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Carolyn K Dong,
Sameer Urgaonkar, Joseph F Cortese,
Francisco-Javier Gamo,
Jose F Garcia-Bustos,
Maria J Lafuente,
Vishal Patel,
Leila Ross,
Bradley I Coleman,
Emily R Derbyshire,
Clary B Clish,
Adelfa E Serrano,
Mandy Cromwell,
Robert H Barker,
Jeffrey D Dvorin,
Manoj T Duraisingh,
Dyann F Wirth,
Jon Clardy,
Ralph Mazitschek
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ABSTRACT: Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc(1) complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc(1) activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.
Chemistry & biology 12/2011; 18(12):1602-10. · 6.52 Impact Factor
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Daria Van Tyne,
Daniel J Park,
Stephen F Schaffner,
Daniel E Neafsey,
Elaine Angelino, Joseph F Cortese,
Kayla G Barnes,
David M Rosen,
Amanda K Lukens,
Rachel F Daniels, [......],
Nicholas A Furlotte,
Eleazar Eskin,
Hyun Min Kang,
Daniel L Hartl,
Bruce W Birren,
Roger C Wiegand,
Eric S Lander,
Dyann F Wirth,
Sarah K Volkman,
Pardis C Sabeti
[show abstract]
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ABSTRACT: The Plasmodium falciparum parasite's ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (∼ 1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.
PLoS Genetics 04/2011; 7(4):e1001383. · 8.69 Impact Factor
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Robert H Barker,
Sameer Urgaonkar,
Ralph Mazitschek,
Cassandra Celatka,
Renato Skerlj, Joseph F Cortese,
Erin Tyndall,
Hanlan Liu,
Mandy Cromwell,
Amar Bir Sidhu, [......],
Jeffrey D Klinger,
Mark Bree,
Edward Lee,
Mikaela Levine,
Roger C Wiegand,
Benito Munoz,
Dyann F Wirth,
Jon Clardy,
Ian Bathurst,
Edmund Sybertz
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ABSTRACT: This study characterizes aminoindole molecules that are analogs of Genz-644442. Genz-644442 was identified as a hit in a screen of ~70,000 compounds in the Broad Institute's small-molecule library and the ICCB-L compound collection at Harvard Medical School. Genz-644442 is a potent inhibitor of Plasmodium falciparum in vitro (50% inhibitory concentrations [IC₅₀s], 200 to 285 nM) and inhibits P. berghei in vivo with an efficacy of > 99% in an adapted version of Peters' 4-day suppressive test (W. Peters, Ann. Trop. Med. Parasitol. 69:155-171, 1975). Genz-644442 became the focus of medicinal chemistry optimization; 321 analogs were synthesized and were tested for in vitro potency against P. falciparum and for in vitro absorption, distribution, metabolism, and excretion (ADME) properties. This yielded compounds with IC₅₀s of approximately 30 nM. The lead compound, Genz-668764, has been characterized in more detail. It is a single enantiomer with IC₅₀s of 28 to 65 nM against P. falciparum in vitro. In the 4-day P. berghei model, when it was dosed at 100 mg/kg of body weight/day, no parasites were detected on day 4 postinfection. However, parasites recrudesced by day 9. Dosing at 200 mg/kg/day twice a day resulted in cures of 3/5 animals. The compound had comparable activity against P. falciparum blood stages in a human-engrafted NOD-scid mouse model. Genz-668764 had a terminal half-life of 2.8 h and plasma trough levels of 41 ng/ml when it was dosed twice a day orally at 55 mg/kg/day. Seven-day rat safety studies showed a no-observable-adverse-effect level (NOAEL) at 200 mg/kg/day; the compound was not mutagenic in Ames tests, did not inhibit the hERG channel, and did not have potent activity against a broad panel of receptors and enzymes. Employing allometric scaling and using in vitro ADME data, the predicted human minimum efficacious dose of Genz-668764 in a 3-day once-daily dosing regimen was 421 mg/day/70 kg, which would maintain plasma trough levels above the IC₉₀ against P. falciparum for at least 96 h after the last dose. The predicted human therapeutic index was approximately 3, on the basis of the exposure in rats at the NOAEL. We were unable to select for parasites with >2-fold decreased sensitivity to the parent compound, Genz-644442, over 270 days of in vitro culture under drug pressure. These characteristics make Genz-668764 a good candidate for preclinical development.
