<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Mechanisms of chloroquine resistance in Plasmodium falciparum"^^ . "Malaria remains the most prevalent infectious disease in the world, with over 249 million cases and over 608 thousand deaths reported in 2022. Despite enormous efforts, two vaccines have only recently been recommended for use in a handful of countries. Therefore, malaria control has focused on vector control on the one hand, and on prophylaxis and treatment with antimalarials on the other. However, control and eradication programmes have been marked by periods of immense progress followed by lapses of stagnation and relapse. For instance, it has been almost 15 years since resistance to the artemisinins was first reported in Southeast Asia, and nearly a decade since parasites resistant to the partner drug piperaquine emerged, bringing artemisinin-based combination therapies under revision. Frequently, mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) are responsible for partner drug resistance, hampering parasite clearance in treated patients. The protein lies in the digestive vacuole membrane of the parasite, where resistance-conferring mutations allow PfCRT to bind and expel drugs like chloroquine or piperaquine out of the vacuole, where they can no longer exert their antimalarial effects. While efforts are in parallel directed to designing new drugs and developing new vaccines, a further understanding of the mechanisms by which PfCRT handles drugs is necessary to extend the longevity of currently available antimalarials and to prevent the emergence of resistance to new drugs.\r\nThis doctoral thesis studied PfCRT from the point of view of its post-translational modifications and of the mutations that it carries in chloroquine and piperaquine resistant parasites. Through the employment of proteomic approaches like the chemoproteomic profiling of kinase inhibitors ML-7 and H-89, I investigated which of the protein kinases in the parasite are responsible for phosphorylation of the Ser33 residue in the cytosolic N-terminus of PfCRT. In parallel, I employed K-CLASP, a method specifically designed to determine which protein kinase in a cellular lysate can phosphorylate a given phosphosite. Both experiments pointed at PfPKA as a candidate for post-translationally modifying PfCRT at the Ser33 residue. In this work, the recombinant expression of PfPKA was attempted in Escherichia coli BL21 (DE3) pLysS, and in Spodoptera frugiperda Sf9 and Sf21 cells, though the protein was found to be either not expressed or present in the insoluble fraction. Additionally, using an established heterologous expression system, Xenopus laevis oocytes, the effects of mutations H97Y, F145I, M343L and G353V on the PfCRT-mediated drug transport kinetics was studied. The findings offered an explanation to why these mutations confer resistance to the partner drug piperaquine but, in doing so, re-sensitize the parasite to chloroquine. I also probed the substrate binding cavity of PfCRT by combining substrate competition kinetics, information theory for model discrimination, graphical analyses of kinetic data, and computational docking and molecular dynamics simulations. With these, I found that chloroquine and piperaquine have separate, independent binding pockets in PfCRT, though some isoforms can have them competing for the same site. Lastly, based on the obtained results and on a suggestion to use chloroquine and piperaquine together in a combination therapy, I generated an artificial double mutant that outperformed isoforms associated with chloroquine or piperaquine resistance in terms of drug transport. I hypothesize that the protein cavity is very flexible and can evolve in ways to offer solutions to a parasite that faces varying drug challenges."^^ . "2024" . . . . . . . "Guillermo Martín"^^ . "Gómez"^^ . "Guillermo Martín Gómez"^^ . . . . . . "Mechanisms of chloroquine resistance in Plasmodium falciparum (PDF)"^^ . . . "GuillermoMartinGomez_Dissertation.pdf"^^ . . . "Mechanisms of chloroquine resistance in Plasmodium falciparum (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Mechanisms of chloroquine resistance in Plasmodium falciparum (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Mechanisms of chloroquine resistance in Plasmodium falciparum (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Mechanisms of chloroquine resistance in Plasmodium falciparum (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Mechanisms of chloroquine resistance in Plasmodium falciparum (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #34966 \n\nMechanisms of chloroquine resistance in Plasmodium falciparum\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .