Download or read book Structure function Analysis of Plasmodium Falciparum Chloroquine Resistance Transporter in Chloroquine Resistance written by Kit Ying Choy and published by . This book was released on 2013 with total page 464 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Structural Investigation of Plasmodium Falciparum Chloroquine Resistance Transporter in the Context of Anti Malarial Drug Resistance written by Jonathan Young Kim and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: All CQ resistance-conferring PfCRT isoforms share the K76T mutation, which is widely used as a molecular marker for CQ resistance. Despite the significance in the impact of drug-resistant malaria, a detailed understanding of PfCRT physiological function and the molecular basis of PfCRT-mediated drug resistance have been hampered by a lack of high-resolution structural information. This dissertation describes the first structure of PfCRT and reveals the interaction of drugs with the purified and reconstituted protein. We determined the structure of the 49-kDa PfCRT 7G8, a clinically relevant CQ-resistant isoform found in South America, to 3.2 Å resolution by single-particle cryo-electron microscopy (cryo-EM), in complex with a specific antigen-binding fragment (Fab) to overcome current size limitations in cryo-EM. Our PfCRT structure displays an inward-open conformation, consists of 10 transmembrane (TM) helices with an inverted topology, and has unique elements including two juxtamembrane helices and a highly conserved cysteine-rich loop between TM helix 7 and 8.
Download or read book Structure and Functional Differentiation of PfCRT Mutation in Chloroquine Resistance CQR in Plasmodium Falciparum Malaria written by Pratap Parida and published by . This book was released on 2016 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Approximately one million deaths are attributed to malaria every year. Latest reports of multi-drug treatment failure of falciparum malaria underscore the desideratum to understand the molecular substratum of drug resistance. The mutations in the digestive vacuole transmembrane protein Plasmodium falciparum chloroquine resistance transporter (PfCRT) are mainly responsible for chloroquine resistance (CQR) in Plasmodium falciparum. Multiple mutations in the PfCRT are concerned in chloroquine resistance, but the evolution of intricate haplotypes is not yet well understood. P. falciparum resistance to chloroquine is the standard antimalarial drug and is mediated primarily by mutant forms of the PfCRT. In this chapter, we present the mechanism of action of the chloroquine, the structural changes of the gene after the mutations as well as different haplotypes of the PfCRT.
Download or read book Biochemical Characterisation of the Plasmodium Falciparum Chloroquine Resistance Transporter written by Fadi Baakdah and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "The emergence of resistance to commonly used antimalarials significantly hindered global efforts in eliminating malaria and cost the human race losses of lives in millions. Plasmodium falciparum parasites are the most accountable for morbidity and mortality compared to the other species that infect humans. At the present time, artemisinin combination therapies is the approach used in the field to treat malaria infected people and has shown tremendous success. However, resistance to these combinations recently emerged and the pattern of progression and spreading is alarming. Chloroquine once was the first-line drug for treatment of malaria infected people, however, it became in-effective due to the spread of chloroquine resistant strains. Many attributes of chloroquine, at the time when it was effective, were desired and as such the field took on different approaches to revive it. Some people took an approach to withdraw the use of chloroquine for a significant period of time resulting in the emergence of chloroquine sensitive strains. Others looked into modifying the structure of chloroquine in order to make derivatives that would be an improvement on the original. Additionally, others went on to investigate the molecular mechanism by which the parasite confers resistance to chloroquine. Presently, it is well known that mutations in the chloroquine resistance transporter (PfCRT), expressed on the membrane of a lysosome-like organelle in the parasite, the digestive vacuole (DV), are the primary determinants of chloroquine resistance. The physiological role and normal substrates are still matters of speculation but the protein seems to be important for the parasite survival because knockout-PfCRT clones could not be established. The crystal structure was resolved showing the spatial arrangement of the polypeptide chain relative to the juxtaposition of the transmembrane domains forming the central cavity where drugs would interact with PfCRT. Given PfCRT’s