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Antimicrobial Agents and Chemotherapy, February 2007, p. 716-723, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.01144-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

High-Throughput Plasmodium falciparum Growth Assay for Malaria Drug Discovery{triangledown}

Mary Lynn Baniecki,1 Dyann F. Wirth,2 and Jon Clardy1*

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,1 Harvard School of Public Health, Department of Immunology and Infectious Disease, Boston, Massachusetts 021152

Received 12 September 2006/ Returned for modification 19 October 2006/ Accepted 5 November 2006

New therapeutic agents for the treatment of malaria, the world's most deadly parasitic disease, are urgently needed. Malaria afflicts 300 to 500 million people and results in 1 to 2 million deaths annually, and more than 85% of all malaria-related mortality involves young children and pregnant women in sub-Saharan Africa. The emergence of multidrug-resistant parasites, especially in Plasmodium falciparum, has eroded the efficacy of almost all currently available therapeutic agents. The discovery of new drugs, including drugs with novel cellular targets, could be accelerated with a whole-organism high-throughput screen (HTS) of structurally diverse small-molecule libraries. The standard whole-organism screen is based on incorporation of [3H]hypoxanthine and has liabilities, such as limited throughput, high cost, multiple labor-intensive steps, and disposal of radioactive waste. Recently, screens have been reported that do not use radioactive incorporation, but their reporter signal is not robust enough for HTS. We report a P. falciparum growth assay that is technically simple, robust, and compatible with the automation necessary for HTS. The assay monitors DNA content by addition of the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) as a reporter of blood-stage parasite growth. This DAPI P. falciparum growth assay was used to measure the 50% inhibitory concentrations (IC50s) of a diverse set of known antimalarials. The resultant IC50s compared favorably with those obtained in the [3H]hypoxanthine incorporation assay. Over 79,000 small molecules have been tested for antiplasmodial activity using the DAPI P. falciparum growth assay, and 181 small molecules were identified as highly active against multidrug-resistant parasites.

* Corresponding author. Mailing address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, C-643, Boston, MA 02115. Phone: (617) 432-2845. Fax: (617) 432-6424. E-mail: jon_clardy{at}hms.harvard.edu.

{triangledown} Published ahead of print on 20 November 2006.

Antimicrobial Agents and Chemotherapy, February 2007, p. 716-723, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.01144-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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