Purified Plasmodium falciparum multi-drug resistance protein (PfMDR 1) binds a high affinity chloroquine analogue

Abstract

We utilize the recent successful overexpression of recombinant Plasmodium falciparum multidrug resistance transporter, purification and reconstitution of the protein, and a novel high affinity chloroquine analogue to probe hypothesized interaction between the transporter and quinoline drugs. Results suggest that PfMDR1 binding sites for chloroquine, mefloquine, and quinine overlap, that P. falciparum chloroquine resistance transporter has intrinsically higher affinity for chloroquine relative to P. falciparum multidrug resistance transporter, and that there is an isoform specific competition between the two transporters for binding of quinoline antimalarial drugs.P. falciparum multidrug resistance transporter (PfMDR1) is an ATP-Binding Cassette (ABC) transporter, many of which (e.g. human P-glycoprotein; HsMDR1) have been implicated in drug resistance phenomena. PfMDR1 is composed of two homologous halves, each containing 6 transmembrane (TM) helices and 1 nucleotide binding domain (NBD). Precisely how PfMDR1 contributes to a variety of antimalarial multi-drug resistance phenotypes has been a source of considerable controversy for some time [1][2][3][4][5]. PfMDR1 is an integral digestive vacuolar (DV) membrane protein [1], with NBDs cytosolically oriented. If traditional ABC transporter drug-pumping models are invoked, PfMDR1 would paradoxically translocate drugs into the DV, where the principal target for CQ and other drugs is believed to reside (e.g., ferriprotoporphyrin IX [FPIX] released upon hemoglobin catabolism, see [6,7]). How concentrating drugs at the site of action would promote resistance is a puzzle. Also, genetics and a plethora of field studies now show that pfcrt mutations are the main molecular determinants of P. falciparum CQ resistance (CQR) [4,8]. It has therefore been suggested that PfMDR1 only modulates the level of CQR and resistance to other antimalarials in a PfCRT-dependent manner [5,9].We have recently reported the successful heterologous overexpression of several PfMDR1 isoforms (allelic forms) found in either CQS or CQR parasites [10], have characterized unusual ATPase activity for these [10,11], and have identified residues in one PfMDR1 isoform that confer a specific drug-stimulated ATPase phenotype [11]. The 3D7 isoform is found within CQS strain 3D7 and has a single amino acid difference (N86) relative to the Dd2 isoform found in CQR strain Dd2 (Y86), whereas the 7G8 isoform has 4 amino acid substitutions relative to 3D7 (Y184F, S1034C, N1042D, D1246Y). Strain Dd2 exhibits a CQR phenotype that is partially reversible by verapamil (VPL) whereas CQR observed in © 2010 Elsevier B.V. All rights reserved.Correspondence to PDR at: roepep@georgetown.edu; 202 687 -7300; 202 687 -7186 (fax). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it i...

A HECT Ubiquitin-Protein Ligase as a Novel Candidate Gene for Altered Quinine and Quinidine Responses in Plasmodium falciparum

“…This approach follows up on the idea of a possible correlation between quinine resistance and reduced intracellular quinine accumulation, as suggested by the fact that pfcrt , pfmdr1 and pfnhe , all encode transporters that are thought to contribute to quinine resistance by reducing digestive vacuolar drug concentrations below toxic levels [12], [13], [19], [21], [22], [32]–[34]. Here we describe a novel putative quinine response gene, termed MAL7P1.19 (PF3D7_0704600).…”

UV-triggered Affinity Capture Identifies Interactions between the Plasmodium falciparum Multidrug Resistance Protein 1 (PfMDR1) and Antimalarial Agents in Live Parasitized Cells

“…50% inhibition of AzBCQ photolabeling occurred at about 4-fold molar excess of ACT-213615 over the AzBCQ probe, whereas the inactive enantiomer did not show significant probe competition up to 6-fold molar excess over AzBCQ (data not shown). The potency of ACT-213615 for inhibiting AzBCQ photolabeling to PfMDR1 was similar to that of mefloquine, the most potent inhibitor of chloroquine probe binding to PfMDR1 yet measured (13).…”

“…Transport studies with PfENT using Xenopus laevis oocytes injected with mRNA encoding PfENT4, PvENT4, or PfENT1 were performed as described previously (12) except that oocytes were preincubated in transport buffer in the presence of 1 or 10 M compound or solvent control for 15 min. Azido-biotin-chloroquine (AzBCQ) photolabeling of partially purified PfMDR1 protein was done as described previously (13). This method was adapted for ACT-460953 photolabeling by titrating the time of UV exposure and the concentration of photo-reactive probe.…”

“…This method was adapted for ACT-460953 photolabeling by titrating the time of UV exposure and the concentration of photo-reactive probe. All other conditions were as reported previously (13). Specific labeling of PfMDR1 was observed at 40 M probe after 10 min of UV irradiation, as described previously (12).…”

Investigating the activity of quinine analogues versus chloroquine resistant Plasmodium falciparum

“…The explanation for the differential antiplasmodial activity of ortho / para vs. meta substituted derivatives could lie in steric and/or electronic interaction with non-heme drug targets, such as the P. falciparum chloroquine resistance transporter (PfCRT), multidrug resistance transporter (PfMDR1), or sodium-proton exchanger (PfNHE). 38,39,40,41,42,43 These possibilities might also account for the lack of correlation between the two sets of IC 50 values. Indeed, derivatives QD-2 , CD-1 , and CN-4 exhibit significantly decreased Hz inhibition at pH 5.2 but improved antiplasmodial activity against Dd2, suggesting alternative targets may be responsible.…”