Raf dimers and uses thereof

Abstract

Disclosed herein are mutated RAF and KSR nucleic acids and polypeptides. Also disclosed are methods of using the mutated RAF and KSR to inhibit the dimerization of RAF/RAF and RAF/KSR. Also disclosed are methods of using the mutated RAF and KSR to screen for inhibitors of dimerization.

1 . A composition comprising: an aqueous solution of RAF/RAF homodimer. 
     
     
         2 - 4 . (canceled) 
     
     
         5 . A composition comprising: an aqueous solution of RAF/KSR heterodimer. 
     
     
         6 - 40 . (canceled) 
     
     
         41 . A method of detecting the presence of a mutation in a RAF kinase domain, the method comprising:
 a) providing a WT RAF kinase domain and a suspected mutant RAF kinase domain, each domain having a cysteine residue located at its N-terminus;   b) incubating the WT RAF kinase domain and the suspected mutant RAF kinase domain with different cross-linking detectable labels;   c) incubating together equimolar amounts of the labeled WT RAF kinase domain and detecting a signal from the detectable label so as to provide a dimerization reference signal; and   d) incubating equimolar amounts of the labeled suspected mutant B-RAF kinase domain and detecting a signal from the detectable labels, an absent signal or a reduce signal compared to that of the dimerization reference signal being an indication that a mutant B-RAF kinase domain is present.   
     
     
         42 . A method of monitoring the formation of RAF/RAF or RAF/KSR kinase domain dimers to detect mutations inhibiting dimerization or drug-like molecules interfering with dimerization, the method comprising:
 a) using either (i) a RAF kinase domain or (ii) a KSR kinase domain at either of their N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins;   b) expressing the fusion proteins to identify combinations that provide specific BRET signals;   c) introducing dimer interface mutations into either of the labeled fusion proteins;   d) expressing the labeled mutated fusion proteins with WT RAF or KSR kinase domains;   e) measuring the BRET signals, a loss or significant reduction of the BRET signal using dimer interface mutations as opposed to mutations remote from the interface, being an indication that a specific BRET signal which depends on the RAF/RAF or RAF/KSR dimerization interface has been obtained.   
     
     
         43 . The method, according to  claim 42 , in which the BRET donor is  renilla  luciferase variant II or rlucII. 
     
     
         44 . The method, according to  claim 42 , in which the BRET acceptor is GFP10. 
     
     
         45 . The method, according to  claim 42 , in which the acceptor label is Yellow Fluorescent Protein (YFP). 
     
     
         46 . The method, according to  claim 42 , in which the donor labeled fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 42 and SEQ ID NO. 48. 
     
     
         47 . The method, according to  claim 42 , in which the acceptor labeled fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO. 22, SEQ ID NO. 30, SEQ ID NO. 40 and SEQ ID NO. 54. 
     
     
         48 . The method, according to  claim 42 , in which the donor labeled mutated fusion proteins comprise sequences SEQ ID NO. 36 and SEQ ID NO. 50. 
     
     
         49 . The method, according to  claim 42 , in which the acceptor labeled mutated fusion proteins comprises a sequence of SEQ ID NO. 32. 
     
     
         50 . A method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising.
 a) fusing a RAF kinase domain at either of its N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins;   b) expressing the fusion proteins to identify combinations that provide specific BRET signals;   c) introducing dimer interface mutations into either of the labeled fusion proteins;   d) expressing the labeled mutated fusion proteins with WT RAF kinase domains;   e) contacting the interface with the potential inhibitor; and   f) measuring the BRET signals, a loss or significant reduction of the BRET signal for the wild-type RAF/RAF BRET pair being an indication that the inhibitor is specifically bound to the interface.   
     
     
         51 . A method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising:
 a) detectably labeling at least one of the dimerization interface residues to generate a detectably labeled RAF monomer;   b) incubating the detectably labeled RAF monomer with the potential inhibitor and a non-labeled RAF monomer;   c) measuring a signal from the detectable label;   d) contacting the RAF dimerization interface with the inhibitor to determine the ability of the potential inhibitor to inhibit RAF/RAF homodimerization.   
     
     
         52 . The method, according to  claim 51 , in which the interface residues include H449, G450, R481, L487, F488, M489, Y538, A541 or K542. 
     
     
         53 . A method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising:
 a) fusing a RAF kinase domain at either of its N- or C-termini to a BRET donor or a BRET acceptor to produce donor labeled and acceptor labeled fusion proteins;   b) expressing the fusion proteins to identify combinations that provide specific BRET signals;   c) introducing dimer interface mutations into either of the labeled fusion proteins;   d) expressing the labeled mutated fusion proteins with WT RAF kinase domains;   e) contacting the interface with the potential inhibitor; and   f) measuring the BRET signals, a loss or significant reduction of the BRET signal for the wild-type RAF/RAF BRET pair being an indication that the inhibitor is specifically bound to the interface.   
     
     
         54 . A method of identifying compounds that bind to a RAF or a KSR dimerization interface, the method comprising:
 a) contacting the interface with a probe to form a probe: interface complex, the probe being displaceable by a test compound;   b) measuring a signal from the probe so as to establish a reference level;   c) incubating the probe:interface complex with the test compound;   d) measuring the signal from the probe;   e) comparing the signal from step d) with the reference level, a modulation of the signal being an indication that the test compound binds to the BIR domain, wherein the probe is a compound labeled with a detectable label or an affinity label.   
     
     
         55 . A method of identifying a potential inhibitor of RAF/RAF homodimerization, the method comprising:
 a) using the atomic coordinates of at least one of the interface residues to generate a three dimensional structure of a RAF dimerization interface;   b) using the three-dimensional structure to design or select the potential inhibitor;   c) synthesizing the inhibitor; and   d) contacting the RAF dimierization interface with the inhibitor to determine the ability of the potential inhibitor to inhibit RAF/RAF homodimerization.   
     
     
         56 . The method, according to  claim 55 , in which the interface residues are H449, G450, R481, L487, F488, M489, Y538, A541 or K542. 
     
     
         57 . A method of identifying a potential inhibitor of RAF/KSR heterodimerization, the method comprising:
 a) using the atomic coordinates of at least one of interface residues to generate a three dimensional structure of a KSR dimerization interface;   b) using the three-dimensional structure to design or select the potential inhibitor;   c) synthesizing the inhibitor; and   d) contacting the KSR dimerization interface with the inhibitor to determine the ability of the potential inhibitor to inhibit RAF/KSR heterodimerization.   
     
     
         58 . The method, according to  claim 57 , in which the interface residues are H699, G700, R732, L738, F739, M740, Y790, A793 or R794. 
     
     
         59 - 61 . (canceled)