A semi-automated luminescence based standard membrane feeding assay identifies novel small molecules that inhibit transmission of malaria parasites by mosquitoes.

Martijn W Vos,Will J R Stone,Teun Bousema,Koen J Dechering,Ben Van Schaijk,Karin M Koolen,Robert W Sauerwein,Geert-Jan Van Gemert,Didier Leroy

doi:10.1038/srep18704

Abstract

Current first-line treatments for uncomplicated falciparum malaria rapidly clear the asexual stages of the parasite, but do not fully prevent parasite transmission by mosquitoes. The standard membrane feeding assay (SMFA) is the biological gold standard assessment of transmission reducing activity (TRA), but its throughput is limited by the need to determine mosquito infection status by dissection and microscopy. Here we present a novel dissection-free luminescence based SMFA format using a transgenic Plasmodium falciparum reporter parasite without resistance to known antimalarials and therefore unrestricted in its utility in compound screening. Analyses of sixty-five compounds from the Medicines for Malaria Venture validation and malaria boxes identified 37 compounds with high levels of TRA (>80%); different assay modes allowed discrimination between gametocytocidal and downstream modes of action. Comparison of SMFA data to published assay formats for predicting parasite infectivity indicated that individual in vitro screens show substantial numbers of false negatives. These results highlight the importance of the SMFA in the screening pipeline for transmission reducing compounds and present a rapid and objective method. In addition we present sixteen diverse chemical scaffolds from the malaria box that may serve as a starting point for further discovery and development of malaria transmission blocking drugs.

Highlights

Activation in vitro measured by the presence of female and male gametes[11,12,13]
We previously described the use of a parasite line expressing a fusion of the green fluorescent and firefly luciferase proteins (NF54HT-GFP-Luc) in a new standard membrane feeding assay (SMFA) format based on the measurement of luciferase activity in groups of homogenised individual or pooled mosquitoes[15]
The results of the current study demonstrate the versatility of the SMFA for screening compounds with activity against different transmission stages of the malaria parasite, and validate the luminescent assay format in combination with microtiter plate-based processing of mosquitoes for improving the efficiency of the biological gold-standard for assessing Plasmodium transmission in controlled experiments

Summary

Introduction

Activation in vitro measured by the presence of female and male gametes[11,12,13]. Such assays are, not always predictive of blocking transmission of the parasite to the mosquito, and agents with putative TRA are best tested in the standard membrane feeding assay (SMFA). We previously described the use of a parasite line expressing a fusion of the green fluorescent and firefly luciferase proteins (NF54HT-GFP-Luc) in a new SMFA format based on the measurement of luciferase activity in groups of homogenised individual or pooled mosquitoes[15] Very effective, this strain may be sub-optimal for drug screening; introduction of the human dihydrofolate reductase (hDHFR) selection marker during integration of the GFP-Luc expression cassette renders it resistant to antifolates[16] and makes it necessary to maintain exposure to antifolate compound WR99210 during parasite culture to prevent reversion to wild-type (WT), possibly modifying its response to other inhibitors by affecting parasite metabolism. Our more scalable luciferase based read-out increases throughput of the assay and makes compound screening with the SMFA less resource intensive than when performed with microscopy

Methods

Results

Discussion

Conclusion