Metabolomic Profiling of the Malaria Box Reveals Antimalarial Target Pathways

ABSTRACT The threat of widespread drug resistance to frontline antimalarials has renewed the urgency for identifying inexpensive chemotherapeutic compounds that are effective against Plasmodium falciparum, the parasite species responsible for the greatest number of malaria-related deaths worldwide. To aid in the fight against malaria, a recent extensive screening campaign has generated thousands of lead compounds with low micromolar activity against blood stage parasites. A subset of these leads has been compiled by the Medicines for Malaria Venture (MMV) into a collection of structurally diverse compounds known as the MMV Malaria Box. Currently, little is known regarding the activity of these Malaria Box compounds on parasite metabolism during intraerythrocytic development, and a majority of the targets for these drugs have yet to be defined. Here we interrogated the in vitro metabolic effects of 189 drugs (including 169 of the drug-like compounds from the Malaria Box) using ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS). The resulting metabolic fingerprints provide information on the parasite biochemical pathways affected by pharmacologic intervention and offer a critical blueprint for selecting and advancing lead compounds as next-generation antimalarial drugs. Our results reveal several major classes of metabolic disruption, which allow us to predict the mode of action (MoA) for many of the Malaria Box compounds. We anticipate that future combination therapies will be greatly informed by these results, allowing for the selection of appropriate drug combinations that simultaneously target multiple metabolic pathways, with the aim of eliminating malaria and forestalling the expansion of drug-resistant parasites in the field.

[1]  Didier Leroy,et al.  Anti-malarial drugs: how effective are they against Plasmodium falciparum gametocytes? , 2012, Malaria Journal.

[2]  A. Vaidya,et al.  Mitochondrial Electron Transport Inhibition and Viability of Intraerythrocytic Plasmodium falciparum , 2010, Antimicrobial Agents and Chemotherapy.

[3]  Christopher M. Armstrong,et al.  Plasmodium IspD (2-C-Methyl-D-erythritol 4-Phosphate Cytidyltransferase), an Essential and Druggable Antimalarial Target. , 2015, ACS infectious diseases.

[4]  P. Rathod,et al.  Triazolopyrimidine-based dihydroorotate dehydrogenase inhibitors with potent and selective activity against the malaria parasite Plasmodium falciparum. , 2008, Journal of medicinal chemistry.

[5]  K. Trenholme,et al.  Plasmodium falciparum gametocytes: with a view to a kill , 2013, Parasitology.

[6]  Victoria C. Corey,et al.  Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) Confer Multidrug Resistance , 2016, mBio.

[7]  R. Amewu,et al.  Rapid kill of malaria parasites by artemisinin and semi-synthetic endoperoxides involves ROS-dependent depolarization of the membrane potential , 2013, The Journal of antimicrobial chemotherapy.

[8]  R. Sinden,et al.  A Male and Female Gametocyte Functional Viability Assay To Identify Biologically Relevant Malaria Transmission-Blocking Drugs , 2014, Antimicrobial Agents and Chemotherapy.

[9]  Silvia Parapini,et al.  A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection. , 2013, The Journal of antimicrobial chemotherapy.

[10]  Ruili Huang,et al.  Chemical signatures and new drug targets for gametocytocidal drug development , 2014, Scientific Reports.

[11]  G. Siuzdak,et al.  Innovation: Metabolomics: the apogee of the omics trilogy , 2012, Nature Reviews Molecular Cell Biology.

[12]  L. Maes,et al.  Repurposing of the Open Access Malaria Box for Kinetoplastid Diseases Identifies Novel Active Scaffolds against Trypanosomatids , 2015, Journal of biomolecular screening.

[13]  Jeremy N. Burrows,et al.  P. falciparum In Vitro Killing Rates Allow to Discriminate between Different Antimalarial Mode-of-Action , 2012, PloS one.

[14]  Joanne M. Morrisey,et al.  Na+ Influx Induced by New Antimalarials Causes Rapid Alterations in the Cholesterol Content and Morphology of Plasmodium falciparum , 2016, PLoS pathogens.

[15]  James R. Brown,et al.  Thousands of chemical starting points for antimalarial lead identification , 2010, Nature.

[16]  Yesmalie Alemán Resto,et al.  Identification of MMV Malaria Box Inhibitors of Perkinsus marinus Using an ATP-Based Bioluminescence Assay , 2014 .

