Profiling malarial lipid biosynthesis and key acyl-coenzyme A synthetase activities with fatty acid alkynes

ABSTRACT Malaria is a major public health burden, causing over a half million deaths and two hundred million clinical episodes annually. During the pathogenic, asexual erythrocytic stage, the malaria parasite scavenges the fatty acids (FA) it needs for lipid synthesis from the host’s plasma. This metabolic

requirement is a vulnerability that could be exploited to block parasite replication. Two parasite acyl- Coenzyme A synthetases, ACS10 and 11, likely play key roles by activating fatty acids to acyl-CoA thioesters, although little is known about their specific roles. Mutations in both enzymes have been

associated with resistance to anti-malarial compounds, with ACS10 shown to be a direct target, and an ACS inhibitor is currently undergoing phase I clinical trials. Thus, a deeper understanding of the roles of ACS10 and 11 could accelerate drug discovery efforts. We hypothesize that ACS10 and 11 each make important contributions to parasite FA uptake through distinctive specificities. In this

proposal, we will develop a new approach, termed “FA alkyne profiling”, for the systematic analysis of FA uptake and lipid biosynthesis in P. falciparum and will employ it to gain insights into the roles and specificities of ACS10 and 11 and the effects of ACS inhibition. In Aim 1, we will optimize FA alkyne

profiling in P. falciparum using six structurally-diverse fatty acid alkynes that together represent three- quarters of P. falciparum fatty acids. Each FA alkyne probe will be validated in competition assays with natural FAs and in parasite viability assays. These studies will provide a quantitative basis for

exploring the effects of perturbations in parasite fatty acid metabolism. In Aim 2, we will employ FA alkyne probes to interrogate the physiological roles of ACS10 and 11. Parasite lines capable of inducible knockdown of ACS10 or 11 will be profiled to establish the effects of enzyme depletion on FA alkyne utilization. We will also use FA alkyne profiling to investigate the effects of two validated

ACS inhibitors with anti-malarial activity. These studies will provide insights into the physiological roles of specific parasite ACSs and will establish a general framework for investigating he effects of inhibition or knockdown of key lipid metabolic enzymes.