n, m, and k each independently have a value or 1 or greater (e.g., n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some embodiments, the linker is a polypeptide having the formula (G.sub.4S).sub.5(EAK).sub.10(G.sub.4S).sub.5.
[0210] In some instances, the linker is described by Formula V.
[0211] Examples of homobifunctional linkers useful in the preparation of conjugates of the invention include, without limitation, diamines and diols selected from ethylenediamine, propylenediamine and hexamethylenediamine, ethylene glycol, diethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol, cyclohexanediol, and polycaprolactone diol.
[0212] In some embodiments, the linker is a bond or a linear chain of up to 10 atoms, independently selected from carbon, nitrogen, oxygen, sulfur or phosphorous atoms, wherein each atom in the chain is optionally substituted with one or more substituents independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro, hydroxyl, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and wherein any two atoms in the chain may be taken together with the substituents bound thereto to form a ring, wherein the ring may be further substituted and/or fused to one or more optionally substituted carbocyclic, heterocyclic, aryl, or heteroaryl rings. In some embodiments, a linker has the structure of Formula I:
A.sup.1-(B.sup.1).sub.a(C.sup.1).sub.b(B.sup.2).sub.c-(D)-(B.sup.3).sub.d(C.sup.2).sub.e(B.sup.4).sub.f-A.sup.2 Formula I
where A.sup.1 is a bond between the linker and polypeptide targeting moiety; A.sup.2 is a bond between the small molecule moiety and the linker; B.sup.1, B.sup.2, B.sup.3, and B.sup.4 each, independently, is selected from optionally substituted C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, O, S, and NR.sup.N; R.sup.N is hydrogen, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl; C.sup.1 and C.sup.2 are each, independently, selected from carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; a, b, c, d, e, and f are each, independently, 0 or 1; and D is optionally substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10 alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkyl, or a chemical bond linking A.sup.1-(B.sup.1).sub.a(C.sup.1).sub.b(B.sup.2).sub.c to (B.sup.3).sub.d(C.sup.2).sub.e(B.sup.4).sub.f- A.sup.2.
Polypeptides
[0213] Polypeptides include, for example, any of a variety of hematologic agents (including, for instance, erythropoietin, blood-clotting factors, etc.), interferons, colony stimulating factors, antibodies, enzymes, and hormones. The identity of a particular polypeptide is not intended to limit the present disclosure, and any polypeptide of interest can be a polypeptide in the present methods.
[0214] In some embodiments, the polypeptide is an ion channel, such as a leukocyte ion channel (e.g., CRAC or Kv1.3), a TRP channel (e.g., TRPV1), a purinergic receptor (e.g., a P2X or P2Y receptor), or an epithelial sodium channel (ENaC).
[0215] In some embodiments, the polypeptide is a GPCR, such as an adenosine receptor (AR), an endothelin receptor (ETR), a bradykinin receptor (BKR), an angiotensin receptor (AIR or AIIR), a cannabinoid receptor (CNR), a muscarinic receptor, a neurotensin receptor (NTR), a C5a receptor (C5aR), a purinergic receptor (e.g., a P2X or P2Y receptor), calcitonin gene-related peptide receptor (CGRP-R), or glucagon-like peptide 1 receptor (GLP1R).
[0216] In some embodiments, the polypeptide is an enzyme, such an enzyme of the PCSK family (e.g., furin), matriptase, prostasin, MT1-MMP (also called MMP14), a disintegrin and metalloproteinase (ADAMS), Factor Xia, Factor D, transglutaminases 2, cathepsin S, CD73, CD39, or membrane guanylyl cyclase C.
Polypeptide Targeting Moieties
[0217] A reference polypeptide described herein can include a target-binding domain that binds to a target of interest (e.g., binds to an antigen). For example, a polypeptide, such as an antibody, can bind to a transmembrane polypeptide (e.g., receptor) or ligand (e.g., a growth factor). In some embodiments, the extracellular target proteins of the present invention include proteins which have a small molecule binding site (e.g., an active site). Exemplary molecular targets (e.g., antigens) for polypeptides described herein (e.g., antibodies) include GPCRs (e.g., Class A GPCRs such as CXCR4, Class B GPCRs, Class C GPCRs, Class D GPCRs, Class E GPCRs and Class F GPCRs), ion channels (e.g., Trp channels, Nav 1.7 channels, and CRAC channels), and enzymes (e.g., carbonic anhydrases and metalloproteases).
[0218] Antibodies
[0219] An IgG antibody consists of two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds. The first domain located at the amino terminus of each chain is variable in amino acid sequence, providing the antibody binding specificities found in each individual antibody. These are known as variable heavy (VH) and variable light (VL) regions. The other domains of each chain are relatively invariant in amino acid sequence and are known as constant heavy (CH) and constant light (CL) regions. For an IgG antibody, the light chain includes one variable region (VL) and one constant region (CL). An IgG heavy chain includes a variable region (VH), a first constant region (CH1), a hinge region, a second constant region (CH2), and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain includes an additional constant region (CH4).
[0220] Antibodies described herein can include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
[0221] The term antigen binding fragment of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term antigen binding fragment of an antibody include a Fab fragment, a F(ab).sub.2 fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAb fragment (Ward et al., (1989) Nature 341:544-546), and an isolated complementarity determining region (CDR). These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments can be screened for utility in the same manner as are intact antibodies.
[0222] Antibodies or fragments described herein can be produced by any method known in the art for the synthesis of antibodies (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can be produced using the methods described in, e.g., Morrison, 1985, Science 229:1202, and humanized antibodies by methods described in, e.g., U.S. Pat. No. 6,180,370.
[0223] Additional antibodies described herein are bispecific antibodies and multivalent antibodies, as described in, e.g., Segal et al., J. Immunol. Methods 248:1-6 (2001); and Tutt et al., J. Immunol. 147: 60 (1991).
[0224] Other Polypeptide Targeting Moieties
[0225] Other polypeptides may be used as targeting moieties, e.g., antibody-like scaffold proteins or antibody mimetics such as fibronectin type III domains, DARPins, or centyrins. In some embodiments, the targeting moiety is a non-antibody protein scaffold, such as a knottin, an affibody, a green fluorescent protein, an ankryn repreat protein, an SH2 domain, or a PDZ domain. Exemplary protein scaffolds of the invention are described in Stern, L. A., et al., Curr. Opion. in Chem. Engineering, 2013, 2:425-432; Banta, S. et al., Annu. Rev. Biomed. Eng., 2013, 15:93-113; Skrlec, K., Trends in Biotechnology, 2015, 33(7):408-418; and Huang, H. et al., Methods Mol. Biol. 2017, 1555:225-254, each of which is incorporated herein with respect to the protein scaffolds therein.