role in chloroquine resistance, we thought if chloroquine was slightly modified it would bypass PfCRT resistance mechanism. The first experimental manuscript thesis, we examined the antimalarial activity of 16 novel chloroquine derivatives against chloroquine-sensitive and -resistant Plasmodium falciparum strains. Only two compounds (e.g., AQ-13 and AQ-129) showed effects that surpassed chloroquine’s effect on chloroquine resistant strains that were examined previously but not to the extent of their relationship with PfCRT. Our results demonstrate that AQ-13 and AQ-129 are poor substrates of PfCRT and thus more effective against chloroquine resistant parasites. In the 2nd manuscript, we describe the high resolution characterisation of an antiserum raised against the full-length C-terminal domain of PfCRT. An IgG pool that recognises a de-phosphorylated Ser411 epitope was extracted and used as a tool to monitor the phosphorylation status of residue Ser411. This pool of IgG`s identified the presence of an Ser411 de-phosphorylated homodimer form of PfCRT that does not localise to the DV membrane as does the monomer PfCRT. We also show that PfCRT monomer in chloroquine-sensitive strain (3D7) is significantly more phosphorylated than in chloroquine-resistant strain (Dd2-H) at Ser411, suggesting a possible functional role for this residue in drug resistance. In the last manuscript, we describe the adoption of mammalian HEK-293F cells as a heterologous system to study PfCRT function. Using HEK-293F cells stably expressing PfCRT wild-type and mutants, we show mutant-PfCRT to cause a significant acidification of the lysosomes, relative to wild-type PfCRT. We also provide direct evidence that acidification was mediated through mutant-PfCRT, since using a proline-165-modified mutant-PfCRT clone restored the acidification of lysosomes to wild-type PfCRT levels. Thus, results of this study show for the first time the role of Pro165 in mutant-PfCRT function"--
Download or read book To Elucidate the Structure and Function of P Falciparum Chloroquine Resistance Transporter PfCRT Using the Substituted cysteine Accessibility Method SCAM written by Guoqing Liang and published by . This book was released on 2010 with total page 189 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Analysis of Plasmodium Falciparum Chloroquine Resistance Transporters in Saccharomyces Cerevisiae written by Nicholas Kyle Baro and published by . This book was released on 2012 with total page 492 pages. Available in PDF, EPUB and Kindle. Book excerpt: This study demonstrates the use of a model eukaryotic heterologous system to elucidate key features of a resistance protein from malaria parasites and highlights the potential of using this system to characterize its endogenous substrate.
Download or read book ANALYSIS OF PLASMODIUM FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER ISOFORMS written by Paul Samuel Callaghan and published by . This book was released on 2015 with total page 430 pages. Available in PDF, EPUB and Kindle. Book excerpt: Finally, I also carry out a large-scale chemogenetic screen of the Saccharomyces cerevisiae deletion mutant library to identify yeast genes relevant for resistance and sensitivity to CQ. This approach reveals overlapping gene pathways relevant to CQ toxicity, some of which have also been previously identified in related chemogenomic screens vs other quinoline drugs (e.g. quinine). Significantly enriched gene ontology (GO) terms persisted across increasingly stringent subsets of phenotypic grouping down to
Download or read book Molecular Characterisation of the Plasmodium Falciparum Pfcrt Gene Involved in Chloroquine Resistance written by and published by . This book was released on 2008 with total page 168 pages. Available in PDF, EPUB and Kindle. Book excerpt: Chloroquine (CQ) resistant P. falciparum was first reported in the 1960s at the Thai-Cambodia border. Gradually CQ resistance has spread and is found in all regions where P. falciparum transmission occurs. The emergence of CQ resistance due to excessive CQ selection pressure on the parasite populations has become an important issue because of the higher mortality and morbidity associated with drug resistance. Currently CQ is no longer recommended to treat falciparum malaria. CQ resistance in P. falciparum has been attributed to a single amino acid substitution on the P. falciparum chloroquine resistant transporter (pfcrt) at position 76 where Iysine is substituted with threonine (K76T). Molecular studies showed that CQ resistance emerged independently at five different geographical locations namely: Southeast Asia, two sites in South America, Papua New Guinea and the Philippines. Further analysis of resistant isolates revealed 22 additional non-silent amino acid substitutions on the pfcrt gene with a new amino acid substitution detected in the study reported here.
