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.
