Online analysis of in vitro resistance to antimalarial drugs through nonlinear regression

Self Cite

v Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC 50 s) were determined by a modified sigmoid E max model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC 50 s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

High-Throughput Analysis of Antimalarial Susceptibility Data by the WorldWide Antimalarial Resistance Network (WWARN) In Vitro Analysis and Reporting Tool

Woodrow

Dahlström

Cooksey

et al. 2013

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“…Parasite susceptibility was tested in parallel against chloroquine diphosphate (Sigma-Aldrich) and artesunate (Holly Pharmaceuticals Co. Ltd.), as previously described (17). Doseresponse curves and IC 50 s using duplicate-well data for each drug concentration were determined using ICESTIMATOR (http://www.antimalarial -icestimator.net/MethodIntro.htm) (19,20).…”

Section: Methodsmentioning

confidence: 99%

Histone Methyltransferase Inhibitors Are Orally Bioavailable, Fast-Acting Molecules with Activity against Different Species Causing Malaria in Humans

Malmquist

Sundriyal

Caron

et al. 2015

l Current antimalarials are under continuous threat due to the relentless development of drug resistance by malaria parasites. We previously reported promising in vitro parasite-killing activity with the histone methyltransferase inhibitor BIX-01294 and its analogue TM2-115. Here, we further characterize these diaminoquinazolines for in vitro and in vivo efficacy and pharmacokinetic properties to prioritize and direct compound development. BIX-01294 and TM2-115 displayed potent in vitro activity, with 50% inhibitory concentrations (IC 50 s) of <50 nM against drug-sensitive laboratory strains and multidrug-resistant field isolates, including artemisinin-refractory Plasmodium falciparum isolates. Activities against ex vivo clinical isolates of both P. falciparum and Plasmodium vivax were similar, with potencies of 300 to 400 nM. Sexual-stage gametocyte inhibition occurs at micromolar levels; however, mature gametocyte progression to gamete formation is inhibited at submicromolar concentrations. Parasite reduction ratio analysis confirms a high asexual-stage rate of killing. Both compounds examined displayed oral efficacy in in vivo mouse models of Plasmodium berghei and P. falciparum infection. The discovery of a rapid and broadly acting antimalarial compound class targeting blood stage infection, including transmission stage parasites, and effective against multiple malaria-causing species reveals the diaminoquinazoline scaffold to be a very promising lead for development into greatly needed novel therapies to control malaria.T he continuous evolution of antimalarial drug resistance by Plasmodium parasites is a major impediment to the elimination of this devastating disease. Artemisinin combination therapies (ACTs) are the current mainstay of malaria chemotherapy, but the development of artemisinin resistance in parasites was reported in 2008 and 2009 along the Thai-Cambodian border (1). This underscores the need to validate new antimalarial targets within the parasite and to develop new antimalarial treatments based on novel scaffolds with desirable characteristics, such as fast killing activity against multiple parasite life stages; efficacy against multidrug-resistant strains and multiple species of human malaria parasites, including Plasmodium vivax; and favorable pharmacokinetics to allow oral administration.Epigenetic gene regulation mediated by histone-modifying enzymes has been shown to play an important role in malaria parasite transcriptional regulation, including the control of virulence genes involved in immune evasion (2, 3). Histone lysine methyltransferase (HKMT) enzymes present a novel potential target class for the development of antimalarials due to the association of histone methylation at distinct lysine positions with both overall transcriptional activation (H3K4me) and multicopy gene family transcriptional repression (H3K9me) (4, 5). Indeed, half of the identified Plasmodium falciparum HKMT enzymes were recently shown to be refractory to genetic disruption (6). The essential and important re...

“…The plates were incubated at ambient temperature for 30 min, and SYBR green I fluorescence was quantified using a FLUOstar OPTIMA instrument (BMG Labtech, Cary, NC). The IC 50 , defined as the drug concentration at which the SYBR green I signal was 50% of that measured from drug-free control wells, was calculated from the inhibitory sigmoid maximum-effect (E max ) model (ICEstimator, version 1.2; http://www.antimalarial-icestimator.net/Method Intro.htm) (21).…”

Section: Methodsmentioning

confidence: 99%

Ex Vivo Susceptibility of Plasmodium falciparum to Antimalarial Drugs in Western, Northern, and Eastern Cambodia, 2011-2012: Association with Molecular Markers

Lim

Dek

Try

et al. 2013

iIn 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC 50 s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC 50 s (GMIC 50 s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P < 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC 50 s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC 50 and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance.