Malaria remains a major threat to public health and economic development in the tropical and sub- tropical regions of the globe, and to travellers from the developed world. Insecticide-impregnated bednets and artemisinins have significantly reduced malaria infections and deaths, but history shows that the malaria parasite Plasmodium and the Anopheles mosquitoes that transmit malaria can eventually evade control efforts. Continual development of new drugs and vaccines is essential to ensure that malaria control is maintained to avoid unnecessary illness and loss of life. Biochemical and molecular genetic studies funded by NIAID revealed that malaria parasites use a two-enzyme biochemical pathway, indirect aminoacylation, for protein synthesis in the apicoplast, a plastid-like organelle. Both enzymes, glutamyl-tRNA synthetase (PfGluRS) and glutamyl-tRNA amidotransferase (PfGluAdT), are nuclear-encoded but imported into the apicoplast. Chemical inhibition or genetic ablation of this pathway is lethal to blood stages of the malaria parasite P. falciparum, suggesting that new drugs that target indirect aminoacylation would be useful for malaria treatment or prevention. This project will develop assays for use in high-throughput screens (HTSs) to identify chemical compounds that inhibit these enzymes, serve as chemical probes, and provide novel starting points for drug discovery. These assays could also be used to interrogate the biochemical properties of the two enzymes that comprise the pathway, providing chemical insights into the enzyme's mechanism of action and molecular structure that can be exploited for drug development. Once the assays have been developed and validated, pilot screens of 5,000 - 20,000 compounds from the FDA collection, LOPAC and diversity sets will be performed to gauge assay performance and provide preliminary data to guide design of future screens of the ~385,000 compound Molecular Libraries Small Molecules Repository (MLSMR). Secondary and tertiary assays that rule out assay artifacts, insufficiently potent or non-selective compounds, will be developed and validated. Ultimately, compounds with significant enzyme-inhibitory activity will be tested to determine whether they can kill malaria parasites in vitro, and the effects of growth-inhibitory compounds on parasite morphology will be examined to understand the biological effects of enzyme inhibition on the parasite's red blood cell stages. At the conclusion of this project, assays suitable for large-scale, high-throughput screening and hit validation campaigns to identify novel potent and selective PfGluRS and PfGluAdT inhibitors will be available, and new insights into the roles played by the indirect aminoacylation pathway in malaria parasites will stimulate further basic and applied research.
There are approximately 200 million malaria cases and 500,000 deaths due to malaria every year. This research will develop and validate a high-throughput laboratory test that can be deployed on a large-scale to identify compounds that inhibit a parasite-specific metabolic pathway that we identified in the malaria parasite's apicoplast. The validated test will be invaluable in subsequent efforts to develop new pharmaceuticals that kill malaria parasites by specifically obstructing the targeted metabolic pathway.
