Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores

Hematin polymerization is a parasite-specific process that enables the deto xification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a uniq ue pharmacological drug target. We have developed a high-throughput in vitr o microassay of hematin polymerization based on the detection of C-14-label ed hematin incorporated into polymeric hemozoin (malaria pigment). The assa y uses 96-well filtration microplates and requires 12 h and a Wallac 1450 M icroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100,000 compounds. Random s creening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the in hibitory activity of a large library of 4-aminoquinolines. Using these meth ods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodi um berghei murine model further identified compounds with actual antimalari al activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.