Publications by authors named "David A Fidock"

202 Publications

Repositioning and Characterization of 1-(Pyridin-4-yl)pyrrolidin-2-one Derivatives as Cytoplasmic Prolyl-tRNA Synthetase Inhibitors.

ACS Infect Dis 2021 Apr 30. Epub 2021 Apr 30.

Medicines for Malaria Venture, ICC, Route de Pré-Bois 20, 1215 Geneva, Switzerland.

Prolyl-tRNA synthetase (PRS) is a clinically validated antimalarial target. Screening of a set of PRS ATP-site binders, initially designed for human indications, led to identification of 1-(pyridin-4-yl)pyrrolidin-2-one derivatives representing a novel antimalarial scaffold. Evidence designates cytoplasmic PRS as the drug target. The frontrunner and its active enantiomer exhibited low-double-digit nanomolar activity against resistant () laboratory strains and development of liver schizonts. No cross-resistance with strains resistant to other known antimalarials was noted. In addition, a similar level of growth inhibition was observed against clinical field isolates of and . The slow killing profile and the relative high propensity to develop resistance (minimum inoculum resistance of 8 × 10 parasites at a selection pressure of 3 × IC) constitute unfavorable features for treatment of malaria. However, potent blood stage and antischizontal activity are compelling for causal prophylaxis which does not require fast onset of action. Achieving sufficient on-target selectivity appears to be particularly challenging and should be the primary focus during the next steps of optimization of this chemical series. Encouraging preliminary off-target profile and oral efficacy in a humanized murine model of malaria allowed us to conclude that 1-(pyridin-4-yl)pyrrolidin-2-one derivatives represent a promising starting point for the identification of novel antimalarial prophylactic agents that selectively target PRS.
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http://dx.doi.org/10.1021/acsinfecdis.1c00020DOI Listing
April 2021

Potent Antimalarials with Development Potential Identified by Structure-Guided Computational Optimization of a Pyrrole-Based Dihydroorotate Dehydrogenase Inhibitor Series.

J Med Chem 2021 May 20;64(9):6085-6136. Epub 2021 Apr 20.

Infectious Diseases Research Collaboration, Kampala, Uganda.

Dihydroorotate dehydrogenase (DHODH) has been clinically validated as a target for the development of new antimalarials. Experience with clinical candidate triazolopyrimidine DSM265 () suggested that DHODH inhibitors have great potential for use in prophylaxis, which represents an unmet need in the malaria drug discovery portfolio for endemic countries, particularly in areas of high transmission in Africa. We describe a structure-based computationally driven lead optimization program of a pyrrole-based series of DHODH inhibitors, leading to the discovery of two candidates for potential advancement to preclinical development. These compounds have improved physicochemical properties over prior series frontrunners and they show no time-dependent CYP inhibition, characteristic of earlier compounds. Frontrunners have potent antimalarial activity against blood and liver schizont stages and show good efficacy in SCID mouse models. They are equally active against and field isolates and are selective for DHODHs versus mammalian enzymes.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00173DOI Listing
May 2021

Genomic and Genetic Approaches to Studying Antimalarial Drug Resistance and Plasmodium Biology.

Trends Parasitol 2021 Jun 11;37(6):476-492. Epub 2021 Mar 11.

Department of Microbiology & Immunology, Department of Medicine, Columbia University Irving Medical Center, New York, NY, USA; Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY, USA. Electronic address:

Recent progress in genomics and molecular genetics has empowered novel approaches to study gene functions in disease-causing pathogens. In the human malaria parasite Plasmodium falciparum, the application of genome-based analyses, site-directed genome editing, and genetic systems that allow for temporal and quantitative regulation of gene and protein expression have been invaluable in defining the genetic basis of antimalarial resistance and elucidating candidate targets to accelerate drug discovery efforts. Using examples from recent studies, we review applications of some of these approaches in advancing our understanding of Plasmodium biology and illustrate their contributions and limitations in characterizing parasite genomic loci associated with antimalarial drug responses.
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http://dx.doi.org/10.1016/j.pt.2021.02.007DOI Listing
June 2021

Plasmodium falciparum LipB mutants display altered redox and carbon metabolism in asexual stages and cannot complete sporogony in Anopheles mosquitoes.

