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Olivo Miotto - One of the best experts on this subject based on the ideXlab platform.
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development of copy number assays for detection and surveillance of piperaquine resistance associated Plasmepsin 2 3 copy number variation in plasmodium falciparum
Malaria Journal, 2020Co-Authors: Megan R Ansbro, Christopher G Jacob, Chanaki Amaratunga, Olivo Miotto, Roberto D'amato, Mihir Kekre, Sokunthea Sreng, Seila Suon, Rick M Fairhurst, Thomas E WellemsAbstract:Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the Plasmepsin 2 and Plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance. To accurately and quickly determine the presence of copy number variations in the Plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of Plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene (pfmdr1), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive Plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of Plasmepsin 2 in Pursat in 2013 to three copies in 2014–2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015. The multiplex TaqMan assay is a robust tool for monitoring both Plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring Plasmepsin 2/3 amplifications. This study shows increasing levels of Plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
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Development of copy number assays for detection and surveillance of piperaquine resistance associated Plasmepsin 2/3 copy number variation in Plasmodium falciparum
Malaria Journal, 2020Co-Authors: Megan R Ansbro, Christopher G Jacob, Chanaki Amaratunga, Olivo Miotto, Roberto D'amato, Mihir Kekre, Sokunthea Sreng, Seila Suon, Rick M Fairhurst, Thomas E WellemsAbstract:Background Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the Plasmepsin 2 and Plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance. Results To accurately and quickly determine the presence of copy number variations in the Plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of Plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene ( pfmdr1 ), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive Plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of Plasmepsin 2 in Pursat in 2013 to three copies in 2014–2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015. Conclusions The multiplex TaqMan assay is a robust tool for monitoring both Plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring Plasmepsin 2/3 amplifications. This study shows increasing levels of Plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
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development of copy number assays for detection and surveillance of piperaquine resistance associated Plasmepsin 2 3 copy number variation in plasmodium falciparum
bioRxiv, 2019Co-Authors: Christopher G Jacob, Megan R Ansbro, Chanaki Amaratunga, Mehul Dhorda, Roberto D'amato, Mihir Kekre, Sokunthea Sreng, Seila Suon, Ranitha Vongpromek, Olivo MiottoAbstract:Abstract Long regarded as the epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified a single nucleotide polymorphism as well as a copy number variation molecular marker that associate with clinical and in vitro resistance. The copy number polymorphism is a duplication of a region containing members of the Plasmepsin multi-gene family of proteases. To accurately and quickly determine the presence of copy number variation in the Plasmepsin 2/3 duplication in field isolates, we developed a quantitative PCR assay using TaqMan probes. We validated copy number estimates using a separate SYBR green-based quantitative PCR assay as well as a novel breakpoint assay to detect the hybrid gene product. Field samples from 2012 – 2015 across 3 sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of Plasmepsin 2/3 gene duplications, as well as pfmdr1. We found high concordance across all methods of copy number detection. For samples derived from dried blood spots we found a greater than 80% success rate in each assay, with more recent samples performing better. We found evidence of extensive copy number amplifications in Pursat (94%, 2015) and Preah Vihear (87%, 2014), and lower levels in Ratanakiri (16%, 2014) in eastern Cambodia. We also see evidence of a shift from two copies of Plasmepsin 2/3 in Pursat 2013 to three copies in 2014-15 (25% to 64%). Pfmdr1 duplications are absent from all samples in 2014 from Preah Vihear and Ratanakiri and 2015 from Pursat. This study shows increasing levels of Plasmepsin 2/3 gene amplifications across Cambodia from 2012 – 2015 and a complete reversion of pfmdr1 mutant parasites in all study locations. The multiplex TaqMan assay is a robust tool for monitoring both Plasmepsin and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring Plasmepsin 2/3 duplications.