[0226] In some embodiments, polypeptide targeting moieties may include monobodies constructed using a fibronectin type III domain (e.g., tenth type III domain of human fibronectin). In some embodiments, the polypeptide targeting moiety is a tenth type III domain of human fibronectin, or a polypeptide variant thereof, having at least 80% sequence identity with SEQ ID NO: 1.
TABLE-US-00001 SEQIDNO:1 VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTV PGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT
[0227] In some embodiments, the polypeptide targeting moiety is an engineered or diversified polypeptide variant of SEQ ID NO: 1. For example, the polypeptide targeting moiety may be an engineered or diversified variant of the fibronectin type III domain of SEQ ID NO: 1, wherein the polypeptide variant includes mutations to one or more amino acids of the peptide (e.g., one or more solvent exposed amino acids of the polypeptide). In particular, loops BC, DE, and FG of the tenth type III domain of human fibronectin (bold in SEQ ID NO. 1) are structurally analogous to the antibody complementarity-determining regions H1, H2, and H3, respectively, and may be diversified or engineered to select for binding to a target (e.g., a polypeptide target). Three additional loops, AB, CD, and EF, are also candidates for engineering or diversification. See, for example, Lipovsek, D. Protein Eng Des Sel. 24(1-2):3-9 (2011).
[0228] In some embodiments, the polypeptide targeting moiety in an engineered or diversified variant of the tenth type III domain of human fibronectin, where the BC, DE, or FG loop regions may include one or more mutations relative to SEQ ID NO: 1. In some embodiments, the polypeptide targeting moiety is an engineered or diversified variant of the tenth type III domain of human fibronectin, having at least 80% sequence identity SEQ ID NO: 2, where X is any amino acid, and m, n, and k each, independently, have a value of 2 or greater (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).
TABLE-US-00002 SEQIDNO:2 VSDVPRDLEVVAATPTSLLISWD(X).sub.mYYRITYGETGGNSPVQEFTVPG (X).sub.nATISGLKPGVDYTITVYAV(X).sub.kSSKPISINYRT
[0229] Libraries of antibody-like scaffold proteins (e.g., fibronectin type III domains) may be generated using molecular display and directed evolution techniques known in the art such as phage display, DNA or RNA display, or yeast surface display.
[0230] Protein Variants
[0231] A protein or polypeptide variant, as described herein, generally has an amino acid sequence that shows significant (e.g., 80% or more, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity with that of a reference polypeptide but includes a limited number of particular amino acid changes (e.g., insertions, deletions, or substitutions, either conservative or non-conservative and/or including one or more amino acid variants or analogs [e.g., D-amino acids, desamino acids]) relative to the reference polypeptide. In certain embodiments, a variant shares a relevant biological activity (e.g., binding to a particular compound or moiety thereof) with the reference polypeptide; in some such embodiments, the variant displays such activity at a level that is not less than about 50% of that of the reference polypeptide and/or is not less than about 0.5 fold below that of the reference polypeptide.
[0232] In some embodiments, a variant polypeptide has an amino acid sequence that differs from that of a reference polypeptide at least (or only) in that the variant has a larger number of cysteine residues and/or has one or more cysteine residues at a position corresponding to a non-cysteine residue in the reference polypeptide. For example, in some embodiments, addition of one or more cysteine residues to the amino or carboxy terminus of any of a polypeptide as described herein can facilitate conjugation of such polypeptide by, e.g., disulfide bonding.
[0233] In some embodiments, amino acid substitutions can be conservative (i.e., wherein a residue is replaced by another of the same general type or group) or non-conservative (i.e., wherein a residue is replaced by an amino acid of another type). In some embodiments, a naturally occurring amino acid can be substituted for a non-naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution), or vice versa.
[0234] Polypeptides made synthetically can include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, an amino acid having an azide-containing side chain, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH.sub.2(CH.sub.2).sub.nCOOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
[0235] Analogs may be generated by substitutional mutagenesis and retain the structure (e.g., a local structure or global structure) of the original protein. Examples of substitutions identified as conservative substitutions are shown in Table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated exemplary substitutions in Table 1, or as further described herein in reference to amino acid classes, are introduced and the products screened.
[0236] Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the protein backbone in the area of the substitution, for example, as a sheet or helical conformation. (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties: [0237] (1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), [0238] (2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr) [0239] (3) acidic/negatively charged: Aspartic acid (Asp), Glutamic acid (Glu) [0240] (4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine (Lys), Arginine (Arg) [0241] (5) residues that influence chain orientation: Glycine (Gly), Proline (Pro); [0242] (6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His), [0243] (7) polar: Ser, Thr, Asn, Gln [0244] (8) basic positively charged: Arg, Lys, His, and; [0245] (9) charged: Asp, Glu, Arg, Lys, His
Other amino acid substitutions are listed in Table 1.
TABLE-US-00003 TABLE 1 Amino acid substitutions Original Exemplary Conservative residue substitution substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, norleucine Leu
[0246] Protein Variants with Altered Reactive Amino Acid Profiles
[0247] In some embodiments, a protein or polypeptide variant may include the addition of one or more reactive amino acid residues (e.g., cysteines) to a protein (e.g., at the amino or carboxy terminus of any of the proteins described herein) can facilitate conjugation of these proteins by, e.g., disulfide bonding. In some embodiments, one or more reactive amino acids (e.g., cysteines) may be removed to decrease the number of possible conjugation sites on a protein. Amino acid substitutions can be conservative (i.e., wherein a residue is replaced by another of the same general type or group) or non-conservative (i.e., wherein a residue is replaced by an amino acid of another type). In addition, a naturally occurring amino acid can be substituted for a non-naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution).