Int J Parasitol 2021 May 11;51(6):441-453. Epub 2021 Mar 11.

Wellcome Centre for Integrative Parasitology, University of Glasgow, Glasgow, United Kingdom; Department of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom. Electronic address:

Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs.
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http://dx.doi.org/10.1016/j.ijpara.2020.10.011DOI Listing
May 2021

3-Hydroxy-propanamidines, a New Class of Orally Active Antimalarials Targeting Plasmodium falciparum.

J Med Chem 2021 Mar 5;64(6):3035-3047. Epub 2021 Mar 5.

Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitaetsstraße 1, Düsseldorf 40225, Germany.

3-Hydroxypropanamidines are a new promising class of highly active antiplasmodial agents. The most active compound exhibited excellent antiplasmodial activity with nanomolar inhibition of chloroquine-sensitive and multidrug-resistant parasite strains of (with IC values of 5 and 12 nM against 3D7 and Dd2 strains, respectively) as well as low cytotoxicity in human cells. In addition, showed strong activity in the mouse model with a cure rate of 66% at 50 mg/kg and a cure rate of 33% at 30 mg/kg in the Peters test after once daily oral administration for 4 consecutive days. A quick onset of action was indicated by the fast drug absorption shown in mice. The new lead compound was also characterized by a high barrier to resistance and inhibited the heme detoxification machinery in .
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http://dx.doi.org/10.1021/acs.jmedchem.0c01744DOI Listing
March 2021

MalDA, Accelerating Malaria Drug Discovery.

Trends Parasitol 2021 Jun 26;37(6):493-507. Epub 2021 Feb 26.

Department of Pediatrics, School of Medicine, University of California, San Diego (UCSD), La Jolla, CA 92093, USA. Electronic address:

The Malaria Drug Accelerator (MalDA) is a consortium of 15 leading scientific laboratories. The aim of MalDA is to improve and accelerate the early antimalarial drug discovery process by identifying new, essential, druggable targets. In addition, it seeks to produce early lead inhibitors that may be advanced into drug candidates suitable for preclinical development and subsequent clinical testing in humans. By sharing resources, including expertise, knowledge, materials, and reagents, the consortium strives to eliminate the structural barriers often encountered in the drug discovery process. Here we discuss the mission of the consortium and its scientific achievements, including the identification of new chemically and biologically validated targets, as well as future scientific directions.
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http://dx.doi.org/10.1016/j.pt.2021.01.009DOI Listing
June 2021

Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in .

J Med Chem 2021 Mar 23;64(5):2739-2761. Epub 2021 Feb 23.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997635PMC
March 2021

Identification and Profiling of a Novel Diazaspiro[3.4]octane Chemical Series Active against Multiple Stages of the Human Malaria Parasite and Optimization Efforts.

J Med Chem 2021 02 12;64(4):2291-2309. Epub 2021 Feb 12.

Wits Research Institute for Malaria, School of Pathology, Department of Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2193, South Africa.

A novel diazaspiro[3.4]octane series was identified from a whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the cyclic amine resistance locus in the mode of resistance.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00034DOI Listing
February 2021

Hemozoin Promotes Lung Inflammation via Host Epithelial Activation.

mBio 2021 02 9;12(1). Epub 2021 Feb 9.

Division of Infectious Diseases, Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, New York, USA.