[17]  S. Hay,et al.  G6PD deficiency: global distribution, genetic variants and primaquine therapy. , 2013, Advances in parasitology.

[18]  Hongshen Ma,et al.  (+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium , 2014, Proceedings of the National Academy of Sciences.

[19]  J. Derisi,et al.  A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis , 2014, Antimicrobial Agents and Chemotherapy.

[20]  X. Su,et al.  Malaria biology and disease pathogenesis: insights for new treatments , 2013, Nature Medicine.

[21]  D. Wirth,et al.  Triaminopyrimidine is a fast-killing and long-acting antimalarial clinical candidate , 2015, Nature Communications.

[22]  A. Björkman,et al.  In vitro recrudescence of Plasmodium falciparum parasites suppressed to dormant state by atovaquone alone and in combination with proguanil. , 2005, Transactions of the Royal Society of Tropical Medicine and Hygiene.

[23]  D. Wright,et al.  Identification of β-hematin inhibitors in the MMV Malaria Box , 2015, International journal for parasitology. Drugs and drug resistance.

[24]  R. Sinden,et al.  The effect of atovaquone (566C80) on the maturation and viability of Plasmodium falciparum gametocytes in vitro. , 1996, Transactions of the Royal Society of Tropical Medicine and Hygiene.

[25]  M. Fry,et al.  Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80). , 1992, Biochemical pharmacology.

[26]  J. Keiser,et al.  Orally Active Antischistosomal Early Leads Identified from the Open Access Malaria Box , 2014, PLoS neglected tropical diseases.

[27]  M. Barrett,et al.  Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics , 2014, PLoS neglected tropical diseases.

[28]  Kellen L. Olszewski,et al.  Host-parasite interactions revealed by Plasmodium falciparum metabolomics. , 2009, Cell host & microbe.

[29]  M. Barrett,et al.  Host Reticulocytes Provide Metabolic Reservoirs That Can Be Exploited by Malaria Parasites , 2015, PLoS pathogens.

[30]  D. Sullivan,et al.  Gametocytocidal Screen Identifies Novel Chemical Classes with Plasmodium falciparum Transmission Blocking Activity , 2014, PloS one.

[31]  J. Richard,et al.  Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5* , 2014, The Journal of Biological Chemistry.

[32]  J. McCarthy,et al.  Effect of antimalarial drugs on Plasmodium falciparum gametocytes. , 2009, The Journal of infectious diseases.

[33]  Elizabeth A. Winzeler,et al.  Mutations in the P-Type Cation-Transporter ATPase 4, PfATP4, Mediate Resistance to Both Aminopyrazole and Spiroindolone Antimalarials , 2014, ACS chemical biology.

[34]  Peter G. Schultz,et al.  In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen , 2008, Proceedings of the National Academy of Sciences.

[35]  S. C. T. P. Rts.  Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial , 2015, The Lancet.

[36]  N. Nelson,et al.  Plasmodium falciparum chloroquine resistance transporter is a H+-coupled polyspecific nutrient and drug exporter , 2015, Proceedings of the National Academy of Sciences.

[37]  Manuel Llinás,et al.  Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond , 2016, PLoS pathogens.

[38]  M. Cassera,et al.  Antiapicoplast and Gametocytocidal Screening To Identify the Mechanisms of Action of Compounds within the Malaria Box , 2013, Antimicrobial Agents and Chemotherapy.

[39]  Robert W. Sauerwein,et al.  Targeting Plasmodium PI(4)K to eliminate malaria , 2013, Nature.

[40]  V. Avery,et al.  Identification of inhibitors of Plasmodium falciparum gametocyte development , 2013, Malaria Journal.

[41]  Kellen L. Olszewski,et al.  Reconstruction and flux-balance analysis of the Plasmodium falciparum metabolic network , 2010, Molecular systems biology.

[42]  Joshua D. Rabinowitz,et al.  A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites , 2006, Journal of the American Society for Mass Spectrometry.

[43]  M. Barrett,et al.  Determination of antiprotozoal drug mechanisms by metabolomics approaches , 2013, Parasitology.

[44]  Stuart L. Schreiber,et al.  High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria , 2016, ACS infectious diseases.

[45]  Kellen L. Olszewski,et al.  Central carbon metabolism of Plasmodium parasites. , 2011, Molecular and biochemical parasitology.

[46]  Stuart L. Schreiber,et al.  High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission , 2016, Cell host & microbe.

