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...

During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns (4,5)P 2 ] in the plasma membrane. Using fluorescently tagged highaffinity probes specific for PtdIns(4,5)P 2 , we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P 2 , stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P 2 is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked selfreinforcing mechanism generates a spatially enriched pool of PtdIns (4,5)P 2 necessary for the membrane anchoring and function of a signaling complex.α-factor | phosphoinositides | scaffold protein | yeast mating response E ukaryotic cells respond to extracellular stimuli and spatial cues via signaling responses that can dramatically alter cell polarity. Ample evidence from diverse cell types indicates that membrane phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] (1), are key regulators of processes, like cytoskeletal remodeling (2) and vesicle-mediated trafficking (3, 4), which are required for polarized growth, cell morphogenesis, and cell division (5). In both mammalian leukocytes and migratory Dictyostelium discoideum amoebae, exposure to chemoattractant results in spatial restriction of PtdIns(3,4,5)P 3 , as well as of the enzymes that catalyze its synthesis (PtdIns 3-kinase) and breakdown [phosphatidylinositol 3-phosphatase and tensin homolog (PTEN)] (6). Likewise, in mammalian kidney cells, localized action of the PtdIns(3,4,5)P 3 phosphatase PTEN plays a key role in determining epithelial cell polarity by defining the content of this lipid in the apical and basolateral membranes (7), thereby affecting the distribution of small GTPases that dictate cell shape (8). Membrane phosphoinositides were also shown to play an important role in driving anchor cell invasion during Caenorhabditis elegans development (9). These examples all indicate that membrane subdomains enriched for specific phosphoinositides are crucial for attracting signaling components specific for establishment and/or maintenance of cell polarity.Likewise, in Saccharomyces cerevisiae, phosphoinositides in the plasma membrane have been implicated in the recruitment of proteins involved in polarized...

Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered G␤␥ freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic ␣-helix (PM motif) are not sufficient for Ste5 action. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388-518) that is essential for its membrane recruitment and function. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Ste5(R407S K411S) is expressed normally, retains G␤␥ and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. R407S K411S loss-of-function mutations abrogate the constitutive activity of gain-of-function Ste5 alleles, including one (P44L) that increases membrane affinity of the PM motif. Thus, the PH domain is essential for stable membrane recruitment of Ste5, and this association is critical for initiation of downstream signaling because it allows Ste5-bound Ste11 (MAPKKK) to be activated by membrane-bound Ste20 (MAPKKKK).[Keywords: Pheromone response; plasma membrane; mutants; yeast; Saccharomyces cerevisiae; Ste20] Supplemental material is available at http://www.genesdev.org.

Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G 1 phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCF Cdc4 ubiquitin ligase and requires phosphorylation by the G 1 cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G 1 Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.

Fungi are nonmotile organisms that obtain carbon from compounds in their immediate surroundings. Confronted with nutrient limitation, the yeast Saccharomyces cerevisiae undergoes a dimorphic transition, switching from spherical cells to filaments of adherent, elongated cells that can invade the substratum. A complex web of sensing mechanisms and cooperation among signaling networks (including a mitogen-activated protein kinase cascade, cyclic adenosine monophosphate–dependent protein kinase, and 5′–adenosine monophosphate–activated protein kinase) elicits the appropriate changes in physiology, cell cycle progression, cell polarity, and gene expression to achieve this differentiation. Highly related signaling processes control filamentation and virulence of many human fungal pathogens.

