Discovery of Potent Small-Molecule Inhibitors of Multidrug-Resistant Plasmodium falciparum Using a Novel Miniaturized High-Throughput Luciferase-Based Assay

Abstract

Malaria is a global health problem that causes significant mortality and morbidity, with more than 1 million deaths per year caused by Plasmodium falciparum. Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites, which necessitates the discovery of new drugs. To identify new antimalarials, we developed an automated 384-well plate screening assay using P. falciparum parasites that stably express cytoplasmic firefly luciferase. After initial optimization, we tested two different types of compound libraries: known bioactive collections (Library of Pharmacologically Active Compounds [LOPAC] and the library from the National Institute of Neurological Disorders and Stroke [NINDS]) and a library of uncharacterized compounds (ChemBridge). A total of 12,320 compounds were screened at 5.5 M. Selecting only compounds that reduced parasite growth by 85% resulted in 33 hits from the combined bioactive collection and 130 hits from the ChemBridge library. Fifteen novel drug-like compounds from the bioactive collection were found to be active against P. falciparum. Twelve new chemical scaffolds were found from the ChemBridge hits, the most potent of which was a series based on the 1,4-naphthoquinone scaffold, which is structurally similar to the FDA-approved antimalarial atovaquone. However, in contrast to atovaquone, which acts to inhibit the bc 1 complex and block the electron transport chain in parasite mitochondria, we have determined that our new 1,4-napthoquinones act in a novel, non-bc 1 -dependent mechanism and remain potent against atovaquone-and chloroquine-resistant parasites. Ultimately, this study may provide new probes to understand the molecular details of the malaria life cycle and to identify new antimalarials.

Identification of MMV Malaria Box Inhibitors of Plasmodium falciparum Early-Stage Gametocytes Using a Luciferase-Based High-Throughput Assay

“…Sexual differentiation was triggered by applying a combination of stress factors known to stimulate gametocytogenesis in vitro, namely, a drop in hct, a high parasitemia, and the consequent hemolysis (31) due to schizont rupture, as well as nutritional stress (32,33). Luciferase-based approaches have previously been shown to be useful for the development of high-throughput assays for compound screening against the asexual stages of P. falciparum (34)(35)(36). Our HTS assay for early gametocytogenesis was developed using the P. falciparum transgenic line NF54…”

A transgenic Plasmodium falciparum NF54 strain that expresses GFP–luciferase throughout the parasite life cycle

“…The ability to easily test potential new interventions on all life cycle stages of the malaria parasite is essential to accelerate translational research. To this end, Plasmodium falciparum parasites expressing luciferase have been created to assess the role of interventions on both asexual blood stage replication and sexual blood stage development [2][3][4][5]. To test interventions on pre-erythrocytic development, a transgenic P. falciparum parasite that is a vigorous gametocyte producer and infects mosquitoes reliably is required and this parasite must then express quantifiable amounts of luciferase.…”

A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3′,4′-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum

“…Of the 25 compounds chosen for our present study, 17 were part of the Sigma-Aldrich LOPAC1280 (Library of Pharmacologically Active Compounds) and the National Institute of Neurological Diseases and Stroke drug library and were previously revealed to possess antimalarial activity (10). Of the remaining eight, mefloquine, halofantrine, artemisinin, and sulfadoxine are conventional malaria chemotherapies and were screened to provide insight into their modes of action.…”