Method for screening inhibitors of Ras
Provided herein are compositions, reactions mixtures, mutant Ras proteins, kits, substrates, and systems for selecting a Ras antagonist, as well as methods of using the same.
1. A method of selecting a Ras antagonist, the method comprising:
(a) combining in a reaction mixture a mutant Ras, a competition probe, and a test compound; and
(b) detecting a decrease in binding between the mutant Ras and the competition probe as compared to binding of the competition probe to the mutant Ras in an absence of the test compound; wherein:
i. the mutant Ras comprises a truncated or full-length sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, that is mutated to have up to 20 mutations including mutation of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 at amino acid residue 62, 92, or 95 to cysteine;
ii. the competition probe is capable of binding and covalently modifying the mutant Ras; and
iii. the decrease in binding between the mutant Ras and the competition probe is indicative of Ras antagonist activity of the test compound.
2. The method of claim 1 , wherein the competition probe competes for binding in a Switch II pocket of the mutant Ras.
3. The method of claim 1 , wherein the competition probe is capable of covalently modifying the mutant Ras by reacting with a cysteine residue of the cysteine mutation.
4. The method of claim 1 , wherein cysteine mutation is not at position 12 or 13 relative to SEQ ID NO: 1.
5. The method of claim 1 , wherein cysteine mutation is at position 12 or 13 relative to SEQ ID NO: 1.
6. The method of claim 1 , wherein the mutant Ras comprises the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29.
7. The method of claim 1 , wherein the mutant Ras is (1) a mutant KRAS comprising mutations of G12D and D92C, or (2) a mutant KRAS comprising mutations of G12D and H95C.
8. The method of claim 1 , wherein the mutant Ras comprises the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35.
9. The method of claim 1 , wherein the mutant Ras comprises the truncated or full-length sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, in which position 62 is C (cysteine) and position 12 is D (aspartic acid).
10. The method of claim 1 , wherein the mutant Ras comprises the truncated or full-length sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, in which position 92 is C (cysteine) and position 12 is D (aspartic acid).
11. The method of claim 1 , wherein the mutant Ras comprises the truncated or full-length sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, in which position 95 is C (cysteine) and position 12 is D (aspartic acid).
12. The method of claim 1 , wherein the mutant Ras comprises the truncated or full-length sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, in which position 92 is C (cysteine) and position 12 is C (cysteine).
13. The method of claim 1 , wherein the mutant Ras comprises the truncated or full-length sequence according to SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, or SEQ ID NO: 4, in which position 62 is C (cysteine) and position 12 is C (cysteine).
14. The method of claim 1 , wherein the mutant Ras has the sequence shown in SEQ ID No: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.
15. The method of claim 1 , wherein the mutant Ras has the sequence shown in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.
16. The method of claim 1 , wherein detecting the decrease in binding comprises measuring the fraction of Ras covalently modified by the competition probe as determined by mass spectrometry.
17. A mutant Ras comprising a truncated or full-length sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, that is mutated to have up to 20 mutations including mutation of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 at amino acid residue 62, 92, or 95 to cysteine, wherein said mutant Ras exhibits the ability to react with a competition probe capable of binding and covalently modifying said mutant Ras.
18. The mutant Ras of claim 17 , wherein the mutant Ras is (1) a mutant KRAS comprising G12D (aspartic acid) at position 12 and C (cysteine) at position 92, or (2) a mutant KRAS comprising (aspartic acid) at position 12 and C (cysteine) at position 95, or (3) a mutant KRAS comprising D (aspartic acid) at position 12 and C (cysteine) at position 62.
19. The mutant Ras of claim 17 , wherein the mutant Ras is (1) a mutant HRAS comprising D (aspartic acid) at position 12 and C (cysteine) at position 92, or (2) a mutant HRAS comprising D (aspartic acid) at position 12 and C (cysteine) at position 95, or (3) a mutant HRAS comprising D (aspartic acid) at position 12 and C (cysteine) at position 62.
20. The mutant Ras of claim 17 , wherein the mutant Ras is (1) a mutant NRAS comprising D (aspartic acid) at position 12 and C (cysteine) at position 92, or (2) a mutant NRAS comprising D (aspartic acid) at position 12 and C (cysteine) at position 95, or (3) a mutant NRAS comprising D (aspartic acid) at position 12 and C (cysteine) at position 62.
21. The mutant Ras of claim 17 , wherein the mutant Ras comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.
22. The mutant Ras of claim 17 , wherein the mutant Ras comprises the truncated or full-length sequence of SEQ ID NO: 1 or SEQ ID NO: 2, in which the amino acid residue 62, 92, or 95 is mutated to cysteine.
23. The mutant Ras of claim 17 , wherein the mutant Ras comprises the sequence shown in SEQ ID No: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.
24. The mutant Ras of claim 17 , wherein the mutant Ras comprises SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.
25. The mutant Ras of claim 17 , wherein a competition probe competes for binding in a Switch II pocket of the mutant Ras.
26. The mutant Ras of claim 17 , wherein the mutant Ras is selected from the group consisting of mutant KRAS, mutant HRAS, mutant NRAS, and any combination thereof.
27. A polynucleotide encoding the mutant Ras of claim 17 .
28. An expression vector comprising the polynucleotide of claim 27 .
29. A host cell comprising the expression vector of claim 28 .
30. The method of claim 1 , wherein the mutant Ras comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.