Download or read book Biochemical and Biophysical Analysis of Recombinant Plasmodium Falciparum Chloroquine Resistance Transporter pfcrt written by Michelle Fortaleza Paguio and published by . This book was released on 2009 with total page 259 pages. Available in PDF, EPUB and Kindle. Book excerpt: Two new methods were also developed: a high throughput fluorescence assay for antimalarial drug screening and a convenient scheme for the purification of recombinant PfCRT. The drug assay has contributed to the efficiency and ease of testing numerous compounds while the optimization of purifying recombinant PfCRT has produced a cleaner and more reliable system to investigate its function. These methods significantly aided in understanding the mechanism of chloroquine resistance.
Download or read book Mutational Analysis of Chloroquine Resistance Transporter crt Gene in Human Plasmodium Falciparum and Plasmodium Vivax Isolated from Sabah written by Lii Lian Tan and published by . This book was released on 2013 with total page 102 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book The Malaria Parasite s Chloroquine Resistance Transporter written by Robert Leon Summers and published by . This book was released on 2017 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The malaria parasite's chloroquine resistance transporter: An exploration of its interactions with drugs and of its evolution as a drug transporter Abstract: Initially identified as the primary determinant of chloroquine resistance in the malaria parasite Plasmodium falciparum, mutations in the 'chloroquine resistance transporter' (PfCRT) can influence the parasite's susceptibility to diverse molecules. The ability of PfCRT to affect the activity of so many compounds is likely to be a product of its location at the membrane of the parasite's digestive vacuole - an acidic compartment in which many types of drugs accumulate, act, and/or are activated. The Xenopus laevis oocyte system enables the functional expression of PfCRT and has been used to demonstrate that a mutant isoform of PfCRT mediates the efflux of chloroquine from the vacuole (i.e., away from its site of action), whereas the wild-type protein lacks this activity. However, the evolution of chloroquine transport activity by PfCRT has yet to be explored, and little is known of how PfCRT interacts with diverse compounds. The overarching aim of this study was to understand how mutations in PfCRT confer chloroquine transport activity and alter the parasite's susceptibility to diverse pharmacons. A kinetic analysis of the inhibition of PfCRT-mediated chloroquine transport by verapamil, a compound which partially restores the activity of chloroquine against drug-resistant parasites, was undertaken in the oocyte system. The findings of this work revealed verapamil to be a partial-mixed-type inhibitor of the transporter, and suggested that the mutations required for chloroquine transport introduce multiple substrate-binding sites into PfCRT. A series of complementary assays were then applied to examine the interactions of PfCRT with a range of compounds to identify, and distinguish between, PfCRT substrates and inhibitors. Using the oocyte system, two new classes of compounds were identified as potent inhibitors of the PfCRT-mediated transport of chloroquine. Transgenic parasite lines that are isogenic except for their pfcrt allele were employed, in conjunction with an assay that indirectly detects the transport of drugs out of the parasite's digestive vacuole, to further characterise these compounds. The resulting data revealed that most of these molecules are not substrates of the mutant transporter. Furthermore, parasite proliferation assays demonstrated that the compounds possessed enhanced activities against parasites harbouring mutant PfCRT. Structure-activity relationships were consistent with these compounds binding to multiple points of attachment within PfCRT via lipophilic and electrostatic interactions. Measurements of chloroquine transport via diverse isoforms of PfCRT (as well as by a series of chimeric proteins) were also undertaken in the oocyte system. These analyses revealed that multiple mutational pathways lead to saturable chloroquine transport via PfCRT. The finding that diverse PfCRT variants are all limited in their capacity to transport chloroquine suggests that resistance could be overcome by re-optimising the chloroquine dosage. Moreover, the results of this study indicated that the remodelling of PfCRT for chloroquine transport required a complex reorganisation of interacting residues. These studies support the idea that, in addition to being a mediator of multidrug resistance, PfCRT is itself a viable drug target. Antimalarial therapies could be formulated to exert opposing selection forces upon PfCRT, thereby exploiting a key resistance mechanism and prolonging drug efficacy against this important human pathogen.
