Hydrogen sulfide biosynthesis is blocked by D-penicillamine action on cystathionine-γ-lyase

Hydrogen sulfide (H₂S) is a gasotransmitter mainly released from L-cysteine following cystathionine-gamma-lyase (CSE) and/or cystathionine-beta-synthase (CBS) enzymatic action [Szabo, 2007; Li et al., 2011]. The pathway leading to H₂S release has been extensively investigated and, with respect to vascular system, CSE seems to represent the prominent enzyme, since it shows deep implications in regulating blood vessels and heart function [Yang et al., 2008; Lavu et al., 2011]. Noteworthy, the majority of work in the field of H₂S research have been achieved by using inhibitors that are not entirely specific or selectivity for CSE and/or CBS [Whiteman et al., 2011]. This aspect highlights the unmet need to develop new selective inhibitors. L-cysteine shows a close structural similarity with D-penicillamine (D-pen), an old drug used in the therapy of rheumatoid arthritis (RA) in \'70s [Golding et al., 1970]. Aim of our study was to investigate if D-pen interferes with H₂S biosynthetic machinery.

In order to pursue our aim, we used a multiple approach by combining isolated organ bath (aorta rings), biochemistry (recombinant enzymes) and in vivo assays (intravital microscopy analysis).

We first observed that D-pen did not induce any substantial vasorelaxation in phenylephrine pre-contracted mouse aorta (<20%), compared to L-cysteine. Next, we observed that vasodilation induced by L-cysteine was significantly blocked when aorta rings were incubated with D-pen prior to L-cysteine addition. Its effect was concentration-dependent (p<0.001, 0.01-1mM; n=6). Furthermore, D-pen inhibited H₂S biosynthesis in aorta homogenated samples in a concentration-dependent manner (p<0.001, 0.01-1mM; n=6). Experiments performed by using human recombinant CSE and CBS showed that D-pen selectively inhibited CSE (IC₅₀ 270µM). The inhibitory effect associated to D-pen was reverted by addition of pyridoxal-5\'-phospate supplementation in both aorta rings and H₂S production assays (p<0.01 and p<0.001, respectively; n=6). Finally, pre-treatment with of D-pen in mice undergoing TNFα-induced mesenteric inflammation resulted in exacerbation of inflammatory conditions determined as leukocyte trafficking (p<0.05; n=5).

In conclusion, our results suggest that D-pen acts as a selective CSE inhibitor, leading to new perspectives in the design and use of specific pharmacological tools for H₂S research field. In addition, inhibition of CSE could account for beneficial effect of D-pen in RA patients, where H₂S has been shown to be detrimental.

References

Szabo, Nat Rev Drug Discov. 2007 Nov;6(11):917-35

Li et al., Annu Rev Pharmacol Toxicol. 2011;51:169-87

Yang et al., Science. 2008 Oct 24;322(5901):587-90

Lavu et al., Clin Sci (Lond). 2011 Mar;120(6):219-29

Whiteman et al., Clin Sci (Lond). 2011 Dec;121(11):459-88

Golding et al., Postgrad Med J., 2007, 46(540):599-605