Malaria elimination has recently been reinstated as a global health priority but current therapies seem to be insufficient for the task. Elimination efforts require new drug classes that alleviate symptoms, prevent transmission and provide a radical cure. To develop these next-generation medicines, public-private partnerships are funding innovative approaches to identify compounds that target multiple parasite species at multiple stages of the parasite life cycle. In this Review, we discuss the cell-, chemistry- and target-based approaches used to discover new drug candidates that are currently in clinical trials or undergoing preclinical testing.

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Antimalarial drug discovery — approaches and progress towards new medicines

Recent advances in malaria drug discovery.

Global Phenotypic Screening for Antimalarials

Malaria and the Neglected Tropical Diseases Human African Trypanosomiasis and Schistosomiasis Mathew Njoroge,† Nicholas M. Njuguna,† Peggoty Mutai,† Dennis S. B. Ongarora,† Paul W. Smith, and Kelly Chibale*,†,‡,§ †Department of Chemistry, ‡Institute of Infectious Disease and Molecular Medicine, and South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town, Rondebosch 7701, South Africa Novartis Institute for Tropical Diseases, Singapore 138670, Singapore

Recent approaches to chemical discovery and development against malaria and the neglected tropical diseases human African trypanosomiasis and schistosomiasis.

Malaria is a disease that still affects a significant proportion of the global human population. Whilst advances have been made in lowering the numbers of cases and deaths, it is clear that a strategy based solely on disease control year on year, without reducing transmission and ultimately eradicating the parasite, is unsustainable. This article highlights the current mainstay treatments alongside a selection of emerging new clinical molecules from the portfolio of Medicines for Malaria Venture (MMV) and our partners. In each case, the key highlights from each research phase are described to demonstrate how these new potential medicines were discovered. Given the increased focus of the community on eradicating the disease, the strategy for next generation combination medicines that will provide such potential is explained.

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Antimalarial drug discovery – the path towards eradication

Malaria is one of the deadliest infectious diseases in the world, with the eukaryotic parasite Plasmodium falciparum causing the most severe form of the disease. Discovery of new classes of antimalarial drugs has become an urgent task to counteract the increasing problem of drug resistance. Screening directly for compounds able to inhibit parasite growth in vitro is one of the main approaches the malaria research community is now pursuing for the identification of novel antimalarial drug leads. Very recently, thousands of compounds with potent activity against the parasite P. falciparum have been identified and information about their molecular descriptors, antiplasmodial potency, and cytotoxicity is publicly available. Now the challenges are how to identify the most promising chemotypes for further development and how best to progress these compounds through a lead optimization program to generate antimalarial drug candidates. We report here the first chemical series to be characterized from one of those screenings, a completely novel chemical class with the generic name cyclopropyl carboxamides that has never before been described as having antimalarial or other pharmacological activities. Cyclopropyl carboxamides are potent inhibitors of drug-sensitive and -resistant strains of P. falciparum in vitro and show in vivo oral efficacy in malaria mouse models. In the present work, we describe the biological characterization of this chemical family, showing that inhibition of their still unknown target has very favorable pharmacological consequences but the compounds themselves seem to select for resistance at a high frequency.

Cyclopropyl Carboxamides, a Chemically Novel Class of Antimalarial Agents Identified in a Phenotypic Screen

