biostudies-literatureSpencer-Smith RBLRD VANCI NIH HHSNIGMS NIH HHS62-68https://www.ebi.ac.uk/biostudies/studies/S-EPMC5193369biostudies-literatureUnknown13(1)RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the ?4-?6-?5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the ?4-?6-?5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.Nature chemical biologyInhibition of RAS function through targeting an allosteric regulatory site.PMC5193369F31 CA192822R01 CA116708R21 CA201717I01 BX002095R01 GM090324O'Bryan JPCobbert JKoide SZhou YJacobs MTherrien MIkura MLavoie HHerrero-Garcia EDowdell ESantana DOkur MNSha FRajakulendran TGupta AEguchi RRSmith MKoide ASicheri FGajwani PDementieva ISpencer-Smith RHancock JFfalseInhibition of RAS function through targeting an allosteric regulatory site.RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the ?4-?6-?5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the ?4-?6-?5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.2017-01-01T00:00:00Z2017 Jan2021-02-19T19:12:27Z2019-03-27T02:32:44ZS-EPMC51933692782080210.1038/nchembio.2231