Protein kinase conjugates and inhibitors
The invention relates to protein conjugates that contain a protein kinase containing a cysteine residue in the ATP binding site and an inhibitor that is covalently and irreversibly bonded to said cysteine residue, such that the activity of the protein kinase is irreversibly inhibited. The invention also relates to compounds that irreversibly inhibit protein kinases.
1. A protein kinase conjugate that has the formula,
X—M—S—CH 2 -Prot
wherein, Prot is a human protein kinase or portion thereof comprising a cysteine residue containing amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 7, 8, 9, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45;
S—CH 2 is the sulfur atom and methylene group from the side chain of said cysteine residue;
M is a modifier moiety that is formed by the covalent bonding of a warhead group with the side chain of said cysteine residue;
X is a chemical moiety that binds in or near the ATP binding site of the protein kinase; and
wherein the protein kinase conjugate is formed in association with a cell.
2. The conjugate of claim 1 , wherein the warhead group is -L-Y, wherein
L is a covalent bond or a bivalent C 1-8 saturated or unsaturated, straight or branched, hydrocarbon chain, wherein one, two, or three methylene units of L are optionally and independently replaced by cyclopropylene, —NR—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO 2 —, —SO 2 N(R)—, —O—, —C(O)—, —OC(O)—, —CH(OH)—, —C(O)O—, —S—, —SO—, —SO 2 —, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N 2 )—;
Y is hydrogen, nitrile, C 1-6 aliphatic optionally substituted with one or more OH, NRxRy, oxo, halogen, NO 2 , or CN, or a 3-10 membered monocyclic or bicyclic, saturated, partially unsaturated, or aryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and wherein said ring is substituted with 1-4 R e groups; and
R, Rx and Ry are independently hydrogen, lower alkyl, lower haloalkyl, or lower cycloalkyl;
each R e is independently selected from -Q-Z, OH, oxo, NO 2 , halogen, CN, a suitable leaving group, or a C 1-6 aliphatic optionally substituted with oxo, halogen, NO 2 , or CN, wherein:
Q is a covalent bond or a bivalent C 1-6 saturated or unsaturated, straight or branched, hydrocarbon chain, wherein one or two methylene units of Q are optionally and independently replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, or —SO 2 —, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO 2 —, or —SO 2 N(R)—; and
Z is hydrogen or C 1-6 aliphatic optionally substituted with oxo, halogen, NO 2 , or CN.
3. The conjugate of claim 1 , wherein the warhead group is
wherein each R e is independently a suitable leaving group, NO 2 , CN, or oxo.
4. The conjugate of claim 1 , wherein the modifier moiety taken together with the side chain Sulfur and methylene of the cysteine residue have a formula selected from the group consisting of
5. The conjugate of claim 1 , wherein Prot is a kinase or portion of a kinase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 7, 8, and 9.
6. The conjugate of claim 1 , wherein Prot is a kinase selected from the group consisting of JAK3, C-KIT, FAK2, FLT3, FES, CDKL4, or wherein Prot is a portion of JAK3 wherein the portion of JAK3 comprises the amino acid sequence of SEQ ID NO:1, a portion of C-KIT wherein the portion of C-KIT comprises the amino acid sequence of SEQ ID NO: 41, a portion of FAK2 wherein the portion of FAK2 comprises the amino acid sequence of SEQ ID NO:6, a portion of FLT3 wherein the portion of FLT3 comprises the amino acid sequence of SEQ ID NO:7 or 41, a portion of FES wherein the portion of FES comprises the amino acid sequence of SEQ ID NO:8, or a portion of CDKL4 wherein the portion of CDLK4 comprises the amino acid sequence of SEQ ID NO:9.
7. The conjugate of claim 1 , wherein Prot is a kinase or portion of a kinase comprising a cysteine residue containing amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28 and 29.
8. The conjugate of claim 1 , wherein Prot is a kinase selected from the group consisting of PFTAIRE1, JNK1, JNK2, JNK3, BMPR2, or wherein Prot is a portion of PFTAIRE1 wherein the portion of PFTAIRE1 comprises the amino acid sequence of SEQ ID NO:26, a portion of JNK1 wherein the portion of JNK1 comprises the amino acid sequence of SEQ ID NO:28, a portion of JNK2 wherein the portion of JNK2 comprises the amino acid sequence of SEQ ID NO:27, a portion of JNK3 wherein the portion of JNK3 comprises the amino acid sequence of SEQ ID NO:28, or a portion of BMPR2 wherein the portion of BMPR2 comprises the amino acid sequence of SEQ ID NO:29.
9. The conjugate of claim 1 , wherein Prot is a kinase or portion of a kinase comprising a cysteine residue containing amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32 and 33.
