P. Falciparum Chloroquine Resistance Transporter Research Articles

Heterologous Expression and ATPase Activity of Mutant versus Wild Type PfMDR1 Protein

Mutation of the P. falciparum chloroquine resistance transporter (PfCRT) causes resistance to chloroquine (CQ) and other antimalarial drugs. Mutation and/or overexpression of one of the multidrug resistance protein homologues found in this malarial parasite (PfMDR1) may further modify or tailor the degree of multidrug resistance. However, considerable controversy surrounds the precise contribution of PfMDR1, in part because no direct biochemical studies of PfMDR1 have yet been possible. Using codon optimization and other principles, we have designed and constructed a yeast optimized version of the wild type pfmdr1 gene and have successfully overexpressed PfMDR1 protein in P. pastoris yeast. The protein is well expressed in either full length form or as two separate half transporters, is well localized to the yeast plasma membrane and is fully functional as evidenced by ATPase activity measurements. We have also expressed mutants that have previously been hypothesized to influence drug resistance in parasites. Using purified plasma membrane fractions, we have analyzed antimalarial drug effects on ATPase activity for wild type versus mutant proteins. Relative to other ABCB transporters involved in drug resistance, PfMDR1 is unusual. It has similar pH, [ATP], and Mg++ dependencies for ATP hydrolysis, yet relatively high Km and Vmax values for ATP hydrolysis, and ATPase activity is only mildly stimulated by antimalarial drugs. The largest measured drug effect is for CQ (to which PfMDR1 is not believed to confer resistance), and it is strongly inhibitory for WT PfMDR1. Drug resistance associated PfMDR1 mutants show either elevated (Dd2 allele encoded) or reduced (7G8 allele) basal ATPase activity and different patterns of drug stimulation or inhibition, relative to WT PfMDR1. The Dd2 PfMDR1 isoform also shows a slightly more alkaline pH optimum. Surprisingly, verapamil alone (1-300 microM) does not significantly affect WT ATPase activity but inhibits the Dd2 isoform at 1 microM. These data should assist ongoing analysis of the contribution of PfMDR1 to antimalarial drug resistance.

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms

The emergence of chloroquine-resistant Plasmodium falciparum malaria imperils the lives of millions of people in Africa, Southeast Asia and South America. Chloroquine resistance is associated with mutations in the P. falciparum chloroquine resistance transporter (PfCRT). We expressed chloroquine-sensitive (HB3) and resistant (Dd2) pfcrt alleles in HEK293 human embryonic kidney cells. PfCRT localized to the lysosomal limiting membrane and was not detected in the plasma membrane. We observed significant acidification of lysosomes containing PfCRT HB3 and Dd2, with Dd2 acidifying significantly more than HB3. A mutant HB3 allele expressing the K76T mutation (earlier found to be key for chloroquine resistance) acidified to the same extent as Dd2, whereas the acidification by a Dd2 allele expressing the T76K “back mutation” was significantly less than Dd2. Thus, the amino acid at position 76 is both an important determinant of chloroquine resistance in parasites and of lysosomal acidification following heterologous expression. PfCRT may be capable of modulating the pH of the parasite digestive vacuole, and thus chloroquine availability. Chloroquine accumulation and glycyl-phenylalanine-2-naphthylamide-induced release of lysosomal Ca2+ stores were unaffected by PfCRT expression. Cytoplasmic domain mutations did not alter PfCRT sorting to the lysosomal membrane. This heterologous expression system will be useful to characterize PfCRT protein structure and function, and elucidate its molecular role in chloroquine resistance.

Assessment of Plasmodium falciparum resistance to ferroquine (SSR97193) in field isolates and in W2 strain under pressure

BackgroundFerroquine (FQ), or SSR97193, is a novel antimalarial drug currently in phase I clinical trials. FQ is a unique organometallic compound designed to overcome the chloroquine (CQ) resistance problem. FQ revealed to be equally active on CQ-sensitive and CQ-resistant Plasmodium falciparum laboratory strains and field isolates. FQ is also curative on rodent malaria parasites. As FQ will be tested in patients, the potential for resistance to this drug was evaluated.MethodsThe relationship between CQ-resistant transporter gene genotype and susceptibility to FQ were studied in 33 Cambodian P. falciparum field isolates previously studied for their in vitro response to CQ. In parallel, the ability of the CQ-resistant strain W2, to become resistant to FQ under drug pressure was assessed.ResultsThe IC50 values for FQ in field isolates were found to be unrelated to mutations occurring in the P. falciparum chloroquine resistance transporter (PfCRT) or to the level of expression of the corresponding mRNA. In vitro, under a drug pressure of 100 nM of FQ, transient survival was observed in only one of two experiments.ConclusionField isolates studies and experimental drug pressure experiments showed that FQ overcomes CQ resistance, which reinforces the potential of this compound as a new antimalarial drug.

Primaquine synergises the activity of chloroquine against chloroquine-resistant P. falciparum

In recent years, resistance to the antimalarial drug, chloroquine, has become widespread. It is, therefore, imperative to find compounds that could replace chloroquine or work synergistically with this drug to overcome chloroquine resistance. We have examined the interaction between chloroquine, a 4-aminoquinoline, and a number of 8-aminoquinolines, including primaquine, a drug that is widely used to treat Plasmodium vivax infections. We find that primaquine is a potent synergiser of the activity of chloroquine against chloroquine-resistant Plasmodium falciparum. Analysis of matched transfectants expressing mutant and wild-type alleles of the P. falciparum chloroquine resistance transporter (PfCRT) indicate that primaquine exerts its activity by blocking PfCRT, and thus enhancing chloroquine accumulation. Our data suggest that a novel formulation of two antimalarial drugs already licensed for use in humans could be used to treat chloroquine-resistant parasites.

DECREASED PREVALENCE OF THE PLASMODIUM FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER 76T MARKER ASSOCIATED WITH CESSATION OF CHLOROQUINE USE AGAINST P. FALCIPARUM MALARIA IN HAINAN, PEOPLE’S REPUBLIC OF CHINA

The use of chloroquine treatment for Plasmodium falciparum malaria was abandoned in China in 1979 because of widespread drug resistance. Subsequent studies found decreases in the prevalence of chloroquine-resistant strains. To evaluate these decreases and assess the current status of chloroquine sensitivity in Hainan, China, we determined the prevalence of the P. falciparum chloroquine resistance transporter (PfCRT) 76T marker in the DNA of blood samples collected from 1978 to 2001. Results showed the presence of PfCRT 76T in 101 of 112 samples (90%) from 1978 to 1981, 30 of 43 samples (70%) from 1986, 22 of 34 samples (65%) from 1997 to 1998, and 37 of 68 samples (54%) from 2001. The prevalence of PfCRT 76T thus progressively decreased after chloroquine was discontinued as a treatment for P. falciparum malaria (chi(2) = 5.2, P < 0.022 [1978-1981 versus 1986]; chi(2) = 7.4, P < 0.006 [1978-1981 versus 1997-1998]; and chi(2) = 28.8, P < 0.0001 [1978-1981 versus 2001]). Reduced prevalence of the PfCRT 76T marker is consistent with greater rates of chloroquine sensitivity from in vitro drug assays of blood samples in 1997 and 2001. Monitoring for continued decreases will provide valuable information for future drug-use policies in China.