Respiratory distress in severe malaria is associated with high mortality, but its pathogenesis remains unclear. The malaria pigment hemozoin (HZ) is abundant in target organs of severe malaria, including the lungs, and is known to be a potent innate immune activator of phagocytes. We hypothesized that HZ might also stimulate lung epithelial activation and thereby potentiate lung inflammation. We show here that airway epithelium stimulated with HZ undergoes global transcriptional reprogramming and changes in cell surface protein expression that comprise an epithelial activation phenotype. Proinflammatory signaling is induced, and key cytoadherence molecules are upregulated, including several associated with severe malaria, such as CD36 and ICAM1. Epithelial and extracellular matrix remodeling pathways are transformed, including induction of key metalloproteases and modulation of epithelial junctions. The overall program induced by HZ serves to promote inflammation and neutrophil transmigration, and is recapitulated in a murine model of HZ-induced acute pneumonitis. Together, our data demonstrate a direct role for hemozoin in stimulating epithelial activation that could potentiate lung inflammation in malaria. Respiratory distress (RD) is a complication of severe malaria associated with a particularly high risk for death in African children infected with the parasite The pathophysiology underlying RD remains poorly understood, and the condition is managed supportively. The parasite-derived factor HZ accumulates in target organs of severe malaria, including the lungs, and is a potent stimulator of immune cells. Our findings demonstrate that HZ causes global activation of lung epithelial cells, a response that directly promotes lung inflammation. HZ stimulates expression of key proinflammatory and cell surface molecules, alters signaling pathways involved in epithelial-matrix remodeling, and promotes neutrophil transmigration and airway inflammation. The lung epithelial activation induced by HZ mimics patterns seen in malarial lung injury and provides new insights into the molecular pathogenesis of RD.
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http://dx.doi.org/10.1128/mBio.02399-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885402PMC
February 2021

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

Sci Rep 2021 Jan 22;11(1):2121. Epub 2021 Jan 22.

Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, 10032, USA.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.
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http://dx.doi.org/10.1038/s41598-021-81486-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822874PMC
January 2021

Artemisinin-resistant K13 mutations rewire Plasmodium falciparum's intra-erythrocytic metabolic program to enhance survival.

Nat Commun 2021 01 22;12(1):530. Epub 2021 Jan 22.

Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY, USA.

The emergence and spread of artemisinin resistance, driven by mutations in Plasmodium falciparum K13, has compromised antimalarial efficacy and threatens the global malaria elimination campaign. By applying systems-based quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we provide evidence that K13 mutations alter multiple aspects of the parasite's intra-erythrocytic developmental program. These changes impact cell-cycle periodicity, the unfolded protein response, protein degradation, vesicular trafficking, and mitochondrial metabolism. K13-mediated artemisinin resistance in the Cambodian Cam3.II line was reversed by atovaquone, a mitochondrial electron transport chain inhibitor. These results suggest that mitochondrial processes including damage sensing and anti-oxidant properties might augment the ability of mutant K13 to protect P. falciparum against artemisinin action by helping these parasites undergo temporary quiescence and accelerated growth recovery post drug elimination.
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http://dx.doi.org/10.1038/s41467-020-20805-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822823PMC
January 2021

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

Sci Rep 2021 Jan 21;11(1):1888. Epub 2021 Jan 21.

Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, UK.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance.
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http://dx.doi.org/10.1038/s41598-021-81343-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820608PMC
January 2021

Expansion of a Specific Plasmodium falciparum PfMDR1 Haplotype in Southeast Asia with Increased Substrate Transport.

mBio 2020 12 1;11(6). Epub 2020 Dec 1.

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, Braga, Portugal

Artemisinin-based combination therapies (ACTs) have been vital in reducing malaria mortality rates since the 2000s. Their efficacy, however, is threatened by the emergence and spread of artemisinin resistance in Southeast Asia. The multidrug resistance protein 1 (PfMDR1) transporter plays a central role in parasite resistance to ACT partner drugs through gene copy number variations (CNV) and/or single nucleotide polymorphisms (SNPs). Using genomic epidemiology, we show that multiple copies encoding the N86 and 184F haplotype are prevalent across Southeast Asia. Applying genome editing tools on the Southeast Asian Dd2 strain and using a surrogate assay to measure transporter activity in infected red blood cells, we demonstrate that parasites harboring multicopy N86/184F PfMDR1 have a higher Fluo-4 transport capacity compared with those expressing the wild-type N86/Y184 haplotype. Multicopy N86/184F PfMDR1 is also associated with decreased parasite susceptibility to lumefantrine. These findings provide evidence of the geographic selection and expansion of specific multicopy PfMDR1 haplotypes associated with multidrug resistance in Southeast Asia. Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of polymorphisms.
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http://dx.doi.org/10.1128/mBio.02093-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733942PMC
December 2020

Plasmodium berghei K13 Mutations Mediate Artemisinin Resistance That Is Reversed by Proteasome Inhibition.

mBio 2020 11 10;11(6). Epub 2020 Nov 10.

