Periodic Reporting for period 2 - ProTeCT (Proteasome as a target to combat trichomoniasis)
Periodo di rendicontazione: 2021-11-01 al 2022-10-31
Trichomoniasis caused by Trichomonas vaginalis, a human protozoan parasite, is the most common non-viral sexually transmitted disease in the world. Estimates of the worldwide prevalence of trichomoniasis range from 170-180 million cases annually. Exact numbers are difficult to obtain because the infection is not nationally reportable, and many infections are asymptomatic. In woman the infection can result in serious consequences, such as infertility, pelvic inflammatory disease, premature rupture of placental membranes, preterm delivery and low-birth-weight infants. In men, it can cause epididymitis, prostatitis, and decrease sperm cell motility.
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.
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 outgoing phase at the University of San Diego I was focused on proteasome purification, and I have optimized the purification scheme for proteasomes from T. vaginalis. In parallel, I have developed a purification scheme for human proteasome from HeLa cells. It is important to isolate proteasome from human cells to ensure that proteasome inhibitors that are developed for T. vaginalis are selective for the pathogen enzyme over the human enzyme. proteasome over human counter. The optimized protocol has subsequently been used by students in the O’Donoghue lab for isolating proteasome from Giardia lamblia, Plasmodium falciparum, Trypanosoma brucei, Schistosoma mansoni, Ixodes ricinus, Babesia divergens and Candida albicans. Using the purified T. vaginalis proteasome, I identified subunit selective inhibitors to inactivate one, two or all three catalytically active subunits and used these tools to perform multiplex substrate profiling by mass spectrometry. In the substrate profiling assay I discovered peptide substrates that are selectively cleaved by each catalytic subunit and from these data, I developed and validated a set of new fluorogenic reporter substrates. These substrates are excellent tools for inhibitor screening. In parallel, with collaboration with Prof. Lars Eckmann, I have screened over 700 inhibitors from commercial libraries on live parasites and counter-screened against HeLa cells. The most promising compounds showed selectivity index ~50 (ratio of human cytotoxicity over trichomonacidal activity). I subsequently evaluated these inhibitors in the enzyme kinetic assay using the subunit-specific reporter substrates and confirmed that they a potent inhibitors of T. vaginalis proteosome while being weak inhibitors of the human proteasome. The top two compounds are being tested in the Eckmann lab in a murine model of Trichomonas infection.
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.
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.
The project results to date are in line with the timeline with the Annex 1 to the Grant Agreement. 1) successful purification protocol for isolating the Tv proteasome has been designed, 2) specific substrates for the three active sites of the proteasome (beta 1, beta 2 and beta 5) have been designed and synthesized, 3) toxicity has been determined for over 700 inhibitors from commercially available proteasome libraries. These fulfilled objectives lead to the development of a novel class of trichomonacidal agents.
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.
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.