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 - 277 . (canceled)
278 . A compound, or a pharmaceutically acceptable salt thereof, comprising:
a presenter protein binding moiety that binds to the cyclophilin protein,
and a cross-linking group that reacts with an amino acid of the KRAS protein to form a covalent bond between the compound and the KRAS protein, wherein the cross-linking group has a structure of
or
the cross-linking group has a structure of formula Ij or Ik:
wherein the wavy line illustrates the point of attachment of the cross-linking group to the remainder of the compound;
X E is absent;
R M is hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl, optionally substituted C 2 -C 6 alkynyl, 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 heteroaryl, optionally substituted C 2 -C 9 heteroaryl 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 N , R O , and R P are independently hydrogen, hydroxyl, 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 heteroaryl, optionally substituted C 2 -C 9 heteroaryl 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.
279 . The compound of claim 278 , or a pharmaceutically acceptable salt thereof, wherein the cross-linking group forms a covalent bond with a cysteine residue of the KRAS protein.
280 . The compound of claim 278 , or a pharmaceutically acceptable salt thereof, wherein the cyclophilin protein is cyclophilin A.
281 . The compound of claim 278 , or a pharmaceutically acceptable salt thereof, wherein the KRAS protein is a KRAS variant protein having at least 80% sequence identity with respect to wild-type KRAS protein.
282 . The compound of claim 281 , or a pharmaceutically acceptable salt thereof, wherein the KRAS variant protein has at least 99% sequence identity with respect to wild-type KRAS protein.
283 . A method of covalently modulating a KRAS protein, the method comprising contacting the KRAS protein with a presenter protein/compound complex, wherein
the presenter protein is a member of the cyclophilin family, and
the compound comprises
a presenter protein binding moiety that binds to the cyclophilin protein,
and a cross-linking group that reacts with an amino acid of the KRAS protein to form a covalent bond between the compound and the KRAS protein, wherein the cross-linking group has a structure of
or
the cross-linking group has a structure of formula Ij or Ik:
wherein the wavy line illustrates the point of attachment of the cross-linking group to the remainder of the compound;
X E is absent;
R M is hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl, optionally substituted C 2 -C 6 alkynyl, 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 heteroaryl, optionally substituted C 2 -C 9 heteroaryl 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 N , R O , and R P are independently hydrogen, hydroxyl, 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 heteroaryl, optionally substituted C 2 -C 9 heteroaryl 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.
284 . The method of claim 283 , wherein the cross-linking group forms a covalent bond with a cysteine residue of the KRAS protein.
285 . The method of claim 283 , wherein the cyclophilin protein is cyclophilin A.
286 . The method of claim 283 , wherein the KRAS protein is a KRAS variant protein having at least 80% sequence identity with respect to wild-type KRAS protein.
287 . The method of claim 286 , wherein the KRAS variant protein has at least 99% sequence identity with respect to wild-type KRAS protein.
288 . A method of identifying a compound capable of covalently binding to a KRAS protein in the presence of a presenter protein, the method comprising:
(a) providing a sample comprising (i) a compound comprising a presenter protein binding moiety and a cross-linking group; (ii) a KRAS protein; and (iii) a presenter protein, and
(b) determining if the compound and the KRAS protein form a covalent bond via the cross-linking group of the compound in the sample,
wherein a compound is identified as covalently binding to a KRAS protein in the presence of a presenter protein if the compound and the KRAS protein react in the sample, and
the presenter protein is a member of the cyclophilin family.
289 . The method of claim 288 , wherein the cyclophilin protein is cyclophilin A.
290 . The method of claim 288 , wherein the KRAS protein is a KRAS variant protein having at least 80% sequence identity with respect to wild-type KRAS protein.
291 . The method of claim 290 , wherein the KRAS variant protein has at least 99% sequence identity with respect to wild-type KRAS protein.