Institute of Infection, Immunity & Inflammation, Wellcome Centre for Integrative Parasitology, University of Glasgow, Glasgow, United Kingdom

The recent emergence of parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the K13 protein, which enhance survival of early ring-stage parasites treated with the artemisinin active metabolite dihydroartemisinin and associate with delayed parasite clearance However, association of K13 mutations with artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous K13 mutations into the gene of an artemisinin-sensitive rodent model of malaria. Introduction of the orthologous K13 F446I, M476I, Y493H, and R539T mutations into K13 yielded gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-h assay and increased survival in an adapted ring-stage survival assay. Mutant K13 parasites also displayed delayed clearance upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into , while the equivalents of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a -selective proteasome inhibitor strongly synergized dihydroartemisinin action in these K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome artemisinin resistance. Taken together, our findings provide clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins and, most importantly, under conditions. Recent successes in malaria control have been seriously threatened by the emergence of parasite resistance to the frontline artemisinin drugs in Southeast Asia. artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon drug treatment. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating artemisinin resistance under laboratory conditions. Nonetheless, the causal role of these mutations under conditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite, Phenotyping of these K13 mutant parasites provides evidence of their role in mediating artemisinin resistance , which supports artemisinin resistance observations. However, we were unable to introduce some of the K13 mutations (C580Y and I543T) into the corresponding amino acid residues, while other introduced mutations (M476I and R539T equivalents) carried pronounced fitness costs. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins and using the well-characterized model. We also show that inhibition of the proteasome offsets parasite resistance to artemisinins in these mutant lines.
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http://dx.doi.org/10.1128/mBio.02312-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667033PMC
November 2020

Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility.

Nat Commun 2020 09 23;11(1):4813. Epub 2020 Sep 23.

Whitehead Institute for Biomedical Research, Cambridge, MA, USA.

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.
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http://dx.doi.org/10.1038/s41467-020-18624-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511413PMC
September 2020

Molecular Mechanisms of Drug Resistance in Malaria.

Annu Rev Microbiol 2020 09;74:431-454

Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, New York 10032, USA; email:

Understanding and controlling the spread of antimalarial resistance, particularly to artemisinin and its partner drugs, is a top priority. parasites resistant to chloroquine, amodiaquine, or piperaquine harbor mutations in the chloroquine resistance transporter (PfCRT), a transporter resident on the digestive vacuole membrane that in its variant forms can transport these weak-base 4-aminoquinoline drugs out of this acidic organelle, thus preventing these drugs from binding heme and inhibiting its detoxification. The structure of PfCRT, solved by cryogenic electron microscopy, shows mutations surrounding an electronegative central drug-binding cavity where they presumably interact with drugs and natural substrates to control transport. susceptibility to heme-binding antimalarials is also modulated by overexpression or mutations in the digestive vacuole membrane-bound ABC transporter PfMDR1 ( multidrug resistance 1 transporter). Artemisinin resistance is primarily mediated by mutations in Kelch13 protein (K13), a protein involved in multiple intracellular processes including endocytosis of hemoglobin, which is required for parasite growth and artemisinin activation. Combating drug-resistant malaria urgently requires the development of new antimalarial drugs with novel modes of action.
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http://dx.doi.org/10.1146/annurev-micro-020518-115546DOI Listing
September 2020

Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda.

Nat Med 2020 10 3;26(10):1602-1608. Epub 2020 Aug 3.

Malaria Genetics and Resistance Unit, Institut Pasteur, Paris, France.

