Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity

Abstract

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.small G protein | guanine nucleotide exchange | nuclear magnetic resonance | crystal structure | small-molecule inhibitors R as is a small GTP-binding protein that functions as a nucleotide-dependent switch for central growth signaling pathways (1, 2). In response to extracellular signals, Ras is converted from a GDP-bound (Ras GDP ) to a GTP-bound (Ras GTP ) state, as catalyzed by guanine nucleotide exchange factors (GEFs), notably the SOS1 protein. Active Ras GTP mediates its diverse growth-stimulating functions through its direct interactions with effectors including Raf, PI3K, and Ral guanine nucleotide dissociation stimulator. The intrinsic GTPase activity of Ras then hydrolyzes GTP to GDP to terminate Ras signaling. The Ras GTPase activity can be further accelerated by its interactions with GTPase-activating proteins (GAPs), including the neurofibromin 1 tumor suppressor (2).Ras, a human oncogene identified and characterized over 30 y ago, is mutated in more than 20% of human cancers. Among the three Ras isoforms (K, N, and H), KRas is most frequently mutated (2). Mutant Ras has a reduced GTPase activity, which prolongs its activated conformation, thereby promoting Rasdependent signaling and cancer cell survival or growth (1, 2).Mutations of Ras in cancer are associated with poor prognosis (2). Inactivation of oncogenic Ras in mice results in tumor shrinkage. Thus, Ras is widely considered an oncology target of exceptional importance. However, development of small-molecule inhibitors against Ras has thus far proven unsuccessful. Given the picomolar affinity between guanine nucleotides and Ras and the high cytosolic concentration of guanine nucleotides, it is very challenging to develop a conventional inhibitor competitive against nucleotide binding (1, 2). Outside of the nucleotide-binding pocket, the Ras protein does not contain obvious cavities for small-molecule binding. A number of small molecules have been reported to bind to Ras (3-7), but their mechanisms of action and the structural basis to achieve Ras inhibition remain elusive.Fra...

Small Molecule Binding Sites on the Ras:SOS Complex Can Be Exploited for Inhibition of Ras Activation

“…The binding site identified at the interface of HRas and SOS ('site B') corresponds to a site previously described in studies of uncomplexed Ras protein 5,6 ('site 2'). However, while Ras ligands 7 and 8, which were identified in these studies, inhibit SOS-mediated nucleotide exchange by preventing the binding of SOS to Ras, the fragments 5 and 6…”

“…5,6 Comparison of published crystal structures with that of the HRas:SOS:5 complex shows that site B corresponds with the relatively shallow 'binding site 2' of the published fragments 7 and 8 on KRas (Fig. 3d).…”

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

“…Recent reports revealed that ligand binding at a pocket between the core β-sheet and helix 2 of K-Ras stabilizes alternate side-chain conformations at or around switch 2 and thereby affects exchange factor binding (15,16). For instance, the side chains of both Y64 and Y71 were displaced in these ligandbound structures relative to an SOS-bound H-Ras structure (15,16).…”

“…Although the potential therapeutic value and mechanism of action of these compounds are still under investigation, it is clear that they do not directly bind to Ras. Recent efforts by us (13) and others (14)(15)(16) toward direct inhibition of Ras have yielded promising initial results. For instance, using fragment screening, crystallography, and other methods, two groups reported ligands that directly bind Ras and inhibit GEF-dependent nucleotide exchange (15,16).…”

“…Recent efforts by us (13) and others (14)(15)(16) toward direct inhibition of Ras have yielded promising initial results. For instance, using fragment screening, crystallography, and other methods, two groups reported ligands that directly bind Ras and inhibit GEF-dependent nucleotide exchange (15,16). However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active, GTP-loaded mutant Ras.…”