Method for screening inhibitors of Ras

• US 9,810,690 B2
• Filed: 11/02/2016
• Issued: 11/07/2017
• Est. Priority Date: 10/19/2015
• Status: Active Grant

• Current Assignee: Araxes Pharma LLC
• Original Assignee: Araxes Pharma LLC
• Inventors: Patricelli, Matthew P., Peters, Ulf, Li, Liansheng, Ren, Pingda, Liu, Yi
• Primary Examiner(s): Mondesi, Robert
• Assistant Examiner(s): Meah, Md. Younus
• Application Number:
  US15/342,100
• Publication Number:
  US 20170131278A1
• Time in Patent Office: 370 Days
• Field of Search: 435 723, 4352523, 536 232, 4241391, 4241431
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12Y 306/05002 Small monomeric GTPase (3.6.5.2)
• CPC Class Codes: G01N 2333/912 transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 2500/20 cell-free systems
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/574 for cancer

Abstract

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.

Claims

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.

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.

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.