Mae Saldajeno-Concar

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product–inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRAS G12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRAS G12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRAS G12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).

Abstract RAS proteins are small GTPases that drive cell proliferation and survival when bound to GTP. Mutant RAS proteins are found in approximately one-third of human cancers, and exist predominantly in the GTP-bound state, leading to excessive downstream signaling via interaction with effectors such as RAF. A KRAS mutation in which glycine-12 is mutated to cysteine (KRASG12C) is found in 11-12% of non-small cell lung cancers. Recently, multiple potent, covalent inhibitors of KRASG12C have been reported that target the inactive, GDP-bound form of KRASG12C, and thus rely on the residual intrinsic hydrolysis of GTP to cycle KRASG12C proteins through the inactive, GDP-bound state. This mechanism is vulnerable to adaptive responses in cancer cells that can activate RAS by increasing upstream signaling and further increase the relative abundance of KRASG12C(GTP) over KRASG12C(GDP). An inhibitor that directly targets the active, GTP-bound form of KRASG12C would overcome this limitation. Drawing inspiration from natural products like cyclosporine and rapamycin, we have developed tri-complex inhibitors of KRASG12C(GTP) that promote a ternary complex between KRASG12C and the abundant immunophilin cyclophilin A (CypA). These sanglifehrin-inspired inhibitors exploit significant non-covalent interactions in the SWI/SWII region of KRAS combined with an electrophilic cysteine-targeted warhead to potently and irreversibly inhibit KRASG12C(GTP). The inhibitors selectively drive formation of KRASG12C-inhibitor-CypA ternary complexes that are sterically prevented from interacting with the RAS Binding Domain (RBD) of BRAF in biochemical studies. In cellular models, KRASG12C(GTP) inhibitors attenuate both RAS-MAPK signaling and cell viability in cancer cell lines bearing KRASG12C mutations, but not other mutations in RAS or other pathway oncoproteins. In vivo administration of a KRASG12C(GTP) inhibitor drives dose-dependent tumor regressions in the NCI-H358 KRASG12C NSCLC xenograft mouse model and is well-tolerated. Consistent with targeting the KRAS(GTP) state, inhibitory activity in vitro is unaffected by RTK activation, whereas the activity of first generation KRASG12C(GDP) inhibitors is significantly attenuated. In addition, proliferation of NCI-H358 and MIA PaCa-2 cells in vitro is suppressed for a significantly longer duration with KRASG12C(GTP) inhibitor treatment compared to KRASG12C(GDP) inhibitors. The combination of sub-maximal concentrations of a MEK inhibitor and a KRASG12C(GTP) inhibitor drove pronounced cell death. In contrast, the MEK and KRASG12C(GDP) inhibitor combination evoked a modest enhancement of the antiproliferative effects and does not cause cell death. Tri-complex inhibitors that target the active, GTP-bound form of KRAS thus represent a second generation of KRASG12C inhibitor. Chemical modulation of the non-covalent and covalent interactions of our tri-complex inhibitors provides an exciting opportunity to step beyond KRASG12C to target the GTP-bound state of additional RAS variants, and we demonstrate in vitro covalent inhibition of KRASG13C. By directly targeting active RAS-GTP, tri-complex inhibitors have the potential to overcome adaptive resistance mechanisms that emerge following inhibition of aberrant RAS-MAPK pathway activation. Citation Format: Christopher J Schulze, Alun Bermingham, Tiffany J Choy, James J Cregg, Gert Kiss, Abby Marquez, Denise Reyes, Mae Saldajeno-Concar, Caroline E Weller, Daniel M Whalen, Yu C Yang, Elena S Koltun, Robert J Nichols, Mallika Singh, David Wildes, Adrian L Gill, Richard L Hansen, Steve Kelsey, Mark A Goldsmith, Jacqueline A.M. Smith. Tri-complex inhibitors of the oncogenic, GTP-bound form of KRASG12C overcome RTK-mediated escape mechanisms and drive tumor regressions in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR10. doi:10.1158/1535-7163.TARG-19-PR10

