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NDUFA12 as a functional target of the anticancer compound Ertredin in human hepatoma cells revealed by label-free chemical proteomics

ABSTRACT: Many studies have been attempted to develop new agents that target EGFR mutants or regulate downstream players in various cancers. A new small molecule, Ertredin, was discovered through cell-based screening to inhibit EGFRvIII mutant cancer cells. Previous studies have shown that Ertredin effectively inhibits anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII mutant cDNA. However, the underlying mechanism remains to be elucidated. In this study, we investigated the target protein of Ertredin by combining DARTS with LC-MS/MS using label-free Ertredin as a bait and HepG2 cell lysates as a proteome pool. As a result, NADH dehydrogenase 1 alpha subcomplex subunit 12, NDUFA12, was identified as one of the binding proteins of Ertredin responsible for the biological activity of the compound. The interaction between NDUFA12 and Ertredin was validated by DARTS and CETSA methods. In addition, genetic knockdown of the identified target NDUFA12 was validated with respect to its association with cell proliferation. Taken together, NDUFA12 is identified as a biologically relevant target protein of Ertredin to address an anti-tumor activity of the compound and these results provide insights into a role of NDUFA12 as a downstream player of EGFRvIII mutant.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ho Jeong Kwon

PROVIDER: MSV000092332 | MassIVE | Mon Jul 03 01:45:00 BST 2023

REPOSITORIES: MassIVE

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Project description:RRx-001 (also known as ABDNAZ, 1-bromoacetyl-3,3-dinitroazetidine) is a novel aerospace-derived compound currently under investigation in several ongoing Phase II studies. In a Phase I trial, it demonstrated anti-cancer activity and evidence of resensitization to formerly effective therapies in heavily pre-treated patients with relapsed/refractory solid tumors. With a pharmacologically unprecedented dinitroazetidine scaffold, RRx-001 generates reactive oxygen and nitrogen species (ROS and RNS) and nitric oxide (NO), elicits changes in intracellular redox status, modulates tumor blood flow, hypoxia and vascular function and triggers apoptosis in cancer cells. Here, the effect of RRx-001 on the epigenome of SCC VII cancer cells was investigated. RRx-001 at 0.5 and 2 μM significantly decreased global DNA methylation, i.e., 5-methylcytosine levels, in SCC VII cells determined by analysis with an enzyme-linked immunosorbent assay (ELISA). Consistently, 0.5-5 μM RRx-001 significantly decreased Dnmt1 and Dnmt3a protein expression determined using Western blot in a dose- and time-dependent manner. In addition, global methylation profiling identified differentially methylated genes in SCC VII cells treated with 0.5, 2, and 5 μM RRx-001 compared to control cells. Twenty-three target sites were hypomethylated and 22 hypermethylated by >10% in the presence of at least two different concentrations of RRx-001. Moreover, RRx-001 at 2 μM significantly increased global acetylated histone H3 and H4 levels in SCC VII cells after 24 hour treatment determined by a fluorometric assay, suggesting that RRx-001 regulates global acetylation in cancer cells. These results demonstrate that, in contrast to the traditional “one drug one target” paradigm, RRx-001 has multi(epi)target features, which contribute to its anti-cancer activity and may rationalize the resensitization to previously effective therapies observed in clinical trials and serve as a unifying mechanism for its anticancer activity.

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NDUFA12 as a functional target of the anticancer compound Ertredin in human hepatoma cells revealed by label-free chemical proteomics

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