10. The conjugate of claim 1 , wherein Prot is a kinase selected from the group consisting of TNK1, YES, FGR, SRC, LIMK1, or wherein Prot is a portion of TNK1 wherein the portion of TNK1 comprises the amino acid sequence of SEQ ID NO:30, a portion of YES wherein the portion of YES comprises the amino acid sequence of SEQ ID NO:31, a portion of FOR wherein the portion of FOR comprises the amino acid sequence of SEQ ID NO:32, a portion of SRC wherein the portion of SRC comprises the amino acid sequence of SEQ ID NO:31, or a portion of LIMK1 wherein the portion of LIMK1 comprises the amino acid sequence of SEQ ID NO:33.
11. The conjugate of claim 1 , wherein Prot is a kinase or portion of a kinase comprising a cysteine residue containing amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 36 and 37.
12. The conjugate of claim 1 , wherein Prot is a kinase selected from the group consisting of FAK, ALK1, ALK2, or wherein Prot is a portion of FAK wherein the portion of FAK comprises the amino acid sequence of SEQ ID NO:35, a portion of ALK1 wherein the portion of ALK1 comprises the amino acid sequence of SEQ ID NO:36, a portion of ALK2 wherein the portion of ALK2 comprises the amino acid sequence of SEQ ID NO:37.
13. The conjugate of claim 1 , wherein Prot is a kinase or portion of a kinase comprising a cysteine residue containing amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39 and 40.
14. The conjugate of claim 1 , wherein Prot is a kinase selected from the group consisting of ZAP70, CRIK, ERK3, CK1g1, CK1g2, CK1g3, or wherein Prot is a portion of ZAP70 wherein the portion of ZAP70 comprises the amino acid sequence of SEQ ID NO:38, a portion of CRIK wherein the portion of CRIK comprises the amino acid sequence of SEQ ID NO:39, a portion of ERK3 wherein the portion of ERK3 comprises the amino acid sequence of SEQ ID NO:40, a portion of CK1g1 wherein the portion of CK1g1 comprises the amino acid sequence of SEQ ID NO:38, or a portion of CK1g2 wherein the portion of CK1g2 comprises the amino acid sequence of SEQ ID NO:38, or a portion of CK1g3 wherein the portion of CK1g3 comprises the amino acid sequence of SEQ ID NO:38.
15. The conjugate of claim 1 , wherein Prot is a kinase or portion of a kinase comprising a cysteine residue containing amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 42, and 43.
16. The conjugate of claim 1 , wherein Prot is a kinase selected from the group consisting of FMS, RON, FGR, or wherein Prot is a portion of FMS wherein the portion of FMS comprises the amino acid sequence of SEQ ID NO:41, a portion of RON wherein the portion of RON comprises the amino acid sequence of SEQ ID NO:42, or a portion of FGR wherein the portion of FGR comprises the amino acid sequence of SEQ ID NO:43.
17. The conjugate of claim 1 , wherein Prot is a kinase or portion of a kinase comprising a cysteine residue containing amino acid sequence of SEQ ID NO:44.
18. The conjugate of claim 1 , wherein Prot is ALK or a portion thereof that comprises a cystein residue containing amino acid sequence of SEQ ID NO:44.
19. The conjugate of claim 1 , wherein Prot is a kinase or portion of a kinase comprising a cysteine residue containing amino acid sequence of SEQ ID NO:45.
20. The conjugate of claim 1 , wherein Prot is B-RAF or a portion thereof that comprises a cysteine residue containing amino acid sequence of SEQ ID NO:45.
21. The conjugate of claim 1 , wherein, if the protein kinase is JAK3, the cysteine residue is Cys945.
22. The conjugate of claim 1 , wherein Prot is C-KIT or a portion thereof, wherein the C-KIT or a portion thereof comprises the amino acid sequence SEQ ID NO: 41.
23. The conjugate of claim 6 , wherein Prot is C-KIT or a portion thereof, wherein the C-KIT or a portion thereof comprises the amino acid sequence SEQ ID NO: 41.
24. The conjugate of claim 6 , wherein Prot comprises the amino acid sequence SEQ ID NO:41.
25. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:1.
26. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:6.
27. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:7.
28. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:8.
29. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:9.
30. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:26.
31. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:27.
32. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:28.
33. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:29.
34. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:30.
35. The conjugate of claim 1 . wherein the cysteine residue containing amino acid sequence is SEQ ID NO:31.
36. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:32.
37. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:33.
38. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:35.
39. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:36.
40. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:37.
41. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:38.
42. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:39.
43. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:40.
44. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:41.
45. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:42.
46. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:43.
47. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:44.
48. The conjugate of claim 1 , wherein the cysteine residue containing amino acid sequence is SEQ ID NO:45.