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  • Funding sources This study was funded by European Community

    2019-09-19

    Funding sources This study was funded by European Community’s Seventh Framework Programme under grant agreement No. 305662 (Project: Community-based scheduled screening and treatment of malaria in pregnancy for improved maternal and infant health: a cluster-randomized trial ‘COSMIC’).
    Acknowledgements We would like to thank all participants and the health and study staff of the Unité de Recherche centre de Nanoro. We would also like to thank MR4 for providing us with malaria parasites contributed by ATCC®.
    Introduction The global burden of malaria has been steadily declining over recent years, generating hope that the disease can be eradicated (Tanner et al., 2015). A major obstacle to this goal is the reliance on antimalarial chemotherapy to control the parasite, as vector control and vaccination strategies are inadequate. New drugs with novel modes of action (MoA) will be needed to overcome parasite resistance to existing drugs, and SB 366791 clinical to eliminate parasites at different stages in the life-cycle. The targets and MoA of many antimalarials are unknown. Large-scale empirical screening studies have uncovered thousands of novel antimalarial compounds encompassing a wide diversity of chemical structures (Plouffe et al., 2008, Gamo et al., 2010, Guiguemde et al., 2010). The main problem now for antimalarial development is how to triage these numerous compounds for further development to achieve the goal of obtaining a variety of new drugs differing in MoA. The putative targets for some of these compounds have been suggested from similarity in chemical structure to known drugs, and biochemical methods such as candidate enzyme inhibition assays and thermal melt assay with recombinant proteins (Guiguemde et al., 2012). However, it is difficult to be certain of the target specificity of these compounds in vivo, especially for assumed targets that share functional domains with many other proteins, e.g. kinases. Another approach for determining MoA of antimalarial compounds is the genetic approach of induced resistance and whole genome sequencing to identify resistance mutations in genes encoding putative target proteins. The approach has been very successful in identifying a few compounds with novel MoA against parasite targets that would likely not have been found using current biochemical methods, e.g. spiroindolones targeting PfATP4 (Rottmann et al., 2010). However, this genetic method is impractical for a large-scale MoA study. Moreover, induced resistance mutations may still fail to provide clues as to direct targets for some drugs, e.g. artemisinin (Ariey et al., 2013). For these reasons, target-based antimalarial discovery will become increasingly more important to identify drugs with new MoA. Target-based drug discovery entails biochemical screening, e.g. enzyme inhibition assay of SB 366791 clinical libraries. The “hit” compounds from these screens are used as starting points for drug development (Flannery et al., 2013), in which MoA are assumed from the correlation of in vitro and in vivo activities. The next phase of “hit to lead” drug development requires synthesis and testing of structurally similar compounds. It is in this phase that problems of target specificity can arise. An instructive example of this problem is that of triclosan and derivatives, in which it was found that the antimalarial activity of this class of compound is not due to inhibition of enoyl-ACP reductase (FabI), the initially assumed target (Yu et al., 2008). Target-based antimalarial drug discovery would benefit from a method that can demonstrate MoA directly in the parasite, i.e. validate that different, but pharmacologically related, compounds exert their antimalarial effect by inhibiting the same target in vivo. Chemogenomic profiling (reviewed in Nijman, 2015) is another way to identify drug MoA that have been neglected for the study of antimalarials. In a narrow sense, this approach entails perturbing the activities of different genes using forward or reverse genetic methods. The cell lines with different genetic perturbations are then tested with different compounds in a phenotypic assay, e.g. growth. Recently, a forward genetic chemogenomic screen using 71 piggyBac transposon insertion lines of Plasmodium falciparum revealed gene–drug interactions, giving new insight into drug MoA (Pradhan et al., 2015). Chemogenomic data obtained from such forward genetic screens can be difficult to interpret though, since the transposon often inserts in gene flanking regions (Balu et al., 2009). The effect of transposon insertion on neighbouring gene function is thus variable. Furthermore, loss-of-function transposon insertions will rarely be obtained for essential Plasmodium genes owing to the haploid nature of the parasite in asexual stages. Since the majority of antimalarial targets are essential in the asexual stages, forward-genetic chemogenomic profiling screens are less likely to identify the direct target of the drug, but rather modifiers of either the drug itself or the affected pathway. On the other hand, chemogenomic profiling using reverse genetic tools that can give robust reductions in essential gene activities are frequently successful at identifying direct drug targets (Nijman, 2015). Reverse genetic chemogenomic profiling in Plasmodium requires genetic tools for conditional attenuation of gene expression. Of the reverse genetic tools available for Plasmodium spp. (reviewed in de Koning-Ward et al., 2015), the glmS ribozyme tool developed by our group has been demonstrated to give robust attenuation of different P. falciparum genes, including essential genes and drug targets (Prommana et al., 2013, Elsworth et al., 2014, Sleebs et al., 2014, McHugh et al., 2015, Xie et al., 2015, Chisholm et al., 2016). In this work, we explored the glmS ribozyme tool for application in reverse genetic chemogenomic profiling to assess MoA of different antimalarial compounds. Chemogenomic profiling was performed using parasites with attenuated expression of DHFR-TS. We show that our approach is robust and can identify DHFR-TS as the target of antifolate drugs in P. falciparum (in both pyrimethamine-sensitive and -resistant backgrounds), and in Plasmodium berghei. In a chemogenomic screen of the Malaria Box compound library, we identified two compounds as novel DHFR-TS inhibitors.