Abstract
BackgroundChloroquine accumulates in the acidic digestive vacuole of the intraerythrocytic malaria parasite, and prevents the detoxication of haematin released during haemoglobin digestion. Changes in protein PfCRT in the digestive vacuole membrane of growing intra-erythrocytic stages of Plasmodium falciparum are crucial for resistance. Expressed in yeast, PfCRT resembles an anion channel. Depressed anion channel function could increase intralysosomal pH to reduce entry of basic drug, or enhanced function could reduce drug interaction with target haematin. The most important resistance-associated change is from positively-charged lysine-76 to neutral threonine which could facilitate drug efflux through a putative channel. It has been proposed that the resistance-reversing effect of verapamil is due to hydrophobic binding to the mutated PfCRT protein, and replacement of the lost positive charge, which repels the access of 4-aminoquinoline cations, thus partially restoring sensitivity. Desethylamodiaquine, the active metabolite of amodiaquine, which has significant activity in chloroquine-resistance, may also act similarly on its own.MethodsChanges in physicochemical parameters in different CQ-resistant PfCRT sequences are analysed, and correlations with drug activity on lines transfected with different alleles of the pfcrt gene are examined.Results and conclusionsThe results support the idea that PfCRT is a channel which, in resistant parasites, can allow efflux of chloroquine from the digestive vacuole. Activity of the chloroquine/verapamil combination and of desethylamodiaquine both correlate with the mean hydrophobicity of PfCRT residues 72-76. This may partly explain clinical-resistance to amodiaquine found in the first chloroquine-resistant malaria cases from South America and enables tentative prediction of amodiaquine's clinical activity against novel haplotypes of PfCRT.
Highlights
Chloroquine accumulates in the acidic digestive vacuole of the intraerythrocytic malaria parasite, and prevents the detoxication of haematin released during haemoglobin digestion
Correlation of physicochemical characteristics with drug activity in transfectants Since drug response is drastically changed in CQ-resistance it is highly relevant to look at correlations between IC50 values of the four CQ-resistant clones themselves and PfCRT physicochemical properties of their mutable residues, as well as to look at the correlations of these properties with the CQ-sensitive and resistant clones overall
Chloroquine-resistant transfectants (Table 6: Figures 1,2,3,4,5,6,7) Chloroquine (CQ), desethylchloroquine (DCQ) and amodiaquine (AQ) IC 50 values showed no significant correlation with any physicochemical feature of the mutable residues of PfCRT
Summary
Chloroquine accumulates in the acidic digestive vacuole of the intraerythrocytic malaria parasite, and prevents the detoxication of haematin released during haemoglobin digestion. A ClC function, possibly gated by membrane potential, could, by varying the entrance or exit of chargebalancing chloride anion, control intralysosomal pH, thereby regulating access of basic drug to the lysosome [3], or influencing the interaction of drug with its target haematin [12]. Apart from these rather non-specific pH effects, which are rendered unlikely by different interactions of the physico-chemically very similar diastereomers quinine and quinidine with mutated PfCRT [13], there is persuasive evidence that resistance depends on a drug efflux process [14]. Contradictory evidence [15] may be explained by postulating drug efflux from an internal compartment such as the lysosome into the cytoplasm, which after a variable delay, depending on experimental conditions, releases drug back into the culture medium
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