A novel small molecule that induces cytotoxicity in lung cancer cells inhibits disulfide reductases GSR and TXNRD1

High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD. ### Competing Interest Statement H.D, R.S. and H.V. are listed as inventors in the issued US patent #9562019 covering LCS3 and its analogs. W.W.L. is a consultant of Hyperbio Therapeutics. R.S. has received research support from Helsinn Healthcare, LOXO Oncology, Merus and Elevation Oncology Inc. that were unrelated to the current study. U.G. has a clinical trial agreement (CTA) with AstraZeneca and had received research funding from AstraZeneca, Esanex and Aurigene. U.G. is currently an employee of Bristol Myers Squibb.