Mutations in pfmdr1 Modulate the Sensitivity of Plasmodium falciparum to the Intrinsic Antiplasmodial Activity of Verapamil

Abstract

As well as having the ability to reverse chloroquine resistance in the human malaria parasite Plasmodium falciparum, verapamil has itself an innate antiplasmodial activity. We show here that mutations in Pgh1, the product of the malaria parasite's pfmdr1 gene, influence the parasite's susceptibility to the toxic effects of verapamil.Verapamil (VP) is a weak base which, in addition to acting as a reverser of chloroquine resistance (CQR) in the malaria parasite Plasmodium falciparum, has itself an intrinsic antiplasmodial activity (1,12,14,26). This activity is independent of its CQR reversal effect, as the susceptibility of chloroquine (CQ)-sensitive parasites to CQ is unaltered even in the presence of highly toxic concentrations of VP, whereas VP alters the susceptibility of CQ-resistant parasites to CQ at both toxic and nontoxic concentrations (15). The CQR reversal effect of VP has been attributed to an interaction of the compound with the P. falciparum chloroquine resistance transporter (PfCRT) (7,17,23), a member of the drug-metabolite transporter superfamily (13, 25) which is localized to the parasite's internal digestive vacuole and is the key determinant of CQR (8,23).Recent allelic exchange experiments have shown that mutations in the pfmdr1 gene product, P-glycoprotein homologue 1 (Pgh1), modulate sensitivity to a range of antimalarial compounds (20). Pgh1 is localized predominantly to the parasite's digestive vacuole membrane (4, 10). It belongs to the ATPbinding cassette transporter superfamily, members of which couple ATP hydrolysis to the translocation of a diverse range of structurally unrelated solutes across cell membranes (reviewed in reference 11). We report here that polymorphisms in pfmdr1 influence the parasite's susceptibility to the intrinsic antiplasmodial effect of VP.Transfected P. falciparum parasites derived from either D10 (CQ-sensitive) or 7G8 (CQ-resistant) strains with pfmdr1 loci altered as described previously (20) The introduction of either one or three of the four 7G8 mutations into the pfmdr1 gene of the D10 parasite (to make D10-mdr 7G8/1 or D10-mdr 7G8/3 ) significantly increased VP sensitivity with respect to the D10-mdr D10 transfectant, a control transfectant with the wild-type pfmdr1 gene (Table 1). Notably, D10-mdr 7G8/1 , with a single mutation at codon 1246, was more sensitive to VP than D10-mdr 7G8/3 , which carries two additional mutations at codons 1034 and 1042. Replacement of these three mutations in the 7G8 parasite with the corresponding wild-type D10 pfmdr1 sequence (7G8-mdr D10 ) was sufficient to confer full D10-like tolerance to VP. Further analysis shows that the pattern of relative sensitivity of these transfectants to VP correlates significantly with the pattern of sensitivity to mefloquine (MQ) and halofantrine (HF) obtained in a previous study (20) (r 2 ϭ 0.82 and P ϭ 0.035 in both cases). This result suggests that specific mutations in pfmdr1 mediate sensitivity to these compounds via a common mechanism. There is no difference in the levels of Pgh1 ...

pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum

“…Nevertheless, in the 3BA6 background the CDY mutations appeared to have a small effect in lowering the CQ IC 50 values in the presence of VP. We also observed that VP alone exerted a slightly greater inhibitory activity against the CDY 3BA6 as compared with the SND 3BA6 lines (data not shown), in agreement with a recent report indicating that pfmdr1 ‐modified CDY mutant lines were more susceptible to VP than were wild‐type SND lines (Hayward et al ., 2005).…”

“…These compounds were tested in duplicate five, four and six independent times for MFQ, HF and ART. *P < 0.05, **P < 0.01, ***P < 0.001. susceptible to VP than were wild-type SND lines (Hayward et al, 2005).…”

Asymptomatic falciparum malaria and genetic polymorphisms of Pfcrt K76T and Pfmdr1 N86Y among almajirai in northeast Nigeria

“…Point mutations at codons of Pfcrt and Pfmdr1 genes are associated with CQ resistance in P. falciparum [20,21,42]. Low levels of mutant alleles (Pfcrt 76T and Pfmdr1 86Y) detected in the present study indicated high CQ sensitivity among the population.…”

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum

“…Indeed, all progeny displaying a food vacuolar staining phenotype have the Dd2 pfmdr1 allele whereas all progeny with a diffuse staining pattern inherited the HB3 pfmdr1 allele. Although verapamil, at a concentration of 30 μM and within a 40 min incubation time, did not affect the Fluo‐4 phenotype, proliferation assays have found that polymorphisms within Pgh‐1 can influence the parasite's susceptibility to verapamil (Hayward et al , 2005), suggesting a possible interaction between verapamil and Pgh‐1.…”