Searching for inhibitors of human H 2 S-synthesizing enzymes by orthogonal methods

Hydrogen sulfide (H2S), a key signalling molecule in human patho-physiology, is produced by the human enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST). Increasing evidence indicates a correlation between increased H2S production and human diseases, such as several cancer types and amyotrophic lateral sclerosis. Identifying selective and potent inhibitors may be beneficial for future therapeutic strategies. Here, recombinant CBS, CSE and MST were expressed and purified, and 31 pyridine derivatives were synthesized and screened for their ability to bind and inhibit these enzymes. Using a combination of orthogonal methodologies, such as differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), circular dichroism (CD) spectropolarimetry, and activity assays based on fluorimetric and colorimetric H2S detection, two compounds sharing the same molecular scaffold showed a weak inhibition of both CBS and CSE. The present work, while presenting a robust methodological platform for screening putative inhibitors of the three human H2S-synthesizing enzymes, highlights the importance of employing complementary methodologies to identify possible false positives in compound screening campaigns.