US2015346204A1PendingUtilityA1

MEK Mutations Conferring Resistance to MEK Inhibitors

49
Individually held — no corporate assignee on recordPriority: Dec 10, 2008Filed: Jun 10, 2015Published: Dec 3, 2015
Est. expiryDec 10, 2028(~2.4 yrs left)· nominal 20-yr term from priority
Inventors:Levi A. GarrawayCaroline EmeryCarlos Garcia-Echeverria
G01N 2333/912A61K 31/4184A61P 35/02G01N 2500/04A61K 38/00G01N 33/573C12N 9/1205A61P 35/00A61P 43/00
49
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Claims

Abstract

The present invention relates to methods, compositions and kits concerning resistance to treatment with an anti-cancer agent, specifically an inhibitor of MEK. In particular embodiments, the invention concerns mutations in a MEK sequence that confer resistance to a MEK inhibitor. Identification of such mutations in a MEK sequence allows the identification and design of second-generation MEK inhibitors. Methods and kits for detecting the presence of a mutant MEK sequence in a sample are also provided.

Claims

exact text as granted — not AI-modified
What is claimed: 
     
         1 . A method of identifying a compound that is useful as a MEK inhibitor that inhibits a mutant MEK polypeptide having MEK activity, comprising:
 a) providing a cell comprising a mutant MEK polypeptide having MEK activity, the mutant MEK polypeptide comprising an amino acid sequence having at least one amino acid substitution as compared to a wild type MEK1 polypeptide (SEQ ID NO: 2) or a MEK2 polypeptide (SEQ ID NO:4), the at least one amino acid substitution occurring at one or more amino acid positions selected from the group consisting of Q56, I99, I103, K104, I111, R113, L115, H119, E120, P124, G128, F129, F133, E203, V211, L215, G237, V258, G276, G286, M302, P326, and H399 of SEQ ID NO: 2 or Q60, I103, I107, K108, I115, R117, L119, H123, E124, P128, G132, F133, F137, E207, V215, L219, G241, V262, G280, M310, and P334 of SEQ ID NO: 4;   b) contacting the cell with a test compound; and   c) determining the effect of the compound on MEK activity or determining the effect of the compound on cell proliferation or cell viability;   wherein downmodulation of the MEK activity or reduction in cell proliferation or a reduction in cell viability as compared to a suitable control identifies the compound as a compound that is useful as a MEK inhibitor that inhibits the mutant MEK polypeptide having MEK activity.   
     
     
         2 . The method according to  claim 1 , wherein the at least one amino acid substitution occurs at one or more amino acid positions selected from the group consisting of Q56P, I99T, I103, K104N, I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D, F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E, M302R, P326L, and H399D of SEQ ID NO: 2 or Q60P, I103T, I107N, K108N, I115N, R117S, L119R, L119P, H123P, E124D, P128L, P128S, G132D, F133L, F137L, E207K, V215D, L219P, G241V, V262G, G280E, M310R, and P334L of SEQ ID NO: 4. 
     
     
         3 . The method according to  claim 1 , wherein the at least one amino acid substitution occurs at one or more amino acid positions selected from the group consisting of Q56, I99, I103, K104, I111, R113, L115, H119, E120, P124, G128, F129, F133, E203, V211, L215, G237, V258, G276, G286, M302, P326, and H399 of SEQ ID NO: 2. 
     
     
         4 . The method according to  claim 1 , wherein the at least one amino acid substitution occurs at one or more amino acid positions selected from the group consisting of Q56P, I99T, I103, K104N, I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D, F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E, M302R, P326L, and H399D of SEQ ID NO: 2. 
     
     
         5 . The method of  claim 1 , wherein the downmodulation of MEK activity comprises downmodulation of phosphorylation of an ERK1 or ERK2 substrate. 
     
     
         6 . The method of  claim 1 , comprising determining the effect of the compound on cell proliferation. 
     
     
         7 . The method of  claim 6 , wherein a shift in the GI50 value of at least 2 fold identifies the compound as a compound that is useful as a MEK inhibitor that inhibits the mutant MEK polypeptide having MEK activity. 
     
     
         8 . An expression vector comprising an isolated nucleic acid molecule encoding a mutant MEK polypeptide having MEK activity, the mutant MEK polypeptide comprising an amino acid sequence having at least one amino acid substitution as compared to a wild type MEK1 polypeptide (SEQ ID NO: 2) or a MEK2 polypeptide (SEQ ID NO:4), the at least one amino acid substitution occurring at one or more amino acid positions selected from the group consisting of Q56, I99, I103, K104, I111, R113, L115, H119, E120, P124, G128, F129, F133, E203, V211, L215, G237, V258, G276, G286, M302, P326, and H399 of SEQ ID NO: 2 or Q60, I103, I107, K108, I115, R117, L119, H123, E124, P128, G132, F133, F137, E207, V215, L219, G241, V262, G280, M310, and P334 of SEQ ID NO: 4. 
     
     
         9 . The expression vector according to  claim 8 , wherein the at least one amino acid substitution occurs at one or more amino acid positions selected from the group consisting of Q56P, I99T, I103, K104N, I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D, F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E, M302R, P326L, and H399D of SEQ ID NO: 2 or Q60P, I103T, I107N, K108N, I115N, R117S, L119R, L119P, H123P, E124D, P128L, P128S, G132D, F133L, F137L, E207K, V215D, L219P, G241V, V262G, G280E, M310R, and P334L of SEQ ID NO: 4. 
     