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genetic markers associated with dihydroartemisinin piperaquine failure in plasmodium falciparum malaria in cambodia a genotype phenotype association study
Lancet Infectious Diseases, 2017Co-Authors: Richard D Pearson, Chanaki Amaratunga, Olivo Miotto, Jacob Almagrogarcia, Roberto D'amatoAbstract:Summary Background As the prevalence of artemisinin-resistant Plasmodium falciparum malaria increases in the Greater Mekong subregion, emerging resistance to partner drugs in artemisinin combination therapies seriously threatens global efforts to treat and eliminate this disease. Molecular markers that predict failure of artemisinin combination therapy are urgently needed to monitor the spread of partner drug resistance, and to recommend alternative treatments in southeast Asia and beyond. Methods We did a genome-wide association study of 297 P falciparum isolates from Cambodia to investigate the relationship of 11 630 exonic single-nucleotide polymorphisms (SNPs) and 43 copy number variations (CNVs) with in-vitro piperaquine 50% inhibitory concentrations (IC 50 s), and tested whether these genetic variants are markers of treatment failure with dihydroartemisinin–piperaquine. We then did a survival analysis of 133 patients to determine whether candidate molecular markers predicted parasite recrudescence following dihydroartemisinin–piperaquine treatment. Findings Piperaquine IC 50 s increased significantly from 2011 to 2013 in three Cambodian provinces (2011 vs 2013 median IC 50 s: 20·0 nmol/L [IQR 13·7–29·0] vs 39·2 nmol/L [32·8–48·1] for Ratanakiri, 19·3 nmol/L [15·1–26·2] vs 66·2 nmol/L [49·9–83·0] for Preah Vihear, and 19·6 nmol/L [11·9–33·9] vs 81·1 nmol/L [61·3–113·1] for Pursat; all p≤10 −3 ; Kruskal-Wallis test). Genome-wide analysis of SNPs identified a chromosome 13 region that associates with raised piperaquine IC 50 s. A non-synonymous SNP (encoding a Glu415Gly substitution) in this region, within a gene encoding an exonuclease, associates with parasite recrudescence following dihydroartemisinin–piperaquine treatment. Genome-wide analysis of CNVs revealed that a single copy of the mdr1 gene on chromosome 5 and a novel amplification of the Plasmepsin 2 and Plasmepsin 3 genes on chromosome 14 also associate with raised piperaquine IC 50 s. After adjusting for covariates, both exo-E415G and Plasmepsin 2–3 markers significantly associate (p=3·0 × 10 −8 and p=1·7 × 10 −7 , respectively) with decreased treatment efficacy (survival rates 0·38 [95% CI 0·25–0·51] and 0·41 [0·28–0·53], respectively). Interpretation The exo-E415G SNP and Plasmepsin 2–3 amplification are markers of piperaquine resistance and dihydroartemisinin–piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into other countries of the Greater Mekong subregion, and elucidate the mechanism of piperaquine resistance. Since Plasmepsins are involved in the parasite's haemoglobin-to-haemozoin conversion pathway, targeted by related antimalarials, Plasmepsin 2–3 amplification probably mediates piperaquine resistance. Funding Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and UK Department for International Development.
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Comparative genome-wide analysis and evolutionary history of haemoglobin-processing and haem detoxification enzymes in malarial parasites
Malaria Journal, 2016Co-Authors: Patrath Ponsuwanna, Theerarat Kochakarn, Kesinee Chotivanich, Prapon Wilairat, Nicholas J. White, Duangkamon Bunditvorapoom, Krittikorn Kumpornsin, Thomas D Otto, Chase Ridenour, Olivo MiottoAbstract:Background Malaria parasites have evolved a series of intricate mechanisms to survive and propagate within host red blood cells. Intra-erythrocytic parasitism requires these organisms to digest haemoglobin and detoxify iron-bound haem. These tasks are executed by haemoglobin-specific proteases and haem biocrystallization factors that are components of a large multi-subunit complex. Since haemoglobin processing machineries are functionally and genetically linked to the modes of action and resistance mechanisms of several anti-malarial drugs, an understanding of their evolutionary history is important for drug development and drug resistance prevention. Methods Maximum likelihood trees of genetic repertoires encoding haemoglobin processing machineries within Plasmodium species, and with the representatives of Apicomplexan species with various host tropisms, were created. Genetic variants were mapped onto existing three-dimensional structures. Genome-wide single nucleotide polymorphism data were used to analyse the selective pressure and the effect of these mutations at the structural level. Results Recent expansions in the falcipain and Plasmepsin repertoires are unique to human malaria parasites especially in the Plasmodium falciparum and P. reichenowi lineage. Expansion of haemoglobin-specific Plasmepsins occurred after the separation event of Plasmodium species, but the other members of the Plasmepsin family were evolutionarily conserved with one copy for each sub-group in every Apicomplexan species. Haemoglobin-specific falcipains are separated from invasion-related falcipain, and their expansions within one specific locus arose independently in both P. falciparum and P. vivax lineages. Gene conversion between P. falciparum falcipain 2A and 2B was observed in artemisinin-resistant strains. Comparison between the numbers of non-synonymous and synonymous mutations suggests a strong selective pressure at falcipain and Plasmepsin genes. The locations of amino acid changes from non-synonymous mutations mapped onto protein structures revealed clusters of amino acid residues in close proximity or near the active sites of proteases. Conclusion A high degree of polymorphism at the haemoglobin processing genes implicates an imposition of selective pressure. The identification in recent years of functional redundancy of haemoglobin-specific proteases makes them less appealing as potential drug targets, but their expansions, especially in the human malaria parasite lineages, unequivocally point toward their functional significance during the independent and repetitive adaptation events in malaria parasite evolutionary history.