Pneurnocystis carinii causes a serious pulmonary infection in immunocompromised hosts. Although several treatment and prophylactic regimens are available, anti-Pneumocystts drugs are not fully successful and often induce adverse effects, particularly in AIDS patients. In vitro drug evaluation systems suitable for a large number of compounds should facilitate the identification and development of new therapies. Thc present work describes three assays for in vitro evaluation of anti-Pneumocystis drugs adapted to microplate format: incorporation of radiolabelled methionine into proteins newly synthesised, measurement of ATP intracellular levels and incorporation of the vital dye neutral red. Reproducibility and sensitivity were compared. MATERIALS AND METHODS. Source of P.carinii. Corticosteroid-treated Wistar rats were used as the source of P.carinii organisms. Parasite extraction was performed as previously described [l]. Briefly, lungs were removed aseptically and minced in sterile Dulbecco modified Eagle's medium (DMEM). Parasites were extracted by agitation of the lung pieces with a magnetic stirrer, and poured successively through sterile gauze and two stainless steel meshes of 250 and 63 pm respectively. A Ficoll-Hypaque gradient (Histopaque1077, Sigma) was used to purify P.carinii from the homogenate. Gradients were centrifuged at 1,000 g for 15 min at 4 C. Although the metabolic assays evaluated in this work are not able to discern between contaminating mammalian cells and parasites, the contribution of lung cells appears to be minimal as evaluated by Giemsa staining. Microplate assay for incorporation of [\"s/ methionine. Evidence of in vitro protein biosynthesis based on the uptake and incorporation of radiolabelled amino acids has been demonstrated by rat-derived Pneumocystis carinii [ 2 , 3 ] . Proteins newly synthesised can be measured quantitatively by scintillation counting permitting assessment of proliferation and viability of the culture over time. The assay is performed in 96 flat-bottom-well microtiter plates in a volume of I50 p1 of methionine-free MEM medium supplemented with 10% of FCS plus 100 U/ml penicillin and 100 p@ml streptomycin, and the corresponding dilution of drug to be tested. P.carinii was inoculated to give a final concentration of 5 x107 parasites per ml and incubated in the presence of drugs at 37 C in a humidified atmosphere of 5% CO, for 8-48 hours (depending on the compound). Following incubation, organisms were pulsed overnight with 5 pCi/ml of [\"S] methionine, harvested on glass fibre filters using a cell harvester (Tomtec) and after adding scintillation liquid, counted in a microbeta scintillation counter (Wallac). Studies were performed by triplicate or quadruplicate and positive (organisms in medium with the corresponding concentration of the vehicle used to solve the drugs) and negative (boiled P.carinii inoculum) controls were included. ATP is the universal energy unit in all living cells, being rapidly degraded by ubiquitous ATPases upon cell death. Consequently, it is possible to evaluate drug activity based on the percentage decrease in ATP levels comparing to control cultures. The assay employs the luciferidluciferase ATP-dependent bioluminescence reaction to measure intracellular ATP. Cushion et a1 have applied this method to evaluate the viability of P.carinii from rats [4] and recently have ATP bioluminescence assay in microplate format. reported the anti-Pneumocystis activity of a wide range of drugs using this technique [ j ] , We have adapted the assay to microplate format. 96-well microtiter plates were prepared with DMEM medium supplemented with 10% FCS, 100 U/ml penicillin, 100 pg/ml streptomycin and 5 x107 parasites per ml. After incubation with the corresponding drug, 75 pI of extractant (7.8% TCA and 51 mM EDTA in water) were added to each well and the plates were shacked 15 min at room temperature. Following incubation, samples were diluted 1/20 in water and 40 pl of this diluted extract were transferred to another plate previously prepared containing 100 pI of Tris-acetate buffer (70 mM Tris-acetate, 2 mM EDTA pH 7.75, 60 pM DTT, BSA 0.125%) and 20 pl of 100 mM Magnesium acetate. After addition of 40 p1 of luciferin-luciferase the light generated was measured by a microplate luminometer (Wallac) during 6 seconds. Results were expressed as percentages of the control. Neutral red assay. Uptake and retention of vital dyes has long been used for the study of viability of eukaryotic cells. Neutral red, a water soluble vital dye, passes through the intact membrane of viable P. carinii and becomes concentrated in lysosomes [6]. The dye is not retained in the vacuoles of the damaged organisms and the plasma membrane is not able to keep it within the cell. Microtiter plates were prepared with medium, drugs and parasites as is described in the ATP bioluminescence assay. After incubation with drugs plates were centrifuged in a microplate rotor at 666 g for 10 min at room temperature. Fresh medium supplemented with 40 p g m l of neutral red to stain viable parasites was added to each well. After 3 hours of incubation plates were centrifuge again, wells were rinsed with PBS and the dye extracted with 1% acetic a c i d 50% ethanol. Plates were finally readed at 540 nm using a microplate reader. RESULTS AND DISCUSSION. We describe three in vitro assays for screening of new agents with potential anti-Pneumocystis activity adapted to microplate format.

M. Jesus Almela

Papers

Cyclopropyl Carboxamides, a Chemically Novel Class of Antimalarial Agents Identified in a Phenotypic Screen

Microplate Assays for in Vitro Evaluation of Anti-Pneumocystis Drugs.