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 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 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 Molecular and Biochemical Characterisation of the Electron Transport Chain of Plasmodium Falciparum written by Thomas Antoine and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Characterisation of the Drug Transport Properties of the Plasmodium Falciparum Chloroquine Resistance Transporter Through Expression in Xenopus Laevis Oocytes written by Anurag Dave and published by . This book was released on 2011 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:

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 Biochemical Characterization of Plasmodium Falciparum Heat Shock Protein 70 written by Tonderayi Sylvester Matambo and published by . This book was released on 2003 with total page 0 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 The Synthesis and Development of Novel Inhibitors of the Plasmodium Falciparum Chloroquine Resistance Transporter written by Karen Joy Deane and published by . This book was released on 2014 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The work detailed in this thesis has been directed towards the synthesis of inhibitors of the Plasmodium falciparum chloroquine resistance transporter (PfCRT). Mutations in this protein are responsible for resistance of the malaria parasite to the antimalarial drug, chloroquine. Inhibitors of PfCRT are known as resistance reversers, and include the antihistamine chlorpheniramine, which has been used as the basis of all the structures that have been developed in this work. Chapter 1 provides an introduction to the disease malaria, caused by Plasmodium parasites. A brief review of antimalarial drugs is presented, with a focus on the most successful of these, chloroquine, and the development of resistance to this drug. Chapter 2 reports the synthetic route to the synthesis of 30 analogues of chlorpheniramine, which includes the preparation and characterisation of 29 previously unreported compounds. Analogues were assayed for and showed the ability to inhibit chloroquine transport by PfCRT, which was correlated to the ability to lower the IC50 of chloroquine in resistant strains of Plasmodium falciparum. The development and synthesis of 10 novel reversed chloroquines is presented in Chapter 3. The targeted structures were analogues of a hybrid structure based on chloroquine and chlorpheniramine, and possessed potent antimalarial activity in addition to their strong activity as inhibitors of PfCRT. 32 new compounds were prepared and characterised. A second generation approach is detailed in the synthesis of 10 reversed sontochins in Chapter 4. The sontochin/chlorpheniramine hybrids showed enhanced activity as inhibitors of PfCRT compared to the previous series. 25 new compounds were synthesised and characterised en route. Chapter 5 describes the synthesis of two chlorpheniramine analogues that have been adapted for inclusion in a reversed tetraoxane. These structures showed no improvement to the activity of chlorpheniramine at inhibiting PfCRT, but possess features for incorporation into an endoperoxide antimalarial. The preparation and characterisation of 20 new compounds is reported.

Download or read book Malaria written by Institute of Medicine and published by National Academies Press. This book was released on 1991-02-01 with total page 312 pages. Available in PDF, EPUB and Kindle. Book excerpt: Malaria is making a dramatic comeback in the world. The disease is the foremost health challenge in Africa south of the Sahara, and people traveling to malarious areas are at increased risk of malaria-related sickness and death. This book examines the prospects for bringing malaria under control, with specific recommendations for U.S. policy, directions for research and program funding, and appropriate roles for federal and international agencies and the medical and public health communities. The volume reports on the current status of malaria research, prevention, and control efforts worldwide. The authors present study results and commentary on the: Nature, clinical manifestations, diagnosis, and epidemiology of malaria. Biology of the malaria parasite and its vector. Prospects for developing malaria vaccines and improved treatments. Economic, social, and behavioral factors in malaria control.

Download or read book Biochemical Characterization of the Malaria Parasite Plasmodium Falciparum CLpB Homologue PfClpB1 Localized to the Apicoplast written by Fabrice Ngansop and published by . This book was released on 2013 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: ClpB is a molecular chaperone that is essential for infectivity and pathogen survival in a host. It belongs to the AAA+ protein family, which cooperates with the DnaK chaperone system to reactivate aggregated proteins. In this study, we purified and then studied the biochemical properties of the apicoplast targeted ClpB isoform from the malaria parasite Plasmodium falciparum: PfClpB1. Plasmodium falciparum is the parasite responsible for the most severe form of malaria. In contrast to the parasitophorous vacuole targeted PfClpB2 from Plasmodium falciparum which contains all characteristic AAA+ sequence motifs, PfClpB1 also includes a 52-residue long non-conserved insert in the middle domain. The ATPase activity study shows that PfClpB1 hydrolyzes ATP in presence of Poly-lysine and [alpha]-casein. Similar to most AAA+ ATPases, addition of ATP induces hexamer formation in PfClpB1. Lastly, PfClpB1 reactivates aggregated firefly luciferase. However, PfClpB1 is unable to efficiently reactivated luciferase in the presence of the E. coli DnaK chaperone system or human Hsp70 and Hsp40 (Hdj1). This can be explained by the extra middle domain sequence of PfClpB1. Our data may suggest that PfClpB1 activity is essential for Plasmodium falciparum survival by preserving the activity of apicoplast proteins.