Artemisinin resistance (delayed P. falciparum clearance following artemisinin-based combination therapy), is widespread across Southeast Asia but to date has not been reported in Africa. Here we genotyped the P. falciparum K13 (Pfkelch13) propeller domain, mutations in which can mediate artemisinin resistance, in pretreatment samples collected from recent dihydroarteminisin-piperaquine and artemether-lumefantrine efficacy trials in Rwanda. While cure rates were >95% in both treatment arms, the Pfkelch13 R561H mutation was identified in 19 of 257 (7.4%) patients at Masaka. Phylogenetic analysis revealed the expansion of an indigenous R561H lineage. Gene editing confirmed that this mutation can drive artemisinin resistance in vitro. This study provides evidence for the de novo emergence of Pfkelch13-mediated artemisinin resistance in Rwanda, potentially compromising the continued success of antimalarial chemotherapy in Africa.
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http://dx.doi.org/10.1038/s41591-020-1005-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541349PMC
October 2020

Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria.

Nature 2020 06 22;582(7810):104-108. Epub 2020 Apr 22.

Center for International Health Research, Rhode Island Hospital, Brown University Medical School, Providence, RI, USA.

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.
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http://dx.doi.org/10.1038/s41586-020-2220-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372601PMC
June 2020

Local emergence in Amazonia of C580Y mutants associated with artemisinin resistance.

Elife 2020 05 12;9. Epub 2020 May 12.

Laboratoire de parasitologie, Centre Nationale de Référence du Paludisme, World Health Organization Collaborating Center for surveillance of antimalarial drug resistance, Institut Pasteur de la Guyane, Cayenne, French Guiana.

Antimalarial drug resistance has historically arisen through convergent mutations in parasite populations in Southeast Asia and South America. For the past decade in Southeast Asia, artemisinins, the core component of first-line antimalarial therapies, have experienced delayed parasite clearance associated with several mutations, primarily C580Y. We report that mutant has emerged independently in Guyana, with genome analysis indicating an evolutionary origin distinct from Southeast Asia. C580Y parasites were observed in 1.6% (14/854) of samples collected in Guyana in 2016-2017. Introducing C580Y or R539T mutations by gene editing into local parasites conferred high levels of artemisinin resistance. growth competition assays revealed a fitness cost associated with these variants, potentially explaining why these resistance alleles have not increased in frequency more quickly in South America. These data place local malaria control efforts at risk in the Guiana Shield.
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http://dx.doi.org/10.7554/eLife.51015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217694PMC
May 2020

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity.

Cell Chem Biol 2020 07 30;27(7):806-816.e8. Epub 2020 Apr 30.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.
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http://dx.doi.org/10.1016/j.chembiol.2020.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369637PMC
July 2020

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13.

PLoS Pathog 2020 04 20;16(4):e1008482. Epub 2020 Apr 20.

Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY, United States of America.

The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment.
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http://dx.doi.org/10.1371/journal.ppat.1008482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192513PMC
April 2020

Turning the tide: targeting PfCRT to combat drug-resistant P. falciparum?

Nat Rev Microbiol 2020 05;18(5):261-262

Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY, USA.

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http://dx.doi.org/10.1038/s41579-020-0353-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269106PMC
May 2020

Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway.

Nat Commun 2020 04 14;11(1):1780. Epub 2020 Apr 14.

Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, CA, 92093, USA.

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing.
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http://dx.doi.org/10.1038/s41467-020-15440-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156427PMC
April 2020

Structure-Based Screening of Glutathione S-Transferase Identifies CB-27 as a Novel Antiplasmodial Compound.

Front Pharmacol 2020 17;11:246. Epub 2020 Mar 17.

Department of Microbiology and Medical Zoology, University of Puerto Rico School of Medicine, San Juan, PR, United States.

