Photoaffinity Labeling of the Plasmodium falciparum Chloroquine Resistance Transporter with a Novel Perfluorophenylazido Chloroquine

Abstract

Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein.

Overcoming Chloroquine Resistance in Malaria: Design, Synthesis, and Structure-Activity Relationships of Novel Hybrid Compounds

“…It has been shown previously that a tertiary amine in this position impacts the activity of CQ analogues, as it influences the diffusion of the compounds in the digestive vacuole (19,20). Ethyl amine compound 21a was first connected to a C 3 linker, using chloroiodopropane (compound 33), leading to chloropro- Table S2 in the supplemental material for SEM and statistics).…”

“…3). The 4-aminoquinoline scaffold has been shown to be essential for the binding of CQ to free heme (18), and both the terminal tertiary amino group and the basicity of the quinolyl nitrogen are key elements (19,20). Ideally, the length of the aliphatic side chain should be 2 to 4 carbons; however, the methyl group has little influence on the activity and was thus not retained.…”

Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

“…An earlier study identified the vacuolar loop between putative transmembrane domains 9 and 10 as part of the PfCRT-binding site for AzBCQ, a CQ analog and photoaffinity label (42). Binding of the affinity label was competitively inhibited by QN, suggesting overlap in their respective binding sites.…”

The Biochemistry of Quinoline Antimalarial Drug Resistance

“…Scatchard analysis indicated a single-drug binding site in PfCRT, and, surprisingly, that CQS and CQR isoforms of PfCRT have similar affinity for CQ (K d ¼ 435 and 385 nM, respectively). A recent follow-up study of CQ binding used covalent attachment of a perfluoroazidotagged CQ probe to quantify CQ probe binding versus other quinoline antimalarials and to further define the drug binding site in PfCRT, which is predicted to be disposed towards the DV side of the DV membrane (Lekostaj et al 2008). Satisfyingly, this binding site can also easily place the quinoline ring of CQ near mutations in PfCRT isoforms that are known to modulate response to drugs (Fidock et al 2000b;Cooper et al 2002).…”

“…Satisfyingly, this binding site can also easily place the quinoline ring of CQ near mutations in PfCRT isoforms that are known to modulate response to drugs (Fidock et al 2000b;Cooper et al 2002). It is clear at this point that both wild-type (CQS) and mutant (CQR) isoforms of PfCRT bind CQ at a single-drug binding site, that related quinoline drugs such as MQ and QN compete with CQ for binding to this site, and that chemoreversal agents such as VPL inhibit CQ binding only for some PfCRT isoforms (Lekostaj et al 2008).…”