Download or read book Characterization of Plasmodium Falciparum Resistance to Novel Drugs written by Sonia Edayé and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "Plasmodium falciparum is the deadly protozoan parasite responsible for malaria. Malaria is one of the most important infectious diseases that has been raging for millennia and affecting almost half of the world's population. The treatment regimen that was based on quinoline drugs such as chloroquine (CQ), was efficient for decades. Nowadays, the use of this class of drugs is doomed to failure due to the emergence of quinoline-resistant parasites. Today, artemisinin-based combination therapies (ACTs) are the first-line drugs for uncomplicated falciparum malaria treatment. ACTs improve the cure rate of malaria and thus are seen as efficient treatment against uncomplicated forms of the disease. Despite their efficiency, these drugs are currently facing the development of resistance. PfCRT and PfMDR1, which are membrane transporters, have been shown to be involved in malaria parasites drug resistance. To tackle the inefficiency of existing drugs in regard to the development of resistance, alternative therapies must be discovered. In this thesis, antimalarial activity of novel potential drugs against P. falciparum is assessed and the interaction of these drugs with PfCRT and PfMDR1 is determined. Furthermore, because many ABC transporter genes play a key role in drug resistance, the characterization of an ABC transporter member of the ABCG family in Plasmodium is addressed and its role in drug resistance investigated.In the first part of this thesis, MK571 (a quinoline analogue) activity against P. falciparum parasites is investigated. MK571 is found to be more toxic to most of the CQ-resistant strains than to the CQ-sensitive strains. In addition, we determine that MK571 is not a substrate of PfCRT as are other quinoline drugs, but is instead a substrate of PfMDR1. Therefore, it can be a good complement to existing quinoline drugs in the treatment of uncomplicated malaria. In the second part, novel compound analogues of chloroquine are tested for their antimalarial activity against CQ-sensitive and -resistant parasites. Although chloroquine analogues tested possess the quinoline ring structure of chloroquine, they are less efficient than chloroquine and are not substrates of PfCRT. One of the analogues (3-ICQ) reverses the resistance of CQ-resistant strains to chloroquine and therefore, could be used in combination with chloroquine in cases of CQ-resistant malaria. In the third part of the thesis we conduct the characterization of PfABCG, the sole member of the P. falciparum ABCG family. The characterization study demonstrates that PfABCG is localized on the parasite plasma membrane and is expressed throughout the asexual life cycle of the parasite. In addition, PfABCG is differentially expressed in various Plasmodium strains. This expression does not correlate with the resistance to chloroquine but to the sensitivity of the parasite to an antihistaminic drug named ketotifen. Overall, this thesis sheds light on challenges and understanding of the complex resistance machinery deployed by the P. falciparum parasite from novel drug discovery to characterization of proteins. " --

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 Characterization of Plasmodium Falciparum Choline Transporters written by and published by . This book was released on 2005 with total page 54 pages. Available in PDF, EPUB and Kindle. Book excerpt: Plasmodium falciparum is the causative agent of severe human malaria. The rapid multiplication of the parasite within human red blood cells requires an active synthesis of new membranes. Choline analogs are potent antimalarial drugs. Although Choline transport has been suggested to be the target of these compounds, their exact mode of action is unknown. We identified two genes PfGAT and PfCTL1 as potential targets of these compounds. Here we report evidence that PfGAT encodes a Glycerol-3-phosphate acyltransferase enzyme responsible for the initial step of synthesis of P. falciparum membranes. Genetic data suggest that this pathway is essential for parasite survival and is a good target for development of new antimalarial drugs. We have also initiated a thorough genetic and biochemical characterization of PfCTL. Our data suggest that membrane proteins of the CTL family are not choline transporters. We generated transgenic parasites lacking PfCTL1, and showed that PfCTL is not essential for parasite intraerythrocytic development and survival. Finally we provide biochemical and genetic data suggesting that choline analogs act by specifically inhibiting phospholipid me%tabolism and the ability of the parasite to generate new membranes.

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 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 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.