parasites are increasingly drug-resistant, requiring the search for novel antimalarials with distinct modes of action. Enzymes in the glutathione pathway, including glutathione S-transferase (GST), show promise as novel antimalarial targets. This study aims to better understand the biological function of GST, assess its potential as a drug target, and identify novel antiplasmodial compounds using the rodent model . By using reverse genetics, we provided evidence that GST is essential for survival of intra-erythrocytic stages and is a valid target for drug development. A structural model of the glutathione S-transferase (PbGST) protein was generated and used in a structure-based screening of 900,000 compounds from the ChemBridge Hit2Lead library. Forty compounds were identified as potential inhibitors and analyzed in parasite drug susceptibility assays. One compound, CB-27, exhibited antiplasmodial activity with an EC of 0.5 μM toward and 0.9 μM toward multidrug-resistant Dd2 clone B2 parasites. Moreover, CB-27 showed a concentration-dependent inhibition of the PbGST enzyme without inhibiting the human ortholog. A shape similarity screening using CB-27 as query resulted in the identification of 24 novel chemical scaffolds, with six of them showing antiplasmodial activity ranging from EC of 0.6-4.9 μM. Pharmacokinetic and toxicity predictions suggest that the lead compounds have drug-likeness properties. The antiplasmodial potency, the absence of hemolytic activity, and the predicted drug-likeness properties position these compounds for lead optimization and further development as antimalarials.
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http://dx.doi.org/10.3389/fphar.2020.00246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090221PMC
March 2020

A Phase 1, Placebo-controlled, Randomized, Single Ascending Dose Study and a Volunteer Infection Study to Characterize the Safety, Pharmacokinetics, and Antimalarial Activity of the Plasmodium Phosphatidylinositol 4-Kinase Inhibitor MMV390048.

Clin Infect Dis 2020 12;71(10):e657-e664

Medicines for Malaria Venture, Geneva, Switzerland.

Background: MMV390048 is the first Plasmodium phosphatidylinositol 4-kinase inhibitor to reach clinical development as a new antimalarial. We aimed to characterize the safety, pharmacokinetics, and antimalarial activity of a tablet formulation of MMV390048.

Methods: A 2-part, phase 1 trial was conducted in healthy adults. Part 1 was a double-blind, randomized, placebo-controlled, single ascending dose study consisting of 3 cohorts (40, 80, 120 mg MMV390048). Part 2 was an open-label volunteer infection study using the Plasmodium falciparum induced blood-stage malaria model consisting of 2 cohorts (40 mg and 80 mg MMV390048).

Results: Twenty four subjects were enrolled in part 1 (n = 8 per cohort, randomized 3:1 MMV390048:placebo) and 15 subjects were enrolled in part 2 (40 mg [n = 7] and 80 mg [n = 8] cohorts). One subject was withdrawn from part 2 (80 mg cohort) before dosing and was not included in analyses. No serious or severe adverse events were attributed to MMV390048. The rate of parasite clearance was greater in subjects administered 80 mg compared to those administered 40 mg (clearance half-life 5.5 hours [95% confidence interval {CI}, 5.2-6.0 hours] vs 6.4 hours [95% CI, 6.0-6.9 hours]; P = .005). Pharmacokinetic/pharmacodynamic modeling estimated a minimum inhibitory concentration of 83 ng/mL and a minimal parasiticidal concentration that would achieve 90% of the maximum effect of 238 ng/mL, and predicted that a single 120-mg dose would achieve an adequate clinical and parasitological response with 92% certainty.

Conclusions: The safety, pharmacokinetics, and pharmacodynamics of MMV390048 support its further development as a partner drug of a single-dose combination therapy for malaria.

Clinical Trials Registration: NCT02783820 (part 1); NCT02783833 (part 2).
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http://dx.doi.org/10.1093/cid/ciaa368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744986PMC
December 2020

Probing the Open Global Health Chemical Diversity Library for Multistage-Active Starting Points for Next-Generation Antimalarials.

ACS Infect Dis 2020 04 4;6(4):613-628. Epub 2020 Mar 4.

School of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States.