[47]  E. Winzeler,et al.  Using genetic methods to define the targets of compounds with antimalarial activity. , 2013, Journal of medicinal chemistry.

[48]  Michelle F Clasquin,et al.  LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine. , 2012, Current protocols in bioinformatics.

[49]  R. Sinden,et al.  Gametocyte and gamete development in Plasmodium falciparum , 1978, Proceedings of the Royal Society of London. Series B. Biological Sciences.

[50]  David M. Shackleford,et al.  3,5-Diaryl-2-aminopyridines as a novel class of orally active antimalarials demonstrating single dose cure in mice and clinical candidate potential. , 2012, Journal of medicinal chemistry.

[51]  L. Tilley,et al.  Iron and heme metabolism in Plasmodium falciparum and the mechanism of action of artemisinins. , 2013, Current opinion in microbiology.

[52]  I. V. Ogungbe,et al.  Screening and Identification of Inhibitors of Trypanosoma brucei Cathepsin L with Antitrypanosomal Activity , 2016, Chemical biology & drug design.

[53]  C. Lambros,et al.  Synchronization of Plasmodium falciparum erythrocytic stages in culture. , 1979, The Journal of parasitology.

[54]  Hai Fang,et al.  supraHex: An R/Bioconductor package for tabular omics data analysis using a supra-hexagonal map , 2014, Biochemical and biophysical research communications.

[55]  C. Wilson,et al.  Point mutations in the dihydrofolate reductase-thymidylate synthase gene and pyrimethamine and cycloguanil resistance in Plasmodium falciparum. , 1995, Molecular and biochemical parasitology.

[56]  Joanne M. Morrisey,et al.  Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum , 2007, Nature.

[57]  Kellen L. Olszewski,et al.  Metabolic QTL Analysis Links Chloroquine Resistance in Plasmodium falciparum to Impaired Hemoglobin Catabolism , 2014, PLoS genetics.

[58]  H. Vial,et al.  Quantitative assessment of DNA replication to monitor microgametogenesis in Plasmodium berghei , 2009, Molecular and biochemical parasitology.

[59]  J. Jensen,et al.  Oxygen- and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in culture , 1985, Antimicrobial Agents and Chemotherapy.

[60]  Isabel M. Vincent,et al.  Metabolomic-Based Strategies for Anti-Parasite Drug Discovery , 2015, Journal of biomolecular screening.

[61]  Victoria C. Corey,et al.  Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL), a Resistance Mechanism for Two Distinct Compound Classes , 2016, ACS infectious diseases.

[62]  Bruce Russell,et al.  Spiroindolones, a Potent Compound Class for the Treatment of Malaria , 2010, Science.

[63]  D. Mancama,et al.  Discovering New Transmission-Blocking Antimalarial Compounds: Challenges and Opportunities. , 2016, Trends in parasitology.

[64]  Jonathan Diep,et al.  Identification of Plasmodium falciparum specific translation inhibitors from the MMV Malaria Box using a high throughput in vitro translation screen , 2016, Malaria Journal.

[65]  A. Vaidya,et al.  Atovaquone, a Broad Spectrum Antiparasitic Drug, Collapses Mitochondrial Membrane Potential in a Malarial Parasite* , 1997, The Journal of Biological Chemistry.

[66]  Donelly A van Schalkwyk,et al.  The Inhibitory Effect of 2-Halo Derivatives of d-Glucose on Glycolysis and on the Proliferation of the Human Malaria Parasite Plasmodium falciparum , 2008, Journal of Pharmacology and Experimental Therapeutics.

[67]  M. Grogl,et al.  Antileishmanial Activity of Compounds Derived from the Medicines for Malaria Venture Open Access Box against Intracellular Leishmania major Amastigotes , 2016, The American journal of tropical medicine and hygiene.

[68]  Jeremy N. Burrows,et al.  The Open Access Malaria Box: A Drug Discovery Catalyst for Neglected Diseases , 2013, PloS one.

[69]  D. Mancama,et al.  Nowhere to hide: interrogating different metabolic parameters of Plasmodium falciparum gametocytes in a transmission blocking drug discovery pipeline towards malaria elimination , 2015, Malaria Journal.

[70]  A F Slater,et al.  Chloroquine: mechanism of drug action and resistance in Plasmodium falciparum. , 1993, Pharmacology & therapeutics.

[71]  M. Mather,et al.  Genetic investigation of tricarboxylic acid metabolism during the Plasmodium falciparum life cycle. , 2015, Cell reports.

[72]  M. Cassera,et al.  Determination of the active stereoisomer of the MEP pathway-targeting antimalarial agent MMV008138, and initial structure-activity studies. , 2015, Bioorganic & medicinal chemistry letters.

[73]  David W. Gray,et al.  A novel multiple-stage antimalarial agent that inhibits protein synthesis , 2015, Nature.

[74]  Michael J. Dagley,et al.  Using metabolomics to dissect host-parasite interactions. , 2016, Current opinion in microbiology.

[75]  S. Ralph,et al.  Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action , 2016, Antimicrobial Agents and Chemotherapy.

[76]  Shailesh N Mistry,et al.  Screening the Medicines for Malaria Venture "Malaria Box" against the Plasmodium falciparum Aminopeptidases, M1, M17 and M18 , 2015, PloS one.

[77]  D. Leroy,et al.  Comparison and Optimization of Different Methods for the In Vitro Production of Plasmodium falciparum Gametocytes , 2012, Journal of parasitology research.

[78]  Christian Scheurer,et al.  Identification of an antimalarial synthetic trioxolane drug development candidate , 2004, Nature.

[79]  G. Ward,et al.  Identification of Cryptosporidium parvum Active Chemical Series by Repurposing the Open Access Malaria Box , 2014, Antimicrobial Agents and Chemotherapy.

[80]  Farah El Mazouni,et al.  A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria , 2015, Science Translational Medicine.

[81]  J. Coppee,et al.  Molecular basis for resistance to myxothiazol, mucidin (strobilurin A), and stigmatellin. Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae. , 1989, The Journal of biological chemistry.

[82]  D. Fidock,et al.  Identification of MMV Malaria Box Inhibitors of Plasmodium falciparum Early-Stage Gametocytes Using a Luciferase-Based High-Throughput Assay , 2013, Antimicrobial Agents and Chemotherapy.

[83]  Christian Scheurer,et al.  Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria , 2011, Proceedings of the National Academy of Sciences.

[84]  W. Milhous,et al.  PS-15: a potent, orally active antimalarial from a new class of folic acid antagonists. , 1993, The American journal of tropical medicine and hygiene.

[85]  Erika L. Flannery,et al.  Next-Generation Sequencing of Plasmodium vivax Patient Samples Shows Evidence of Direct Evolution in Drug-Resistance Genes. , 2015, ACS infectious diseases.

[86]  P. Shaw,et al.  Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling. , 2016, International journal for parasitology.

[87]  D. Kwiatkowski,et al.  Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance , 2015, Science.

[88]  Peter G. Schultz,et al.  A Chemical Genomic Analysis of Decoquinate, a Plasmodium falciparum Cytochrome b Inhibitor , 2011, ACS chemical biology.

[89]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[90]  Patrick Hunziker,et al.  Transmission-blocking strategies: the roadmap from laboratory bench to the community , 2016, Malaria Journal.

[91]  Yongyuth Yuthavong,et al.  Malarial dihydrofolate reductase as a paradigm for drug development against a resistance-compromised target , 2012, Proceedings of the National Academy of Sciences.

[92]  Elizabeth A. Winzeler,et al.  Na+ Regulation in the Malaria Parasite Plasmodiumfalciparum Involves the Cation ATPase PfATP4 and Is a Target of the Spiroindolone Antimalarials , 2013, Cell host & microbe.

[93]  K. Kuhen,et al.  KAF156 Is an Antimalarial Clinical Candidate with Potential for Use in Prophylaxis, Treatment, and Prevention of Disease Transmission , 2014, Antimicrobial Agents and Chemotherapy.

[94]  Anang A. Shelat,et al.  Chemical genetics of Plasmodium falciparum , 2010, Nature.

[95]  Peter G. Kremsner,et al.  Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. , 2015 .

[96]  P. Rathod,et al.  High-throughput Screening for Potent and Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase* , 2005, Journal of Biological Chemistry.

[97]  L. Tilley,et al.  Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum , 2013, BMC Biology.

[98]  J. Hemingway,et al.  Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria , 2012, Proceedings of the National Academy of Sciences.

[99]  R. Aebersold,et al.  A uniform proteomics MS/MS analysis platform utilizing open XML file formats , 2005, Molecular systems biology.

[100]  S. Ralph,et al.  Metabolic Dysregulation Induced in Plasmodium falciparum by Dihydroartemisinin and Other Front-Line Antimalarial Drugs. , 2016, The Journal of infectious diseases.