Antimicrobial Agents and Chemotherapy 03/2011; 55(6):2612-22. · 4.84 Impact Factor
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Michael L Booker,
Cecilia M Bastos,
Martin L Kramer,
Robert H Barker,
Renato Skerlj,
Amar Bir Sidhu,
Xiaoyi Deng,
Cassandra Celatka, Joseph F Cortese,
Jose E Guerrero Bravo, [......],
Shahid M Khan,
Manoj Duraisingh,
Bradley Coleman,
Elizabeth J Goldsmith,
Margaret A Phillips,
Benito Munoz,
Dyann F Wirth,
Jeffrey D Klinger,
Roger Wiegand,
Edmund Sybertz
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ABSTRACT: Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED(50) values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.
Journal of Biological Chemistry 10/2010; 285(43):33054-64. · 4.77 Impact Factor
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ABSTRACT: The development of a concise strategy to access 2-amino-3-hydroxy-indoles, which are disclosed as novel antimalarials with potent in vivo activity, is reported. Starting from isatins the target compounds are synthesized in 2 steps and in good yields via oxoindole intermediates by employing tert-butyldimethylsilyl amine (TBDMSNH(2)) as previously unexplored ammonia equivalent.
Organic Letters 09/2010; 12(18):3998-4001. · 5.86 Impact Factor
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ABSTRACT: A high-throughput screening program identified two piperazine sulfonamides with activity against Plasmodium falciparum. Both screening positives had three structural features with potential liabilities: furanyl, thiourea and nitrophenyl groups. The furan could be replaced with no loss of activity, replacement of the nitrophenyl led to some loss of activity, and any attempt to replace the thiourea led to a significant decrease in activity, which implicates this reactive functional group's role in the antiplasmodial activity of this compound class.
Bioorganic & medicinal chemistry letters 10/2009; 20(1):218-21. · 2.65 Impact Factor
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Derek C Martyn,
Amarjit Nijjar,
Cassandra A Celatka,
Ralph Mazitschek, Joseph F Cortese,
Erin Tyndall,
Hanlan Liu,
Maria M Fitzgerald,
Thomas J O'Shea,
Sanjay Danthi,
Jon Clardy
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ABSTRACT: Two sets of diaminopyrimidines, totalling 45 compounds, were synthesized and assayed against Plasmodium falciparum. The SAR was relatively shallow, with only the presence of a 2-(pyrrolidin-1-yl)ethyl group at R(2) significantly affecting activity. A subsequent series addressed high LogD values by introducing more polar side groups, with the most active compounds possessing diazepine and N-benzyl-4-aminopiperidyl groups at R(1)/R(2). A final series attempted to address high in vitro microsomal clearance by replacing the C6-Me group with CF(3), however antiplasmodial activity decreased without any improvement in clearance. The C6-CF(3) group decreased hERG inhibition, probably as a result of decreased amine basicity at C2/C4.
Bioorganic & medicinal chemistry letters 10/2009; 20(1):228-31. · 2.65 Impact Factor
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ABSTRACT: A 43-member 1,2-dioxolane library was synthesized by coupling a 1,2-dioxolane-3-acetic acid derivative to a range of amines. Ten compounds had EC(50)s30nM against Plasmodium falciparum 3D7 and Dd2 strains, and another 15 compounds had EC(50)s50nM against both 3D7 and Dd2. The library was then subjected to a range of in vitro DMPK assays, which revealed that side chains with a heteroatom were required for favorable solubility, LogD and membrane permeability. CYP450 inhibition was isoform dependent, with 2C19 and 3A4 particularly susceptible, and the majority of compounds tested against rat and human microsomes were metabolized rapidly.
