Development of subunit selective substrates forTrichomonas vaginalisproteasome

Abstract

The protozoan parasite, Trichomonas vaginalis (Tv) causes trichomoniasis, the most common, non-viral, sexually transmitted infection in the world. Only two closely related drugs are approved for its treatment. The accelerating emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health. There is an urgent need for novel effective anti-parasitic compounds. The proteasome is a critical enzyme for T. vaginalis survival and was validated as a drug target to treat trichomoniasis. However, to develop potent inhibitors of the T. vaginalis proteasome, it is essential that we understand which subunits should be targeted. Previously, we identified two fluorogenic substrates that were cleaved by T. vaginalis proteasome, however after isolating the enzyme complex and performing an in-depth substrate specificity study, we have now designed three fluorogenic reporter substrates that are each specific for one catalytic subunit. We screened a library of peptide epoxyketone inhibitors against the live parasite and evaluated which subunits are targeted by the top hits. Together we show that targeting of the β5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of β5 plus either β1 or β2 results in improved potency.

Clickable Probes for Pathogen Proteasomes: Synthesis and Applications

“…Notably, the background was less pronounced with LJL-1 and LJL-2 compared to that with Me 4 BodipyFL-Ahx 3 L 3 VS. Previous research using purified Tv20S identified the electrophoretic migration order of the β subunits as β2, β1, and β5, from highest to lowest molecular weight . Accordingly, we wished to employ inhibitors that selectively inactivate one or other β subunit such that the uninhibited subunit(s) would be available to bind to the probe.…”

“…Lysate (16 μg) was preincubated with (1) DMSO, (2) 20 μM E64, (3) 2 mM PMSF or (4) 10 μM MZB, prior to addition of probe. (B) Schematic of inhibitor or inhibitor combinations known to inhibit one or more Tv20S β subunits . (C) Fluorescence imaging of Tv20S β subunits after incubation with β subunit-specific inhibitors or combinations of inhibitors, and enrichment from parasite lysate using the biotin functionality of LJL-2.…”

“…A number of 20S inhibitors and inhibitor combinations have been shown to preferentially inhibit one or more Tv20S catalytic β subunits . The carmaphycin B analog, CP-17 selectively inhibits β5 at 1 μM.…”

“…Over the past decade, the research tools available to characterize the proteasome have advanced on various fronts, and include the use of optimized peptidyl fluorogenic substrates to measure the activity of each of the three catalytic β subunits,, methodologies to purify the proteasome, ,, and the availability of cryoEM structures to understand how small molecules engage the target. ,, Also, active site-directed probes have been developed to define cellular localization, facilitate imaging in small animal models and understand the specificities of small molecule proteasome inhibitors. − …”

“…In terms of active site-directed probes with which to visualize c20S ( e.g ., after native or denaturing gel electrophoresis), the most commonly used is the irreversible, fluorescent, peptidyl vinyl sulfone, Me 4 BodipyFL-Ahx 3 L 3 VS (Figure A), a derivative of the originally described MV151 (Bodipy TMR-Ahx 3 L 3 VS). , During our efforts to characterize parasite 20S molecules as potential drug targets, we identified limitations to this probe. Specifically, it binds irreversibly to cysteine proteases (SI 1), which are often highly expressed in parasites, , and sometimes labels just two of the three catalytic β subunits , even though the genomic/proteomic evidence indicates the presence of all three. Lastly, Me 4 BodipyFL-Ahx 3 L 3 VS lacks the functionality to enable isolation of the catalytic β subunits from protein extracts which would then facilitate their identification via proteomics.…”