Download or read book World Malaria Report 2018 written by World Health Organization and published by World Health Organization. This book was released on 2019-02-12 with total page 210 pages. Available in PDF, EPUB and Kindle. Book excerpt: This year s report shows that after an unprecedented period of success in global malaria control progress has stalled. Data from 2015?2017 highlight that no significant progress in reducing global malaria cases was made in this period. There were an estimated 219 million cases and 435 000 related deaths in 2017. The World malaria report 2018 draws on data from 90 countries and areas with ongoing malaria transmission. The information is supplemented by data from national household surveys and databases held by other organizations.
Download or read book Functional Studies on the Chloroquine Resistance Transporter PfCRT and the HECT E3 Ubiquitin protein Ligase PfUT in Plasmodium Falciparum written by Monika Jankowska-Döllken and published by . This book was released on 2019* with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Posttranslational modifications (PTMs) affect fundamental cellular functions of the human malaria parasite Plasmodium falciparum, including regulation of protein stability, metabolism, proliferation, apoptosis and signal transduction. This study investigates the importance of phosphorylation and ubiquitination in modulating the parasite intraerythrocytic development, as well as their implication in antimalarial drug resistance. The chloroquine resistance transporter PfCRT is a drug-metabolite carrier annotated as a prominent determinant of parasite's reduced susceptibility to quinoline drugs. PfCRT is posttranslationally modified by phosphorylation and palmitoylation. However, the role of these PTMs in regulation of PfCRT function is not fully resolved. Chemical and genetic approaches employed in the current study revealed the relevance of PfCRT phosphorylation at serine 33 in regulating the drug resistance-mediating function of this transporter and in vitro fitness of the parasite. The PfCRT allelic exchange mutants, in which serine 33 was replaced by alanine showed increased sensitivity to chloroquine and quinine. Moreover, PfCRT serine 33 substitution by phospho-mimicking amino acids, glutamic and aspartic acid, could respectively partially and fully, restore the resistance phenotype. The fitness variation between the PfCRT mutants was linked to differences in merozoite numbers and their invasion efficiencies, with alanine mutants displaying a significant advantage in this regard. Identification of a kinase implicated in this phenomenon is desired, as its inhibition in combination with chloroquine could reduce or prevent the further spread of resistance. A HECT E3 ubiquitin ligase, termed ubiquitin transferase PfUT, is a novel candidate gene for multifactorial resistance to quinine. PfUT was shown to localize to the parasite's ER/Golgi complex, but its role in reduced susceptibility to quinine and its physiological function remain unclear. Characterization of PfUT was attempted by a glmS ribozyme-based conditional knockdown of encoding it gene, generated using the CRISPR-Cas9 genome editing technology. Unexpectedly, integration of the glmS sequence resulted in a 2-fold increase in PfUT transcripts correlated with protein levels. PfUT overexpression, in turn, led to S phase-associated lengthening of parasite's cycle, reflected in impaired growth. Glucosamine-induced incomplete downregulation partially restored the wild type phenotype. Moreover, the transgenic parasites exhibited an enhanced susceptibility to quinine and quinidine. An alternative disruption of the pfut locus via a selection-linked integration (SLI-TGD) strategy was unsuccessful, despite multiple attempts. These results underline the importance of PfUT in parasite proliferation and survival. However, a direct proof of PfUT's association with quinine resistance and identification of its biological substrates await further investigation.
