P98 Tetracyclin antibiotics as inhibitors of H 2 S synthesis

Hydrogen sulfide (H2S) has emerged as a gaseous signaling molecule with important roles in neuromodulation, blood pressure regulation, angiogenesis, cardioprotection, cellular energetics and apoptosis. H2S is produced endogenously by three enzymes in the sulfur metabolic network, namely cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Although several compounds that inhibit CSE are available, no selective inhibitors for CBS or 3MST currently exist. In the present study we screened the LOPAC library for inhibitors of H2S-producing enzymes. The methylene blue assay was used to measure H2S production from recombinant enzymes. HCT116 and HT29 cells were used for MTT-based proliferation assays. Screening of the LOPAC library yielded several hit compounds that blocked H2S-synthesizing enzymes to varying extents. Among those, doxycyclin and demeclocycline were found to inhibit 3MST by approximately 50% at 100 uM, with doxycyclin being a more potent 3MST inhibitor than demeclocycline in the in vitro activity assay. Tetracyclin, meclocyclin, oxycyclin, methacyclin, tigecyclin and minocyclin did not affect 3MST activity when used up to 300 uM. From the above-mentioned tetracyclines, tigecyclin at 100 uM caused only a slight inhibition of both CBS and CSE; tigecyclin additionally inhibited CSE. In a cell-based assay, demeclocyclin, but not doxycyclin, inhibited the growth of HCT116 and HT29, two colon cancer cell lines in which CBS is highly expressed and its product, H2S supports cell proliferation. We conclude that based on its selectivity towards 3MST, demeclocyclin could serve as a lead compound that after undergoing optimization could yield future selective 3MST inhibitors.