Characterization of a small molecule inhibitor of disulfide reductases that induces oxidative stress and lethality in lung cancer cells

Abstract

Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss 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 therapeutic utility of inhibiting disulfide reductases in LUAD.

Discovery and Mechanistic Elucidation of NQO1‐Bioactivatable Small Molecules That Overcome Resistance to Degraders

“…Several small molecules with a degree of chemical analogy to RBS-10 have recently been reported and linked by some means to NQO1, in some cases as potential inhibitors. [56,[58][59] Our findings with RBS-10 support the study of the mechanistic role that NQO1 may play in facilitating the toxicity of those small molecules through redox-activation.…”

Tandem mass tag-based thermal proteome profiling for the discovery of drug-protein interactions in cancer cells

“… 8 The protocol below describes the specific steps for performing TPP on H23 lung adenocarcinoma cells treated with a small molecule compound (2 μM LCS3), that we previously used successfully to identify two disulfide reductase effector targets of this drug. 1 We describe how to perform TPP and the adaptations made for the specific purpose of discovering LCS3 effector targets, as a representative example for identifying ligand-protein interactions using TPP in human cancer cell lines. We present a detailed description of the preparation of cell lysates, treatment, and temperature challenge.…”

“…Ultimately, two proteins (GSR and TXNRD1) were found to mediate crucial biological properties of LCS3. 1 The depth of proteome coverage will be dependent on the size of the proteome of the organism under investigation, the offline fractionation scheme, if used, the LC-MS conditions, and the performance of the mass spectrometer used.…”

“…There are very few published examples of TPP being used to identify primary effector targets. 1 , 18 , 19 , 20 , 21 A recently published review comprehensively describes recent developments in TPP applications and details all protein targets, including off-targets, that have been identified by TPP to-date. 22 …”

A Shift in Thinking: Cellular Thermal Shift Assay-Enabled Drug Discovery

“…Therefore, combining transcriptomics, proteomics, and cellular response with proteome-wide CETSA MS can be a powerful tool to understand the mode of action of a compound. This was the case with a small molecule previously identified as selectively inhibiting growth of human lung cancer cell lines, that was further characterized by Johnson et al Detailed transcriptome profiling pointed toward oxidative stress and apoptosis as probable cellular mechanisms responsible for the observed cellular response. Based on the results from the CETSA MS experiment, the identified targets involved in oxidation–reduction were selected for further validation in orthogonal methods such as biochemical enzymatic assays and cytotoxicity activity.…”