Abstract From a screening of 27 106 covalent DNA-encoded compound library, a novel KRAS G12C chemical series has been identified. Original hit series compound displayed low µM covalent binding activity to KRAS G12C under GDP form associated with a k(inact)/Ki of 1,03 M-1.s-1, a stable electrophilic covalent warhead (T1/2 in 5mM GSH > 24h), +12°C stabilization Delta Tm in DSF assay and 3 µM IC50 value in GEF assay, while no detectable covalent binding to KRAS G12C under GTP form and to KRAS WT or KRAS G12D under GDP form. From 3 µM, it also induced significant pERK inhibition in H358 KRAS-G12C but not in A549 KRAS G12S NSCLC cell lines. Unique binding mode to SII pocket was demonstrated by Xray crystallography. Multi-parametric and structural biology guided chemical optimization yielded potent KRAS G12C lead compounds which combine k(inact)/Ki > 500 M-1.s-1, Delta Tm=20°C, 10 nM range pERK IC50 values with correlated direct KRAS G12C selective covalent modification. They also exhibited 100 nM range KRAS G12C allele-specific anti-proliferative activity and triggered significant apoptosis induction. In vivo treatment of mice bearing H358 tumour xenografts through oral route with optimized candidates showed a marked and prolonged inhibition of both pERK and DUSP6 mRNA as well as consistent direct KRAS-G12C covalent modification as evidenced by LC-MS in tumor samples. Altogether, these data demonstrate the power of DNA-encoded library approach to deliver potential drug candidates with selective cysteine-targeted irreversible mechanism of action on challenging oncology targets such as KRAS. Citation Format: Gary McCort, Rosalia Arrebola, Loreley Calvet, Baptiste Ronan, Fabrice Vergne, Francis Duffieux, Alexey Rak, Isabelle Meaux, David Papin, Florence Fassy, Cecile Delorme, Magali Matthieu, Jean-Paul Nicolas, Christophe Marcireau, Valerie Steier, Pierre-Yves Abecassis, Valerie Czepczor, Heather A. Thomson, Christopher D. Hupp, J.P. Guilinger, Ying Zhang, Anthony D. Keefe, John W. Cuozzo, Julie Liu, Laurent Debussche. Discovery of novel potent allele-selective KRAS-G12C covalent inhibitors stemming from DNA-encoded library [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3070.