Libraries
[0248] In some embodiments, the compounds, small molecule moieties, and/or polypeptide targeting moieties of the invention are members of libraries. Libraries including the compounds, small molecule moieties, and/or polypeptide targeting moieties of the invention may be tagged to assist in identification of library members. For example, the libraries may be tagged using any method known in the art such as DNA display libraries (also known as DNA-encoded libraries), RNA display libraries, yeast display libraries, or phage display libraries. Tagged libraries offer advantages such as allowing the screening of large libraries in one pot. In some embodiments, the compounds, small molecule moieties, and/or polypeptide targeting moieties of the invention are members of large libraries (e.g., libraries including at least 10.sup.5 members). In some embodiments, a library of the invention includes between about 10.sup.2 to 10.sup.20 complexes (e.g., about 10.sup.2 to 10.sup.3, 10.sup.2 to 10.sup.4, 10.sup.2 to 10.sup.5, 10.sup.2 to 10.sup.6, 10.sup.2 to 10.sup.7, 10.sup.2 to 10.sup.8, 10.sup.2 to 10.sup.6, 10.sup.2 to 10.sup.10, 10.sup.2 to 10.sup.11, 10.sup.2 to 10.sup.12, 10.sup.2 to 10.sup.13, 10.sup.2 to 10.sup.14, 10.sup.2 to 10.sup.15, 10.sup.2 to 10.sup.16, 10.sup.2 to 10.sup.17, 10.sup.2 to 10.sup.18, 10.sup.2 to 10.sup.16, 10.sup.4 to 10.sup.5, 10.sup.4 to 10.sup.6, 10.sup.4 to 10.sup.7, 10.sup.4 to 10.sup.8, 10.sup.4 to 10.sup.6, 10.sup.4 to 10.sup.10, 10.sup.4 to 10.sup.11, 10.sup.4 to 10.sup.12, 10.sup.4 to 10.sup.13, 10.sup.4 to 10.sup.14, 10.sup.4 to 10.sup.15, 10.sup.4 to 10.sup.16, 10.sup.4 to 10.sup.17, 10.sup.4 to 10.sup.18, 10.sup.4 to 10.sup.16, 10.sup.4 to 10.sup.20, 10.sup.5 to 10.sup.6, 10.sup.5 to 10.sup.7, 10.sup.5 to 10.sup.8, 10.sup.5 to 10.sup.6, 10.sup.5 to 10.sup.10, 10.sup.5 to 10.sup.11, 10.sup.5 to 10.sup.12, 10.sup.5 to 10.sup.13, 10.sup.5 to 10.sup.14, 10.sup.5 to 10.sup.15, 10.sup.5 to 10.sup.16, 10.sup.5 to 10.sup.17, 10.sup.5 to 10.sup.18, 10.sup.5 to 10.sup.19, or 10.sup.5 to 10.sup.20 members.
[0249] Library Generation
[0250] Libraries useful in the methods of the invention may be prepared using any method known in the art. In some embodiments, libraries of polypeptide targeting moieties (e.g., fibronectin type III domains) may be prepared by a designed sitewise diversification strategy utilizing high throughput evolution and bioinformatics. In some embodiments, the members of the library may be prepared with only one reactive amino acid residue or with one optimal reactive amino acid residue. For example, libraries of polypeptide targeting moieties may be prepared using methods similar to those described in Woldring et al. PLoS One 10:e0138956 (2015), the methods of which are herein incorporated by reference. In some embodiments, the library of polypeptide targeting moieties is a synthetic antibody library. Synthetic antibody libraries may be prepared using any method known in the art. For example, a synthetic antibody library may be prepared by utilizing a CDR randomization method such as those described in Chen et al. Methods in Mol. Biol. 1131:113-131 (2014) and Mandrup et al. PLoS One 8(10):e76834 (2013), the methods of which are herein incorporated by reference. The libraries of polypeptide targeting moieties may be DNA or RNA display libraries, yeast display libraries, or phage display libraries.
[0251] Libraries of polypeptide targeting moieties may be conjugated to one or more small molecule moieties to prepare a library of bifunctional compounds for use in the methods of the invention. The polypeptide targeting moieties may be conjugated to the one or more small molecule moieties via a linker (e.g., a polyethylene-containing linker). In some embodiments, all of the polypeptide targeting moieties in the library are conjugated to the same small molecule moiety. In some embodiments, all of the polypeptide targeting moieties in the library are conjugated to the small molecule moiety with the same linker. In some embodiments, the polypeptide targeting moieties in the library are conjugated to a small molecule moiety with different linkers.
[0252] Library Screening
[0253] The libraries of bifunctional compounds of the invention may be screened for the ability to modulate the activity of a target protein using any method known in the art. In some embodiments, libraries of bifunctional compound may be screened for activity using flow cytometry methods, magnetic selection methods, or pull down methods. In some embodiments, the libraries of bifunctional compounds may be screened for activity using fluorescence based competition assays. For example, libraries of bifunctional compounds may be screened using methods described in Cho et al. Protein Eng. Des. Sel. 23:567-577 (2010); Tillotson, et al. Methods 60:27-37 (2013); Wang et al. J. Immunol. Methods 304:30-42 (2005); Wang et al. Nat. Methods 4:143-145 (2007); or Hoogenboom Nature Biotech. 23:1105-1116 (2005), the methods of each of which are herein incorporated by reference. The identity of the bifunctional compounds in the library with optimal activity may be determined utilizing any method known in the art. For example, if the bifunctional compound library is a DNA or RNA display library, the identity of the bifunctional compounds may be determined by sequencing the DNA or RNA tag on each compound. If the bifunctional compound library is a yeast display library, the identity of the bifunctional compound may be determined by sequencing the enriched yeast plasmid.
Target Proteins
[0254] A target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein) is any protein whose activity is desirably altered. In one example, a target protein mediates a disease condition or a symptom of a disease condition. As such, a desirable therapeutic effect can be achieved by modulating (inhibiting or increasing) its activity.
[0255] Target proteins can be naturally occurring, e.g., wild type. Alternatively, a target protein can vary from the wild type protein but still retain biological function, e.g., as an allelic variant, a splice mutant or a biologically active fragment. In some embodiments, a target protein is a transmembrane protein.
