Inhibitors of the 20S proteasome β5 subunit as potent and selective agents against <i>Trichomonas vaginalis</i>

<i>Trichomonas vaginalis</i> is the causative agent of the most prevalent, non-viral sexually transmitted infectious disease, yet treatment options are limited to three nitroheterocyclic antimicrobials of the same drug class, and resistance to these agents is a manifest clinical problem. The 20S proteasome is a validated new drug target against <i>T. vaginalis,</i> but inhibitors of the proteasome complex have so far only shown modest selectivity over human cells. We screened a library of 373 diverse peptide inhibitors with different reactive warheads against diverse <i>T. vaginalis</i> strains in a growth and survival assay and identified several compounds with potencies in the 10-20 nM range. Notably, these compounds were up to 200-fold selective for <i>T. vaginalis</i> over mammalian cells and could overcome metronidazole resistance. Removal of the epoxide or the adjacent methyl group in the inhibitors carrying an epoxyketone functionality abolished activity, underlining the functional importance of this warhead. Biochemical and whole-cell assays of inhibitory activity against each of the three catalytically active subunits (β1, β2, and β5) of the proteasome showed that inhibition of β5 was sufficient to mediate activity against <i>T. vaginalis</i>. Furthermore, the high selectivity of the best compounds came from their preferential inhibition of <i>T. vaginalis</i> β5 over human β5. Docking calculations of the most selective inhibitor to the <i>T. vaginalis</i> substrate-binding pocket revealed the formation of two stabilizing hydrogen bonds with a threonine residue that is absent in the human proteasome. These results encourage the therapeutic development of highly potent and selective proteasome inhibitors against <i>T. vaginalis</i>.