Molecular basis for the mechanism of action and resistance to artemisinin combination therapy in Plasmodium falciparum

Abstract: Malaria, caused by the Plasmodium falciparum parasite, remains a leading cause of death among children in Africa. To improve treatment efficacy and delay development and spread of antimalarial drug resistance artemisinin artemisinin-based combination therapy (ACT) is now globally recommended as first-line treatment of uncomplicated P. falciparum malaria as a cornerstone in modern malaria control. The aim of this thesis is to improve the understanding of the molecular basis of potential evolution of P. falciparum resistance to ACT. After the worldwide introduction of ACT several reports demonstrate that the multidrug resistance protein 1 (pfmdr1) and chloroquine resistance transporter (pfcrt) genes are under selective pressure. This thesis describes the in vivo selective process for pfmdr1 haplotype coding for aminoacids 86N, 184F 1246D in reinfections after artemether-lumefantrine treatment. The selective window is within 35 days after treatment during the elimination phase of the partner drug. PFMDR1 homologue model structures unveiled the functional interference of 86N, 184F and 1246D in antimalarial drug transport. This was further supported by in vitro susceptibility of P. falciparum pfmdr1 transfectants clones to aminoquinolines indicating that PFMDR1 may act as a vacuolar importer. Since the resistance mechanisms of P. falciparum to the major ACTs are largely unknown other candidate genes were analysed. Therefore the multidrug resistance-associated 1 (pfmrp1) gene diversity in P. falciparum and its potential contribution to decreased ACT sensitivity was studied. Some 21 nonsynonymous and 6 synonymous single nucleotide polymorphisms were identified. The polymorphism I876V appears to be significantly (P<0.05) selected in reinfections after artemether-lumefantrine. The structural role of I876V polymorphism and impact for PFMRP1 transport was then studied in bacterial ABC transporter homologue, MsbA, and shown to be related to the nucleotide binding region of ABC transporters. To investigate mechanism of action of artemisinins in P. falciparum, parasite’s calcium homeostasis was studied using techniques of live single cell imaging and flow cytometry. Our work suggests that artemisinin triggers Ca2+ signalling- dependent cell death in P. falciparum. Parasite cell death was partially rescued (31%) by the Ca2+ chelator Bapta. In conclusion, P. falciparum is adapting to the new ACTs. Complex mechanisms of pfmdr1/pfcrt are being selected by partner drugs and may represent entry points towards alarming evolution of tolerance and resistance to ACT.