[0256] In some embodiments, the target protein is a GPCR. In some embodiments, the target protein is a Rhodopsin-like receptor such as a protein encoded by the gene CCR1; CCR2; CCR3; CCR4; CCR5; CCR8; CCRL2; XCR1; CX3CR1; GPR137B; CCRL1; CCR6; CCR7; CCR9; CCR10; CXCR3; CXCR4; CXCR5; CXCR6; CXCR7; IL8RA; IL8RB; GPR182; DARC; GPER; AGTR1; AGTR2; AGTRL1; BDKRB1; BDKRB2; GPR15; GRP25; OPRD1; OPRK1; OPRM1; OPRL1; SSTR1; SSTR2; SSTR3; SSTR4; SSTR5; NPBWR1; NPBWR2; GPR1; GALR1; GALR2; GALR3; CYSLTR1; CYSLTR2; LTB4R; LTB4R2; RXFP1; RXFP2; RXFP3; RXFP4; KISS1R; MCHR1; UTS2R; CCKAR; CCKBR; NPFFR1; NPFFR2; HCRTR1; HCRTR2; AVPR1A; AVPR1B; AVPR2; GNRHR; QRFPR; GPR22; GPR176; BRS3; NMBR; GRPR; EDNRA; EDNRB; GPR37; NMUR1; NMUR2; NTSR1; NTSR2; TRHR; GHSR; GPR39; MLNR; C3AR1; C5AR1; CMKLR1; FPR1; FPRL1; FPRL2; MAS1; MAS1L; GPR1; GPR32; GPR44; GPR77; MTNR1A; MTNR1B; TACR1; TACR2; TACR3; NPYR; NPY2R; PPYR1; NPYSR; PRLHR; PROKR1; PROKR2; GPR19; GPR50; GPR75; GPR83; FSHR; LHCGR; TSHR; LGR4; LGR5; LGR6; FFAR1; FFAR2; FFAR3; GPR42; P2RY1; P2RY2; P2RY4; P2RY6; P2RY8; P2RY11; HCAR1; HCAR2; HCAR3; GPR31; GPR82; OXFR1; SUCNR1; P2RY12; P2RY13; P2RY14; GPR34; GPR87; GPR171; PTAFR; CNR1; CNR2; LPAR1; LPAR2; LPAR3; S1PR1; S1PR2; S1PR3; S1PR4; S1PR5; MC1R; MC3R; MC4R; MC5R; MC2R; GPR3; GPR6; GPR12; PTGDR; PTGER1; PTGER2; PTGER3; PTGER4; PTGFR; PTGIR; TBXA2R; LPAR4; LPAR5; LPAR6; P2RY10; F2RL1; F2RL2; F2RL3; GPR183; GPR4; GPR65; GPR68; GPR17; GPR18; GPR20; GPR35; GPR55; F2R; RHO; OPN1SW; OPN1MW; OPN1LW; OPN3; OPN4; OPN5; RGR; RRH; 5-HT; HT2RA; HT2RB; HT2RC; HTR6; ADRA1A; ADRA1B; ADRA1D; ADRA2A; DARA2B; ADRA2C; ADRB1; ADRB2; ADRB3; DRD1; DRD2; DRD3; DRD4; DRD5; TAAR1; TAAR2; TAAR3; TAAR5; TAAR6; TAAR8; TAAR9; HRH2; HRH1; HRH3; HRH4; ADORA1; ADORA2A; ADORA2B; ADORA3; CHRM1; CHRM2; CHRM3; CHRM4; CHRM5; GPR21; GPR27; GPR45; GPR52; GPR61; GPR63; GPR78; GPR84; GPR85; GPR88; GPR101; GPR161; GPR173; HTR1A; HTR1B; HTR1D; HTR1E; HTR1F; HTR4; HTR5A; HTR7; VN1R1; VN1R2; VN1R3; VN1R4; or VN1R5. In some embodiments, the target protein is a Secretin receptor such as a protein encoded by the gene ADCYAP1R1; CALCR; CRHR1; CRHR2; GIPR; GCGR; GLP1R; GLP2R; GHRHR; PTHR1; PTHR2; SCTR; VIPR1; VIPR2; BAI1; BAI2; BAI3; CD97; CELSR1; CELSR2; CELSR3; EMR1; EMR2; EMR3; EMR4; CPR56; CPR64; GPR97; GPR110; GPR111; GPR112; GPR113; GPR114; GPR115; GPR123; GPR125; GPR126; GPR128; GPR133; GPR144; GPR157; ELTD1; LPHN1; LPHN2; LPHN3; GPR116; HCTR-5; HCTR-6; KPG 006; or KPG 008. In some embodiments, the target protein is a metabotropic glutamate receptor such as a protein encoded by the gene GRM1; GRM5; GRM2; GRM3; GRM4; GRM6; GRM7; or GRM8. In some embodiments, the target protein is a cyclic AMP receptor. In some embodiments, the target protein is a Frizzled receptor family such as a protein encoded by the gene FZD1; FZD2; FZD3; FZD4; FZD5; FZD6; FZD7; FZD8; FZD9; or FZD10. In some embodiments, the target protein is smoothened.
[0257] In some embodiments, the target protein is an ion channel. In some embodiments, the target protein is a calcium activated potassium channel such as a protein encoded by the gene KCNMA1; KCNC1; KCNN2; KCNN3; KCNN4; KCNT1; KCNT2; or KCNU1. In some embodiments, the target protein is a CatSper and Two-pore channel such as a protein encoded by the gene CATPSER1; CATSPER2; CATSPER3; CATSPER4; TPCN1; or TPCN2. In some embodiments, the target protein is a cyclic nucleotide-regulated channels such as a protein encoded by the gene CNGA1; CNGA2; CNGA3; CNGA4; CNGB1; CNGB3; HCN1; HCN2; HCN3; or HCN4. In some embodiments, the target protein is an inwardly rectifying potassium channel such as a protein encoded by the gene KCNJ1; KCNJ2; KCNJ12; KCNJ4; KCNJ14; KCNJ3; KCNJ6; KCNJ9; KCNJ5; KCNJ10; KCNJ15; KCNJ16; KCNJ8; KCNJ11; or KCNJ13. In some embodiments, the target protein is a ryanodine receptor such as a protein encoded by the gene RYR1; RYR2; or RYR3. In some embodiments, the target protein is a transient receptor potential channel such as a protein encoded by the gene TRPAA1; TRPC1; TRPC2; TRPC3; TRPC4; TRPC5; TRPC6; TRPC7; TRPM1; TRPM2; TRPM3; TRPM4; TRPM5; TRPM6; TRPM7; TRPM8; MCOLN1; MCOLN2; MCOLN3; PKD2; PKD2L1; PDK2L2; TRPV1; TRPV2; TRPV3; TRPV4; TRPV5; or TRPV6. In some embodiments, the target protein is a two-P potassium channel such as a protein encoded by the gene KCNK1; KCNK2; KCNK3; KCNK4; KCNK5; KCNK6; KCNK7; KCNK9; KCNK10; KCNK12; KCNK13; KCNK15; KCNK16; KCNK17; or KCNK18. In some embodiments, the target protein is a voltage-gated calcium channel such as a protein encoded by the gene CACNA1S; CACNA1C; CACNA1D; CACNA1F; CACNA1A; CACNA1B; CACNA1E; CACNA1G; CACNA1H; or CACNA1I. In some embodiments, the target protein is a voltage-gated potassium channel such as a protein encoded by the gene KCNA1; KCNA2; KCNA3; KCNA4; KCNA5; KCNA6; KCNA7; KCNA10; KCNB1; KCNB2; KCNC1; KCNC2; KCNC3; KCNC4; KCND1; KCND2; KCND3; KCNF1; KCNG1; KCNG1; KCNG2; KCNG3; KCNG4; KCNQ1; KCNA2; KCNA3; KCNA4; KCNQ5; KCNV1; KCNV2; KCNS1; KCNS2; KCNS3; KCNH1; KCNH5; KCNH2; KCNH6; KCNH7; KCNH8; KCNH3; or KCNH4. In some embodiments, the target protein is a voltage-gated proton channel such as a protein encoded by the gene HVCN1. In some embodiments, the target protein is a voltage-gated sodium channel such as a protein encoded by the gene SCN1A; SCN2A; SCN3A; SCN4A; SCN5A; SCN8A; SCN9A; SCN10A; or SCN11A.
