Date

6-2014

Document Type

Dissertation

Degree Name

Ph.D.

Department

Department of Physiology and Pharmacology

Institution

Oregon Health & Science University

Abstract

Malaria is a devastating parasitic disease that affects more than 200 million people and is a leading cause of mortality in the developing world. Although many potent antimalarial therapies exist, the effectiveness of these compounds has been limited by the emergence of drug resistant Plasmodium parasites, the cost of multi-day treatment, and restrictive mechanisms of action, which often fail to protect against the sexual or liver stage parasites that are responsible for disease transmission. A major priority for antimalarial drug development, and his project, is the identification of multi-stage therapies that are broadly effective as treatments, prophylactics, and transmission blocking agents following a single, oral dose.

This project focuses specifically on the cytochrome bc1 complex (cyt bc1) as a target for single-dose, multi-stage therapy. Biologically, cyt bc1 is essential for the regeneration of ubiquinone, which plays an essential role in major metabolic processes for P. falciparum, including heme and pyrimidine biosynthesis. While various cyt bc1 inhibitors, including atovaquone (ATV) and ELQ-300, are effective single-dose prophylactics, they have not previously demonstrated single-dose, curative activity against blood-stage parasites. For that reason, I first evaluated the 4(1H)-quinolone compound, ELQ-400, as a more potent, in vivo alternative to ATV and ELQ-300.

In single-dose murine models, ELQ-400 rapidly reduced blood-stage parasitemia and prevented recrudescence at low doses of 1 mg/kg. In order to understand the mechanism of the single-dose response to ELQ-400, I next assessed how 4(1H)-quinolones interacted with cyt bc1. I used drug sensitivity screens against P. falciparum clones containing selective mutations at either the oxidative (Qo) or reductive (Qi) sites of cyt bc1 to create a structure activity (SAR) profile of Qo vs. Qi targeting for the ELQs. I found that, while 5 and 7-position substituents were broadly associated with Qo-targeting, aryl side chains and 6-halogens contributed to Qi site inhibition. Intriguingly, ELQ-400 contained both Qo and Qi directing features, suggesting a role as potential dual-site inhibitor of cyt bc1. Accordingly, ELQ-400 demonstrated signs of both Qi and Qo site inhibition in cytochrome b reduction assays conducted in P. denitrificans.

Strikingly, I found that the single-dose activity of ELQ-400 could be effectively replicated by co-administration of the respective Qo and Qi site inhibitors, ATV and ELQ-300. Acute assessment of ATV vs. ELQ-300 therapy also revealed several specific differences that may be attributable to general Qo vs. Qi site inhibition. While ATV therapy initiated more rapid parasite clearance, ELQ-300 treatment was associated with both a lower resistance propensity and a cumulative multi-day dosing effect. Importantly, both ELQ-400 and ATV:ELQ-300 combination therapy combined the desirable clinical features of ATV and ELQ-300 monotherapy, suggesting that dual-site inhibition of cyt bc1 is a viable strategy for multi-stage, single-dose antimalarial treatment.

Identifier

doi:10.6083/M4Z31XDG

School

School of Medicine

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