Colin Berry - One of the best experts on this subject based on the ideXlab platform.
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generation of an affinity matrix useful in the purification of natural inhibitors of Plasmepsin ii an antimalarial drug target
Biotechnology and Applied Biochemistry, 2009Co-Authors: Angel Ramirez, Judith Mendiola, Yasel Guerra, Anabel Otero, Aida Hernandezzanui, Beatriz Garcia, Colin Berry, Maria A ChavezAbstract:: An affinity matrix containing the antimalarial drug target Plm II (Plasmepsin II) as ligand was generated. This enzyme belongs to the family of Plasmodium (malarial parasite) aspartic proteinases, known as Plms (Plasmepsins). The procedure established to obtain the support has two steps: the immobilization of the recombinant proenzyme of Plm II to NHS (N-hydroxysuccinimide)-activated Sepharose and the activation of the immobilized enzyme by incubation at pH 4.4 and 37 degrees C. The coupling reaction resulted in a high percentage immobilization (95.5%), and the matrices obtained had an average of 4.3 mg of protein/ml of gel. The activated matrices, but not the inactive ones, were able to hydrolyse two different chromogenic peptide substrates and haemoglobin. This ability was completely blocked by the addition of the general aspartic-proteinase inhibitor, pepstatin A, to the reaction mixture. The matrices were useful in the affinity purification of the Plm II inhibitory activity detected in marine invertebrates, such as Xestospongia muta (giant barrel sponge) and the gorgonian (sea-fan coral) Plexaura homomalla (black sea rod), with increases of 10.2- and 5.9-fold in the specific inhibitory activity respectively. The preliminary K(i) values obtained, 46.4 nM (X. muta) and 1.9 nM (P. homomalla), and the concave shapes of the inhibition curves reveal that molecules are reversible tight-binding inhibitors of Plm II. These results validated the use of the affinity matrix for the purification of Plm II inhibitors from complex mixtures and established the presence of Plm II inhibitors in some marine invertebrates.
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the activity and inhibition of the food vacuole Plasmepsin from the rodent malaria parasite plasmodium chabaudi
Acta Tropica, 2006Co-Authors: Tiago M Martins, Ana Domingos, Colin Berry, David WyattAbstract:The rodent malaria parasite Plasmodium chabaudi encodes one food vacuole Plasmepsin—the aspartic proteinases important in haemoglobin degradation. A recombinant form of this enzyme was found to cleave a variety of peptide substrates and was susceptible to a selection of naturally occurring and synthetic inhibitors, displaying an inhibition profile distinct from that of aspartic proteinases from other malaria parasites. In addition, inhibitors of HIV proteinase that kill P. chabaudi in vivo were also inhibitors of this new Plasmepsin. P. chabaudi is a widely used model for human malaria species and, therefore, the characterisation of this Plasmepsin is an important contribution towards understanding its biology.
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activity and inhibition of Plasmepsin iv a new aspartic proteinase from the malaria parasite plasmodium falciparum
FEBS Letters, 2002Co-Authors: David Wyatt, Colin BerryAbstract:A new aspartic proteinase from the human malaria parasite Plasmodium falciparum is able to hydrolyse human haemoglobin at a site known to be the essential primary cleavage site in the haemoglobin degradation pathway. Thus, Plasmepsin IV may play a crucial role in this critical process which yields nutrients for parasite growth. Furthermore, synthetic inhibitors known to inhibit parasite growth in red cells in culture are able to inhibit the activity of this enzyme in vitro. As a result, Plasmepsin IV appears to be a potential target for the development of new antiparasitic drugs.
