Screening Pyridine Derivatives against Human Hydrogen Sulfide-synthesizing Enzymes by Orthogonal Methods

Abstract

Biosynthesis of hydrogen sulfide (H2S), a key signalling molecule in human (patho)physiology, is mostly accomplished by the human enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST). Several lines of evidence have shown a close correlation between increased H2S production and human diseases, such as several cancer types and amyotrophic lateral sclerosis. Identifying compounds selectively and potently inhibiting the human H2S-synthesizing enzymes may therefore prove beneficial for pharmacological applications. Here, the human enzymes CBS, CSE and MST were expressed and purified from Escherichia coli, and thirty-one pyridine derivatives were synthesized and screened for their ability to bind and inhibit these enzymes. Using differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), circular dichroism spectropolarimetry (CD), and activity assays based on fluorimetric and colorimetric H2S detection, two compounds (C30 and C31) sharing structural similarities were found to weakly inhibit both CBS and CSE: 1 mM C30 inhibited these enzymes by approx. 50% and 40%, respectively, while 0.5 mM C31 accounted for CBS and CSE inhibition by approx. 40% and 60%, respectively. This work, while presenting a robust methodological platform for screening putative inhibitors of the human H2S-synthesizing enzymes, highlights the importance of employing complementary methodologies in compound screenings.

Targeting Glutathione and Cystathionine β-Synthase in Ovarian Cancer Treatment by Selenium–Chrysin Polyurea Dendrimer Nanoformulation

“…The ability of SeChry to inhibit the human H 2 S-synthesizing enzymes was evaluated by activity assays carried out with isolated recombinant cystathionine β-synthase (tCBS, a catalytically competent truncated version lacking the s -adenosyl- l -methionine-responsive regulatory C-terminal domain), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MST). Proteins were produced and isolated as reported [56]. The H 2 S-synthesizing assays were performed, employing both the fluorometric probe 7-azido-4-methylcoumarin (AzMc) and the colorimetric methylene blue (MB) method [56].…”

“…Proteins were produced and isolated as reported [56]. The H 2 S-synthesizing assays were performed, employing both the fluorometric probe 7-azido-4-methylcoumarin (AzMc) and the colorimetric methylene blue (MB) method [56]. Fluorescence and absorbance readings were recorded in a FLUOstar Optima (BMG Labtech) plate reader.…”

N-Acetylcysteine Serves as Substrate of 3-Mercaptopyruvate Sulfurtransferase and Stimulates Sulfide Metabolism in Colon Cancer Cells

“…Activity assays on isolated recombinant human MST were adapted from [19,33,69]. The assays were carried out in 96-well black plates, using the H 2 S-selective fluorescent probe 7AzC and a Thermo Scientific Appliskan plate reader.…”

“…Several human pathologies, including neurodegenerative, cardiovascular and oncological diseases, are known to be associated with altered H 2 S metabolism (reviewed in [2,6]), and the enzymes responsible for H 2 S biosynthesis are currently recognized as potential pharmacological targets [31,32,33]. H 2 S and related species, including sulfane sulfur species, seem to play a major role in cancer biology [34,35].…”

Screening of Heteroaromatic Scaffolds against Cystathionine Beta-Synthase Enables Identification of Substituted Pyrazolo[3,4-c]Pyridines as Potent and Selective Orthosteric Inhibitors

“…A series of different scaffolds showing a promising inhibitory potential against CBS have been reported so far, with many of them being structurally unrelated synthetic molecules or natural products including flavonoids, coumarins and marine metabolites [ 2 , 33 , 34 , 35 , 36 ]. Yet, the most potent CBS inhibitor is a remarkably simple molecule, aminoacetic acid (AOAA), believed to react with PLP toward the formation of a new external aldimine [ 3 , 28 , 37 ].…”