Abstract Genetic and pharmacologic evidence has shown that SHP2, a non-receptor protein tyrosine phosphatase (PTP) and scaffold protein encoded by the PTPN11 gene, is a convergent signal transduction node that integrates growth factor signals from multiple receptors to promote activation of RAS and its downstream effectors. Guided by structural insights from X-ray data, we describe a strategy aimed at the identification of a highly potent and selective allosteric SHP2 inhibitor series. Our efforts led to the discovery of RMC-4550, a potent and selective SHP2 inhibitor which exhibits a high quality, drug-like preclinical profile. RMC-4550 inhibits purified, activated full length human SHP2 with an IC50 of 1.55 nM, and has cellular IC50 of 39 nM in PC9 cells with a pERK readout. RMC-4550 has no detectable inhibitory activity up to 10 µM against the catalytic domain of SHP2, a panel of 14 additional protein phosphatases, and a panel of 468 protein kinases. RMC-4550 exhibits low to moderate cross species in vitro intrinsic clearance (3.6-24 µL/min/million cells) in hepatocytes, a high passive permeability (458 nm/s) and efflux ratio of 1. The ADME properties translate into favorable pharmacokinetic profiles in preclinical species. RMC-4550 has moderate to high bioavailability and has a half-life amenable for once daily oral administration. In the EGFR-driven KYSE-520 human esophageal cancer xenograft model, we observed a dose dependent efficacy consistent with target modulation, assessed by phospho-ERK inhibition in tumors. RMC-4550 is well tolerated at doses that achieved maximal and sustained efficacy in this model. RMC-4550 was synthesized in 5 linear (6 total) steps from the readily accessible or commercially available intermediates. The chemical structure and synthesis of RMC-4550, along with detailed structure-activity relationships will be presented. In summary, RMC-4550 exemplifies a novel class of potent allosteric inhibitors of SHP2 with an excellent drug like property profile. Citation Format: Elena S. Koltun, Naing Aay, Andreas Buckl, Ashutosh S. Jogalekar, Gert Kiss, Abby Marquez, Kevin T. Mellem, Kasia Mordec, Mae Saldajeno-Concar, Chris M. Semko, Nidhi Tibrewal, Christos Tzitzilonis, Walter Won, Jacqueline A. Smith, Susan E. Wilson, Robert J. Nichols, Zhengping Wang, David Wilds, Mallika Singh, Adrian L. Gill. RMC-4550, an allosteric inhibitor of SHP2: Synthesis, structure, and anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4878.

Abstract The guanine nucleotide exchange factor (GEF) protein SOS1 activates RAS by promoting its conversion from the GDP-bound RAS(OFF) state to the GTP-bound RAS(ON) state. SOS1 catalyzes or accelerates this nucleotide exchange reaction in response to upstream signals conveyed by a range of growth factor receptors. It acts by promoting the release of tightly bound GDP and thereby facilitating the binding of GTP, which is present at higher intracellular concentrations than GDP, to generate RAS(ON). SOS1 itself is activated by RAS through the binding of RAS(ON) to an allosteric site on the SOS1 protein, which leads to a positive feedback loop between SOS1 and RAS that increases the amplitude and duration of RAS signaling. As a result, there is considerable potential for amplification of RAS signals by SOS1. For this reason, and because SOS1 is a convergent node downstream of RTK signaling, SOS1 represents an attractive therapeutic target in RAS driven cancers. We have developed a collection of novel, proprietary small molecule inhibitors of SOS1. Here we describe the preclinical profile of a potent, selective, and orally bioavailable in vivo tool compound, RM-023, which disrupts the critical interaction between KRAS and SOS1. By preventing formation of the KRAS-SOS1 complex, these inhibitors block reloading of KRAS with GTP, and thereby inhibit RAS pathway signaling and RAS-driven cancer cell growth in vitro. Oral administration of RM-023 produced a dose-dependent suppression of tumor RAS pathway activation in vivo and inhibited tumor growth in preclinical xenograft models of diverse RAS-addicted cancers at well-tolerated doses. Enhanced anti-tumor activity in RAS-addicted cancer models was observed when RM-023 was administered in combination with other RAS pathway inhibitors. We believe SOS1 inhibition represents an attractive companion for combination with RAS-directed inhibitors and may have unique utility in select RAS-addicted tumor types. Citation Format: Andreas Buckl, Elsa Quintana, Grace J. Lee, Nataliya Shifrin, Mengqi Zhong, Lindsay S. Garrenton, David C. Montgomery, Carlos Stahlhut, Frances Zhao, Dan M. Whalen, Severin K. Thompson, Arlyn Tambo-ong, Micah Gliedt, John E. Knox, James J. Cregg, Naing Aay, Jong Choi, Bao Nguyen, Atti Tripathi, Ruiping Zhao, Mae Saldajeno-Concar, Abby Marquez, Daphne Hsieh, Laura L. McDowell, Elena S. Koltun, Alun Bermingham, David Wildes, Mallika Singh, Zhengping Wang, Richard Hansen, Jan A. Smith, Adrian L. Gill. Discovery of a potent, selective, and orally bioavailable SOS1 inhibitor, RMC-023, an in vivo tool compound that blocks RAS activation via disruption of the RAS-SOS1 interaction [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 1273.