US2018246102A1PendingUtilityA1

Method for screening inhibitors of ras

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Assignee: ARAXES PHARMA LLCPriority: Oct 19, 2015Filed: Sep 22, 2017Published: Aug 30, 2018
Est. expiryOct 19, 2035(~9.3 yrs left)· nominal 20-yr term from priority
Inventors:Matthew PatricelliUlf PetersLiansheng LiPingda RenYi Liu
G01N 33/575G01N 33/573G01N 2500/04G01N 2500/20C12Y 306/05002G01N 2333/912G01N 33/542C12N 9/14
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Claims

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

exact text as granted — not AI-modified
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 mutated sequence, full-length or truncated, of KRAS isoform a (SEQ ID NO: 1), KRAS isoform b (SEQ ID NO: 2), HRAS (SEQ ID NO: 3), NRAS (SEQ ID NO: 4), MRAS (SEQ ID NO: 5), ERAS (SEQ ID NO: 6), RRAS2 isoform a (SEQ ID NO: 7), RALA (SEQ ID NO: 8), RALB (SEQ ID NO: 9), RIT1 isoform 2 (SEQ ID NO: 10), RRAS2 isoform b (SEQ ID NO: 45), RRAS2 isoform c (SEQ ID NO: 46), RIT1 isoform 1 (SEQ ID NO: 47), or RIT1 isoform 3 (SEQ ID NO: 48), wherein said full-length or truncated sequence is mutated to include one or more cysteine residues at one or more positions selected from the group consisting of 62, 92, 95, and a corresponding position of 62, 92, 95 in MRAS, ERAS, RRAS2 isoform a, RALA, RALB, RIT1 isoform 2, RRAS2 isoform b, RRAS2 isoform c, RIT1 isoform 1, or RIT1 isoform 3; 
 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 the cysteine residue of a cysteine mutation. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the cysteine mutation is not at position 12 or 13 relative to SEQ ID NO: 1. 
     
     
         6 . The method of  claim 1 , wherein the cysteine mutation is at position 12 or 13 relative to SEQ ID NO: 1. 
     
     
         7 . The method of  claim 1 , wherein the mutant Ras comprises SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. 
     
     
         8 . 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. 
     
     
         9 . The method of  claim 1 , wherein the mutant Ras comprises SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. 
     
     
         10 . The method of  claim 1 , wherein the mutant Ras comprises a mutated sequence, full-length or truncated, of 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). 
     
     
         11 . The method of  claim 1 , wherein the mutant Ras comprises a mutated sequence, full-length or truncated, of 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). 
     
     
         12 . The method of  claim 1 , wherein the mutant Ras comprises a mutated sequence, full-length or truncated, of 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). 
     
     
         13 . The method of  claim 1 , wherein the mutant Ras comprises a mutated sequence, full-length or truncated, of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, in which the residue corresponding to residue 62 of SEQ ID NO: 1 is C (cysteine). 
     
     
         14 . The method of  claim 1 , wherein the mutant Ras comprises a mutated sequence, full-length or truncated, of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, in which the residue corresponding to residue 92 of SEQ ID NO: 1 is C (cysteine). 
     
     
         15 . The method of  claim 1 , wherein the mutant Ras comprises a mutated sequence, full-length or truncated, of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, in which the residue corresponding to residue 95 of SEQ ID NO: 1 is C (cysteine). 
     
     
         16 . The method of  claim 1 , wherein the mutant Ras comprises a mutated sequence, full-length or truncated, of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, in which the residue corresponding to residue 62 of SEQ ID NO: 1 is C (cysteine), and the residue corresponding to residue 92 of SEQ ID NO: 1 is C (cysteine). 
     
     
         17 . 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. 
     
     
         18 . A mutant Ras comprising a mutated sequence, full-length or truncated, of KRAS isoform a (SEQ ID NO: 1), KRAS isoform b (SEQ ID NO: 2), HRAS (SEQ ID NO: 3), NRAS (SEQ ID NO: 4), MRAS (SEQ ID NO: 5), ERAS (SEQ ID NO: 6), RRAS2 isoform a (SEQ ID NO: 7), RALA (SEQ ID NO: 8), RALB (SEQ ID NO: 9), RIT1 isoform 2 (SEQ ID NO: 10), RRAS2 isoform b (SEQ ID NO: 45), RRAS2 isoform c (SEQ ID NO: 46), RIT1 isoform 1 (SEQ ID NO: 47), or RIT1 isoform 3 (SEQ ID NO: 48), wherein said full-length or truncated sequence is mutated to include one or more cysteine residues at one or more positions selected from the group consisting of 62, 92, 95, and a corresponding position of 62, 92, 95 in MRAS, ERAS, RRAS2 isoform a, RALA, RALB, RIT1 isoform 2, RRAS2 isoform b, RRAS2 isoform c, RIT1 isoform 1, or RIT1 isoform 3. 
     
