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Antimicrobial Agents and Chemotherapy, June 2009, p. 2564-2568, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01466-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Colorimetric High-Throughput Screen for Detection of Heme Crystallization Inhibitors{triangledown}

Margaret A. Rush,1,2 Mary Lynn Baniecki,2 Ralph Mazitschek,3 Joseph F. Cortese,3 Roger Wiegand,3 Jon Clardy,2 and Dyann F. Wirth1*

Harvard School of Public Health, Department of Immunology and Infectious Disease, Boston, Massachusetts 02115,1 Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, Massachusetts 02115,2 Infectious Disease Initiative, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 021423

Received 3 November 2008/ Returned for modification 24 January 2009/ Accepted 14 March 2009

Malaria infects 500 million people annually, a number that is likely to rise as drug resistance to currently used antimalarials increases. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum, metabolizes hemoglobin and releases toxic heme, which is neutralized by a parasite-specific crystallization mechanism to form hemozoin. Evidence suggests that chloroquine, the most successful antimalarial agent in history, acts by disrupting the formation of hemozoin. Here we describe the development of a 384-well microtiter plate screen to detect small molecules that can also disrupt heme crystallization. This assay, which is based on a colorimetric assay developed by Ncokazi and Egan (K. K. Ncokazi and T. J. Egan, Anal. Biochem. 338:306-319, 2005), requires no parasites or parasite-derived reagents and no radioactive materials and is suitable for a high-throughput screening platform. The assay's reproducibility and large dynamic range are reflected by a Z factor of 0.74. A pilot screen of 16,000 small molecules belonging to diverse structural classes was conducted. The results of the target-based assay were compared with a whole-parasite viability assay of the same small molecules to identify small molecules active in both assays.

* Corresponding author. Mailing address: Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Avenue, Building I, Room 705, Boston, MA 02115. Phone: (617) 432-1563. Fax: (617) 432-4766. E-mail: dfwirth{at}hsph.harvard.edu

{triangledown} Published ahead of print on 23 March 2009.

Antimicrobial Agents and Chemotherapy, June 2009, p. 2564-2568, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01466-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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