METHODS AND REAGENTS FOR ANALYZING PROTEIN-PROTEIN INTERFACES
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.