Periodic Reporting for period 2 - ProTeCT (Proteasome as a target to combat trichomoniasis)
Reporting period: 2021-11-01 to 2022-10-31
Trichomoniasis treatment now relies on two antibiotic drugs – metronidazole and tinidazol but the accelerating emergence of resistance to current antibiotics and no alternative treatment options pose an increasing threat to public health, resulting in an urgent need for novel effective antiparasitic compounds.
Proteasomes are multisubunit, energy-dependent, proteolytic complexes that are essential for protein homeostasis in mammalian cells and in protozoa. Unlike mammalian cells parasitic organisms express a single proteasome that is essential for their survival.
In a murine model of vaginal trichomonad infection, proteasome inhibitors eliminated or significantly reduced parasite burden upon topical treatment without any apparent adverse effects. These findings validated the proteasome of T. vaginalis as a therapeutic target for development of a novel class of trichomonacidal agents.
The overall objective of my project “Proteasome as a target to combat trichomoniasis” is to functionally and structurally characterize proteasome from the parasite Trichomonas vaginalis and develop potent and selective inhibitors as potential chemotherapeutics for trichomoniasis treatment.
During the return phase at Institute of Organic Chemistry and Biochemistry CAS I learnt new cloning, expression, purification and structural determination techniques. I was able to recombinantly express active Tv20S proteasome, which consists of 28 subunits. The recombinant proteasome was purified and two structures were solved using CryoEM technology each with a different covalently bound inhibitor bound. Inhibitor A, binds to all 3 catalytically active sites, whereas the inhibitor B (the inhibitor with the best selectivity of Tv versus the human proteasome) binds to two catalytically active sites. Structure solving is now in progress and the information obtained will be used to design new specific inhibitors to achieve greater selectivity.
The new insights we have gained over the past 3 years in the collaboration between IOCB and UCSD will be published in 3 peer-reviewed journals.
So far, the optimized methods mentioned above can be applied to the study of other protozoan proteasomes such as proteasome from Giardia sp., Trypanosoma sp. etc. Socioeconomic status and health are closely related, in addition to educating the general public the successful discovery of new drugs against trichominiasis will have a positive socioeconomic impact.
During the return phase, we met the objectives mentioned in Anex 1 for the return phase in Dr. Boura's laboratory. Since the proteasome isolated from the parasites was not pure enough for CryoEM analysis, we produced Tv20S recombinantly. This is the first parasite 20S proteasome that has been expressed recombinantly. We confirmed the biochemical activity of rTv20S and were able to obtain two 3D structures of Tv20S with a specific covalently bound inhibitor.