The Parasite Reduction Ratio (PRR) Assay Version 2: Standardized Assessment of Plasmodium falciparum Viability after Antimalarial Treatment In Vitro

Abstract

With artemisinin-resistant Plasmodium falciparum parasites emerging in Africa, the need for new antimalarial chemotypes is persistently high. The ideal pharmacodynamic parameters of a candidate drug are a rapid onset of action and a fast rate of parasite killing or clearance. To determine these parameters, it is essential to discriminate viable from nonviable parasites, which is complicated by the fact that viable parasites can be metabolically inactive, whilst dying parasites can still be metabolically active and morphologically unaffected. Standard growth inhibition assays, read out via microscopy or [3H] hypoxanthine incorporation, cannot reliably discriminate between viable and nonviable parasites. Conversely, the in vitro parasite reduction ratio (PRR) assay is able to measure viable parasites with high sensitivity. It provides valuable pharmacodynamic parameters, such as PRR, 99.9% parasite clearance time (PCT99.9%) and lag phase. Here we report the development of the PRR assay version 2 (V2), which comes with a shorter assay duration, optimized quality controls and an objective, automated analysis pipeline that systematically estimates PRR, PCT99.9% and lag time and returns meaningful secondary parameters such as the maximal killing rate of a drug (Emax) at the assayed concentration. These parameters can be fed directly into pharmacokinetic/pharmacodynamic models, hence aiding and standardizing lead selection, optimization, and dose prediction.

Next Generation Chemiluminescent Probes for Antimalarial Drug Discovery

“…As detailed in the Materials and Methods section, we thus recommend including a control that quantifies residual β-galactosidase activity derived from nonviable parasites. The most recent PRR protocol (version 2) requires a 14-day regrowth phase using the [ 3 H]-hypoxanthine incorporation readout . Here, we used the same streamlined protocol for the chemiluminescence-based readout.…”

“…Parasite Reduction Ratio Assay. PRR assays were conducted according to the new PRR assay version 2 protocol published by Walz et al, 47 which was adapted from Sanz et al 30 The [ 3 H]-and β-gal SENSOR -based PRR assays were performed using transparent and white 96-well plates, respectively. Compound stock solutions were diluted to 100× the final assay concentration using CM.…”

“…PRR assays were conducted according to the new PRR assay version 2 protocol published by Walz et al, which was adapted from Sanz et al . The [ 3 H]- and β-gal SENSOR -based PRR assays were performed using transparent and white 96-well plates, respectively.…”

Translation inhibition efficacy does not determine thePlasmodium bergheiliver stage antiplasmodial efficacy of protein synthesis inhibitors

“…Cultures are then monitored for several weeks to detect parasite recrudescence and calculate the number of viable parasites present. When compounds are tested at equivalent effective concentrations, the PRR assay reveals compound specific differences that are masked in 48- or 72 h growth assays, where compounds often appear equally effective ( 36 ). This is also true for standard liver stage assays that quantify EEF biomass ( 37, 38 ), typically after 48 h of continuous treatment starting about the time of sporozoite addition, where any compound active against the EEF at the earliest stage of development will present a similarly dramatic reduction in biomass.…”

Accelerating antimalarial drug discovery with a new high-throughput screen for fast-killing compounds

“…However, SERCs characteristically require fast-killing, as seen in next-generation lead compounds such as MMV688533, INE963, ZY19489, and GSK484, the subjects of ongoing clinical trials [10][11][12][13] . The standard method employed to determine the speed of killing of parasites, the parasite reduction ratio (PRR), first was described in 2012; since then PRR has been utilized widely to assess the killing properties of candidate compounds 14,15 . The conventional PRR assay has a relatively low throughput due to the complexity of the protocol, requiring at least one month to yield results.…”