Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action

Abstract

e High-throughput phenotypic screening of chemical libraries has resulted in the identification of thousands of compounds with potent antimalarial activity, although in most cases, the mechanism(s) of action of these compounds remains unknown. Here we have investigated the mode of action of 90 antimalarial compounds derived from the Malaria Box collection using high-coverage, untargeted metabolomics analysis. Approximately half of the tested compounds induced significant metabolic perturbations in in vitro cultures of Plasmodium falciparum. In most cases, the metabolic profiles were highly correlated with known antimalarials, in particular artemisinin, the 4-aminoquinolines, or atovaquone. Select Malaria Box compounds also induced changes in intermediates in essential metabolic pathways, such as isoprenoid biosynthesis (i.e., 2-C-methyl-D-erythritol 2,4-cyclodiphosphate) and linolenic acid metabolism (i.e., traumatic acid). This study provides a comprehensive database of the metabolic perturbations induced by chemically diverse inhibitors and highlights the utility of metabolomics for triaging new lead compounds and defining specific modes of action, which will assist with the development and optimization of new antimalarial drugs. Malaria remains a major global health problem, and there is a pressing need to discover and develop new antimalarials. Traditional antimalarial drugs have been severely undermined by the emergence of drug-resistant strains and current treatments rely heavily on artemisinin-based combination therapies. Alarmingly, artemisinin resistance has arisen in Southeast Asia during the last decade, highlighting the urgent need for new antimalarials (1). Extensive efforts to produce a malaria vaccine have met limited success and a pipeline of new antimalarial drugs will be required to mitigate extensive morbidity and mortality from malaria for the foreseeable future (2).High-throughput phenotypic screening of chemical libraries against the malaria parasite, Plasmodium falciparum, has provided a major step forward in the discovery of novel antimalarial compounds. Almost 30,000 compounds that selectively inhibit growth of cultured P. falciparum asexual red blood cell (RBC) stages have been identified in these screens, providing excellent starting points for the discovery of new antimalarial drugs (3-5). A prioritized collection of these "hit" compounds, known as the Malaria Box, has been assembled by the Medicines for Malaria Venture (MMV) and provided free to the research community to facilitate this drug development pipeline (6).A key limitation to the further optimization of many of these compounds is the lack of information on their mode of action. While not essential for registration, information on the mode of action of inhibitors discovered in phenotypic screens will significantly accelerate drug development by allowing structure-based drug design, monitoring of activity, toxicity and resistance, and facilitation of rational clinical usage in combination with other medicines. Furthermore, in...

Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate

“…Nevertheless, the specific metabolic profile observed in both the RhopH2 knockdown and furosemide-treated parasites (i.e decreased folate and phosphoenolpyruvate, increased threonine, histidine, asparagine, serine and argininosuccinate) supports a common impact on parasite biochemistry. This metabolic profile was not observed following treatment with 100 other antimalarial compounds using the same metabolomics methodology (Creek et al, 2016), suggesting this profile is specific for NPP inhibition and it is consistent with the increased threonine and histidine levels reported for other NPP inhibitors (where folate, phosphoenolpyruvate, serine and argininosuccinate were not assayed) (Dickerman et al, 2016). …”

“…The mechanism responsible for the observed down-regulation of pyrimidine synthesis and glycolysis is not clear, but may be secondary to a starvation response or compromised viability. Metabolites in these two pathways are particularly susceptible to depletion in parasites exposed to a range of antimalarial compounds (Creek et al, 2016). The observed increase in amino acids in the RhopH2 knockdown could be due to a reduced efflux of excess amino acids produced by haemoglobin digestion (Krugliak et al, 2002), which may otherwise render the infected cells susceptible to osmotic challenge or, alternatively, increased protein degradation to survive nutrient starvation, in a manner analogous to that observed following isoleucine starvation (Babbitt et al, 2012).…”

A high parasite density environment induces transcriptional changes and cell death in Plasmodium falciparum blood stages

“…A Dionex RSLC U3000 LC system (Thermo) coupled with a high‐resolution, Q‐Exactive MS (Thermo) was used for analysis of medium components, as described elsewhere . Briefly, 10 μL sample was separated on a ZIC‐pHILIC column (5 μm, 4.6 by 150 mm; Merck, Kenilworth, NJ, USA) over a 32 min gradient using 20 m m ammonium carbonate (A) and acetonitrile (B) as mobile phases.…”

Cholesterol-dependent enrichment of understudied erythrocytic stages of humanPlasmodiumparasites

“…The P. falciparum genome is poorly annotated (40% of genes) compared to many model organisms (85% of genes for S. cerevisiae), making metabolomics an attractive method for unbiased study of parasite biology (7,8). Indeed, Plasmodium metabolomics has become increasingly popular in recent years for investigation of parasite biology and antimalarial drug action (9)(10)(11)(12)(13)(14)(15)(16)(17)(18).…”