Background: Ras is a nucleotide-dependent switch that converts from an inactive GDP-bound state to an active GTP-bound state when activated by guanine nucleotide exchange factors, such as SOS. Active RasGTP then binds to and activates downstream signaling effectors. Ras is the most frequently mutated oncogene and hyperactive mutant Ras constitutively signals to effectors to promote cell survival, proliferation and metastasis. Thus, Ras oncoprotein has been considered by the cancer community to be one of the most important oncology drug targets. Despite the enormous interest and extensive exploratory efforts in industry and academia, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. We describe in this abstract the identification and characterization of small-molecule inhibitors of the Ras oncoprotein. Materials and Methods: To explore a new means of directly targeting Ras, we used a fragment-based lead discovery approach via an NMR-based screen. Hits from the fragment screen were characterized for their interactions with Ras by NMR and X-ray crystallography and for their effects on Ras activation and signaling in reconstituted biochemical assays in vitro and in cellular assays in vivo. Results: From the fragment-based screen, we identified a group of small molecules that each bind to a common site adjacent to the switch I/II regions in the Ras protein. X-ray crystallography studies of three compound-Ras complexes indicate that the binding site can be expanded upon ligand binding. Nucleotide exchange factors, notably SOS, are required to convert inactive RasGDP to active RasGTP. We determined that the compound-binding site is located at the interface of Ras and SOS. A subset of our Ras-binding molecules indeed inhibited SOS-mediated nucleotide exchange. Further mechanistic studies revealed that through steric hindrance the compounds block the formation of the Ras-SOS complex, a key intermediate of the exchange reaction. At the cellular level, our compounds inhibit the formation of active RasGTP and prevent Ras signaling to downstream effectors. To define the potential clinic utility of these compounds, we performed biological characterization of Ras-driven tumors and identified a subset of Ras mutant tumors that depend on nucleotide exchange factors for the activation of Ras, suggesting a specific profile for the use of exchange inhibitors. Conclusions: We conclude that the compounds act as competitive inhibitors of nucleotide exchange to prevent the activation of Ras. The discovery of a binding pocket on Ras with functional significance represents a breakthrough finding that will offer a new direction for therapeutic intervention of the Ras oncoprotein. Our findings provide new opportunities to target the “undruggable” Ras oncoprotein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4759. doi:1538-7445.AM2012-4759

The KRASG12C mutation is found in 11% of non-small cell lung cancers, 4% of colorectal cancers, and 2% of pancreatic cancers in the U.S., and drives these cancers by shifting the cellular equilibrium of KRAS towards the GTP-bound, active state, KRASG12C(ON). The resulting increased levels of KRASG12C(ON) in turn increase signaling output to initiate and support the oncogenic state. In recent years, a class of KRASG12C(OFF) inhibitors has transformed the treatment landscape for patients with cancers bearing KRASG12C. These inhibitors work via sequestration of the GDP-bound, inactive state, KRASG12C(OFF), starving cancer cells of their oncogenic driver, KRASG12C(ON). Recent reports on the nature of resistance to KRASG12C(OFF) inhibitors suggest this class of drugs can be overcome through reactivation of KRASG12C to the ON form. Direct inhibition of KRASG12C(ON) with a first in class, potent, orally bioavailable, selective, tri-complex inhibitor RMC-6291, represents a more robust approach and presents the possibility that RMC-6291 will be a ‘best-in-class’ inhibitor of tumors harboring KRASG12C. RMC-6291 is a potent covalent inhibitor of KRASG12C(ON) that forms a tri-complex within tumor cells between KRASG12C(ON) and cyclophilin A (CypA), a highly abundant immunophilin. The assembled tri-complex prevents KRASG12C(ON) from signaling via steric blockade of RAS effector binding. In cells, kinetic analyses demonstrate near-immediate disruption of RAS effector binding and extinction of KRASG12C(ON) signaling. Oral administration of RMC-6291 produces deep and durable suppression of RAS pathway activity in KRASG12C tumor models and drives profound tumor regressions in vivo at well-tolerated doses. In a mouse clinical trial consisting of multiple patient- and cell line-derived xenograft models of KRASG12C NSCLC, RMC-6291 outperformed adagrasib, a KRASG12C(OFF) inhibitor, by increasing the number of responses, the depth of tumor regressions, and the durability of responses. Combination treatment with RMC-6291 and SHP2 or SOS1 inhibitors was well tolerated in preclinical models and further increased anti-tumor activity, likely by preventing reactivation of wild-type RAS proteins that cooperate with KRASG12C to fuel cancer growth. RMC-6291 also combined well with immune checkpoint inhibitors, sensitizing KRASG12C-bearing cancer models to anti-tumor immunity. RMC-6291 is a next-generation, mutant-selective inhibitor of KRASG12C(ON) that overcomes limitations of first-generation KRASG12C(OFF) inhibitors in preclinical models by directly targeting the active form of this important oncogenic driver. Citation Format: Robert J. Nichols, Y.C. Yang, Jim Cregg, Chris J. Schulze, Zhican Wang, Richa Dua, Jingjing Jiang, Lindsay S. Garrenton, Nicole Nasholm, Alun Bermingham, John E. Knox, Kyle Seamon, Michael Longhi, Kang-Jye Chou, Shaoling Li, David P. Wildes, Mallika Singh, Elena S. Koltun, Adrian L. Gill, Jacqueline A.M. Smith. RMC-6291, a next-generation tri-complex KRASG12C(ON) inhibitor, outperforms KRASG12C(OFF) inhibitors in preclinical models of KRASG12C cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3595.

