Genetically-encoded fluorescent ATP sensors for mode of action analyses of antiparasitic compounds in Plasmodium falciparum

Abstract

This dissertation is based on publications that have established the usage of novel methods for adenosine triphosphate (ATP) and pH determinations in Plasmodium falciparum (P. falciparum). Based on these and complementary methods, mode of action (MoA) analyses of established drugs and novel antiparasitic compounds were conducted to support drug development. The deadliest form of malaria, malaria tropica, caused by the intracellular parasite P. falciparum, claims more than half a million casualties annually. With resistances against each established antimalarial drug class against P. falciparum found in the field, the development of novel and improved antiparasitic compounds poses a matter of highest concern for global health. To this end, understanding of the underlying mechanisms is crucial. Therefore, within the scope of this work, genetically-encoded fluorescence-based ATP and pH sensors were stably integrated into the genome of P. falciparum. Within the adult blood stages of the parasite, this allowed an analysis of its energy metabolism, which is central for parasite survival. Based on this system, MoA analyses of a panel of antiparasitic compounds were carried out. In the case of a promising drug candidate, these were complemented with additional methods for elucidation of its mechanism. Thereby, two different variants of ATP sensors, ATeam1.03nD/nA and ATeam1.03YEMK, as well as an improved pH sensor, sfpHluorin, were established and characterized via plate reader spectrofluorometry and epifluorescence microscopy, for the first time in P. falciparum. In vitro and in cellulo characterization of the sensors demonstrated their proof of principle and unveiled advantages of the ATeam1.03YEMK sensor cell line in respect to fluorescence intensity and pH stability. Based on that, the effects of a selection of antiparasitic compounds on the sensor readouts were determined. This revealed characteristic response patterns caused by 4-aminoquinolines, arylamino alcohols, redox cyclers, as well as dihydroartemisinin, doxycycline, atovaquone, and cycloheximide. In a next step, the effects of the drug candidate arylmethylamino steroid compound 1o (1o) on the sensor readouts were determined. The investigation uncovered parallels to arylamino alcohols such as mefloquine. Based on these parallels, subsequent analyses via transmission electron microscopy (TEM), the parasitederived heme species distribution, as well as light microscopic morphology of stage-specific 1o incubations suggest that the parasite-killing activity could be based on interference with the parasite’s hemoglobin uptake. The results of this work benefit the understanding of P. falciparum’s parasite biology and the MoA of antiparasitic compounds to support drug development. Furthermore, the established system is now available for the malaria community to address a broad range of research questions.