Project Summary. Malaria puts at risk 50% of the world’s population and is responsible for nearly 600,000 yearly deaths, mostly in children under the age of five in Africa. While a large portfolio of anti-malarial agents has been used to combat the disease, drug resistance has compromised the effectiveness of most clinically approved drugs, and the recent identification of resistance alleles against the current front line artemisinin combinations in Africa threatens current disease control programs. Thus, the identification of new drugs to combat drug resistant malaria is essential to continued progress against the disease. Our group in collaboration with Medicines for Malaria Venture (MMV) validated dihydroorotate dehydrogenase as a clinically valuable drug target for the treatment of malaria through studies on triazolopyrimidine DSM265, which advanced to phase II clinical development before the project was stopped due to discovery of off-target toxicity in preclinical species. In this current proposal we are working to identify new generation DHODH inhibitors by focusing on a different chemical series from DSM265, thus not expected to share an overlapping toxicity profile. Secondly, we plan structure-based approaches to identify inhibitors that will have reduced resistance risk compared to DSM265, which selected for resistance in 2 patients treated in the Phase II study. In aim one we plan to complete lead optimization of three related pyrazole-based DHODH inhibitor series, identified by scaffold hop using computational approaches (in collaboration with Schrödinger) from a pyrrole series we completed work on during the current fund period. Compounds from our pyrazole series have demonstrated high potency (sub nanomolar to low micromolar), and a reduced propensity to select for resistant parasites in vitro. We have a strong understanding of the SAR around these series, including the potency drivers, and the metabolic hot spots, and we plan mix and match chemistry to identify compounds with improved metabolic stability that will support human half-life (>100 h) and dosing targets (< 500 mg) set out by MMV. In aims 2 and 3 we use a combination of experimental and computational approaches (Schrödinger) to define the enzyme:ligand kinetic and thermodynamic binding properties that are associated with reduced resistance risk, as well as to correlate resistance risk to compound physical chemical properties. Computational models and measured thermodynamic/kinetic parameters will inform design and synthesis of new compounds predicted to have reduced resistance risk. The DHODH program is ideally suited to study the contribution of binding energetics to resistance propensity, as we have a wealth of structural information over three different chemical series with different physical chemical properties and alternative binding modes to the enzyme active site. Successful completion of these aims will allow identification of the strongest DHODH candidate for fu...