High-Throughput Plasmodium falciparum Growth Assay for Malaria Drug Discovery

Malaria has afflicted humans since the introduction of agriculture some 10,000 years ago, and inherited diseases, such as sickle cell anemia and the thalassemias, testify to its profound selection signature on the human genome (4). Malaria continues to be a major public health problem in large regions of the world, especially the developing world. The vast majority of malaria deaths are caused by Plasmodium falciparum, and young children and pregnant mothers in sub-Saharan Africa are the primary victims. Because most cases of malaria occur in medically underserved areas, completely accurate figures for the disease's impact are difficult to obtain, but by any measure, malaria is a pernicious disease. Some insight into its impact can be gained by using disability-adjusted life years, which measure the disease's toll on both longevity and quality of life, and the World Health Organization (WHO) estimates the malaria burden at 42 million disability-adjusted life years (20a). Success in preventing and treating malaria varies. Only 50 years ago, WHO and others were optimistic that malaria could be eradicated through a combination of effective therapeutic agents to treat the existing disease, insecticides to eliminate its mosquito vector, and various public health measures for prevention. Optimism, not the disease, disappeared in the face of a perfect storm of factors: the disengagement of developed countries from regions where malaria is endemic, brought about by the independence of former colonies; the cessation of armed conflicts by developed countries in regions where malaria is endemic; the collapse of public health institutions in many developing nations; the lack of financial incentives for pharmaceutical companies to develop antimalarial agents; and the spread of resistance in the parasite (especially to chloroquine) and the mosquito vector (especially to dichlorodiphenyltrichloroethane). This combination of factors has led to the resurgence and spread of malaria in large parts of the developing world. Resistance to the most commonly used and approved antimalarial drugs has been reported throughout Africa and Asia, and new drugs are needed. The range of current antimalarials is narrow. There are only four classes of compounds: those based on quinine (chloroquine, mefloquine, amodiaquine, and halofantrine) or other aminoquinolines (primaquine and tafenoquine), the antifolate compounds (pyramethamine, proguanil, cyocloguanil, dapsome, and sulfadoxine), the artemisinin derivatives (artesunate, artemether/arteether, Co-artem, and others), and, most recently, the hydroxynapthoquinone atovaquone. This lack of structural diversity means that previously developed therapeutic alternatives, really modifications of the same basic molecular templates, might prime new drug candidates for the rapid emergence of resistance. Current antimalarials also have a limited range of cellular targets: only four to six processes in these sophisticated eukaryotic organisms with complex life cycles. Most drugs target either metabolism or the detoxification of hemoglobin, the parasite's food source. Drugs that target hemoglobin detoxification include the 4-aminoquinolines (chloroquine and amodiaquine) and the amino alcohols (quinine, mefloquine, halofantrine, and lumefantrine). The parasite's folate metabolism is targeted by the antifolates (sulfadoxine-pyrimethamine and dapsone), and its mitochondria are targeted by atovaquone (6). In addition to these targets, artemisinin, or more likely an artemisinin-derived molecule, probably targets the sarco- and/or endoplasmic reticulum Ca2+ ATPase (11). The P. falciparum genome has been sequenced, revealing over 5,500 genes of P. falciparum that surely contain a large, though admittedly unknown, number of targets that are suitable for small-molecule intervention (9). The discovery of new targets, especially those that can be affected by small molecules, can be most easily approached with a high-throughput screening (HTS) campaign with structurally diverse small-molecule libraries. Conducting a HTS campaign to identify small molecules that might lead to drug candidates obviously requires a robust HTS, and such a screen does not currently exist. Previously, the only means of detecting parasite growth was microscopic examination of blood smears using a modified Wright-Giemsa stain or the [3H]hypoxanthine incorporation assay (7). While the latter method could in principle be automated, it is not well suited for HTS, as it requires radioactive materials that pose safety and disposal problems and has multiple steps that are technically demanding. Recently, new nonradioactive screens have emerged, using DNA stains as a reporter to measure parasite growth (1, 5, 16, 17). The use of DNA stains to detect parasite DNA has greatly aided the ease of drug susceptibility testing. However, these methods are not sufficiently robust and are therefore not amenable to HTS of large compound libraries. As part of a larger effort to develop antimalarial agents, we set about developing a HTS based on DNA staining with blood-stage P. falciparum that would identify inhibitory small molecules from structurally diverse small-molecule libraries. A whole-organism method allows all relevant blood-stage targets to be screened simultaneously and ensures that screening positives have at least minimal levels of desirable pharmacokinetic properties, such as cell permeability and activity in a cellular context. Such a screen can also be readily expanded to other parasite strains to study resistance profiles or to include variables, such as culture time, that would distinguish fast- from slow-acting compounds. The choice of a 4′,6-diamidino-2-phenylindole (DAPI) DNA stain led to a robust and reproducible reporter of parasite growth that responded to chemically and mechanistically diverse known antimalarial inhibitors. In addition, it is well suited for HTS performed using a 384-well microtiter plate, as demonstrated by its performance in a screen of approximately 79,000 compounds.