Tetracycline antibiotics as inhibitors of hydrogen sulphide production in rat liver in vitro

Hydrogen sulphide (H2S) is generated by three enzymatic routes (1). Two enzymes are able to use pyridoxal phosphate-dependent metabolism of L-cysteine, CBS (cystathionine β-synthase) and CSE (cystathionine γ-lyase). The third pathway involves cysteine aminotransferase to generate 3-mercaptopyruvate, which is utilised by MPST (3-mercaptopyruvate sulphurtransferase), a pyridoxal phosphate-independent enzyme. At present, there is a dearth of inhibitors available to target any of these enzymes with any selectivity. A recently published study by Asimakopoulou et al. (2) described the use of a screen for inhibitors which suggested that selected tetracycline antibiotics were able to inhibit human recombinant MPST activity at high concentrations (100 µM and above) with little impact on CBS/CSE activities. We set out to confirm whether these compounds were effective against the rat liver H2S-synthesing enzymes.

Production of H2S using rat liver cytosolic fractions (n=3/4) was assessed in the presence of either L-cysteine or mercaptopyruvate using the methylene blue method (3). Data were analysed for statistical significance using repeated measures ANOVA followed by Dunnett’s multiple comparison test.

Using 10 mM L-cysteine as substrate, screening of antibiotics at a concentration of 1 mM failed to identify any significant inhibition: demeclocycline (121 ± 2 % control); doxycycline (130 ± 5); methacycline (95 ± 3); minocycline (129 ± 5); oxytetracycline (132 ± 7); and tetracycline (133 ± 5). In contrast, when mercaptopyruvate was used as substrate a number of these compounds evoked significant inhibitions: demeclocycline (53 ± 8 %, P<0.05); doxycycline (39 ± 3, P<0.01); methacycline (57 ± 9, P<0.01); minocycline (157 ± 14, P<0.01); oxytetracycline (71 ± 9); and tetracycline (40 ± 4, P<0.05). Further analysis of the concentration-dependence of antibiotic inhibition of mercaptopyruvate-derived H2S production allowed calculation of apparent pIC50 values and a rank order of apparent potency: tetracycline (3.3 ± 0.02), doxycycline (3.1 ± 0.1), methacycline (3.1 ± 0.1) > oxytetracycline (2.2 ± 0.3).

In contrast to the previous study using human recombinant MPST (2), tetracycline was the most potent of the antibiotics in inhibiting rat liver MPST activity, although doxycycline and demeclocycline appeared to be of similar potency in the two assays. Whether this difference reflects species differences in the enzyme’s sensitivity to these antibiotics or whether this is a feature of native vs recombinant expression remains to be determined.

In summary, we have confirmed that the tetracycline antibiotics show some selectivity for MPST over L-cysteine-metabolising enzymes. The low potency displayed, however, limits their usefulness in functional assays, but may allow the development of future, more potent and selective inhibitors of these enzymes.

1. Papapetropoulos A et al. (2015). Br J Pharmacol 172: 1633-1637. PM:24909294

2. Asimakopoulou A et al. (2014). Nitric Oxide, 39: S45.

3. Rashid S et al. (2013). Br J Pharmacol 168: 1902-1910. PM:23215842