Investigating drug targets in infectious diseases with genetically engineered yeast systems

Abstract

Communicable diseases are the cause of an estimated 10 million deaths yearly, and low-income regions are heavily affected. Malaria cases and deaths are on the rise again after decades of decline, and several new epidemics have emerged in the 21st century, including the devastating covid-19 pandemic; new diseases emerge, others are neglected, and pathogens become resistant. Taken together, there is a persistent need to invent and develop new drugs. In this thesis, we investigate several drug targets in infectious diseases using genetically modified yeast and provide multiple tools in the drug discovery pipeline. We designed a yeast reporter to detect inhibitors of the SARS-CoV-2 Main protease, and scaled it to an automated high-throughput drug screen, identifying boron-containing inhibitors that were previously only found in virtual screens. Moreover, we establish target-based platforms in yeast to identify inhibitors of two proteins in Plasmodium spp., the causative agents of malaria. In one setting, we focus on the P. vivax deoxyhypusine synthase (DHS), which catalyzes the first step in a unique post-translational modification of the eukaryotic translation factor 5A. Here, we apply both a virtual screen and a chemical library screen in yeast and identify three compounds that preferably reduce growth of the P. vivax DHS expressing yeast strain but not the human counterpart. In another setting, we set out to explore novel targets for antimalarials and find the proteasome subunit Rpn11 to be a suitable candidate. We attempt to validate essentiality by constructing transgenic blood stage P. falciparum parasites with knockdown elements, and establish target-based platforms in yeast and in vitro that enable us to identify selective inhibitors of P. falciparum Rpn11. Lastly, using directed evolution experiments in yeast, we can characterize the cellular adaptation to Rpn11 inhibition with limited acquired resistance.