Overcoming EGFR Resistance by Monovalent and Bident Inhibitors Targeting Cys775

Abstract Covalent targeting of EGFR cysteine 797 by osimertinib is one of the most successful breakthroughs in targeted therapy, fundamentally transforming the treatment landscape for non-small-cell lung cancer (NSCLC) patients. However, resistance driven by the mutation of C797 remains a major clinical challenge. Developing novel covalent strategies beyond C797 targeting presents a compelling opportunity for next-generation EGFR inhibitors. We first demonstrated that cysteine 775, located deep within the ATP-binding pocket, is accessible by a rationally designed covalent molecule ZNL-3, which as the first-in-class covalent cysteine 775 inhibitor exhibited strong efficacy in osimertinib-resistant mouse models. To further enhance resilience to resistance-causing mutations, we developed a dual-warhead, bident compound YNW-1 which covalently targets both cysteines 775 and 797 simultaneously. YNW-1 is the first intramolecular lock to exhibit balanced reactive efficiency on both cysteines, rendering single-site mutations ineffective at conferring resistance. This study establishes the therapeutic potential of an EGFR covalent inhibitor through unprecedented targeting of cysteine 775. It also provides the first evidence that dual-cysteine engagement offers superior efficacy to conventional covalent inhibitors by delaying the emergence of resistance. Further optimization for clinical translation is currently ongoing in our laboratory.