Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaign are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for a more in-depth downstream evaluation influence the future direction of projects, so cytotoxicity profiling of the screening libraries at early stage is essential for increasing the likelihood of candidate success. In this study, we analyzed results from a cell-based cytotoxicity screening campaign, comprising nearly 10,000 compounds in NCATS annotated libraries, and over 100,000 compounds in a diversity library, evaluated in four cell lines (HEK 293, NIH 3T3, CRL-7250 and HaCat) and a cancer cell line (KB 3-1, a HeLa subline) with overall screening outcomes, hit rates, pan-activity and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile large libraries for cytotoxicity in both normal and cancer cell lines. In summary, the results generated here constitutes a valuable resource for the scientific community and provides insight on identifying cytotoxic compounds with particular mechanisms of action, and prioritizing compounds with suitable cytotoxicity profile for further evaluation.