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the aspartic proteinase from the rodent parasite plasmodium berghei as a potential model for Plasmepsins from the human malaria parasite plasmodium falciparum
FEBS Letters, 1999Co-Authors: Michelle J Humphreys, Sylvia D Fowler, Annette Klinder, Richard P. Moon, Katharina Rupp, Robert G Ridley, Colin BerryAbstract:The gene encoding an aspartic proteinase precursor (proPlasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei Plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of Plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proPlasmepsin gene in P. berghei. Two Plasmepsins have previously been reported in P. falciparum. Here, we describe two further proPlasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of Plasmepsin inhibitors is discussed.
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high level expression and characterisation of Plasmepsin ii an aspartic proteinase from plasmodium falciparum
FEBS Letters, 1994Co-Authors: Jeffrey Hill, Lorraine Tyas, Lowri H Phylip, Ben M Dunn, Colin BerryAbstract:DNA encoding the last 48 residues of the propart and the whole mature sequence of Plasmepsin II was inserted into the T7 dependent vector pET 3a for expression in E. coli. The resultant product was insoluble but accumulated at ∼20 mg/l of cell culture. Following solubilisation with urea, the zymogen was refolded and, after purification by ion-exchange chromatography, was autoactivated to generate mature Plasmepsin II. The ability of this enzyme to hydrolyse several chromogenic peptide substrates was examined; despite an overall identity of ∼35% to human renin, Plasmepsin II was not inhibited significantly by renin inhibitors.
Roberto D'amato - One of the best experts on this subject based on the ideXlab platform.
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Development of copy number assays for detection and surveillance of piperaquine resistance associated Plasmepsin 2/3 copy number variation in Plasmodium falciparum
Malaria Journal, 2020Co-Authors: Megan R Ansbro, Christopher G Jacob, Chanaki Amaratunga, Olivo Miotto, Roberto D'amato, Mihir Kekre, Sokunthea Sreng, Seila Suon, Rick M Fairhurst, Thomas E WellemsAbstract:Background Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the Plasmepsin 2 and Plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance. Results To accurately and quickly determine the presence of copy number variations in the Plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of Plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene ( pfmdr1 ), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive Plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of Plasmepsin 2 in Pursat in 2013 to three copies in 2014–2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015. Conclusions The multiplex TaqMan assay is a robust tool for monitoring both Plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring Plasmepsin 2/3 amplifications. This study shows increasing levels of Plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
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development of copy number assays for detection and surveillance of piperaquine resistance associated Plasmepsin 2 3 copy number variation in plasmodium falciparum
Malaria Journal, 2020Co-Authors: Megan R Ansbro, Christopher G Jacob, Chanaki Amaratunga, Olivo Miotto, Roberto D'amato, Mihir Kekre, Sokunthea Sreng, Seila Suon, Rick M Fairhurst, Thomas E WellemsAbstract:Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the Plasmepsin 2 and Plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance. To accurately and quickly determine the presence of copy number variations in the Plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of Plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene (pfmdr1), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive Plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of Plasmepsin 2 in Pursat in 2013 to three copies in 2014–2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015. The multiplex TaqMan assay is a robust tool for monitoring both Plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring Plasmepsin 2/3 amplifications. This study shows increasing levels of Plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
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development of copy number assays for detection and surveillance of piperaquine resistance associated Plasmepsin 2 3 copy number variation in plasmodium falciparum
bioRxiv, 2019Co-Authors: Christopher G Jacob, Megan R Ansbro, Chanaki Amaratunga, Mehul Dhorda, Roberto D'amato, Mihir Kekre, Sokunthea Sreng, Seila Suon, Ranitha Vongpromek, Olivo MiottoAbstract:Abstract Long regarded as the epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified a single nucleotide polymorphism as well as a copy number variation molecular marker that associate with clinical and in vitro resistance. The copy number polymorphism is a duplication of a region containing members of the Plasmepsin multi-gene family of proteases. To accurately and quickly determine the presence of copy number variation in the Plasmepsin 2/3 duplication in field isolates, we developed a quantitative PCR assay using TaqMan probes. We validated copy number estimates using a separate SYBR green-based quantitative PCR assay as well as a novel breakpoint assay to detect the hybrid gene product. Field samples from 2012 – 2015 across 3 sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of Plasmepsin 2/3 gene duplications, as well as pfmdr1. We found high concordance across all methods of copy number detection. For samples derived from dried blood spots we found a greater than 80% success rate in each assay, with more recent samples performing better. We found evidence of extensive copy number amplifications in Pursat (94%, 2015) and Preah Vihear (87%, 2014), and lower levels in Ratanakiri (16%, 2014) in eastern Cambodia. We also see evidence of a shift from two copies of Plasmepsin 2/3 in Pursat 2013 to three copies in 2014-15 (25% to 64%). Pfmdr1 duplications are absent from all samples in 2014 from Preah Vihear and Ratanakiri and 2015 from Pursat. This study shows increasing levels of Plasmepsin 2/3 gene amplifications across Cambodia from 2012 – 2015 and a complete reversion of pfmdr1 mutant parasites in all study locations. The multiplex TaqMan assay is a robust tool for monitoring both Plasmepsin and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring Plasmepsin 2/3 duplications.
