New tools to facilitate drug discovery for pathogen proteasomes

Summary Parasitic diseases associated with poverty, including the neglected tropical diseases (NTDs), comprise a diverse group of conditions that afflict over a billion of the world's poorest people with devastating health, social and economic consequences. Current drugs have limitations, including insufficient efficacy, toxicity, prolonged treatment durations, and the emergence or reality of drug resistance; yet, drug R&D for NTDs has struggled historically due to a lack of financial incentives. The need for new drugs remains. An exciting drug target to emerge in the last decade for the treatment of parasitic infections is the proteasome, a multi-subunit, cytosolic protein that regulates protein homeostasis by degrading misfolded and senescent proteins. The 20S proteasome core comprises two stacked rings of seven β subunits sandwiched between two rings of α subunits. Three of the seven β subunits (β1, β2 and β5) are catalytically active, and small molecules that inhibit one or more of these subunits are now in clinical trials for treatment of leishmaniasis. Two key bottlenecks exist in advancing research regarding parasite proteasomes. First, the isolation of parasite 20S proteasome relies on purifying native enzyme from cell extracts, a time-consuming, multi-step procedure that yields limited enzyme, often with purity <50%, and that requires restrictive biosafety level 2 environments. A second bottleneck is the lack of activity-based probes (ABPs) with which the catalytic β subunits of parasite proteasomes can be detected, identified and characterized. Our proposal will address these bottlenecks with two independent scientific aims. First, using baculovirus and Sf9 insect cells, we will recombinantly express and purify, in one step, the 20s proteasome of three divergent and medically important parasites, namely, the apicomplexan protozoan, Plasmodium falciparum, the trypanosomatid protozoan, Trypanosoma brucei, and the platyhelminth bloodfluke, Schistosoma mansoni. Second, we will design and synthesize ABPs that react with all three catalytic β subunits of these parasite 20S proteasomes. We will use click chemistry to attach fluorescent and biotin reporters to these probes which will allow us to visualize the catalytic β subunits on protein gels and then enrich them for their formal identification by proteomics. These probes will be used to show on-target efficacy of proteasome inhibitors and will, therefore, aid in future medicinal chemistry campaigns. In summary, the proposed project will provide new methods and tools for the development of proteasome inhibitors as anti- parasitic drugs, and will stimulate wider interest in this emerging research field. Project Number: 1R21AI188258-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Anthony O'Donoghue (+1 co-PI) | Institution: UNIVERSITY OF CALIFORNIA, SAN DIEGO, LA JOLLA, CA | Award Amount: $438,500 | Activity Code: R21 | Study Section: Anti-Infective Resistance and Targets Study Section [AIRT] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI18825801A1