[101]  Daniel Amador-Noguez,et al.  Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer. , 2010, Analytical chemistry.

[102]  Manuel Llinás,et al.  Kinetic Flux Profiling Elucidates Two Independent Acetyl-CoA Biosynthetic Pathways in Plasmodium falciparum* , 2013, The Journal of Biological Chemistry.

[103]  G. McFadden,et al.  Parasites resistant to the antimalarial atovaquone fail to transmit by mosquitoes , 2016, Science.

[104]  Wenyun Lu,et al.  Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry. , 2006, Journal of chromatography. A.

[105]  L. Cui,et al.  Translational regulation during stage transitions in malaria parasites , 2015, Annals of the New York Academy of Sciences.

[106]  Mark C. Field,et al.  Pyrimidine Salvage in Trypanosoma brucei Bloodstream Forms and the Trypanocidal Action of Halogenated Pyrimidines , 2013, Molecular Pharmacology.

[107]  Improved methods for magnetic purification of malaria parasites and haemozoin , 2010, Malaria Journal.

[108]  Kyoungmi Kim,et al.  Effects of imputation on correlation: implications for analysis of mass spectrometry data from multiple biological matrices , 2016, Briefings Bioinform..

[109]  Yuexin Li,et al.  Quinolone-3-Diarylethers: A New Class of Antimalarial Drug , 2013, Science Translational Medicine.

[110]  Joachim Müller,et al.  In Vitro Screening of the Open-Source Medicines for Malaria Venture Malaria Box Reveals Novel Compounds with Profound Activities against Theileria annulata Schizonts , 2016, Antimicrobial Agents and Chemotherapy.

[111]  K. Dechering,et al.  A simple and predictive phenotypic High Content Imaging assay for Plasmodium falciparum mature gametocytes to identify malaria transmission blocking compounds , 2015, Scientific Reports.

[112]  José A. Fernández Robledo,et al.  Identification of MMV Malaria Box Inhibitors of Perkinsus marinus Using an ATP-Based Bioluminescence Assay , 2014, PloS one.

[113]  Kiaran Kirk,et al.  Diverse chemotypes disrupt ion homeostasis in the malaria parasite , 2014, Molecular microbiology.

[114]  J. Crowley,et al.  Malaria Parasites Produce Volatile Mosquito Attractants , 2015, mBio.

[115]  Isabel M. Vincent,et al.  Untargeted Metabolomics To Ascertain Antibiotic Modes of Action , 2016, Antimicrobial Agents and Chemotherapy.

[116]  Kellen L. Olszewski,et al.  Extraction of hydrophilic metabolites from Plasmodium falciparum-infected erythrocytes for metabolomic analysis. , 2013, Methods in molecular biology.

[117]  S. Oliver,et al.  Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi , 2016, PLoS neglected tropical diseases.

[118]  Teun Bousema,et al.  A semi-automated luminescence based standard membrane feeding assay identifies novel small molecules that inhibit transmission of malaria parasites by mosquitoes , 2015, Scientific Reports.

[119]  W. Trager,et al.  Human malaria parasites in continuous culture. , 1976, Science.

[120]  A. Kaiser,et al.  Target evaluation of deoxyhypusine synthase from Theileria parva the neglected animal parasite and its relationship to Plasmodium. , 2014, Bioorganic & medicinal chemistry.

[121]  K. Kirk,et al.  The malaria parasite cation ATPase PfATP4 and its role in the mechanism of action of a new arsenal of antimalarial drugs , 2015, International journal for parasitology. Drugs and drug resistance.

[122]  A. Calderón,et al.  Preliminary LC-MS Based Screening for Inhibitors of Plasmodium falciparum Thioredoxin Reductase (PfTrxR) among a Set of Antimalarials from the Malaria Box , 2016, Molecules.

[123]  P. Fokou,et al.  Repurposing the Open Access Malaria Box To Discover Potent Inhibitors of Toxoplasma gondii and Entamoeba histolytica , 2014, Antimicrobial Agents and Chemotherapy.

[124]  Yuming Guo,et al.  The effect of air pollution on human physiological function in China: a longitudinal study , 2015, The Lancet.

[125]  H. Balaram,et al.  Metabolic Fate of Fumarate, a Side Product of the Purine Salvage Pathway in the Intraerythrocytic Stages of Plasmodium falciparum* , 2011, The Journal of Biological Chemistry.