A Cell-based HTS for Delayed Death Inhibitors of the Malarial Parasite Plastid

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DESCRIPTION (provided by applicant): Plasmodium falciparum, an Apicomplexan parasite and the causal agent of severe malaria, causes disease in over 500 million individuals and kills over one million children yearly. Existing drugs suffer from parasite resistance, high cost or toxicity, and the pre-clinical pipeline is woefully thin. Recent antimalarial drug discovery efforts have focused on the apicoplast, an essential organelle phylogenetically related to cyanobacterial plastids. This organelle is the site of fatty acid, isoprenoid and heme biosynthesis, and contains an estimated 400 nuclear-encoded proteins, many of unknown function. Detailed studies with antibiotics that target apicoplast ribosomes (such as azithromycin) reveal that apicoplast inhibitors manifest a unique delayed death phenotype, whereby only the progeny of drug-treated parasites die. Why the apicoplast is essential for P. falciparum and what factors mediate its critical biological functions are intriguing questions that can be experimentally addressed with novel apicoplast-specific probes. We propose to develop assays that identify inhibitors of apicoplast development by taking advantage of this delayed death phenotype. Our primary screen will expose cultured P. falciparum to compounds for one or two generations (48 or 96 hr). Parasite growth will be measured using the dsDNA-intercalating SYBR Green dye. Compounds will be tested first in 96 hr assays, and only active compounds will be retested in 48 hr assays. Compounds that manifest increased potency only in the second generation will be retained for further screening. This will include repeat assays, dose-response assays to identify well- behaved inhibitors, and counterscreens to select against generally cytotoxic inhibitors. One secondary screen will use [3H]-hypoxanthine as an alternative measure of growth inhibition. Another, based on whole cell imaging, will use a transgenic line expressing GFP-labeled apicoplasts to visually identify inhibitors of apicoplast development. Assay development will be guided using the reference compounds azithromycin, chloroquine and isoniazid (representing delayed death, fast-acting and inactive compounds respectively) and configured for HTS using a library of 400 FDA-approved drugs. Later studies will include optimization on the Tecan HTS automation platform at the Molecular Libraries Screening Center Network Center at Columbia University, in collaboration with members of this center. In addition to their therapeutic potential, apicoplast inhibitors will be invaluable in allowing us to directly address questions about key apicoplast processes and proteins. We propose to develop a high throughput assay to screen chemical libraries for compounds that inhibit the development of a compartment called the apicoplast in Plasmodium falciparum malaria parasites. Inhibition of the apicoplast is characterized by a type of delayed death whereby compounds only kill the progeny of drug-treated parasites inside infected red blood cells. We intend to identify new inhibitors that can be used to understand apicoplast biology and the reasons for this delayed death, and that have potential for the development of new therapeutics.