T6496 targeting EGFR mediated by T790M or C797S mutant: machine learning, virtual screening and bioactivity evaluation study.

Abstract

Acquired resistance to EGFR is a major impediment in lung cancer treatment, highlighting the urgent need to discover novel compounds to overcome EGFR drug resistance. In this study, we utilized in silico methods and bioactivity evaluation for drug discovery to identify novel active anticancer agents targeting EGFR^T790M/L858R and EGFR^{T790M/C797S/L858R}. Firstly, we employed ROC-guided machine learning to retrieve nearly 7,765 compounds from a collection of three libraries (comprising over 220,000 compounds). Next, virtual screening, cluster analysis, and binding model analysis were employed to identify six potential compounds. Additionally, the kinase assay revealed that these six compounds demonstrated higher sensitivity to EGFR than c-Met. Among these compounds, T6496 inhibited both EGFR^T790M/L858R and EGFR^{T790M/C797S/L858R} kinases, with an IC₅₀ of 3.30 and 8.72 μM. Furthermore, we evaluated the antitumor effects of the six selected compounds, and compound T6496 exhibited the strongest anticancer activity against H1975 cell lines, with an IC₅₀ value of 2.7 μM. These results suggest that T6496 may mitigate EGFR resistance caused by T790M or C797S mutations. Moreover, the AO staining assay, JC-1 staining, ROS experiment and hemolytic toxicity evaluation revealed that T6496 could induce apoptosis in H1975 cell lines in a time-dependent and concentration-dependent manner, and is a potential compound for further structural optimization.