Inhibition of heme crystal growth by antimalarials and other compounds: Implications for drug discovery

During intraerythrocytic infection, Plasmodium falciparum parasites crystallize toxic heme released during hemoglobin catabolism. The proposed mechanism of quinoline inhibition of crystal growth is either by a surface binding or a substrate sequestration mechanism. The kinetics of heme crystal growth was examined in this work using a new high-throughput crystal growth determination assay based on the differential solubility of free vs. crystalline FP in basic solutions. Chloroquine (IC₅₀=4.3μM) and quinidine (IC₅₀=1.5μM) showed a previously not recognized reversible inhibition of FP crystal growth. This inhibition decreased by increasing amounts of heme crystal seed, but not by greater amounts of FP substrate. Crystal growth decreases as pH rises from 4.0 to 6.0, except for a partial local maxima reversal from pH 5.0 to 5.5 that coincides with increased FP solubility. The new crystal growth determination assay enabled a partial screen of existing clinical drugs. Nitrogen heterocycle cytochrome P450 inhibitors also reversibly blocked FP crystal growth, including the azole antifungal drugs clotrimazole (IC₅₀=12.9μM), econazole (IC ₅₀=19.7μM), ketoconazole (IC₅₀=6.5μM), and miconazole (IC₅₀=21.4μM). Fluconazole did not inhibit. Both subcellular fractionation of parasites treated with subinhibitory concentrations of ketoconazole and in vitro hemozoin growth assays demonstrated copurification of hemozoin and ketoconazole. The chemical diversity of existing CYP inhibitor libraries that bind FP presents new opportunities for the discovery of antimalarial drugs that block FP crystal growth by a surface binding mechanism and possibly interfere with other FP-sensitive Plasmodium pathways.