[0258] In some embodiments, the target protein is an enzyme. In some embodiments, the target protein is a oxidoreductase, dehydrogenase, luciferase, DMSO reductase, alcohol dehydrogenase (NAD), alcohol dehydrogenase (NADP), homoserine dehydrogenase, aminopropanol oxidoreductase, diacetyl reductase, glycerol dehydrogenase, propanediol-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase (NAD+), D-xylulose reductase, L-xylulose reductase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, HMG-CoA reductase, glucose oxidase, L-gulonolactone oxidase, thiamine oxidase, xanthine oxidase, acetaldehyde dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate dehydrogenase, oxoglutarate dehydrogenase, biliverdin reductase, protoporphyrinogen oxidase, monoamine oxidase, dihydrofolate reductase, methylenetetrahydrofolate reductase, sarcosine oxidase, dihydrobenzophenanthridine oxidase, NADH dehydrogenase, urate oxidase, nitrite reductase, nitrate reductase, glutathione reductase, thioredoxin reductase, sulfite oxidase, cytochrome c oxidase, coenzyme Q-cytochrome c reductase, catechol oxidase, laccase, cytochrome c peroxidase, catalase, myeloperoxidase, thyroid peroxidase, glutathione peroxidase, 4-hydroxyphenylpyruvate dioxygenase, renilla-luciferin 2-monooxygenase, cypridina-luciferin 2-monooxygenase, firefly luciferase, watasenia-luciferin 2-monooxygenase, oplophorus-luciferin 2-monooxygenase, cytochrome P450 oxidase, cytochrome P450, aromatase, a protein encoded by the gene CYP2D6, CYP2E1, or CYP3A4, cytochrome P450 oxidase, nitric oxide dioxygenase, nitric oxide synthase, aromatase, a protein encoded by the gen CYP2D6, CYP2E1, or CYP3A4, phenylalanine hydroxylase, tyrosinase, superoxide dismutase, ceruloplasmin, nitrogenase, deiodinase, glutathione S-transferase, Catechol-O-methyl transferase, DNA methyltransferase, histone methyltransferase, ornithine transcarbamoylase, aminolevulinic acid synthase, choline acetyltransferase, Factor XIII, gamma glutamyl transpeptidase, transglutaminase, hypoxanthine-guanine phosphoribosyltransferase, thiaminase, alanine transaminase, aspartate transaminase, butyrate kinase, hydrolytic enzyme, nuclease, endonuclease, exonuclease, acid hydrolase, phospholipase A, acetylcholinesterase, cholinesterase, lipoprotein lipase, ubiquitin carboxy-terminal hydrolase L1, phosphatase, alkaline phosphatase, fructose bisphosphatase, phospholipase C, cGMP specific phosphodiesterase type 5, phospholipase D, restriction enzyme Type 1, deoxyribonuclease I, RNase H, ribonuclease, amylase, sucrase, chitinase, lysozyme, maltase, lactase, beta-galactosidase, hyaluronidase, adenosylmethionine hydrolase, S-adenosyl-L-homocysteine hydrolase, alkenylglycerophosphocholine hydrolase, alkenylglycerophosphoethanolamine hydrolase, cholesterol-5,6-oxide hydrolase, hepoxilin-epoxide hydrolase, isochorismatase, leukotriene-A4 hydrolase, limonene-1,2-epoxide hydrolase, microsomal epoxide hydrolase, trans-epoxysuccinate hydrolase, alanine aminopeptidase, angiotensin converting enzyme, serine protease, chymotrypsin, trypsin, thrombin, Factor X, plasmin, acrosin, Factor VII, Factor IX, prolyl oligopeptidase, Factor XI, elastase, Factor XII, proteinase K, tissue plasminogen activator, Protein C, separase, pepsin, rennet, renin, trypsinogen, plasmepsin, matrix metalloproteinase, metalloendopeptidase, urease, beta-lactamase, arginase, adenosine deaminase, GTP cyclohydrolase I, nitrilase, helicase, DnaB helicase, RecQ helicase, ATPase, NaKATPase, ATP synthase, kynureninase, ornithine decarboxylase, uridine monophosphate synthetase, aromatic-L-amino-acid decarboxylase, rubisCO, carbonic anhydrase, tryptophan synthase, phenylalanine ammonia-lyase, cystathionine gamma-lyase, cystathionine beta-lyase, leukotriene C4 synthase, dichloromethane dehalogenase, halohydrin dehalogenase, adenylate cyclase, guanylate cyclase, phenylalanine racemase (ATP-hydrolysing), serine racemase, mandelate racemase, UDP-glucose 4-epimerase, methylmalonyl CoA epimerase, a protein encoded by the gene FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP5, FKBP6, FKBP8, FKBP9, FKBP10, FKBP52, or FKBPL, cyclophilin, parvulin, prolyl isomerase, 2-chloro-4-carboxymethylenebut-2-en-1,4-olide isomerase, beta-carotene isomerase, farnesol 2-isomerase, furylfuramide isomerase, linoleate isomerase, maleate isomerase, maleylacetoacetate isomerase, maleylpyruvate isomerase, parvulin, photoisomerase, prolycopene isomerase, prolyl isomerase, retinal isomerase, retinol isomerase, zeta-carotene isomerase, enoyl CoA isomerase, protein disulfide isomerase, phosphoglucomutase, muconate cycloisomerase, 3-carboxy-cis,cis-muconate cycloisomerase, tetrahydroxpteridine cycloisomerase, inositol-3-phosphate synthase, carboxy-cis,cis-muconate cyclase, chalcone isomerase, chloromuconate cycloisomerase, (+)-bornyl diphosphate synthase, cycloeucalenol cycloisomerase, alpha-pinene-oxide decyclase, dichloromuconate cycloisomerase, copalyl diphosphate synthase, ent-copalyl diphosphate synthase, syn-copalyl-diphosphate synthase, terpentedienyl-diphosphate synthase, halimadienyl-diphosphate synthase, (S)-beta-macrocarpene synthase, lycopene epsilon-cyclase, lycopene beta-cyclase, prosolanapyrone-III cycloisomerase, D-ribose pyranase, topoisomerase, 6-carboxytetrahydropterin synthase, FARSB, glutamine synthetase, argininosuccinate synthetase, CTP synthase, pyruvate carboxylase, acetyl-CoA carboxylase, or DNA ligase.