The KRASG12C mutation is found in 11% of non-small cell lung cancers and 4% of colorectal cancers. Recently, a class of KRASG12C(OFF) inhibitors has shown promising activity in patients whose cancers bear KRASG12C. These data validate KRASG12C as an oncogenic driver, as well the mechanism of action of the KRASG12C(OFF) inhibitor class – sequestration of inactive, GDP-bound KRASG12C(OFF) proteins. Previous work has demonstrated this mechanism of action is vulnerable to adaptive tumor cell responses that activate KRASG12C by increasing upstream signaling and driving the cellular pool of KRASG12C towards the RAS(ON) state. These escape mechanisms, in which KRASG12C can be reactivated in the presence of a KRASG12C(OFF) inhibitor, highlight the potential for an inhibitor that directly targets and disables the KRASG12C(ON) form. Using structure-based drug design, we have discovered RM-032, a potent covalent inhibitor of KRASG12C(ON) that forms a tri-complex between KRASG12C(ON) and cyclophilin A (CypA), a highly abundant immunophilin. The assembled tri-complex prevents KRASG12C(ON) from signaling via steric blockade of RAS effector binding. In cells, kinetic analyses demonstrate near-immediate disruption of RAS effector binding and extinction of KRASG12C(ON) signaling. RM-032 is dual selective for KRASG12C(ON) and NRASG12C(ON). In vitro, RM-032 drives increased durability of inhibition of both RAS pathway signaling and cell proliferation in KRASG12C tumor cells compared with KRASG12C(OFF) inhibition. RM-032 displays attractive drug-like properties including cross-species oral bioavailability, and is predicted to achieve adequate exposures following oral dosing in humans. Oral administration of RM-032 produces deep and durable suppression of RAS pathway activity in KRASG12C tumor models and drives profound tumor regressions in vivo at well-tolerated doses. Across multiple tumor xenograft models, advanced KRASG12C(ON) inhibitors, including RM-032, appear to outperform KRASG12C(OFF) inhibitors. RM-032 permits a broad array of combination opportunities for treating KRASG12C mutant cancer types where single agent KRASG12C(ON) inhibition may be insufficient, for example with agents targeting nodes both upstream (e.g., SHP2 and SOS1) and downstream (e.g., MEK and ERK) of RAS, as well as parallel pathways (e.g., mTORC1). RM-032 is a next generation mutant-selective inhibitor of KRASG12C(ON) that may overcome liabilities of first-generation KRASG12C(OFF) inhibitors and provide additional benefit to patients by directly targeting the active form of this important oncogenic driver mutation. Citation Format: Robert J. Nichols, Jim Cregg, Christopher J. Schulze, Zhican Wang, Kevin Yang, Jingjing Jiang, Daniel M. Whalen, Rich Hansen, Lindsay S. Garrenton, Alun Bermingham, John E. Knox, Tiffany Choy, Denise Reyes, Mayra Rios, Kyle Seamon, Michael Longhi, Kang-Jye Chou, Shaoling Li, David P. Wildes, Mallika Singh, Elena S. Koltun, Adrian L. Gill, Jacqueline A. M. Smith. A next generation tri-complex KRASG12C(ON) inhibitor directly targets the active, GTP-bound state of mutant RAS and may overcome resistance to KRASG12C(OFF) inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1261.