S-3-Carboxypropyl-l-cysteine specifically inhibits cystathionine γ-lyase–dependent hydrogen sulfide synthesis

Abstract

Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H2S synthesis and is a potential target for modulating H2S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ± 3 and 180 ± 15 μm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine β-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H2S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80–90%, as monitored by the transfer of radiolabel from [35S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H2S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H2S biogenesis inhibitors.

Inhibitors of bacterial H 2 S biogenesis targeting antibiotic resistance and tolerance

“…Most hCSE inhibitors act by targeting the coenzyme pyridoxal phosphate (PLP) and therefore can interfere with the activity of other PLP enzymes (32,33,35). In addition, PAG can covalently modify a residue in the active site of hCSE (30).…”

Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations

“…Lysates from human (HepG2, HEK293 and SH-SY5Y) and murine (bEnd3) cell lines, were prepared as described previously (49). Red blood cell lyastes were prepared as described previously (30) and the concentration of hemoglobin in each sample was assessed by the absorption at 415 nm using and extinction coefficient of 128 mM -1 cm -1 (50).…”

Mechanism-Based and Computational Modeling of Hydrogen Sulfide Biogenesis Inhibition - Interfacial Inhibition

“…Altogether, our results suggest than D-pen is a mixed inhibitor of cystathionine γ-lyase ( Scheme S1 ). Interestingly, despite its with weak potency to impair both cystathionine and cysteine cleavage activity of CSE, the inhibitory mode adopted by D-pen is quite peculiar as it differs from the ones reported for trifluoroalanine (43), propargylglycine (36, 43) and S-3-carboxypropyl-L-cysteine (32) that are suicide inhibitors. It also contrasts with the inhibitory mode detected for aminoethoxyvinyglycine, a slow and tight binding inhibitor of CSE (43), and the mode of action of I157172 (29) and aurintricarboxylic acid (30) or (R) -2-oxo- N -(prop-2-yn-1-yl)thiazolidine-4-carboxamide (31), respectively competitive cofactor and substrate inhibitors.…”

“…The scarcity of selective CSE inhibitors has prompted recent interest in this area with high-throughput (virtual) screening (HTS) of compound libraries that identified iminoquinolinone derivative, N-propargyl D-cysteine derivative, aurintricarboxylic acid, isoxsuprine and 2-arylidene hydrazinecarbodithioates as selective modulators of CSE activity (26-30). In addition, a semi-rational in silico drug screening study combined with chemical synthesis (31) and a screening of substrate analogs (32) reported (R) -2-oxo- N -(prop-2-yn-1-yl)thiazolidine-4-carboxamide and S-3-carboxypropyl-L-cysteine as potent selective inhibitors of CSE, with the later inducing stable aminoacrylate formation. Finally, D-penicillamine (D-pen), a drug already used in the management of rheumatoid arthritis and Wilson’s disease, recently repurposed in cancer treatment and responsible for the reversible inhibition of catalase, has also been reported to selectively inhibit CSE but the molecular bases for such inhibitory effect have not been investigated (33-35).…”