There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc 1 . Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.T he discovery of atovaquone 20 years ago validated the malaria parasite's mitochondrial electron transport chain (ETC) as an exploitable drug target. Atovaquone targets the ETC at the level of the bc 1 complex (1), with inhibition preventing proton pumping, resulting in a loss of mitochondrial membrane potential (2) and eventual organelle dysfunction, an important function of which is to provide intermediates for pyrimidine synthesis (3, 4). The bc 1 complex requires reducing equivalents provided by ubiquinol, which in turn is generated by membrane-bound dehydrogenases upstream in the ETC that catalyze redox reactions by reducing ubiquinone. The parasite lacks the canonical protonmotive NADH dehydrogenase (Complex I) but instead harbors a bacterial-like type II NADH:ubiquinone oxidoreductase, Plasmodium falciparum NDH2 (PfNDH2) (5). Based on these key observations, we undertook a drug-discovery initiative to develop costeffective inhibitors capable of inhibiting PfNDH2 with the goal of providing antimalarials that overcome the limitations of the expensive atovaquone. Although our initial drug-discovery efforts were focused on optimization of activity versus PfNDH2, we found, during hit-to-lead development, that optimized structures with single-digit nanomolar activity versus the primary target ...

SummaryIt is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQsensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energydependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.

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