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genetic markers associated with dihydroartemisinin piperaquine failure in plasmodium falciparum malaria in cambodia a genotype phenotype association study
Lancet Infectious Diseases, 2017Co-Authors: Richard D Pearson, Chanaki Amaratunga, Olivo Miotto, Jacob Almagrogarcia, Roberto D'amatoAbstract:Summary Background As the prevalence of artemisinin-resistant Plasmodium falciparum malaria increases in the Greater Mekong subregion, emerging resistance to partner drugs in artemisinin combination therapies seriously threatens global efforts to treat and eliminate this disease. Molecular markers that predict failure of artemisinin combination therapy are urgently needed to monitor the spread of partner drug resistance, and to recommend alternative treatments in southeast Asia and beyond. Methods We did a genome-wide association study of 297 P falciparum isolates from Cambodia to investigate the relationship of 11 630 exonic single-nucleotide polymorphisms (SNPs) and 43 copy number variations (CNVs) with in-vitro piperaquine 50% inhibitory concentrations (IC 50 s), and tested whether these genetic variants are markers of treatment failure with dihydroartemisinin–piperaquine. We then did a survival analysis of 133 patients to determine whether candidate molecular markers predicted parasite recrudescence following dihydroartemisinin–piperaquine treatment. Findings Piperaquine IC 50 s increased significantly from 2011 to 2013 in three Cambodian provinces (2011 vs 2013 median IC 50 s: 20·0 nmol/L [IQR 13·7–29·0] vs 39·2 nmol/L [32·8–48·1] for Ratanakiri, 19·3 nmol/L [15·1–26·2] vs 66·2 nmol/L [49·9–83·0] for Preah Vihear, and 19·6 nmol/L [11·9–33·9] vs 81·1 nmol/L [61·3–113·1] for Pursat; all p≤10 −3 ; Kruskal-Wallis test). Genome-wide analysis of SNPs identified a chromosome 13 region that associates with raised piperaquine IC 50 s. A non-synonymous SNP (encoding a Glu415Gly substitution) in this region, within a gene encoding an exonuclease, associates with parasite recrudescence following dihydroartemisinin–piperaquine treatment. Genome-wide analysis of CNVs revealed that a single copy of the mdr1 gene on chromosome 5 and a novel amplification of the Plasmepsin 2 and Plasmepsin 3 genes on chromosome 14 also associate with raised piperaquine IC 50 s. After adjusting for covariates, both exo-E415G and Plasmepsin 2–3 markers significantly associate (p=3·0 × 10 −8 and p=1·7 × 10 −7 , respectively) with decreased treatment efficacy (survival rates 0·38 [95% CI 0·25–0·51] and 0·41 [0·28–0·53], respectively). Interpretation The exo-E415G SNP and Plasmepsin 2–3 amplification are markers of piperaquine resistance and dihydroartemisinin–piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into other countries of the Greater Mekong subregion, and elucidate the mechanism of piperaquine resistance. Since Plasmepsins are involved in the parasite's haemoglobin-to-haemozoin conversion pathway, targeted by related antimalarials, Plasmepsin 2–3 amplification probably mediates piperaquine resistance. Funding Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and UK Department for International Development.
Chanaki Amaratunga - One of the best experts on this subject based on the ideXlab platform.
