A MICROTITRE-BASED METHOD FOR MEASURING THE HEME POLYMERIZATION INHIBITORY ACTIVITY (HPIA) OF ANTIMALARIAL-DRUGS

The malaria parasite metabolizes haemoglobin and detoxifies the result ing haem by polymerizing it to form haemozoin (malaria pigment). A pol ymer identical to haemozoin, beta-haematin, can be obtained in vitro f rom haematin at acidic pH. Quinoline-containing antimalarials (e.g. ch loroquine) inhibit the formation of either polymer. Haem polymerizatio n is an essential and unique pharmacological target. To identify molec ules with haem polymerization inhibitory activity (HPIA) and quantify their potency, we developed a simple, inexpensive, quantitative in-vit ro spectrophotometric microassay of haem polymerization. The assay use s 96-well U-bottomed polystyrene microplates and requires 24 h and a m icroplate reader. The relative amounts of polymerized and unpolymerize d haematin are determined, based on solubility in DMSO, by measuring a bsorbance at 405 nm in the presence of test compounds as compared with untreated controls. The final product (a solid precipitate of polymer ized haematin) was validated using infrared spectroscopy and the assay proved reproducible; in this assay, activity could be partly predicte d based on the compound's chemical structure. Both water-soluble and w ater-insoluble compounds can be quantified by this method. Although th e throughput of this assay is lower than that of radiometric methods, the assay is easier to set up and cheaper, and avoids the problems rel ated to radioactive waste disposal.