Methods of Preparation
[0259] The compounds of the invention may be prepared using methods known in the art. For example, a small molecule moiety may be conjugated to a linker which includes a cross-linking group (e.g., a maleimide) to produce a compound of Formula VII:
A-L-B Formula VII
[0260] wherein A includes a small molecule moiety; [0261] L is a linker; and [0262] B is a cross-linking moiety.
[0263] The compound of Formula VII may then be reacted with a polypeptide targeting moiety including one or more reactive amino acid residues (e.g., a free cysteine, a lysine, or a non-natural amino acid). In some embodiments, the small molecule moiety is conjugated to the polypeptide targeting moiety in a site-specific manner. In some embodiments, the site of conjugation is a solvent exposed amino acid of the polypeptide targeting moiety. In some embodiments, the site of conjugation is a solvent exposed amino acid located within the polypeptide chain (e.g., a solvent exposed amino acid residue located in a solvent exposed loop). In some embodiments, the site of conjugation is located at or near the terminus (e.g., within 10 amino acid residues of the C-terminus or within 10 amino acid residues of the N-terminus) of the polypeptide. In some embodiments, the polypeptide targeting moiety has been modified to include a reactive amino acid residue at a specific site. In some embodiments, the polypeptide targeting moiety has been modified to include no more than one reactive amino acid residue or no more than one reactive residue of a particular type (e.g., no more than one cysteine, no more than one lysine). In some embodiments, when the polypeptide targeting moiety is an antibody, the small molecule moiety is conjugated via a reactive amino acid residue in a CDR (e.g., CDR1) of the antibody. In some embodiments, the small molecule moiety is conjugated to the polypeptide targeting moiety via a glycosylation site. In some embodiments, the small molecule moiety is conjugated to the polypeptide targeting moiety via a framework residue distinct from the antigen binding site.
[0264] In some embodiments, a free cysteine utilized to conjugate a small molecule moiety to a polypeptide targeting moiety is produced by reducing the polypeptide targeting moiety under conditions sufficient to reduce at least one disulfide bond (e.g., a disulfide bond in the CDR or a loop region of the polypeptide targeting moiety). For example, the small molecule moiety may be conjugated to the polypeptide targeting moiety using methods similar to those described in Badescu, et al. Bioconjug. Chem. 25(3):460-469 (2014); Badescu et al. Bioconjug. Chem. 25(6):1124-1136 (2014); Bryant et al. Mol. Pharm. 12(6):1872-1879 (2015); Schumacher et al. Org. Biomol. Chem. 7261-7269 (2014); or Bryden et al. Bioconjug. Chem. 611-617 (2014), the methods of each of which are herein incorporated by reference.
[0265] In some embodiments, a small molecule is conjugated to a polypeptide (e.g., via a linker) by enzymatic ligation. Enzymatic ligation may be performed by various techniques known to one in the art. For example, enzymatic ligation may be performed by (a) prenylation of CaaX motifs with protein farnesyltransferase (e.g., a described in Rose, M. W., et al., Biopolymers, 2005, 80, 164-171; and Hurwitz, H. I.; Casey, P. J. Curr. Topics Membr. 2002, 52, 531-550); (b) conjugation of [a]-LPETG and GGG-[b] with sortase A (e.g., as described in Levary, D., et al., PLOS One, 2011, 6, e18342); (c) ligation with lipoic acid ligase (Lpl) at Lpl acceptor peptide (e.g., as described in Fernndez-Surez M, et al., Nat Biotech. 2007, 25, 1483-1487); or (d) using a transglutaminase reaction (e.g., as described in Strop, P. et al., Chemistry and Biology, 2013, 20, 161-167).
Uses
[0266] Treatment of Diseases or Disorders
[0267] Compounds described herein may be useful in the methods of treating diseases or disorders related to the target proteins described herein, and, while not bound by theory, are believed to exert their desirable effects through their ability to modulate (e.g., positively or negatively modulate) the activity of a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein).
[0268] Pharmaceutical Compositions
[0269] For use as treatment of human and animal subjects, the compounds of the invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desirede.g., prevention, prophylaxis, or therapythe compounds are formulated in ways consonant with these parameters. A summary of such techniques is found in Remington: The Science and Practice of Pharmacy, 21.sup.st Edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.
[0270] Compounds described herein may be present in amounts totaling 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intravesicular, intraurethral, intrathecal, epidural, aural, or ocular administration, or by injection, inhalation, or direct contact with the nasal, genitourinary, reproductive or oral mucosa. Thus, the pharmaceutical composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.
[0271] In general, for use in treatment, compounds described herein may be used alone, or in combination with one or more other active agents. An example of other pharmaceuticals to combine with the compounds described herein would include pharmaceuticals for the treatment of the same indication. Another example of a potential pharmaceutical to combine with compounds described herein would include pharmaceuticals for the treatment of different yet associated or related symptoms or indications. Depending on the mode of administration, compounds will be formulated into suitable compositions to permit facile delivery. Each compound of a combination therapy may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Desirably, the first and second agents are formulated together for the simultaneous or near simultaneous administration of the agents.
[0272] Compounds of the invention may be prepared and used as pharmaceutical compositions comprising an effective amount of a compound described herein and a pharmaceutically acceptable carrier or excipient, as is well known in the art. In some embodiments, a composition includes at least two different pharmaceutically acceptable excipients or carriers.
[0273] Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. A formulation will generally include a diluents as well as, in some cases, adjuvants, buffers, preservatives and the like. Compounds can be administered also in liposomal compositions or as microemulsions.
[0274] For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.
