Monobodies specific for the dimerization interface of RAS inhibit RAS activation and signaling.

  • Major finding: Monobodies specific for the dimerization interface of RAS inhibit RAS activation and signaling.

  • Concept: The α4–α5 interface mediates the dimerization and nanoclustering of membrane-bound RAS.

  • Impact: Monobody inhibitors are a potential strategy to suppress oncogenic RAS in human tumors.

Activating mutations in the RAS family of small GTPases, which regulate a number of cellular signaling pathways, occur in almost a third of tumors and drive transformation and tumor-promoting signal transduction. Multiple approaches have been employed to identify RAS inhibitors, such as targeting the switch-1 (SW1) and SW2 regions, located on the effector lobe of RAS, to inhibit the conversion of RAS–GDP to RAS–GTP via the nucleotide exchange process and thus ablate RAS activation. To ascertain whether other regions of RAS may be potential targets for RAS inhibition, Spencer-Smith and colleagues generated monobodies, which are synthetic binding proteins that can function as genetically encoded intracellular inhibitors, specific for RAS. The monobody NS1 specifically bound to HRAS with high affinity and KRAS with moderate affinity, but did not bind to NRAS, and inhibited RAS-mediated signaling and transformation in vitro without affecting nucleotide exchange on HRAS. Analysis of the crystal structure of NS1 in complex with HRAS–GDP showed that NS1 interacted with the α4, β6­, and α5 regions of HRAS, which are present on the allosteric lobe distal to SW1/SW2 regions on the effector lobe. Further, analysis of over a hundred HRAS crystal structures confirmed that RAS dimerized in the active conformation via the α4–α5 interface. Consistent with these findings, NS1 inhibited the dimerization and nanoclustering of both HRAS and KRAS and ablated RAS-driven CRAF–BRAF heterodimerization and subsequent RAF kinase activity. Taken together, these results provide additional evidence that the α4–α5 interface mediates RAS dimerization and signaling, identify a targetable site in RAS that may potently inhibit RAS-mediated cellular signaling, and show that monobody inhibitors represent another potential class of RAS inhibitors.

Spencer-Smith R, Koide A, Zhou Y, Eguchi RR, Sha F, Gajwani P, et al. Inhibition of RAS function through targeting an allosteric regulatory site. Nat Chem Biol 2016 Nov 7 [Epub ahead of print].

©2016 American Association for Cancer Research.
2016
American Association for Cancer Research.