Mechanism of cystathionine-β-synthase inhibition by disulfiram: The role of bis(N,N-diethyldithiocarbamate)-copper(II)

Abstract

Background: Hydrogen sulfide (H₂S) is an endogenous mammalian gasotransmitter. Cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) are the principal enzymes responsible for its biogenesis. A recent yeast screen suggested that disulfiram (a well-known inhibitor of aldehyde dehydrogenase and a clinically used drug in the treatment of alcoholism) may inhibit CBS in a cell-based environment. However, prior studies have not observed any direct inhibition of CBS by disulfiram. We investigated the potential role of bioconversion of disulfiram to bis(N,N-diethyldithiocarbamate)-copper(II) complex (CuDDC) in the inhibitory effect of disulfiram on H₂S production and assessed its effect in two human cell types with high CBS expression: HCT116 colon cancer cells and Down syndrome (DS) fibroblasts.

Methods: H₂S production from recombinant human CBS, CSE and 3-MST was measured using the fluorescent H₂S probe AzMC. Mouse liver homogenate (a rich source of CBS) was also employed to measure H₂S biosynthesis. The interaction of copper with accessible protein cysteine residues was evaluated using the DTNB method. Cell proliferation and viability were measured using the BrdU and MTT methods. Cellular bioenergetics was evaluated by Extracellular Flux Analysis.

Results: While disulfiram did not exert any significant direct inhibitory effect on any of the H₂S-producing enzymes, its metabolite, CuDDC was a potent inhibitor of CBS and CSE. The mode of its action is likely related to the complexed copper molecule. In cell-based systems, the effects of disulfiram were variable. In colon cancer cells, no significant effect of disulfiram was observed on H₂S production or proliferation or viability. In contrast, in DS fibroblasts, disulfiram inhibited H₂S production and improved proliferation and viability. Copper, on its own, failed to have any effects on either cell type, likely due to its low cell penetration. CuDDC inhibited H₂S production in both cell types studied and exerted the functional effects that would be expected from a CBS inhibitor: inhibition of cell proliferation of cancer cells and a bell-shaped effect (stimulation of proliferation at low concentration and inhibition of these responses at higher concentration) in DS cells. Control experiments using a chemical H₂S donor showed that, in addition to inhibiting CBS and CSE, part of the biological effects of CuDDC relates to a direct reaction with H₂S, which occurs through its complexed copper.

Conclusions: Disulfiram, via its metabolite CuDDC acts as an inhibitor of CBS and a scavenger of H₂S, which, in turn, potently suppresses H₂S levels in various cell types. Inhibition of H₂S biosynthesis may explain some of the previously reported actions of disulfiram and CuDDC in vitro and in vivo. Disulfiram or CuDDC may be considered as potential agents for the experimental therapy of various pathophysiological conditions associated with H₂S overproduction.

Keywords: Bioenergetics; Cancer; Disulfiram; Down syndrome; Gasotransmitters; H(2)S; Mitochondria.