     
         19 . The mutant Ras of  claim 18 , wherein the mutant Ras is (1) a mutant KRAS, mutant HRAS, or mutant NRAS, in which position 62 is C (cysteine); (2) a mutant MRAS in which position 72 is C (cysteine); (3) a mutant ERAS in which position 100 is C (cysteine); (4) a mutant RRAS2 or a mutant RALA, in which position 73 is C (cysteine); or (5) a mutant RIT1 in which position 80 is C (cysteine). 
     
     
         20 . The mutant Ras of  claim 18 , wherein the mutant Ras is (1) a mutant KRAS, mutant HRAS, or mutant NRAS, in which position 92 is C (cysteine); (2) a mutant MRAS in which position 102 is C (cysteine); (3) a mutant ERAS in which position 130 is C (cysteine); (4) a mutant RRAS2 or a mutant RALA, in which position 103 is C (cysteine); or (5) a mutant RIT1 in which position 110 is C (cysteine). 
     
     
         21 . The mutant Ras of  claim 18 , wherein the mutant Ras is (1) a mutant KRAS, mutant HRAS, or mutant NRAS, in which position 95 is C (cysteine); (2) a mutant MRAS in which position 105 is C (cysteine); (3) a mutant ERAS in which position 133 is C (cysteine); (4) a mutant RRAS2 or a mutant RALA, in which position 106 is C (cysteine); or (5) a mutant RIT1 in which position 113 is C (cysteine). 
     
     
         22 . The mutant Ras of  claim 18 , wherein the mutant Ras contains a mutation at D92C and an additional mutation at G12D relative to SEQ ID NO: 1. 
     
     
         23 . The mutant Ras of  claim 18 , wherein the mutant Ras contains a mutation at H95C and an additional mutation at G12D relative to SEQ ID NO: 1. 
     
     
         24 . The mutant Ras of  claim 18 , wherein the mutant Ras comprises a sequence shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. 
     
     
         25 . The mutant Ras of  claim 18 , wherein the mutant Ras comprises SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. 
     
     
         26 . The mutant Ras of  claim 18 , wherein a competition probe competes for binding in a Switch II pocket of the mutant Ras. 
     
     
         27 . The mutant Ras of  claim 18 , wherein the mutant Ras is selected from the group consisting of mutant KRAS, mutant MRAS, mutant ERAS, mutant RRAS2, mutant RALA, mutant RALB, mutant RIT1, and any combination thereof. 
     
     
         28 . A polynucleotide encoding the mutant Ras of  claim 18 . 
     
     
         29 . An expression vector comprising the polynucleotide of  claim 28 . 
     
     
         30 . A host cell comprising the expression vector of  claim 29 . 
     
     
         31 . A mutant Ras comprising a mutated sequence, full-length or truncated, of MRAS (SEQ ID NO: 5), ERAS (SEQ ID NO: 6), RRAS2 isoform a (SEQ ID NO: 7), RALA (SEQ ID NO: 8), RALB (SEQ ID NO: 9), RIT1 isoform 2 (SEQ ID NO: 10), RRAS2 isoform b (SEQ ID NO: 45), RRAS2 isoform c (SEQ ID NO: 46), RIT1 isoform 1 (SEQ ID NO: 47), or RIT1 isoform 3 (SEQ ID NO: 48), wherein said full-length or truncated sequence is mutated to include a cysteine residue at position 23, or a corresponding position of 23 in MRAS, ERAS, RRAS2 isoform a, RALA, RALB, RIT1 isoform 2, RRAS2 isoform b, RRAS2 isoform c, RIT1 isoform 1, or RIT1 isoform 3. 
     
     
         32 . A method of selecting a Ras antagonist, the method comprising:
 (a) combining in a reaction mixture the mutant Ras of  claim 31 , 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 competition probe is capable of binding and covalently modifying the mutant Ras; and 
 ii. the decrease in binding between the mutant Ras and the competition probe is indicative of Ras antagonist activity of the test compound.

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