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development of copy number assays for detection and surveillance of piperaquine resistance associated Plasmepsin 2 3 copy number variation in plasmodium falciparum
Malaria Journal, 2020Co-Authors: Megan R Ansbro, Christopher G Jacob, Chanaki Amaratunga, Olivo Miotto, Roberto D'amato, Mihir Kekre, Sokunthea Sreng, Seila Suon, Rick M Fairhurst, Thomas E WellemsAbstract:Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the Plasmepsin 2 and Plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance. To accurately and quickly determine the presence of copy number variations in the Plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of Plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene (pfmdr1), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive Plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of Plasmepsin 2 in Pursat in 2013 to three copies in 2014–2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015. The multiplex TaqMan assay is a robust tool for monitoring both Plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring Plasmepsin 2/3 amplifications. This study shows increasing levels of Plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
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Development of copy number assays for detection and surveillance of piperaquine resistance associated Plasmepsin 2/3 copy number variation in Plasmodium falciparum
Malaria Journal, 2020Co-Authors: Megan R Ansbro, Christopher G Jacob, Chanaki Amaratunga, Olivo Miotto, Roberto D'amato, Mihir Kekre, Sokunthea Sreng, Seila Suon, Rick M Fairhurst, Thomas E WellemsAbstract:Background Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the Plasmepsin 2 and Plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance. Results To accurately and quickly determine the presence of copy number variations in the Plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of Plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene ( pfmdr1 ), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive Plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of Plasmepsin 2 in Pursat in 2013 to three copies in 2014–2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015. Conclusions The multiplex TaqMan assay is a robust tool for monitoring both Plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring Plasmepsin 2/3 amplifications. This study shows increasing levels of Plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
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development of copy number assays for detection and surveillance of piperaquine resistance associated Plasmepsin 2 3 copy number variation in plasmodium falciparum
bioRxiv, 2019Co-Authors: Christopher G Jacob, Megan R Ansbro, Chanaki Amaratunga, Mehul Dhorda, Roberto D'amato, Mihir Kekre, Sokunthea Sreng, Seila Suon, Ranitha Vongpromek, Olivo MiottoAbstract:Abstract Long regarded as the epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified a single nucleotide polymorphism as well as a copy number variation molecular marker that associate with clinical and in vitro resistance. The copy number polymorphism is a duplication of a region containing members of the Plasmepsin multi-gene family of proteases. To accurately and quickly determine the presence of copy number variation in the Plasmepsin 2/3 duplication in field isolates, we developed a quantitative PCR assay using TaqMan probes. We validated copy number estimates using a separate SYBR green-based quantitative PCR assay as well as a novel breakpoint assay to detect the hybrid gene product. Field samples from 2012 – 2015 across 3 sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of Plasmepsin 2/3 gene duplications, as well as pfmdr1. We found high concordance across all methods of copy number detection. For samples derived from dried blood spots we found a greater than 80% success rate in each assay, with more recent samples performing better. We found evidence of extensive copy number amplifications in Pursat (94%, 2015) and Preah Vihear (87%, 2014), and lower levels in Ratanakiri (16%, 2014) in eastern Cambodia. We also see evidence of a shift from two copies of Plasmepsin 2/3 in Pursat 2013 to three copies in 2014-15 (25% to 64%). Pfmdr1 duplications are absent from all samples in 2014 from Preah Vihear and Ratanakiri and 2015 from Pursat. This study shows increasing levels of Plasmepsin 2/3 gene amplifications across Cambodia from 2012 – 2015 and a complete reversion of pfmdr1 mutant parasites in all study locations. The multiplex TaqMan assay is a robust tool for monitoring both Plasmepsin and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring Plasmepsin 2/3 duplications.
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Plasmepsin ii iii copy number accounts for bimodal piperaquine resistance among cambodian plasmodium falciparum
Nature Communications, 2018Co-Authors: Selina Bopp, Charles J. Woodrow, Angana Mukherjee, Wesley P Wong, Stephen F Schaffner, Chanaki Amaratunga, Pamela Magistrado, Mehul Dhorda, Elizabeth A. AshleyAbstract:Multidrug resistant Plasmodium falciparum in Southeast Asia endangers regional malaria elimination and threatens to spread to other malaria endemic areas. Understanding mechanisms of piperaquine (PPQ) resistance is crucial for tracking its emergence and spread, and to develop effective strategies for overcoming it. Here we analyze a mechanism of PPQ resistance in Cambodian parasites. Isolates exhibit a bimodal dose–response curve when exposed to PPQ, with the area under the curve quantifying their survival in vitro. Increased copy number for Plasmepsin II and Plasmepsin III appears to explain enhanced survival when exposed to PPQ in most, but not all cases. A panel of isogenic subclones reinforces the importance of Plasmepsin II–III copy number to enhanced PPQ survival. We conjecture that factors producing increased parasite survival under PPQ exposure in vitro may drive clinical PPQ failures in the field.
