The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be “undruggable,” between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.
Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRAS G12C inhibitors. To date, KRAS G12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRAS G12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRAS G12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.
Natural products have proven to be a rich source of molecular architectures for drugs. Here, an integrated approach to natural product screening is proposed, which uncovered eight new natural product scaffolds for KRAS—the most frequently mutated oncogenic driver in human cancers, which has remained thus far undrugged. The approach combines aspects of virtual screening, fragment‐based screening, structure‐activity relationships (SAR) by NMR, and structure‐based drug discovery to overcome the limitations in traditional natural product approaches. By using our approach, a new “snugness of fit” scoring function and the first crystal‐soaking system of the active form of KRASG12D, the protein–ligand X‐ray structures of a tricyclic indolopyrrole fungal alkaloid and an indoloisoquinolinone have been successfully elucidated. The natural product KRAS hits discovered provide fruitful ground for the optimization of highly potent natural‐product‐based inhibitors of the active form of oncogenic RAS. This integrated approach for screening natural products also holds promise for other “undruggable” targets.
KRASG12C mutations are predominantly found in non-small cell lung cancer (NSCLC, 13%), in colorectal cancer (CRC, 3%), and with a lower prevalence in pancreatic ductal adenocarcinoma (PDAC, 1%). The amino acid exchange at position 12 from glycine to cysteine renders RAS insensitive to GAP-catalyzed hydrolysis but not to intrinsic hydrolysis and consequently, KRASG12C is still dependent on GEF stimulation to achieve full activation. The active GTP-loaded form of KRASG12C is favored and leads to activation of downstream signaling and proliferation. A number of recent publications has shown that targeting this mutant form of KRAS, using several covalent KRASG12C inhibitors binding to the inactive GDP-KRASG12C form, leads to anti-proliferative effects and induction of apoptosis in KRASG12C mutant cancer cell lines, CDX and PDX models. Early clinical data for AMG 510 and MRTX849 revealed a response rate of 35-45% in NSCLC and of 7-17% in CRC patients. Here, we show that BI 1823911 has potent anti-proliferative activity in a panel of KRASG12C mutant cancer cell lines with higher or similar potency compared to these two most advanced compounds in clinical development. In a panel of KRASG12C NSCLC cell lines, treatment with BI 1823911 results in downregulation of MAPK pathway-responsive genes, such as DUSP6 and CCND1, and the extent of pathway modulation correlates with sensitivity. Likewise, we observe strong and sustained inactivation of the MAPK pathway at the protein level using p-ERK as a pharmacodynamic (PD) biomarker. A MIA PaCa-2 cell line-derived pancreatic cancer xenograft model was selected for extensive PK/PD/efficacy analyses in vivo. Briefly, BI 1823911 tested at 60 mg/kg showed similar anti-tumor activity compared to both competitor compounds dosed at clinically relevant exposures. Results of the ongoing in-depth PK/PD/efficacy analysis will be shared. Furthermore, BI 1823911 was tested with a daily oral dose of 60 mg/kg in a panel of NSCLC or CRC CDX or PDX mouse models and showed comparable efficacy to AMG 510 and MRTX849, respectively. Preclinical and clinical data suggest that monotherapy with a KRASG12C inhibitor will not be sufficient to achieve durable response. Combination therapy of a KRASG12C inhibitor may therefore lead to enhanced anti-tumor efficacy and may address adaptive resistance mechanisms. Therefore, we selected a panel of KRASG12C mutant cancer cell lines and tested a large set of compounds in combination with BI 1823911 to identify synergistic anti-proliferative activity. Among other MAPK and PI3K pathway inhibitors, a SOS1::KRAS inhibitor was confirmed as promising combination partner. We will show results from in vitro and in vivo combination studies in NSCLC and CRC tumor models that show deep and durable responses upon combination of BI 1823911 with SOS1::KRAS inhibitor BI 1701963 providing a strong rationale for clinical investigation of this combination. Citation Format: Fabio Savarese, Andreas Gollner, Dorothea Rudolph, Jesse Lipp, Johannes Popow, Marco H. Hofmann, Heribert Arnhof, Jörg Rinnenthal, Francesca Trapani, Michael Gmachl, Daniel Gerlach, Joachim Broeker, Peter Ettmayer, Andreas Mantoulidis, Jason Phan, Christian A. Smethurst, Matthias Treu, Alex G. Waterson, Hengyu Lu, Annette Machado, Joseph Daniele, Stephan W. Fesik, Christopher P. Vellano, Timothy P. Heffernan, Joseph R. Marszalek, Darryl B. McConnell, Mark Petronczki, Norbert Kraut, Irene C. Waizenegger. In vitro and in vivo characterization of BI 1823911 - a novel KRASG12C selective small molecule inhibitor [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 1271.
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