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Abstract
Hydrogen sulfide is an essential catabolite that intervenes in the pathophysiology of several diseases from hypertension to stroke, diabetes and pancreatitis. It 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). Research in this field is currently impaired by the lack of pharmacological tools such as selective enzymatic inhibitors that could target specifically only one of these pathways. We used a novel approach based on a hybrid method that includes drug design, synthetic biology, metabolomics and pharmacological assays to rationally design a new inhibitor selective for the CSE enzyme. The identification of this compound opens new frontiers towards a better understanding of the role of CSE over CBS in the pathophysiology of diseases where a role for the H2S pathway has been proposed and the development of new lead compounds that could target the CSE enzyme.
Highlights
Structure flexibility of AVG makes it difficult to rationalise the mode of binding of this compound until a crystal structure of its complex with CSE becomes available
We first carried out a preliminary in silico analysis of the structures of the two enzymes to understand the geometric properties of the active sites
We found that, during the reaction, a predominant quantity of cysteine is converted into cystine with a minor quantity of lanthionine
Summary
During our NMR based metabolomics studies we observed something peculiar: when PAG was used in the NMR assay, no appreciable formation of pyruvate was observed, as expected, but the resonances of L-Cys surprisingly decreased and new product resonances, different from pyruvate, appeared and progressively increased (Fig. 5A) This behaviour was confirmed in a 13C 2D HSQC spectrum acquired at the end of the kinetics (Fig. 5B): in the presence of PAG, the cysteine resonances at 3.90 ppm (CαH and 55 ppm in the carbon spectrum not shown) and at 3.05 ppm (CβH2 and 25 ppm) were absent at the end of the reaction. The cavity which contains PAG in the active site, which is where the substrate cysteine should bind, is big enough to accommodate compound 2a (Fig. 7) This can form multiple interactions with the surrounding groups and hydrogen bond with the side chains of Ser[63], Tyr[114], Gly[116], Arg[119], Asn[241], and Ser[358]. We can conclude that we have identified a new compound which has all features to optimally fit in CSE and inhibit selectively its function
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