ACCESS TO HEMATIN - THE BASIS OF CHLOROQUINE RESISTANCE

The saturable uptake of chloroquine by parasites of Plasmodium falcipa rum has been attributed to specific carrier-mediated transport of chlo roquine. It is suggested that chloroquine is transported in exchange f or protons by the parasite membrane Na+/H+ exchanger [J Biol Chem 272: 2652-2658 (1997)]. Once inside the parasite, it is proposed that chlor oquine inhibits the polymerization of hematin, allowing this toxic hem oglobin metabolite to accumulate and kill the cell [Pharmacol Ther 57: 203-235 (1993)]. To date, the contribution of these proposed mechanism s to the uptake and antimalarial activity of chloroquine has not been assessed. Using sodium-free medium, we demonstrate that chloroquine is not directly exchanged for protons by the plasmodial Na+/H+ exchanger . Furthermore, we show that saturable chloroquine uptake at equilibriu m is due solely to the binding of chloroquine to hematin rather than a ctive uptake: using Ro 40-4388, a potent and specific inhibitor of hem oglobin digestion and, by implication, hematin release, we demonstrate a concentration-dependent reduction in the number of chloroquine bind ing sites. An equal number of chloroquine binding sites are found in b oth resistant and susceptible clones, but the apparent affinity of chl oroquine binding is found to correlate with drug activity (r(2) = 0.93 , p < 0.0001). This completely accounts for both the reduced drug accu mulation and activity observed in resistant clones and the ''reversal' ' of resistance produced by verapamil. The data presented here reconci le most of the available biochemical data from studies of the mode of action of chloroquine and the mechanism of chloroquine resistance. We show that the activity of chloroquine and amodiaquine is directly depe ndent on the saturable binding of the drugs to hematin and that the in hibition of hematin polymerization may be secondary to this binding. T he chloroquine-resistance mechanism regulates the access of chloroquin e to hematin. Our model is consistent with a resistance mechanism that acts specifically at the food vacuole to alter the binding of chloroq uine to hematin rather than changing the active transport of chloroqui ne across the parasite plasma membrane.