Thioredoxin reductase is inhibited by the carbamoylating activity of the anticancer sulfonylhydrazine drug laromustine

Abstract

The thioredoxin system facilitates proliferative processes in cells and is upregulated in many cancers. The activities of both thioredoxin (Trx) and its reductase (TrxR) are mediated by oxidation/reduction reactions among cysteine residues. A common target in preclinical anticancer research, TrxR is reported here to be significantly inhibited by the anticancer agent Laromustine. This agent, which has been in clinical trials for acute myelogenous leukemia and glioblastoma multiforme, is understood to be cytotoxic principally via interstrand DNA crosslinking that originates from a 2-chloroethylating species generated upon activation in situ. The spontaneous decomposition of Laromustine also yields methyl isocyanate, which readily carbamoylates thiols and primary amines. Purified rat liver TrxR was inhibited by Laromustine with a clinically relevant IC50value of 4.65 µM. A derivative of Laromustine that lacks carbamoylating activity did not appreciably inhibit TrxR while another derivative, lacking only the 2-chloroethylating activity, retained its inhibitory potency. Furthermore, in assays measuring TrxR activity in murine cell lysates, a similar pattern of inhibition among these compounds was observed. These data contrast with previous studies demonstrating that glutathione reductase, another enzyme that relies on cysteine-mediated redox chemistry, was not inhibited by methylcarbamoylating agents when measured in cell lysates. Mass spectrometry of Laromustine-treated enzyme revealed significant carbamoylation of TrxR, albeit not on known catalytically active residues. However, there was no evidence of 2-chloroethylation anywhere on the protein. The inhibition of TrxR is likely to contribute to the cytotoxic, anticancer mechanism of action for Laromustine.

Small molecule inhibitors of mammalian thioredoxin reductase as potential anticancer agents: An update

“…The purified rat liver TrxR was inhibited by 258 with a clinically relevant IC 50 value of 4.65 μM. Mass spectrometry of laromustine‐treated enzyme revealed significant carbamoylation of TrxR . In addition, the authors showed that 101MDCE ( 259 , Figure ), an analog of 258 that generates only methyl isocyanate, decreased the activity of TrxR1 significantly.…”

“…In addition, the authors showed that 101MDCE ( 259 , Figure ), an analog of 258 that generates only methyl isocyanate, decreased the activity of TrxR1 significantly. Compound 259 inhibited TrxR1 with an IC 50 value of 9.90 μM …”

pH‐dependent general base catalyzed activation rather than isocyanate liberation may explain the superior anticancer efficacy of laromustine compared to related 1,2‐bis(methylsulfonyl)‐1‐(2‐chloroethyl)hydrazine prodrugs

“…Bis(sulfonyl)hydrazines with segregated carbamoylating and chloroethylating activities have been used in an analogous manner to successfully dissect the contributions of these activities toward various processes such as DNA cross‐linking/nicking, enzyme inhibition, and cytotoxicity in a number of in vitro studies . It is realized that these in vivo experiments involve many additional variables that can greatly complicate interpretation compared to in vitro studies.…”

Evaluation ofN-acetyl-S-(p-chlorophenylcarbamoyl)cysteine as an irreversible inhibitor of mammalian thioredoxin reductase1

“…In this work, we found that the anticancer agent NACC was a TrxR1 inhibitor. In vitro, the inhibition of mammalian TrxR1 by NACC was comparable to some of the existing TrxR inhibitors, such as Butyltin (IV) Benzoates 3 , quinone compound 4 and anticancer drug laromustine 22 . The K i value of MGd, the TrxR inhibitor undergoing clinical trial, is even higher.…”