Novel tools for the elucidation of physio-pathological role of H2S

Hydrogen sulphide is endogenously synthesized mainly by two pyridoxal-5′-phosphate-dependent enzymes involved in L-cysteine metabolism: cystathionine-ß-synthase (CBS) and cystathionine-γ- lyase (CSE). The H2S pathway has been proposed to be involved in the pathophysiology of several diseases. At the present stage the research in this field is impaired by the lack of pharmacological tools such as selective enzymatic inhibitors. The goal of our study is the development compounds that selectively regulate enzymatic activity. Here we present the synthesis and the activity of a selective CSE inhibitor. We preliminarily selected and tested commercially available cysteine surrogates because of the unavailability of a pharmacophoric model as a lead for rational design of targeted enzyme inhibitors. The catalytic profiles of recombinant CBS and CSE were assessed in the presence of the selected compounds. On the basis of the results obtained were designed new compounds aiming to obtain novel molecular entities embodying the structural features of both cysteine and the well known CSE inhibitor, DL-propargylglycine. The synthetically modified compounds, obtained in our laboratory, were tested in vitro by using rat aortic rings. The compound showing maximal inhibitory effects in this test was an oxothiazolidine derivative, dubbed compound VII. The effects of this compound on the enzyme kinetics was further tested on the purified enzymes using a metabolomic approach based on nuclear magnetic resonance techniques. These studies clearly showed that compound VII is a potent enzyme inhibitor of CSE, without affecting the CBS kinetics. The identification of this highly selective CSE inhibitor may help to better define the role of CSE vs CBS in the pathophysiology of the diseases where a role for the H2S pathway has been proposed