Tropical malaria, caused by the parasite Plasmodium falciparum, is responsible for nearly one million deaths each year. Since the parasite develops resistance against most clinically relevant drugs, novel antimalarial drugs are urgently needed. Glucose-6-phosphate dehydrogenase (G6PD) is a novel target for antimalarial drug design based on observations that humans with a genetic deficiency in this enzyme are protected against malaria. G6PD catalyzes the initial step in the pentose phosphate pathway, yielding NADPH, an essential reducing equivalent to detoxify oxidative stress in red blood cells (RBCs). The malaria parasite is susceptible to oxidative stress in the RBC stage. Naturally occurring G6PD deficiency leads to a lack of reducing equivalents, an increase in oxidative stress, and, as a consequence, to a protection against malaria. NADPH in parasite- infected RBCs is generated by human G6PD, but also by a parasite enzyme with G6PD activity, called P. falciparum glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (PfGluPho). PfGluPho knockdown and knockout leads to growth arrest and death of the parasite. Therefore, our overall objective is to develop PfGluPho inhibitors to kill the parasite and treat malaria. We were the first to clone and express recombinant PfGluPho, established a high-throughput screening assay, conducted medicinal chemistry, in vitro ADME and rodent pharmacokinetic studies and identified two probes, ML276 and ML304, that selectively inhibit PfGluPho (IC50 <1 M), but not human G6PD, which is critical for avoiding hemolytic toxicity. Both probes inhibit the growth of chloroquine-sensitive and -resistant parasites with IC50s in the low M range, but show limitations in microsomal stability and rodent pharmacokinetics. Based on these promising and extensive preliminary results, we now aim to continue our successful team approach to advance the two probes towards the development of novel antimalarial drugs. Specific Aim 1 designs and synthesizes novel analogs of ML276 and ML304. Specific Aim 2 determines their in vitro potency and selectivity, tests their in cellulo activity and toxicity, and evaluates their physicochemical properties. Specific Aim 3 determines their rodent pharmacokinetics and potential off-target effects. Specific Aim 4 assesses their potential hemolytic risk in human RBCs and executes proof-of-concept studies in a malaria mouse model. Results from the assays in Specific Aims 2 to 4 will feedback into the chemical design and synthesis process for further compound optimization described in Specific Aim 1. Complimentary skillsets, established collaborations and unparalleled resources foreshadow a proficient execution of the proposed aims with the goal to generate a potent, selective and stable PfGluPho inhibitor with in vitro, in cellulo and in vivo activity against malaria parasites. This approach has the potential to generate high impact results towards developing novel and desperately needed antimalarial drugs and to help treat and eradicate one of the most deadly diseases in the world.