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  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. Wirth 1 * 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 ; and Infectious Disease Initiative, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142 3 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. Malaria poses an enormous public health burden, causing over 1 million fatalities annually worldwide, with the majority of morbidity and mortality attributed to Plasmodium falcipa- rum malaria. Chloroquine (CQ) served as the main chemo- therapeutic for several decades, but the emergence and spread of drug resistance has limited its current usefulness. After the introduction of every new antimalarial drug, with the exception of artemisinin, resistant malaria parasites have emerged (25). Hence, the continued development of new antimalarial drugs is necessary to continue to successfully treat malaria infection. The malaria parasite cycles between two hosts, namely, mos- quitoes (Anopheles sp.) and humans. In the human host, after a brief liver stage, P. falciparum resides exclusively inside red blood cells, where it feeds on hemoglobin, reproduces, and then releases progeny, which invade new red blood cells. Dur- ing this intraerythrocytic stage, proteases digest hemoglobin within the food vacuole, a lysosome-like organelle (9). As he- moglobin is digested, heme molecules, containing redox-active iron centers, are released. The parasite overcomes the oxida- tive stress thus produced through the crystallization of free heme molecules into hemozoin (20). Hemozoin crystals are formed in an enzyme-independent reaction that is essential for parasite survival and therefore an excellent target for antima- larial chemotherapy. CQ, the most successful antimalarial agent to date, accumulates in the food vacuole, where it inhib- its heme crystallization and prevents parasite proliferation (18). Small-molecule disruption of hemozoin formation has been proposed as a mechanism of action of many other anti- malarial agents, including mefloquine (MQ), amodiaquine (AMQ), quinine, and quinidine, since each of these drugs successfully inhibits heme crystallization in in vitro assays, and they are all structurally related to CQ. We believe that the development of antimalarial agents based on the physicochemical process of heme crystallization could identify molecules that are less likely to generate resis- tance, like CQ. Drug resistance usually entails expression changes or mutations in the target protein or similar changes in pumps in order to expel the antimalarial agent (22, 26). Since heme crystallization inhibitors do not target a protein but a physicochemical process, resistance can occur only through the latter approach. For example, CQ resistance is achieved through multiple mutations in the P. falciparum CQ resistance transporter 1 (pfcrt1) allele, which encodes a transmembrane protein that, when mutated, significantly reduces the concen- tration of CQ in the food vacuole (8). Since the heme crystal- lization pathway remains unaltered in resistant parasites, it is still possible to exploit parasite heme crystallization while avoiding cross-resistance with CQ. In this study, we have adapted the pyridine hemichrome inhibition of -hematin (Phi) assay described by Ncokazi and Egan for high-throughput screening (HTS) (17). This assay recapitulates in vivo heme crystallization by solubilizing hema- tin and then allowing it to spontaneously form crystalline -he- matin (synthetically identical to hemozoin) within a 384-well plate (1). Pyridine is used as a developing reagent to monitor heme crystallization, as pyridine molecules coordinated with the iron centers of free heme molecules produce a concentra- tion-dependent color change, with a strong absorption at 405 * Corresponding author. Mailing address: Department of Immunol- ogy 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@hsph.harvard .edu.  Published ahead of print on 23 March 2009. 2564