PL02 Modulation of H2S levels in cells and tissues using pharmacological agents: Advances and pitfalls

Abstract

The biological roles of endogenous H 2 S are multiple and rapidly expanding. Hydrogen sulfide exerts its effects in most organ systems including the central and peripheral nervous system, the cardiovascular, gastrointestinal and respiratory systems; H 2 S also participates in the regulation of cellular metabolism and immunological/inflammatory responses. Much of the enzymatically generated H 2 S is derived from two pyridoxal-5′-phosphate (PLP)-dependent enzymes responsible for the metabolism of l -cysteine: cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE); a third pathway that catalyzes the production of H 2 S from l -cysteine via the combined action of 3-mercaptopyruvate sulphurtransferase and cysteine aminotransferase (3MST/CAT) has also been described. CSE and CBS catalyze several H 2 S-generating reactions using cysteine and/or homocysteine as substrates. CBS is the predominant H 2 S-generating enzyme in the brain and nervous system and is highly expressed in liver and kidney, while CSE is mainly expressed in the liver and in vascular and non-vascular smooth muscle. To study the role of this novel gasotransmitter, investigators have relied on genetic models, knock down of H 2 S-producing enzymes by siRNA, and on pharmacological inhibitors. As genetic and siRNA approaches require additional expertise and access to specialized facilities, most researchers prefer to use pharmacological inhibitors. The most commonly used agents to inhibit H 2 S biosynthesis include propargylglycine (PAG), β -cyanoalanine (BCA), aminooxyacetic acid (AOAA), trifluoroalanine and hydroxylamine. Using recombinant human H 2 S-producing enzymes we have found that PAG exhibits selectivity towards CSE vs. CBS, but its potency is lower than that of l -aminoethoxyvinylglycine (AVG). BCA, although selective for CSE, it also inhibits CBS when used at high concentrations. In contrast, none of the compounds tested exhibited significant selectivity towards CBS. In addition, the above-mentioned compounds did not inhibit 3MST. It should also be stressed that even the inhibitors that were found to be selective for CSE have been shown to inhibit other PLP-dependent enzymes. Inhibiting CSE or CBS activity will affect the levels of several metabolites and intermediates in the transulfuration pathway. For example, CBS inhibition leads to increased homocysteine levels. To avoid this unwanted side effect when long term inhibition of CBS or CSE is desired (such as in conditions where H 2 S production is up regulated contributing to disease progression), scavenging of H 2 S would be the preferred approach. Our efforts to develop H 2 S scavengers will be presented during the meeting. We conclude that design and development of new inhibitors for each of the H 2 S-synthesizing enzymes, as well as H 2 S scavengers is an unmet need for the field, so that the role of H 2 S-regulated pathways in the development and treatment of disease can be evaluated.