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.
