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.Chloroquine has for decades been the primary chemotherapeutic means of malaria treatment and control (1). This safe and inexpensive 4-aminoquinoline compound accumulates inside the digestive vacuole of the infected red blood cell, where it is believed to form complexes with toxic heme moieties and interfere with detoxification mechanisms that include heme sequestration into an inert pigment called hemozoin (2-4). Chloroquine resistance (CQR) was first reported in Southeast Asia and South America and has now spread to the vast majority of malaria-endemic countries (1). pfcrt was recently identified as a candidate gene for CQR after the analysis of a genetic cross between a chloroquine-resistant clone (Dd2, Indochina) and a chloroquine-sensitive clone (HB3, Honduras) (5-7). The PfCRT protein localizes to the digestive vacuole membrane and contains 10 putative transmembrane domains (7,8). Point mutations in PfCRT, including the Lys 76 → Thr (K76T) mutation in the first predicted transmembrane domain, show an association with CQR in field isolates and clinical studies (7,9,10). Episomal complementation assays demonstrated a low-level, atypical CQR phenotype in chloroquine-selected, transformed, pseudo-diploid parasite lines that coexpressed the Dd2 form of pfcrt (containing eight point mutations; Table 1), under the control of a heterologous promoter, with the wild-type endogenous allele (7).To address whether mutations in pfcrt are sufficient to confer CQR, we implemented an allelic exchange approach to replace the endogenous pfcrt allele of a chloroquine-sensitive line (GC03) with pfcrt alleles from chloroquine-resistant lines of Asian, African, or South American origin (Table 1) (fig. S1) (11). This approach maintained the endogenous promoter and terminator regulatory elements for correct stage-specific expression and did not use chloroquine during the selection procedure. As a result of this gene's highly interrupted nature * To whom correspondence should be addressed. dfidock@aecom.yu.edu. Wootton et al. (39) recently presented compelling evidence for rapid evolutionary sweeps of mutant pfcrt sequences throughout malaria-endemic areas, starting from a limited number of initi...

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.

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. [ 3 H]hypoxanthine incorporation assays demonstrated up to a 40% decrease in chloroquine with or without verapamil IC 50 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.

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.

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