This study shows an association between the pfcrt T76 mutation in P. falciparum and the development of chloroquine resistance during the treatment of malaria. This mutation can be used as a marker in surveillance for chloroquine-resistant falciparum malaria.

Recent reports of increased tolerance to artemisinin derivatives-the last widely effective class of antimalarials -bolster the medical need for new treatments. The spirotetrahydro-β-carbolines, or spiroindolones, are a new class of fast-acting and potent schizonticidal drugs displaying low nanomolar potency against Plasmodium falciparum and Plasmodium vivax clinical isolates. Spiroindolones rapidly diminish protein synthesis in P. falciparum, an effect that is ablated in parasites bearing non-synonymous mutations in the gene encoding the P-type cation-transporter ATPase4 (PfATP4). The optimized spiroindolone NITD609 shows an acceptable safety profile and pharmacokinetic properties compatible with once-daily oral dosing; and demonstrates singledose efficacy in a rodent malaria model. Collectively, these data demonstrate that NITD609 possesses a pharmacological profile suitable for a new drug candidate for the treatment of malaria.Globally, 3.3 billion people are exposed to malaria, a devastating disease that causes over 800,000 deaths each year and kills more under five-year-olds than any other infectious agent (1). Fifty years ago, malaria had been eliminated from many areas of the world through effective antimalarial drug treatments, vector control interventions and disease prevention # Corresponding authors (Winzeler@scripps.edu and Thierry.diagana@novartis.com). * These authors equally contributed to this work One-sentence summary We describe the pharmacological profile of a new antimalarial drug candidate-the spiroindolone NITD609-which through a novel mechanism of action rapidly clears a Plasmodium infection upon administration of a single oral dose in a malaria mouse model. NIH Public Access Author ManuscriptScience. Author manuscript; available in PMC 2011 September 3. (2). However, the global spread of drug resistance resulted, by the 1980s, in a substantial increase in disease incidence and mortality. Today, some encouraging epidemiological data suggest that the introduction of new drugs (notably the artemisinin-based combination therapies or ACTs) may have reversed that trend (3). Derivatives of the endoperoxide artemisinin constitute the only antimalarial drugs that remain effective in all malariaendemic regions, but recent reports suggest that decades of continuous use as monotherapies might have fostered the emergence of resistance (4-6). This realization has triggered a concerted search for new drugs that could be deployed if artemisinin resistance were to spread.Many of the therapies currently in development utilize known antimalarial pharmacophores (e.g. aminoquinolines and/or peroxides) chemically modified to overcome the liabilities of their predecessors (7). While these compounds may prove to be important in the treatment of malaria, it would be preferable to discover novel chemotypes with a distinct mechanism of action (8). However, despite significant advances in our understanding of Plasmodium genome biology, the identification and validation of new drug targets has proven challengi...

SummaryThe determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K76I mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.

The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites.

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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...

BACKGROUND Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)–propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale. METHODS We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci. RESULTS We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas — one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China — with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay. CONCLUSIONS No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral.

The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K761 mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.