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.

Figure 1

Purification of recombinant CSE and CBS. Representative SDS-PAGE of fractions after different purification steps of recombinant GST-CSE (A) and GST-CBS (B) from bacterial cell lysates. Lanes: M, protein marker. 1(A) cell lysate before purification; 1(B) PBS after GSTrap column calibration; 2(A) PBS after GSTrap column calibration; 2(B) cell lysate before purification; (3) cell lysate after purification; (4,5) first and second wash with binding buffer respectively; (6) eluted GST-CSE or GST-CBS; (7) elution buffer. (C) UV absorption spectra of CBS in 10 mM phosphate buffer pH = 8.2. The peak at 430 nm indicates the presence of haem in CBS.

Figure 2

H₂S production from purified CSE (grey bars) and CBS (black bars). H₂S generated: (A) by different amounts of CSE and CBS with 1 mM L-cys as substrate; (B) at different time intervals using 5 μg of either CSE or CBS; and (C) at different concentrations of PLP; (D) by purified (5 μg) CSE at the indicated concentrations of L-cys; and (E) by purified CBS (5 μg) in the absence or presence of the indicated concentrations of L-cys and homocysteine (Hcy). Data are presented as mean ± SEM; n = 6; *P < 0.05 versus control CSE, ^#P < 0.05 versus control CBS (for A and B); *P < 0.05 versus –PLP (C); *P < 0.05 versus (–) L-cys or –L-cys/Hcy (for D and E).

Figure 3

Inhibition of H₂S production from purified CSE by different inhibitors. Each reaction was performed in the presence of 100 μL sodium phosphate buffer containing 5 μg of CSE, 10 μM PLP and 1 mM of L-cys as substrates. Inhibitors were added 15 min before the L-cys at the indicated concentration and reactions allowed to proceed for 60 min (A–F). Data are presented as mean ± SEM; n = 9; *P < 0.05 versus control.

Figure 4

Inhibition of H₂S production from purified CBS by different inhibitors. Each reaction was performed in the presence of 100 μL sodium phosphate buffer containing 5 μg of CBS, 10 μM PLP, 1 mM of L-cys and 1 mM homocysteine as substrates. Inhibitors (A–F) were added 15 min before the substrates at the indicated concentration and reactions allowed to proceed for 60 min. Data are presented as mean ± SEM; n = 9; *P < 0.05 versus control.

Figure 5

Effect of DEA/NO on CSE and CBS activity. Reactions were performed as detailed in Figures 3 and 4 in the presence of the NO donor DEA/NO. Data are presented as mean ± SEM; n = 9; *P < 0.05 versus control.

Figure 6

AOAA inhibits CSE biological activity in aortic tissue. (A) Representative Western blots depicting expression of CSE and CBS in rat tissues. (B,C) Phenylephrine contracted aortic rings were exposed to D- or L-cys in the absence (B) or presence (C) of inhibitors H₂S synthesis, inhibitors AOAA and PAG (5 mM each). Data are presented as mean ± SEM; n = 5; *P < 0.05 versus control.

Figure 7

Chemical structures of compounds that block CSE selectively or preferentially.