Proximity-triggered, mutant-specific modification of K-Ras(G12C) with local polypharmaceutical release

The K-Ras(G12C) mutation (glycine 12 to cysteine) is a key oncogenic driver in tumorigenesis identified in around 13% of non-small cell lung cancers and 3% of colorectal cancers and is the target of the FDA-approved covalent inhibitors, sotorasib, and adagrasib. Unfortunately, the efficacy of these therapies is impeded by limited progression-free survival rates, partial response, and the development of resistance. While combination therapies involving both sotorasib and adagrasib are being explored to enhance treatment outcomes, the inherent challenge of heightened toxicity when employing multiple agents in the clinical setting persists. We aim to address this challenge through a novel targeted approach for K-Ras(G12C) mutation-driven cancers, designed to intrinsically act on multiple pathways concurrently through a single but multi-functional prodrug, conceptually a targeted unimolecular combination therapy. These prodrug molecules are derivatives of cytotoxic natural products that transfer molecular fragments to K-Ras(G12C). This phenomenon, known as group transfer, results in the covalent labeling of cysteine 12 in K-Ras(G12C). We posit that the group transfer of E3 ligase recruiting fragments could engender the targeted degradation of K-Ras(G12C). The therapeutic impact is enhanced by liberating the cytotoxic jerantinine alkaloids inherent in our prodrugs, which in themselves target multiple pathways (polypharmaceuticals). The ultimate goal is to demonstrate selective group transfer within the complex cellular milieu and the synergistic effects of targeted protein degradation coupled with the release of cytotoxic polypharmaceuticals. Aim 1: Determine the binding location of jerantinine alkaloids to K-Ras(G12C) and the selectivity of jerantinine alkaloid-mediated group transfer to K-Ras(G12C). Following the synthesis of a library of hybrid ligands, designed to interrogate the effect of electrophile type and steric bulk on group transfer, we will test these compounds for covalent labeling of K-Ras(G12C), and selectivity for K-Ras(G12C) using Whole Protein Mass Spectrometry (MS). LC/MS-MS-based assays will be used to evaluate covalent labeling of K-Ras(G12C) in cells. Once compounds demonstrating covalent labeling of K-Ras in cells are identified, we will employ activity-based protein profiling (ABPP) to evaluate their selectivity towards a specific subset of cysteines within the proteome. Aim 2: Determine if jerantinine alkaloid mediated group transfer leads to K-Ras degradation and identify any synergistic effects of targeted degradation and concomitant release of cytotoxic natural product. Hybrid molecules containing E3 ligase recruiting functionality will be synthesized. Covalent modification of K- Ras(G12C) by these molecules and selectivity will be confirmed using the methods outlined in Aim 1. K- Ras(G12C) degradation will be evaluated using western blot, and the efficacy and synergistic effects of degradation and polypharmaceutical release will be determined by screening against suitable 2D cell lines. Serial passage experiments will indicate if these molecules can evade the development of resistance.