[0275] Various sustained release systems for drugs have also been devised. See, for example, U.S. Pat. No. 5,624,677, which is herein incorporated by reference.
[0276] Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for compounds of the invention. Suitable forms include syrups, capsules, and tablets, as is understood in the art.
[0277] Each compound of a combination therapy, as described herein, may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately.
[0278] The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include, but are not limited to, kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to subjects, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one subject, multiple uses for a particular subject (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple subjects (bulk packaging). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.
[0279] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with nontoxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.
[0280] Two or more compounds may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned. In one example, the first compound is contained on the inside of the tablet, and the second compound is on the outside, such that a substantial portion of the second compound is released prior to the release of the first compound.
[0281] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluents (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.
[0282] Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.
[0283] The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
[0284] Generally, when administered to a human, the oral dosage of any of the compounds of the combination of the invention will depend on the nature of the compound, and can readily be determined by one skilled in the art. Typically, such dosage is normally about 0.001 mg to 2000 mg per day, desirably about 1 mg to 1000 mg per day, and more desirably about 5 mg to 500 mg per day. Dosages up to 200 mg per day may be necessary.
[0285] Administration of each drug in a combination therapy, as described herein, can, independently, be one to four times daily for one day to one year, and may even be for the life of the subject. Chronic, long-term administration may be indicated.
EXAMPLES
Example 1. Determination of Optimal Binding Sites, Linkers, and Small Molecule Moieties
[0286] A non-binding fibronectin type III domain is mutated to contain a single cysteine at specific positions (e.g., G79, T28, R30, R78, D80, S53, T71, K63, or L19). The fibronectin type III domains are displayed on the surface of a yeast cell. For example, using the methods described in Chen et al. Methods Enzymol. 523:303-326 (2013) and/or Boder et al. Nat. Biotechnol. 15:553-557 (1997).
[0287] Small molecule moieties (e.g., sulfonamide-containing small molecule moieties) are conjugated to the mutated fibronectin type III domains via a cross-linking group (e.g., a maleimide). If more than one small molecule moiety is used, the bifunctional compounds with different small molecule moieties or different linkers may be separated.
[0288] The bifunctional compounds are combined with a target protein (e.g., a carbonic anhydrase such as carbonic anhydrase 9 or carbonic anhydrase 2) and the binding of the bifunctional compound to the target is determined, e.g., using a fluorescence based competition assay.
Example 2. Preparation of Bifunctional Compound Libraries
[0289] Bifunctional compounds identified in Example 1 which are determined to bind to the target protein are utilized as starting points for libraries.
[0290] A library of fibronectin type III domains identified in Example 1 as resulting in bifunctional compounds capable of binding the target protein is prepared by a designed sitewise diversification strategy of the remainder of the paratope (e.g., diversification of any amino acid except the added cysteine) developed through high throughput evolution and bioinformatics. For example, diversification is conducted using methods described in Woldring et al. PLoS One 10:e0138956 (2015).
[0291] The library of fibronectin type III domains is introduced into the yeast display system by homologous recombination. The displayed fibronectin type III domains are conjugated to the small molecule moieties (e.g., via a cross-linking moiety such as a maleimide-containing cross-linking moiety).
Example 3. Screening of Bifunctional Compound Against Target Protein
[0292] Bifunctional compounds are screened for activity against target proteins, e.g., using flow cytometry, magnetic selection, or yeast pull down assays. For example, bifunctional compounds are screened with flow cytometry with fluorescently labeled human lysate or yeast pull-down on adherent human cell monolayers as described in Cho et al. Protein Eng. Des. Sel. 23:567-577 (2010); Tillotson, et al. Methods 60:27-37 (2013); Wang et al. J. Immunol. Methods 304:30-42 (2005); or Wang et al. Nat. Methods 4:143-145 (2007).
[0293] The identity of bifunctional compounds which bind to the target protein are determined by sequencing enriched yeast plasmid.
Example 4. Identification of Bifunctional Compounds which Bind Carbonic Anhydrase
[0294] Bifunctional compounds comprising a fibronectin type III domain (Fn) conjugated via a linker to acetazolamide (AAZ) were identified by screening yeast-displayed Fn-AAZ libraries for their ability to bind either carbonic anhydrase 9 (CA9) or carbonic anhydrase 2 (CA2). Selection of an Fn-AAZ library against CA9 produced Fn-AAZ clones that bind with high affinity to CA9, including Fn-AAZ clones that bind to CA9 with higher affinity than either Fn or AAZ, alone. Selected Fn-AAZ clones also showed increased specificity for CA9 over related carbonic anhydrase 2 (CA2). Selection of an Fn-AAZ library against CA2 produced Fn-AAZ clones that bind with high affinity to CA2.
[0295] Target Biotinylation
[0296] 5 M recombinant His-tagged human carbonic anhydrase 9 (CA9, Sino Biological) was incubated with 500 M EZ-link NHS-Biotin (Thermo) in PBS, pH 7.4 for 4 hours at room temperature. Approximately 2 biotin molecules conjugated per CA9 molecule was confirmed by MALDI-TOF mass spectrometry. Carbonic anhydrase 2 (CA2, Sino Biological) was biotinylated in the same manner at 15 M with 135 M NHS-Biotin, and conjugation was again confirmed by MALDI-TOF. Unreacted biotin was removed by two sequential desaltings using Zeba 7K desalting spin columns (Thermo).
[0297] Yeast Display of Fibronectin Clones
[0298] Plasmids encoding single clones pCT-FnR (a hydrophilic fibronectin domain binder to rabbit IgG) and pCT-FnR-T28C were transformed into EBY100 yeast following the EZ yeast method (Zymo Research). Plasmids were generated using standard cloning techniques. Transformed yeast were grown by shaking at 30 C. in SD-CAA (16.8 g/L sodium citrate dihydrate, 3.9 g/L citric acid, 20.0 g/L dextrose, 6.7 g/L yeast nitrogen base, 5.0 g/L casamino acids) media and induced by transferring to SG-CAA (10.2 g/L sodium phosphate dibasic heptahydrate, 8.6 g/L sodium phosphate monobasic monohydrate, 19.0 g/L galactose, 1.0 g/L dextrose, 6.7 g/L yeast nitrogen base, 5.0 g/L casamino acids) media and shaking for at least 4 hours at 30 C.
