Elsevier

Biochemical Pharmacology

Volume 55, Issue 7, 1 April 1998, Pages 987-994
Biochemical Pharmacology

Original Articles
Mechanisms of Inhibition of the Thioredoxin Growth Factor System by Antitumor 2-Imidazolyl Disulfides

https://doi.org/10.1016/S0006-2952(97)00597-2Get rights and content

Abstract

The interactions of a series of 2-imidazolyl disulfide antitumor compounds with the thioredoxin reductase(TR)/thioredoxin (hTrx) redox system have been studied. Disulfides III-2 (n-butyl 2-mercaptoimidazolyl disulfide) and VI-2 (ethyl 2-mercaptoimidazolyl disulfide) were substrates for reduction by TR with Km values of 43 and 48 μM. Disulfides IV-2 (1-methylpropyl 2-mercaptoimidazolyl disulfide) and DLK-36 (benzyl 2-mercaptoimidazolyl disulfide) were competitive inhibitors of the reduction of hTrx by TR with Ki values of 31 μM. None of the disulfides were substrates for reduction by human glutathione reductase. The disulfides caused reversible thioalkylation of hTrx at the redox catalytic site as shown by the fact that there was no thioalkylation of a mutant hTrx where both the catalytic site Cys32 and Cys35 residues were replaced by Ser. In addition, the disulfides caused a slower irreversible inactivation of hTrx as a substrate for reduction by TR, with half-lives for III-2 of 30 min, for IV-2 of 4 hr, and for IX-2 (t-butyl 2-mercaptoimidazolyl disulfide) of 24 hr. This irreversible inactivation of hTrx occurred at concentrations of the disulfides an order of magnitude below those that inhibited TR, and involved the Cys73 of hTrx, which is outside the conserved redox catalytic site, as shown by the resistance to inactivation of a mutant hTrx where Cys73 was replaced by Ser. Electrophoretic and mass spectral analyses of the products of the reaction between the disulfides and hTrx show that modification of 1–3 Cys residues of the protein occurred in a concentration-dependent fashion. The disulfides inhibited the hTrx-dependent proliferation of MCF-7 breast cancer cells with ic50 values for III-2 and IV-2 of 0.2 and 1.2 μM, respectively. The results show that although the catalytic sites of TR and hTrx are reversibly inhibited by the 2-imidazolyl disulfides, it is the irreversible thioalkylation of Cys73 of hTrx by the disulfides that most probably accounts for the inhibition of thioredoxin-dependent cell growth by the disulfides.

Section snippets

Preparation of Thioredoxins

Recombinant hTrx, Cys32 → Ser/Cys35 → Ser mutant hTrx (C32S/C35S) and Cys73 → Ser (C73S) mutant hTrx were prepared and purified as previously described 4, 10. Twenty-five micromolar stock solutions of the hTrxs in 5 mM of DTT were stored at −20°. Before use, the DTT was removed by passing the hTrx solution through a PD-10 desalting column (Pharmacia). The hTrx solution was kept at 4° and used fresh within 2 hr or hTrx was allowed to oxidize by keeping it at room temperature for at least 3 weeks

Cell Growth Inhibition

The effects of the disulfides on the growth of human MCF-7 breast cancer cells were studied (Fig. 1). The ic50 values of III-2 and IV-2 with 10% FBS were 35.0 and 3.2 μM, and in the presence of 1 μM of hTrx the ic50 values were 0.2 and 1.2 μM, respectively. We have found that C73S stimulates MCF-7 cell growth to the same extent as hTrx [4]; however, disulfides III-2 and IV-2 at 1 μM did not inhibit the cell growth caused by 1 μM of C73S (results not shown).

Interaction Between Disulfides and TR

Disulfides III-2 and VI-2 were

Discussion

The 2-imidazolyl disulfides interact with both TR and hTrx. It is postulated that the unbranched alkyl moieties of the disulfides III-2 and VI-2, both of which are substrates for TR, facilitate thiol/disulfide exchange with a Cys residue at the catalytic site of TR and the liberation of 2-mercaptoimidazole (Fig. 6A). There is a subsequent thiol/disulfide exchange to give an oxidized catalytic site followed by reduction by NADPH to regenerate the reduced enzyme. Branching of the alkyl

Acknowledgements

This work was supported by Grants CA 48725, and CA 42286 17094 (G. P.) and the University of Regina (D. L. K.).

References (26)

  • DL Kirkpatrick et al.

    Synthesis and evaluation of imidazolyl disulfides for selective cytotoxicity to hypoxic EMT6 tumor cells in vitro

    Eur J Med Chem

    (1992)
  • A Weichsel et al.

    Crystal structures of reduced, oxidized, and mutated human thioredoxinsEvidence for a regulatory homodimer

    Structure

    (1996)
  • A Holmgren

    Thioredoxin

    Annu Rev Biochem

    (1985)

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