Most phenotypic screens aiming to discover new antimalarial chemotypes begin with low cost, high-throughput tests against the asexual blood stage (ABS) of the malaria parasite life cycle. Compounds active against the ABS are then sequentially tested in more difficult assays that predict whether a compound has other beneficial attributes. Although applying this strategy to new chemical libraries may yield new leads, repeated iterations may lead to diminishing returns and the rediscovery of chemotypes hitting well-known targets. Here, we adopted a different strategy to find starting points, testing ∼70,000 open source small molecules from the Global Health Chemical Diversity Library for activity against the liver stage, mature sexual stage, and asexual blood stage malaria parasites in parallel. In addition, instead of using an asexual assay that measures accumulated parasite DNA in the presence of compound (SYBR green), a real time luciferase-dependent parasite viability assay was used that distinguishes slow-acting (delayed death) from fast-acting compounds. Among 382 scaffolds with the activity confirmed by dose response (<10 μM), we discovered 68 novel delayed-death, 84 liver stage, and 68 stage V gametocyte inhibitors as well. Although 89% of the evaluated compounds had activity in only a single life cycle stage, we discovered six potent (half-maximal inhibitory concentration of <1 μM) multistage scaffolds, including a novel cytochrome bc1 chemotype. Our data further show the luciferase-based assays have higher sensitivity. Chemoinformatic analysis of positive and negative compounds identified scaffold families with a strong enrichment for activity against specific or multiple stages.
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http://dx.doi.org/10.1021/acsinfecdis.9b00482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155171PMC
April 2020

Preclinical Antimalarial Combination Study of M5717, a Plasmodium falciparum Elongation Factor 2 Inhibitor, and Pyronaridine, a Hemozoin Formation Inhibitor.

Antimicrob Agents Chemother 2020 03 24;64(4). Epub 2020 Mar 24.

Global Health Institute of Merck, Eysins, Switzerland

Antimalarial drug resistance in the parasite poses a constant challenge for drug development. To mitigate this risk, new antimalarial medicines should be developed as fixed-dose combinations. Assessing the pharmacodynamic interactions of potential antimalarial drug combination partners during early phases of development is essential in developing the targeted parasitological and clinical profile of the final drug product. Here, we have studied the combination of M5717, a translation elongation factor 2 inhibitor, and pyronaridine, an inhibitor of hemozoin formation. Our test cascade consisted of isobolograms as well as studies in the severe combined immunodeficient (SCID) mouse model. We also analyzed pharmacokinetic and pharmacodynamic parameters, including genomic sequencing of recrudescent parasites. We observed no pharmacokinetic interactions with the combination of M5717 and pyronaridine. M5717 did not negatively impact the rate of kill of the faster-acting pyronaridine, and the latter was able to suppress the selection of M5717-resistant mutants, as well as significantly delay the recrudescence of parasites both with suboptimal and optimal dosing regimens.
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http://dx.doi.org/10.1128/AAC.02181-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179297PMC
March 2020

The Antimalarial Natural Product Salinipostin A Identifies Essential α/β Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites.

Cell Chem Biol 2020 02 23;27(2):143-157.e5. Epub 2020 Jan 23.

Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/β serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.
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http://dx.doi.org/10.1016/j.chembiol.2020.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027986PMC
February 2020

Combining Stage Specificity and Metabolomic Profiling to Advance Antimalarial Drug Discovery.

Cell Chem Biol 2020 02 5;27(2):158-171.e3. Epub 2019 Dec 5.

Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA. Electronic address:

We report detailed susceptibility profiling of asexual blood stages of the malaria parasite Plasmodium falciparum to clinical and experimental antimalarials, combined with metabolomic fingerprinting. Results revealed a variety of stage-specific and metabolic profiles that differentiated the modes of action of clinical antimalarials including chloroquine, piperaquine, lumefantrine, and mefloquine, and identified late trophozoite-specific peak activity and stage-specific biphasic dose-responses for the mitochondrial inhibitors DSM265 and atovaquone. We also identified experimental antimalarials hitting previously unexplored druggable pathways as reflected by their unique stage specificity and/or metabolic profiles. These included several ring-active compounds, ones affecting hemoglobin catabolism through distinct pathways, and mitochondrial inhibitors with lower propensities for resistance than either DSM265 or atovaquone. This approach, also applicable to other microbes that undergo multiple differentiation steps, provides an effective tool to prioritize compounds for further development within the context of combination therapies.
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http://dx.doi.org/10.1016/j.chembiol.2019.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031696PMC
February 2020

Structure and drug resistance of the Plasmodium falciparum transporter PfCRT.

Nature 2019 12 27;576(7786):315-320. Epub 2019 Nov 27.

Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY, USA.

The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.
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http://dx.doi.org/10.1038/s41586-019-1795-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911266PMC
December 2019