[0299] Selection of Sites for Conjugation to Small Molecules
[0300] The structure of Fn was evaluated to select amino acids (e.g., solvent exposed amino acids) for conjugation to a small molecule (AAZ) (
[0301] As shown in
[0302] As shown in
[0303] Single Clone and Library Conjugation to Small Molecules
[0304] Two million induced yeast expressing selected fibronectin clones were washed in PBS, pH 6.5, then incubated in 50 L PBS, pH 6.5 with 0.02, 0.2, or 2 M fluorescein-5-maleimide (Pierce) or maleimide-XPEG-Acetazolamide (AAZ) for 10 minutes, 30 minutes, 1, or 2 hours at room temperature while rocking. Yeast were washed 2 in 1 mL PBS, pH 7.4+1 g/L BSA (PBSA) to remove unconjugated small molecule. Library conjugations were performed on 3.410.sup.9 (15 diversity) transformed and induced yeast expressing the designed libraries in 2 mL PBS, pH 6.5 with 8 M maleimide-XPEG-AAZ for 2 hours at room temperature while rocking. Small molecules with PEG linkers of lengths 2, 3, 5, and 7 were conjugated in separate reactions. After incubation, yeast were washed 4 times in 50 mL PBSA to remove unconjugated small molecule and the 4 conjugated yeast populations were pooled.
[0305] Library Construction
[0306] Oligonucleotides encoding the Fn-Cys yeast-displayed designed libraries were synthesized by IDT DNA Technologies, amplified by overlap extension PCR, and transformed into EBY100 by homologous recombination with the pCT vector following the protocol by Woldring, D. R., et al. PLoS One. 10:e0138956 (2015). As shown in
[0307] Sequence analysis confirms that library construction matches design.
[0308] Magnetic Bead Selection
[0309] 15 diversity, small molecule conjugated libraries were incubated with 10 L streptavidin coated dynabeads (Thermo) for 1 hour at room temperature while tumbling. Unbound yeast were removed and again incubated with 10 L streptavidin coated Dynabeads. Unbound yeast were removed and incubated with 35.5 nM biotinylated CA9 in PBSA for 30 minutes at room temperature. Yeast were washed 4 in 2 mL PBSA, then incubated with 10 L streptavidin coated Dynabeads in 1 mL PBSA for 1 hour at room temperature. Beads were washed 2 in PBSA, resuspended in SD-CAA media and incubated overnight while shaking at 30 C. Beads were then removed and the yeast were induced in SG-CAA for subsequent magnetic bead sorting, sorting by flow cytometry, or analysis.
[0310] Target Binding and Flow Cytometry
[0311] Fluorescein-5-maleimide conjugated to yeast-displayed FN was detected directly on a BD Accuri C6 flow cytometer using the standard equipped 488 nm excitation laser and 533/30 nm emission filter. In binding experiments, yeast-displaying Fn were conjugated with maleimide-XPEG-AAZ as described above. Yeast were incubated to equilibrium with indicated concentrations of biotinylated target at room temperature in sufficient volume of PBSA to ensure at least 10-fold molar excess of target to Fn. Primary antibody against c-MYC (9E10) was also included in the target incubation. Yeast were then washed in cold PBSA and incubated with streptavidin-AF647 (Thermo) and goat anti-mouse-FITC (Thermo) for 15 minutes at 4 C. Yeast were again washed in cold PBSA and analyzed using a BD Accuri C6 flow cytometer equipped with the standard 488 nm laser and 533/30 nm emission filter for FITC detection and the 640 nm laser and 675/25 nm emission filter for AF647 detection.
[0312] Yeast-Displayed Fn-AAZ is Functional at Multiple Conjugation Sites
[0313] As shown in
[0314] Fn-AAZ Conjugates can be Selected for Binding to Carbonic Anhydrase 9
[0315] As shown in
[0316] As shown in
[0317] As shown in
[0318] Selection of Fn-AAZ Conjugates that Bind Selectively to Carbonic Anhydrase 9
[0319] As shown in
[0320] By contrast,
[0321] Fn-AAZ can be Selected for Binding to Carbonic Anhydrase 2
[0322] As shown in
[0323] Fn-AAZ Conjugate Clones Identified Having High Affinity for Carbonic Anhydrase 9
[0324] Fn-AAZ clones selected for binding to carbonic anhydrase 9 were expressed, purified, and sequenced according to methods well-known to one of skill in the art.
TABLE-US-00004 SEQIDNO:3 MASSSDSPRNLEVTNATPNSLTISWDSYLDCAYYYRITYGETGGNSPSQE FTVPGYTNSVTISGLKPGQDYTITVYAVASSNDVSNPISINYRTEIDKPS QGS SEQIDNO:4 MASSSDSPRNLEVTNATPNSLTISWDDSYCVIYYRITYGETGGNSPSQEF TVPGYTNSVTISGLKPGQDYTITVYAVTDYSKLDPSNPISINYRTEIDKP SQGS SEQIDNO:5 MASSSDSPRNLEVTNATPNSLTISWDYHQNGCAVSYRITYGETGGNSPSQ EFTVPGYYDTYSATISGLKPGQDYTITVYAVTGYNDDSNPISINYRTEID KPSQGS SEQIDNO:6 MASSSDSPRNLEVTNATPNSLTISWDYSYCVLYYRITYGETGGNSPSQEF TVPGYYYSATISGLKPGQDYTITVYAVTDTGDESNPISINYRTEIDKPSQ GS SEQIDNO:7 MASSSDSPRNLEVTNATPNSLTISWDYSYCVLSYRITYGETGGNSPSQEF TVPGYYTSATISGLKPGQDYTITVYAVATIDYKDSNPISINYRTEIDKPS QGS SEQIDNO:8 MASSSDSPRNLEVTNATPNSLTISLDDPSFCVIYYRITYGETGGNSPSQE FTVPGYTNTATISGLKPGQDYTITVYAVASYGYLTSNPISINYRTEIDKP SQGS
Example 5. Identification of Bifunctional Compounds which Bind CXCR4
[0325] Libraries of bifunctional compounds were constructed by site-specific conjugation of a fibronectin type III domain (Fn), at either Cys28 or Cys80, to a cyclic peptide CXCR4 antagonist (CP) via a linker. The yeast-displayed Fn-CP libraries were screened for their ability to bind polypeptide target, CXCR4.
Other Embodiments
[0326] It is to be understood that while the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and alterations are within the scope of the following claims.
[0327] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
[0328] In the claims, articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
[0329] It is also noted that the term comprising is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term comprising is used herein, the term consisting of is thus also encompassed and disclosed.
[0330] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
[0331] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any polynucleotide or protein encoded thereby; any method of production; any method of use) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.