Methods and reagents for analyzing protein-protein interfaces

Abstract

The present disclosure provides methods and reagents useful for analyzing protein-protein interfaces such as interfaces between a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein. In some embodiments, the target and/or presenter proteins are intracellular proteins. In some embodiments, the target and/or presenter proteins are mammalian proteins.

1 . A conjugate comprising a presenter protein binding moiety conjugated to a target protein,
 (i) wherein the presenter protein binding moiety is a cyclophilin binding moiety comprising the structure of Formula III or IV:   
       
         
           
           
               
               
           
         
         wherein Z 3 , Z 4 , Z 5 , and Z 6  are each, independently, hydroxyl, optionally substituted C 1 -C 6  alkyl, optionally substituted C 1 -C 6  heteroalkyl, or Z 3  and Z 4  or Z 5  and Z 6  combine to form, with the atoms to which they are attached, an optionally substituted 10 to 40 member macrocycle; 
         at least one of Z 3 , Z 4 , Z 5 , Z 6 , or R 5  comprises a point of attachment to a cross-linking group; 
         e is 0, 1, 2, 3, or 4; 
         R 5  is optionally substituted C 1 -C 6  alkyl, optionally substituted C 2 -C 6  alkenyl, optionally substituted C 2 -C 6  alkynyl, optionally substituted C 1 -C 6  heteroalkyl, optionally substituted C 2 -C 6  heteroalkenyl, optionally substituted C 2 -C 6  heteroalkynyl, optionally substituted C 3 -C 10  carbocyclyl, optionally substituted C 6 -C 10  aryl, optionally substituted C 6 -C 10  aryl C 1 -C 6  alkyl, optionally substituted C 2 -C 9  heterocyclyl, or optionally substituted C 2 -C 9  heterocyclyl C 1 -C 6  alkyl; 
         R 6  is optionally substituted C 1 -C 6  alkyl; 
         each R 7  is, independently, hydroxyl, cyano, optionally substituted amino, halogen, thiol, optionally substituted C 1 -C 6  alkyl, optionally substituted C 2 -C 6  alkenyl, optionally substituted C 2 -C 6  alkynyl, optionally substituted C 1 -C 6  heteroalkyl, optionally substituted C 2 -C 6  heteroalkenyl, optionally substituted C 2 -C 6  heteroalkynyl, optionally substituted C 3 -C 10  carbocyclyl, optionally substituted C 6 -C 10  aryl, optionally substituted C 6 -C 10  aryl C 1 -C 6  alkyl, optionally substituted C 2 -C 9  heterocyclyl, or optionally substituted C 2 -C 9  heterocyclyl C 1 -C 6  alkyl; and 
         R 8  is hydrogen, optionally substituted C 1 -C 6  alkyl, optionally substituted C 2 -C 6  alkenyl, optionally substituted C 2 -C 6  alkynyl, optionally substituted aryl, C 3 -C 7  carbocyclyl, optionally substituted C 6 -C 10  aryl C 1 -C 6 alkyl, and optionally substituted C 3 -C 7  carbocyclyl C 1 -C 6  alkyl; or 
         (ii) wherein the presenter protein binding moiety is an FKBP binding moiety comprising the structure of Formula IIa: 
       
       
         
           
           
               
               
           
         
         wherein Z 1  and Z 2  are each, independently, optionally substituted C 1 -C 6  alkyl, optionally substituted C 1 -C 6  heteroalkyl, or Z 1  and Z 2  combine to form, with the atoms to which they are attached, an optionally substituted 10 to 40 member macrocycle; 
         at least one of Z 1  or Z 2  comprises a point of attachment to a cross-linking group; 
         X 2  is absent, CH 2 , O, S, SO, SO 2 , or NR 4 ; and 
         each R 4  is, independently, hydrogen, optionally substituted C 1 -C 6  alkyl, optionally substituted C 2 -C 6  alkenyl, optionally substituted C 2 -C 6  alkynyl, optionally substituted aryl, C 3 —C carbocyclyl, optionally substituted C 6 -C 10  aryl C 1 -C 6  alkyl, and optionally substituted C 3 -C 7  carbocyclyl C 1 -C 6  alkyl, 
         wherein the cross-linking group of the presenter protein binding moiety forms a covalent bond with the target protein. 
       
     
     
         2 . The conjugate of  claim 1 , wherein the presenter protein binding moiety is a cyclophilin binding moiety comprising the structure of Formula III. 
     
     
         3 . The conjugate of  claim 2 , wherein the cyclophilin binding moiety is capable of binding PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1. 
     
     
         4 . The conjugate of  claim 1 , wherein the presenter protein binding moiety is a cyclophilin binding moiety comprising the structure of Formula IV. 
     
     
         5 . The conjugate of  claim 4 , wherein the cyclophilin binding moiety is capable of binding PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1. 
     
     
         6 . The conjugate of  claim 1 , wherein the presenter protein binding moiety is an FKBP binding moiety comprising the structure of Formula IIa. 
     
     
         7 . The conjugate of  claim 6 , wherein the FKBP binding moiety is capable of binding FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52. 
     
     
         8 . The conjugate of  claim 1 , wherein the cross-linking group is a mixed disulfide, maleimide, vinyl sulfone, vinyl ketone, alkyl halide, isocyanate, isothiocyanate, sulfonyl chloride, acid halide, active ester, acid anhydride, acylazide, imidoester, haloheteroaryl, diazo compound, carbodiimide, hydrazide, alkoxyamine, azide, or alkyne. 
     
     
         9 . The conjugate of  claim 8 , wherein the cross-linking group forms a covalent bond with the a reactive group of an amino acid side chain of the target protein. 
     
     
         10 . The conjugate of  claim 1 , wherein the target protein is a GTPase, GTPase activating protein, Guanine nucleotide-exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier/remodeler, or a protein with classical protein-protein interaction domains and motifs. 
     
     
         11 . The conjugate of  claim 10 , wherein the target protein is a GTPase. 
     
     
         12 . The conjugate of  claim 11 , wherein the target protein is K-Ras, H-Ras, or N-Ras. 
     
     
         13 . The conjugate of  claim 12 , wherein the target protein is K-Ras. 
     
     
         14 . The conjugate of  claim 13 , wherein the target protein is K-Ras having a G12C mutation. 
     
     
         15 . The conjugate of  claim 13 , wherein the target protein is K-Ras having a S39C mutation. 
     
     
         16 . The conjugate of  claim 12 , wherein the target protein in N-Ras. 
     
     
         17 . The conjugate of  claim 16 , wherein the target protein in N-Ras having a G12C mutation.