Selectivity of commonly used pharmacological inhibitors for cystathionine β synthase (CBS) and cystathionine γ lyase (CSE)

Abstract

Background and Purpose Hydrogen sulfide (H2S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2S are cystathionine β synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS.Experimental Approach To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method.Key Results β-cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14 ± 0.2 μM vs. 40 ± 8 μM respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC50 1.1 ± 0.1 μM); the IC50 for AOAA for inhibiting CBS was 8.5 ± 0.7 μM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC50 for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE.Conclusions and Implications In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.

Impact of endogenous hydrogen sulfide on toll-like receptor pathway in renal ischemia/reperfusion injury in rats

“…PAG is a selective inhibitor for CSE, while HA has broad biological activity. 26 HA inhibits not only CSE and CBS, but also other PLP enzymes. In addition, HA is capable of generating nitric oxide.…”

“…The pyridoxal-5 0 -phosphate (PLP)-dependent enzymes CSE and CBS are two major enzymes for the production of endogenous H 2 S. 26 In this study, PAG and HA were used to suppress H 2 S production through the inhibition of CSE and CBS. PAG is a selective inhibitor for CSE, while HA has broad biological activity.…”

Hydrogen sulfide as an oxygen sensor

“…8D), enhances hypoxic relaxation of rat aortas (14) and perfused trout gills (150). Although inhibitors of H 2 S biosynthesis have a variety of inherent problems (3,156), they have been shown to inhibit hypoxic responses of lamprey aorta, bovine pulmonary arteries, rat aorta, and perfused trout gills and rat lungs (94,125,150) (Fig. 9).…”

“…During hypoxia, CBS migrates from the cytosol to the mitochondrial matrix and in the absence of Lon protease degradation, generates H 2 S from the abundance of cysteine in the matrix. Oxidation of H 2 S in the mitochondria is initiated by an interaction with the enzyme sulfide:quinone oxidoreductase (SQR), producing two SQR persulfides that are then transferred to a mobile sulfide carrier (RSSH), one of which is oxidized to sulfite (SO 3 2 -) by mitochondrial sulfur dioxygenase (ETHE1). Sulfur transferase (ST) transfers the other persulfide and forms thiosulfate (S 2 O 3 2 -).…”

“…With the pK a1 of *6.9 for the reaction H 2 S4HS -+ H + , half of intracellular sulfide and more than two-thirds of extracellular sulfide is also an anion. Although H 2 S freely diffuses across lipid membranes (100), it is conceivable that HS -can also substitute for, or compete with, the main regulatory anions, Cl -and HCO 3 - (Fig. 6B).…”