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

• Published in: Biochemical pharmacology 1998-04, Vol.55 (7), p.987-994
• Main Authors: Kirkpatrick, D.Lynn, Kuperus, Miles, Dowdeswell, Marla, Potier, Noelle, Donald, Lynda J, Kunkel, Mark, Berggren, Margareta, Angulo, Miguel, Powis, Garth
• Format: Article
• Language: English
• Subjects: Antineoplastic agents; Antineoplastic Agents - pharmacology; Biological and medical sciences; Catalysis; disulfides; Disulfides - pharmacology; Electrophoresis, Polyacrylamide Gel; General aspects; growth factor; human thioredoxin; Humans; Imidazoles - pharmacology; Kinetics; Mass Spectrometry; Medical sciences; Oxidation-Reduction; Pharmacology. Drug treatments; thioredoxin reductase; Thioredoxin-Disulfide Reductase - antagonists & inhibitors; Thioredoxins - antagonists & inhibitors; Tumor Cells, Cultured
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/S0006-2952(97)00597-2

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 K m 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 K i 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 Cys 32 and Cys 35 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 Cys 73 of hTrx, which is outside the conserved redox catalytic site, as shown by the resistance to inactivation of a mutant hTrx where Cys 73 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 ic 50 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 Cys 73 of hTrx by the disulfides that most probably accounts for the inhibition of thioredoxin-dependent cell growth by the disulfides.