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genetic markers associated with dihydroartemisinin piperaquine failure in plasmodium falciparum malaria in cambodia a genotype phenotype association study
Lancet Infectious Diseases, 2017Co-Authors: Richard D Pearson, Chanaki Amaratunga, Olivo Miotto, Jacob Almagrogarcia, Roberto D'amatoAbstract:Summary Background As the prevalence of artemisinin-resistant Plasmodium falciparum malaria increases in the Greater Mekong subregion, emerging resistance to partner drugs in artemisinin combination therapies seriously threatens global efforts to treat and eliminate this disease. Molecular markers that predict failure of artemisinin combination therapy are urgently needed to monitor the spread of partner drug resistance, and to recommend alternative treatments in southeast Asia and beyond. Methods We did a genome-wide association study of 297 P falciparum isolates from Cambodia to investigate the relationship of 11 630 exonic single-nucleotide polymorphisms (SNPs) and 43 copy number variations (CNVs) with in-vitro piperaquine 50% inhibitory concentrations (IC 50 s), and tested whether these genetic variants are markers of treatment failure with dihydroartemisinin–piperaquine. We then did a survival analysis of 133 patients to determine whether candidate molecular markers predicted parasite recrudescence following dihydroartemisinin–piperaquine treatment. Findings Piperaquine IC 50 s increased significantly from 2011 to 2013 in three Cambodian provinces (2011 vs 2013 median IC 50 s: 20·0 nmol/L [IQR 13·7–29·0] vs 39·2 nmol/L [32·8–48·1] for Ratanakiri, 19·3 nmol/L [15·1–26·2] vs 66·2 nmol/L [49·9–83·0] for Preah Vihear, and 19·6 nmol/L [11·9–33·9] vs 81·1 nmol/L [61·3–113·1] for Pursat; all p≤10 −3 ; Kruskal-Wallis test). Genome-wide analysis of SNPs identified a chromosome 13 region that associates with raised piperaquine IC 50 s. A non-synonymous SNP (encoding a Glu415Gly substitution) in this region, within a gene encoding an exonuclease, associates with parasite recrudescence following dihydroartemisinin–piperaquine treatment. Genome-wide analysis of CNVs revealed that a single copy of the mdr1 gene on chromosome 5 and a novel amplification of the Plasmepsin 2 and Plasmepsin 3 genes on chromosome 14 also associate with raised piperaquine IC 50 s. After adjusting for covariates, both exo-E415G and Plasmepsin 2–3 markers significantly associate (p=3·0 × 10 −8 and p=1·7 × 10 −7 , respectively) with decreased treatment efficacy (survival rates 0·38 [95% CI 0·25–0·51] and 0·41 [0·28–0·53], respectively). Interpretation The exo-E415G SNP and Plasmepsin 2–3 amplification are markers of piperaquine resistance and dihydroartemisinin–piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into other countries of the Greater Mekong subregion, and elucidate the mechanism of piperaquine resistance. Since Plasmepsins are involved in the parasite's haemoglobin-to-haemozoin conversion pathway, targeted by related antimalarials, Plasmepsin 2–3 amplification probably mediates piperaquine resistance. Funding Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and UK Department for International Development.