Bioorganic & medicinal chemistry letters 09/2009; 19(19):5657-60. · 2.65 Impact Factor
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ABSTRACT: Malaria infects 500 million people annually, a number that is likely to rise as drug resistance to currently used antimalarials increases. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum, metabolizes hemoglobin and releases toxic heme, which is neutralized by a parasite-specific crystallization mechanism to form hemozoin. Evidence suggests that chloroquine, the most successful antimalarial agent in history, acts by disrupting the formation of hemozoin. Here we describe the development of a 384-well microtiter plate screen to detect small molecules that can also disrupt heme crystallization. This assay, which is based on a colorimetric assay developed by Ncokazi and Egan (K. K. Ncokazi and T. J. Egan, Anal. Biochem. 338:306-319, 2005), requires no parasites or parasite-derived reagents and no radioactive materials and is suitable for a high-throughput screening platform. The assay's reproducibility and large dynamic range are reflected by a Z factor of 0.74. A pilot screen of 16,000 small molecules belonging to diverse structural classes was conducted. The results of the target-based assay were compared with a whole-parasite viability assay of the same small molecules to identify small molecules active in both assays.
Antimicrobial Agents and Chemotherapy 04/2009; 53(6):2564-8. · 4.84 Impact Factor
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ABSTRACT: Artemisinin-derived compounds play an integral role in current malaria chemotherapy. Given the virtual certainty of emerging resistance, we have investigated spiro-1,2-dioxolanes as an alternative scaffold. The endoperoxide functionality was generated by the SnCl(4)-mediated annulation of a bis-silylperoxide and an alkene. The first set of eight analogs gave EC(50) values of 50-150 nM against Plasmodium falciparum 3D7 and Dd2 strains, except for the carboxylic acid analog. A second series, synthesized by coupling a spiro-1,2-dioxolane carboxylic acid to four separate amines, afforded the most potent compound (EC(50) approximately 5 nM).
Bioorganic & medicinal chemistry letters 01/2009; 18(24):6521-4. · 2.65 Impact Factor
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Daniel E Neafsey,
Stephen F Schaffner,
Sarah K Volkman,
Daniel Park,
Philip Montgomery,
Danny A Milner,
Amanda Lukens,
David Rosen,
Rachel Daniels,
Nathan Houde, [......],
Marcelo U Ferreira,
Sandra do Lago Moraes,
Aditya P Dash,
Chetan E Chitnis,
Roger C Wiegand,
Daniel L Hartl,
Bruce W Birren,
Eric S Lander,
Pardis C Sabeti,
Dyann F Wirth
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ABSTRACT: An array-based SNP genotyping platform for Plasmodium falciparum is reported together with an analysis of SNP diversity in global population samples.
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Dyann F Wirth,
Pardis C Sabeti,
Eric S Lander,
Bruce W Birren,
Daniel Hartl,
Roger Wiegand,
Chetan E Chitnis,
Aditya P Dash,
Sandra do Lago Moraes,
Marcelo U Ferreira, [......],
Nathan Houde,
Rachel Daniels,
David Rosen,
Amanda Lukens,
Danny Milner,
Philip Montgomery,
Daniel Park,
Sarah K Volkman,
Stephen F Schaffner,
Daniel Neafsey
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ABSTRACT: Organismic and Evolutionary Biology Background: The malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum. Results: Using an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, inter-population differentiation, and the degree to which allele frequencies are correlated between populations. Conclusions: The distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.
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Philip Montgomery,
David Rosen,
Nathan Houde, Joseph F Cortese,
Erin Tyndall,
Casey Gates,
Nicole Stange-Thomann,
Omar Ndir,
Soulyemane Mboup,
Marcelo U Ferreira, [......],
Danny Arnold Milner,
Amanda Kathleen Lukens,
Rachel Daniels,
Ousmane Sarr,
Daouda Ndiaye,
Roger C Wiegand,
Daniel L Hartl,
Eric Steven Lander,
Pardis Christine Sabeti,
Dyann Fergus Wirth
[show abstract]
[hide abstract]
ABSTRACT: Organismic and Evolutionary Biology Background: The malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum. Results: Using an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, interpopulation differentiation, and the degree to which allele frequencies are correlated between populations. Conclusions: The distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.
