Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay

Abstract

BackgroundThe emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs.MethodsParasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined.ResultsThe parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium–high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture ‘Malaria Box’.ConclusionThe quantification of new infections to determine parasite viability offers important advantages over existing methods, and is amenable to medium–high throughput screening. In particular, the parasite viability fast assay allows discrimination of rapidly parasiticidal anti-malarial candidates.

Evaluation of 4-Amino 2-Anilinoquinazolines against Plasmodium and Other Apicomplexan Parasites In Vitro and in a P. falciparum Humanized NOD- scid IL2Rγ null Mouse Model of Malaria

“…The results demonstrate that the 2-anilinoquinazolines 1, 2, and 4 display fast parasite killing kinetics comparable to those of artemisinin and chloroquine. Pyrimethamine and atovaquone showed a slower killing response in this assay, consistent with the findings described in the literature (27). This assay reconfirms existing data that the 2-anilinoquinazoline compound class kills parasites within the first 24 h of invasion of the erythrocyte (8).…”

“…In vitro P. falciparum viability kinetics. To determine the kinetics of the loss of viability in P. falciparum asexual stages following treatment with compounds 1, 2, and 4, we followed the protocol described by Linares et al (27). This assay measures the rate of parasite killing within 24 and 48 h of treatment.…”

New developments in anti-malarial target candidate and product profiles

“…These can be integrated to give a PRR tot [23], as a measure of the power of an individual molecule. The rank order of PRRs can be estimated in vitro [116, 117], or in SCID (severe combined immunodeficient) NOD-SCID IL2Rδ −/− mice [118]. Molecules can be initially ranked as very fast (such as PfATP4 inhibitors), fast (artesunate and other endoperoxides) and medium (as fast as mefloquine).…”

Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL), a Resistance Mechanism for Two Distinct Compound Classes

“…21,22 However, liver and gametocyte-stage screens show that this compound has limited activity against non-asexual stages. 23−28 Due to its drug-like properties, asexual blood stage potency in a drug-resistant line, fast/moderate parasite killing rate, 29 and novel chemical scaffold, we sought to gain insight into benzimidazolyl piperidine mechanisms of action and/or resistance development. We generated MMV007564-resistant (MMV007564 R ) asexual P. falciparum lines using an in vitro selection method (Figure 1) that has been previously used to associate compounds with their targets.…”