Abstract B037: Hit-finding by Cysteine-Scanning (HCS): A method for finding druggable pockets
Authors: van Tienen Laurens Moore , Bayoumi Shadwa , Muneeruddin Khaja , Leymarie Nancy , Kornfilt David , Bian Yuemin , Kesar Devishi , Li Ruitong , Atack Thomas , Popa Alexandra-Mariela , Jandova Zuzana , Trollmann Philipp , Bergner Andreas , Rumpel Klaus , Kessler Dirk , Sellers William R. 
Published: May 2023

Large-scale cancer sequencing efforts coupled with deep functional genomic interrogation of cancer models provides us an increasingly detailed view of the critical cancer drivers required for persistent growth and survival of cancer cells. Targeting such drivers has provided significant therapeutic benefit to patients across a wide range of cancers, yet progress has been, in large part, confined to the targeting of catalytic domains in kinases. To expand the reach of therapeutics into new classes of therapeutic targets, we need to identify and validate specific mechanisms and pockets to enable drug discovery. To enable this, we are developing a method called Hit-finding by Cysteine-Scanning (HCS) for identifying druggable pockets in proteins of interest. Here, we seek to couple the ability to create systematic variant libraries across proteins of interest with increasingly diverse covalent small molecule/fragment libraries. Thus, HCS involves screening pooled cysteine (cys)-variants against libraries of covalent compounds. To demonstrate the utility of this approach, we applied HCS to systematically screen 189 KRAS-G12D variants. We successfully purified KRAS cys-variant recombinant proteins in pools of seven or eight achieving coverage of 184/189 variants. KRAS variant pools were then screened against a pilot library of 47 covalent compounds with diverse reactivity detecting reactive variant-ligand pairs by intact LC-MS. This allowed us to quantify the accessibility and reactivity across the entire KRAS-G12D protein. The data confirmed the known SII-pocket in KRAS and revealed potentially novel ligand-bound conformations of oncogenic KRAS mutant proteins. In parallel, the same KRAS cys-variant library was generated as a barcoded library in a mammalian expression system. Upon screening this library in intact cells we observed that Sotorasib functionally inhibit the relevant KRAS SII-pocket cys-variants, enabling enrichment of these variants in a negative selection screen. Importantly, binding of a panel of SII-pocket compounds to variant H95C was found to be independent of the KRAS mutant context. Our results indicate that HCS could be a useful tool for the development of inhibitors for currently undruggable oncogenic KRAS mutants as well as potentially other drug targets.

Citation Format: Laurens Moore van Tienen, Shadwa Bayoumi, Khaja Muneeruddin, Nancy Leymarie, David Kornfilt, Yuemin Bian, Devishi Kesar, Ruitong Li, Thomas Atack, Alexandra-Mariela Popa, Zuzana Jandova, Philipp Trollmann, Andreas Bergner, Klaus Rumpel, Dirk Kessler, William R. Sellers. Hit-finding by Cysteine-Scanning (HCS): A method for finding druggable pockets [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B037.