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in vitro antimalarial evaluation of piperidine and piperazine based chalcones inhibition of falcipain 2 and Plasmepsin ii hemoglobinases activities from plasmodium falciparum
ChemistrySelect, 2017Co-Authors: Hemandra K Tiwari, Nidhi Jatana, Puran Singh Sijwali, Kailash C Pandey, Nickolay Yu Gorobets, Sandeep Garg, Prashant Kumar, Latha Narayanan, Krishan Kumar, Ben M DunnAbstract:Never-ending effort to develop new treatments for malaria, targeting the hemoglobin-degradation in the food vacuole of the parasite is of particular interest because it appears to be critical for the erythrocytic stage parasite development. The Plasmodium aspartic proteinases Plasmepsins and cysteine proteases falcipains have been shown to be the major hemoglobin-degrading proteases and are proposed as the high priority drug targets by the World Health Organization for developing novel small molecules as inhibitors of hemoglobin degradation. In the present study, several piperidine and piperazine-based chalcones were assessed for anti-malarial activity against the chloroquine-susceptible P. falciparum 3D7 strain and inhibition of Plasmepsin II and falcipain-2. Degradation of hemoglobin by falcipain-2 in the presence or absence of inhibitors was also demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The IC50 values of chalcones for cell growth inhibition were in the range of 1.60-153.51 μM. IC50 values for cytotoxicity of selected chalcones against Michigan Cancer Foundation (MCF) 7 human breast adenocarcinoma cells were in the range of 8.46-15.64 μM. Molecular docking studies revealed the binding orientation of these chalcones in the active sites of falcipain-2. Our results clearly depict the advantage of these chalcones as they kill P. falciparum malaria parasite in culture, most likely via inhibition of falcipain.
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ANALYSIS OF BINDING INTERACTIONS OF PEPSIN INHIBITOR-3 TO MAMMALIAN AND MALARIAL ASPARTIC PROTEASES
2015Co-Authors: Mavis Agb, Ben M DunnAbstract:The nematode Ascaris suum primarily infects pigs, but also causes disease in humans. As part of its survival mechanism in the intestinal tract of the host, the worm produces a number of protease inhibitors, including pepsin inhibitor-3 (PI3), a 17 kDa protein. Recombinant PI3 expressed in E. coli has previously been shown to be a competitive inhibitor of a sub-group of aspartic proteinases: pepsin, gastricsin and cathepsin E. The previously determined crystal structure of the complex of PI3 with porcine pepsin (p. pepsin) showed that there are two regions of contact between PI3 and the enzyme. The first three N-terminal residues (QFL) bind into the prime side of the active site cleft and a polyproline helix (139–140) in the C-terminal domain of PI3 packs against residues 289–295 that form a loop in p. pepsin. Mutational analysis of both inhibitor regions was conducted to assess their contributions to the binding affinity for p. pepsin, human pepsin (h. pepsin) and several malarial aspartic proteases, the Plasmepsins. Overall, the polyproline mutations have a limited influence on the Ki values for all the enzymes tested, with the values for p. pepsin remaining in the low nanomolar range. The largest effect was seen with a Q1L mutant, with a 200-fold decrease in Ki for Plasmepsin 2 from Plasmodium falciparum (PfPM2). Thermodynamic measurements of the binding of PI3 to p
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enzymatic characterization of recombinant food vacuole Plasmepsin 4 from the rodent malaria parasite plasmodium berghei
PLOS ONE, 2015Co-Authors: Peng Liu, Melissa R. Marzahn, Arthur H Robbins, Scott Mcclung, Charles A Yowell, Stanley M Stevens, John B Dame, Ben M DunnAbstract:The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV Plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1’, S2’ and S3’. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV Plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite Plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the Plasmepsin family and provides leads for anti-malarial drug design.
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The inhibition of peptidomimetic compounds on FV Plasmepsins and hcatD.
2015Co-Authors: Peng Liu, Melissa R. Marzahn, Arthur H Robbins, Charles A Yowell, Stanley M Stevens, John B Dame, Scott H. Mcclung, Ben M DunnAbstract:aΨ = -CH2-NH-, nL = norleucine.bPfPM1, PfPM2 and PfPM4, Plasmepsins 1, 2 and 4 from Plasmodium falciparum, respectively; PvPM4, Plasmepsin 4 from P. vivax; PoPM4, Plasmepsin 4 from P. ovalae; and PmPM4, Plasmepsin 4 from P. malariae.cThese dissociation constants were reported in [41].dThese dissociation constants were reported in [42].The inhibition of peptidomimetic compounds on FV Plasmepsins and hcatD.
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Docked image for 6u with Plasmepsin 2.
2015Co-Authors: Anil K. Singh, Ben M Dunn, Sumit Rathore, Yan Tang, Nathan E. Goldfarb, Vinoth Rajendran, Prahlad C. Ghosh, Neelu Singh, N. Latha, Brajendra K. SinghAbstract:Docked image for 6u with Plasmepsin 2.