Thioredoxin reductase is inhibited by the carbamoylating activity of the anticancer sulfonylhydrazine drug laromustine
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 IC50 value 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.
KeywordsThioredoxin reductase Alkylating agents Methyl isocyanate Laromustine
This project was supported by grants from the National Center for Research Resources (5P20RR016463-12 to K.P.R. and P20-RR018787 to S.A.G) and the National Institute of General Medical Sciences (8 P20 GM103423-12 to K.P.R.), of the National Institutes of Health, and from the Colby College Division of Natural Sciences. The authors would also like to acknowledge Alan Sartorelli (Yale University School of Medicine) for furnishing the compounds necessary to begin this project. The authors would like to thank Jeffrey Katz and Nicholas Bizer (Colby College) for providing counsel toward the chemical syntheses.
- 4.Penketh PG, Shyam K, Baumann RP, Remack JS, Brent TP, Sartorelli AC (2004) 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazi ne (VNP40101M): I. Direct inhibition of O6-alkylguanine-DNA alkyltransferase (AGT) by electrophilic species generated by decomposition. Cancer Chemother Pharmacol 53:279–287PubMedCrossRefGoogle Scholar
- 51.Atkinson JM, Shelat AA, Carcaboso AM, Kranenburg TA, Arnold LA, Boulos N, Wright K, Johnson RA, Poppleton H, Mohankumar KM, Feau C, Phoenix T, Gibson P, Zhu L, Tong Y, Eden C, Ellison DW, Priebe W, Koul D, Yung WK, Gajjar A, Stewart CF, Guy RK, Gilbertson RJ (2011) An integrated in vitro and in vivo high-throughput screen identifies treatment leads for ependymoma. Cancer Cell 20:384–399PubMedCrossRefGoogle Scholar
- 57.Raizer J, Rice L, Rademaker A, Chandler J, Levy R, Muro K, Grimm S (2011) A phase I trial of laromustine (VNP40101 M) and temozolomide for patients with malignant gliomas in first relapse or progression. Neuro Oncol 13:60–61Google Scholar