Identification of kinase inhibitors
First Claim
1. A method for identifying a ligand binding to an inactive conformation of a target protein kinase, comprising (a) contacting the inactive conformation of said target protein kinase, which contains or is modified to contain a reactive group at or near a binding site of interest, with one or more ligand candidates capable of covalently bonding to said reactive group thereby forming a kinase-ligand conjugate;
- and (b) detecting the formation of said kinase-ligand conjugate and identifying the ligand present in said kinase-ligand conjugate.
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Accused Products
Abstract
The invention concerns the identification of protein kinase inhibitors that preferentially bind to the inactive conformation of a target protein kinase. The inhibitors are identified by locking the target protein kinase in an inactive conformation, and using Tethering to identify inhibitors preferentially targeting the inactive conformation.
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Citations
63 Claims
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1. A method for identifying a ligand binding to an inactive conformation of a target protein kinase, comprising
(a) contacting the inactive conformation of said target protein kinase, which contains or is modified to contain a reactive group at or near a binding site of interest, with one or more ligand candidates capable of covalently bonding to said reactive group thereby forming a kinase-ligand conjugate; - and
(b) detecting the formation of said kinase-ligand conjugate and identifying the ligand present in said kinase-ligand conjugate. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A method for identifying a ligand that binds to the inactive conformation of a target protein kinase, comprising
(a) obtaining the inactive conformation of said target protein kinase comprising an — - SH group, masked —
SH group, or activated —
SH group;
(b) combining said inactive conformation of said target protein kinase with one or more ligand candidates wherein said ligand candidates each comprises a disulfide bond;
(c) forming a kinase-ligand conjugate wherein at least one ligand candidate binds to the inactive conformation of the target protein kinase under disulfide exchange conditions, and (d) detecting the formation of said kinase-ligand conjugate and identifying the ligand present in said conjugate. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
- SH group, masked —
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47. A method for identifying ligands binding to an inactive conformation of a target protein kinase, comprising
(a) contacting the inactive conformation of said protein kinase having a first and a second binding site of interest and containing or modified to contain a nucleophile at or near the first site of interest with a plurality of ligand candidates, said candidates having a functional group reactive with the nucleophile, under conditions such that a reversible covalent bond is formed between the nucleophile and a candidate that has affinity for the first site of interest, to form a kinase-first ligand complex; -
(b) identifying the first ligand from the complex of (a);
(c) designing a derivative of the first ligand identified in (b) to provide a small molecule extender (SME) having a first functional group reactive with the nucleophile on the kinase and a second functional group reactive with a second ligand having affinity for the binding second site of interest;
(d) contacting the SME with the kinase to form a kinase-SME complex, and (e) contacting the kinase-SME complex with a plurality of second ligand candidates, said candidates having a functional group reactive with the SME in said kinase-SME complex, wherein a candidate that has affinity for said second binding site of interest on said kinase forms a reversible covalent bond with said kinase-SME complex, whereby a second ligand is identified. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)
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62. A method for identifying ligands binding to an inactive conformation of a target protein kinase, comprising
(a) obtaining the inactive conformation of the target protein kinase comprising an — - SH group, masked —
SH group, or activated —
SH group;
(b) combining in a mixture the inactive conformation of the target protein kinase with a plurality of ligand candidates that are each capable of forming a disulfide bond with the —
SH group, masked —
SH group, or activated —
SH group thereby forming at least one kinase-ligand conjugate;
(c) analyzing the mixture by mass spectrometry; and
(d) detecting the most abundant kinase-ligand conjugate that is formed and identifying the ligand thereon.
- SH group, masked —
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63. A method for identifying ligands binding to an inactive conformation of a target protein kinase, comprising
(a) screening a library of ligand candidates with a kinase-ligand conjugate formed by the covalent bonding of the inactive conformation of a kinase comprising a first reactive functionality with a compound that comprises (1) a second reactive functionality and (2) a chemically reactive group, wherein the second reactive functionality of the compound reacts with the first reactive functionality of the inactive conformation of said target protein kinase to form a first covalent bond such that the kinase-ligand conjugate contains a free chemically reactive group, under conditions wherein at least one member of the library forms a second covalent bond with the kinase-ligand conjugate; - and
(b) identifying a further ligand that binds covalently to the chemically reactive group of the kinase-ligand conjugate.
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Specification