Reversible inhibition of human thioredoxin reductase activity by cytotoxic alkyl 2-imidazolyl disulfide analogues

Abstract

The thioredoxin/thioredoxin reductase system is important for several aspects of the regulation of cellular proliferation by both intracellular and extracellular mechanisms. The effects ofn-butyl 2-imidazolyl disulfide (III-2), 1-methylpropyl 2-imidazolyl disulfide (IV-2), andn-decyl 2-imidazolyl disulfide (VII-2) on purified human placental thioredoxin reductase activity were examined. The analogues were competitive inhibitors with DTNB for reduction by thioredoxin reductase, withK _i values for III-2, IV-2, and VII-2 being 3.3, 13.0, and 8.6 μM, respectively. The inhibition was noncompetitive with reduced nicotinamide adenine dinucleotide phosphate (NADPH). None of the analogues was a suicide substrate inhibitor of the flavoenzyme. III-2 and VII-2 were metabolized by thioredoxin reductase at about half the rate of DTNB, whereas IV-2 was not detectably metabolized. The second order rate constants for the reactions of III-2 and IV-2 with reduced GSH were 931 and 91M ⁻¹ s⁻¹, respectively. The lower reactivity of IV-2 with reduced GSH and the lack of the analogue's metabolism by thioredoxin reductase may be due to the more sterically hindered structure of this analogue. The 50% inhibitory concentrations (IC₅₀ values) for the inhibition of serum-dependent cellular proliferation of Swiss 3T3 murine fibroblasts by III-2, IV-2, and VII-2 were 2.0, 3.5, and 4.0 μM, respectively. IV-2 was considerably more potent as an inhibitor of the thioredoxin-dependent cellular proliferation of Swiss 3T3 fibroblasts, showing an IC₅₀ value of 60 nM. Thus, inhibition of cellular proliferation by alkyl 2-imidazolyl disulfide analogues may involve interaction with thioredoxin, thioredoxin reductase, or an alternative target that is redox-regulated by thioredoxin.