Background <p>The introduction of EGFR inhibitors has revolutionized the management of NSCLC, leading to a significant extension of survival in the target patient population. Unfortunately, acquired resistance appears to be unavoidable, which restricts the use of EGFR-TKIs in the clinic. EGFR^C797S represents a novel resistant mutation after treatment with the latest generation of EGFR inhibitor Osimertinib. Consequently, developing next-generation EGFR-TKIs targeting such resistant mutations is strongly demanded.</p> Methods <p>Osimertinib-resistant NSCLC cell lines were constructed by transfecting EGFR^{del19/T790M/C797S} or EGFR^{L858R/T790M/C797S} lentiviral plasmids into NSCLC PC-9 cells, followed by puromycin screening. The drug screening was conducted in a drug repurposing compound library provided by TargetMol. The hit compound (CZC54252) was validated by kinase inhibition, western blot, and molecular docking assays. Its role in overcoming Osimertinib resistance was further evaluated by cellular viability, proliferation, and apoptosis tests. Finally, animal studies were conducted in xenograft models to verify the anti-NSCLC effects of CZC54252 in vivo.</p> Results <p>The cell models resistant to Osimertinib have been successfully established, and CZC54252 was screened as a hit compound that could overcome Osimertinib resistance, with IC₅₀ values of 0.24 ± 0.06 µM and 0.27 ± 0.09 µM on PC-9^{del19/T790M/C797S} and PC-9^{L858R/T790M/C797S} cells, respectively, which were significantly lower than those of Osimertinib (2.1 ± 0.32 µM and 2.8 ± 0.37 µM, respectively). CZC54252 displayed potent inhibitory activity on EGFR^{del19/T790M/C797S} (IC₅₀: 1.66 nM) and EGFR^{L858R/T790M/C797S} (IC₅₀: 2.92 nM), and significantly repressed EGFR phosphorylation in western blot assay. Additionally, the pharmacodynamics studies demonstrated that CZC54252 moderately suppressed cell proliferation and induced cell apoptosis both in vitro and in vivo. Notably, not all drug candidates targeting the same molecular pathways as CZC54252 showed activity in the screening, indicating that CZC54252 may overcome Osimertinib resistance by directly targeting EGFR^C797S triple mutations rather than its original targets.</p> Conclusion <p>These data indicate that CZC54252 represents a promising lead compound for overcoming Osimertinib resistance by targeting EGFR^C797S, which provides a chemical basis for the development of novel fourth-generation EGFR inhibitors.</p> Graphical Abstract <p></p>