ABSTRACT
Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ-resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side-chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug-protein interactions involving this mutant residue.
Subject(s)
Antimalarials/pharmacology , Chloroquine/pharmacology , Drug Resistance/drug effects , Membrane Proteins/metabolism , Plasmodium falciparum/drug effects , Verapamil/pharmacology , Animals , Hemin/metabolism , Membrane Proteins/genetics , Membrane Transport Proteins , Mutation , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Protozoan ProteinsABSTRACT
Microtubule-stabilizing agents are increasingly studied for cancer treatment based largely on the prior success of paclitaxel and docetaxel. In this review, we focus on the clinical development of epothilones and discodermolide, and we discuss salient preclinical and clinical highlights of these two novel natural products. These agents are distinguished by their biochemical features making them poor P-glycoprotein substrates and capable of inducing cytotoxicity in cell lines or in vivo tumor models harboring mutations in tubulin. There is now considerable data regarding the efficacy of the epothilones in human beings and discodermolide holds such promise, as well.
Subject(s)
Microtubules/drug effects , Microtubules/physiology , Mitosis/drug effects , Alkanes/chemistry , Alkanes/pharmacology , Alkanes/therapeutic use , Animals , Carbamates/chemistry , Carbamates/pharmacology , Carbamates/therapeutic use , Cell Line, Tumor , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Disease Models, Animal , Drug Screening Assays, Antitumor/methods , Drug Screening Assays, Antitumor/trends , Epothilones/chemistry , Epothilones/pharmacology , Epothilones/therapeutic use , Humans , Lactones/chemistry , Lactones/pharmacology , Lactones/therapeutic use , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/drug effects , Molecular Structure , Multicenter Studies as Topic , PyronesABSTRACT
Plasmodium falciparum malaria is increasingly difficult to treat and control due to the emergence of parasite resistance to the major antimalarials, notably chloroquine. Recent work has shown that the chloroquine resistance phenotype can be conferred by multiple amino acid mutations in the parasite digestive vacuole transmembrane protein PfCRT. Here, we have addressed whether chloroquine resistance can also be affected by changes in expression levels of this protein. Transient transfection reporter assays revealed that truncation of the pfcrt 3'-untranslated region just prior to putative polyadenylation sites resulted in a 10-fold decrease in luciferase expression levels. Using allelic exchange on a chloroquine-resistant line (7G8 from Brazil), this truncated 3'-untranslated region was inserted downstream of the pfcrt coding sequence, in the place of the endogenous 3'-untranslated region. The resulting pfcrt-modified "knockdown" clones displayed a marked decrease in pfcrt transcription and an estimated 30-40% decrease in PfCRT protein expression levels. [3H]hypoxanthine incorporation assays demonstrated up to a 40% decrease in chloroquine with or without verapamil IC50 levels of pfcrt knockdown clones, relative to the 7G8 parent. Single-cell photometric analyses were consistent with an altered intracellular pH in the knockdown clones, providing further evidence for a relationship between PfCRT, pH regulation, and chloroquine resistance. Genetic truncation of 3'-untranslated regions provides a useful approach for assessing the impact of candidate genes on drug resistance or other quantifiable phenotypes in P. falciparum.
Subject(s)
Chloroquine/pharmacology , Drug Resistance , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Plasmodium falciparum/metabolism , 3' Untranslated Regions , Alleles , Animals , Antimalarials/pharmacology , Blotting, Northern , Blotting, Southern , Blotting, Western , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Genetic Vectors , Hydrogen-Ion Concentration , Luciferases/metabolism , Membrane Transport Proteins , Microscopy, Immunoelectron , Models, Genetic , Mutation , Phenotype , Plasmodium falciparum/drug effects , Protozoan Proteins , Time Factors , TransfectionABSTRACT
Plasmodium falciparum chloroquine resistance is a major cause of worldwide increases in malaria mortality and morbidity. Recent laboratory and clinical studies have associated chloroquine resistance with point mutations in the gene pfcrt. However, direct proof of a causal relationship has remained elusive and most models have posited a multigenic basis of resistance. Here, we provide conclusive evidence that mutant haplotypes of the pfcrt gene product of Asian, African, or South American origin confer chloroquine resistance with characteristic verapamil reversibility and reduced chloroquine accumulation. pfcrt mutations increased susceptibility to artemisinin and quinine and minimally affected amodiaquine activity; hence, these antimalarials warrant further investigation as agents to control chloroquine-resistant falciparum malaria.