Download or read book Analysis of Drug Resistance Mechanisms in Intact Plasmodium Falciparum infected Red Blood Cells written by Sarah Reiling and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "Malaria is a major global health concern, with half of the world's population being at risk of infection. Among the Plasmodium species that infect humans, P. falciparum causes most fatalities. Chloroquine (CQ) was the drug of choice for decades and considered safe, affordable and easy-to-use until resistance emerged. However, the exact mechanism of CQ resistance is not known. CQ is suggested to accumulate in the parasite's digestive vacuole due to its weak base properties, where it exerts its antimalarial action. Several transporters are involved in intracellular distribution of antimalarial drugs. Among them are the P. falciparum chloroquine resistance transporter (PfCRT) and the P. falciparum multidrug resistance 1 transporter (PfMDR1). Both are located in the digestive vacuolar membrane but transport substrates in opposing directions. While PfCRT transports substrates out of the digestive vacuole (DV), PfMDR1 transports substrates into the DV. PfMDR1 contains five polymorphisms that are suggested to be involved in altered drug transport, although the exact role of each amino acid mutation remains unknown. To gain more insight into the transport functions of PfMDR1, variants with different mutation patterns were analyzed using the fluorescent substrate Fluo-4. We found a crucial role for asparagine (N) at residue 1042 in Fluo-4 transport, while substitution with aspartic acid (D) abolished all transport. In addition, we showed an association of the PfMDR1 N1042D mutation with increased mefloquine but decreased quinine sensitivity. Furthermore, competition studies of Fluo-4 with the antimalarial drugs chloroquine, mefloquine and quinine showed distinct transport inhibition patterns for parasites of different genetic background. This can be used as a tool to evaluate parasite susceptibility to antimalarial drugs.Next, we investigated the mechanism of resistance to CQ in more detail. We showed that parasite survival is higher in CQ-resistant strains compared to CQ-sensitive strains in the initial 10 hours after exposure to equally lethal CQ concentrations. Moreover, dark cytosolic structures appeared in CQ-sensitive strains that were later confirmed as hemozoin-containing compartments surrounded by a membrane bilayer. Leakage of hemozoin crystals out of the DV was ruled out since lysis of the digestive vacuolar membrane did not occur during that time frame. These data suggest that CQ resistance is not linked to reduced drug concentrations in the DV alone, and additional regulatory mechanisms in the parasite must play a crucial role during CQ exposure.To pursue these findings, a commercially available fluorescent tagged CQ analogue, LynxTagTM-CQ-GREEN (CQ-GREEN), was examined for its suitability in studying CQ transport and intracellular drug accumulation. While CQ-GREEN was half as effective in parasite killing of CQ-sensitive strains compared to unmodified CQ, no significant changes in parasite killing were observed in CQ-resistant strains. However, live cell imaging showed that CQ-GREEN accumulated in the parasite cytosol and not the DV. These results show for the first time a potential target for a CQ analogue outside the digestive vacuole. Moreover, intracellular CQGREEN uptake rates were reduced in CQ-resistant strains compared to CQ-sensitive strains. This, too, suggests that CQ-resistant strains must have evolved a regulatory mechanism to decrease intracellular CQ accumulation.The results presented in this thesis expand our understanding of substrate transport by PfMDR1. Furthermore, a novel phenotype was described for CQ-sensitive strains upon drug exposure that was not seen in CQ-resistant strains. These data suggest that altered regulatory mechanisms play a role in CQ resistance and are likely located in the parasite cytosol." --
Download or read book The Plasmodium Falciparum Chloroquine Resistance Transporter PFCRT Mediates the Activity of Chloroquine resistance Reversal Agents in the Malaria Parasite written by Kristin Lane and published by . This book was released on 2007 with total page 138 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Investigating the Plasmodium Falciparum Chloroquine Resistance Transporter in a Heterologous Expression System written by David A. Liebelt and published by . This book was released on 2007 with total page 204 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Analysis of Parasite Chloroquine Resistance Transporter Proteins and Their Roles in Drug Resistance Phenomena written by Bryce Riegel and published by . This book was released on 2020 with total page 444 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this thesis, I measure drug transport efficiencies of multiple PvCRT isoforms heterologously expressed in yeast. I also measure drug transport for novel PPQR-associated PfCRT isoforms, and test whether an orthologue of PfCRT found in the closely related apicomplexan parasite Toxoplasma gondii (TgCRT), is a functional transporter of CQ, as part of a broader investigation of the role of TgCRT in the endolysosomal system of T. gondii.