     
         10 . The expression vector according to  claim 8 , wherein the at least one amino acid substitution occurs at one or more amino acid positions selected from the group consisting of Q56P, I99T, I103, K104N, I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D, F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E, M302R, P326L, and H399D of SEQ ID NO: 2. 
     
     
         11 . A host cell comprising the expression vector of  claim 8 . 
     
     
         12 . A cell-based screening method for identifying a test compound as a MEK inhibitor that inhibits a mutant MEK polypeptide having MEK activity, the method comprising:
 contacting a host cell comprising the expression vector of  claim 8  with a test compound,   measuring sensitivity of the host cell to the test compound using an assay selected from the group consisting of a cell proliferation assay, a cell viability assay, and MEK activity assay, wherein a reduction in cell proliferation, cell viability, or MEK activity in the presence of the test compound identifies the host cell as having sensitivity to the test compound and wherein sensitivity of the host cell to the test compound identifies the compound as a MEK inhibitor that inhibits the mutant MEK polypeptide having MEK activity.   
     
     
         13 . The method of  claim 12 , wherein sensitivity of the host cell to the test compound is measured using the MEK activity assay comprising a an ERK phosphorylation assay, wherein a reduction in ERK phosphorylation in the presence of the test compound identifies the compound as a MEK inhibitor that inhibits the mutant MEK polypeptide having MEK activity. 
     
     
         14 . A method of identifying a compound as a MEK inhibitor that inhibits a mutant MEK polypeptide having MEK activity, comprising:
 (a) providing an assay composition comprising a mutant MEK polypeptide having MEK activity, the mutant MEK polypeptide comprising an amino acid sequence having at least one amino acid substitution as compared to a wild type MEK1 polypeptide (SEQ ID NO: 2) or a MEK2 polypeptide (SEQ ID NO:4), the at least one amino acid substitution occurring at one or more amino acid positions selected from the group consisting of Q56, I99, I103, K104, I111, R113, L1115, H119, E120, P124, G128, F129, F133, E203, V211, L215, G237, V258, G276, G286, M302, P326, and H399 of SEQ ID NO: 2 or Q60, I103, I107, K108, I115, R117, L119, H123, E124, P128, G132, F133, F137, E207, V215, L219, G241, V262, G280, M310, and P334 of SEQ ID NO: 4 and a MEK substrate;   (b) contacting the assay composition with a test compound under conditions that permit phosphorylation of the MEK substrate in the absence of the test compound; and   (c) determining the effect of the compound on phosphorylation of the MEK substrate;   wherein downmodulation of phosphorylation of the MEK substrate as compared to a suitable control identifies the compound as a compound that inhibits the mutant MEK polypeptide having MEK activity.   
     
     
         15 . The method according to  claim 14 , wherein the at least one amino acid substitution occurs at one or more amino acid positions selected from the group consisting of Q56P, I99T, I103, K104N, I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D, F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E, M302R, P326L, and H399D of SEQ ID NO: 2 or Q60P, I103T, I107N, K108N, I115N, R117S, L119R, L119P, H123P, E124D, P128L, P128S, G132D, F133L, F137L, E207K, V215D, L219P, G241V, V262G, G280E, M310R, and P334L of SEQ ID NO: 4. 
     
     
         16 . The method according to  claim 14 , wherein the at least one amino acid substitution occurs at one or more amino acid positions selected from the group consisting of Q56P, I99T, I103, K104N, I111N, R113S, L115P, L115P, H119P, E120D, P124L, P124S, G128D, F129L, F133L, E203, V211D, L215P, G237V, V258G, G276E, G286E, M302R, P326L, and H399D of SEQ ID NO: 2. 
     
     
         17 . The method of  claim 14 , wherein the MEK substrate is ERK1 or ERK2. 
     
     
         18 . A method of identifying a compound as a MEK inhibitor that inhibits a mutant MEK polypeptide having MEK activity, comprising:
 a) selecting a potential drug using computer-assisted modeling with a three-dimensional crystal or solution structure of a mutant MEK polypeptide, wherein the mutant MEK polypeptide comprises an amino acid sequence having at least one amino acid substitution as compared to a wild type MEK1 polypeptide (SEQ ID NO: 2) or a MEK2 polypeptide (SEQ ID NO:4), the at least one amino acid substitution conferring resistance to one or more MEK inhibitors on the mutant MEK polypeptide;   b) contacting the potential drug with the mutant MEK polypeptide; and   c) detecting the interaction of the potential drug with the mutant MEK polypeptide;   wherein a compound that is capable of interacting with the mutant MEK polypeptide is identified as a MEK inhibitor that inhibits the mutant MEK polypeptide having MEK activity.   
     
     
         19 . The method of  claim 18 , wherein the at least one amino acid substitution occurring at one or more amino acid positions selected from the group consisting of Q56, I99, I103, K104, I111, R113, L115, H119, E120, P124, G128, F129, F133, E203, V21, L215, G237, V258, G276, G286, M302, P326, and H399 of SEQ ID NO: 2 or Q60, I103, I107, K108, I115, R117, L119, H123, E124, P128, G132, F133, F137, E207, V215, L219, G241, V262, G280, M310, and P334 of SEQ ID NO: 4. 
     
     
         20 . The method of  claim 18 , wherein the test compound is a member of a library of compounds.

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