Molecular analysis of antimalarial therapies : from drug development to mode of action

• Main Author: Dziekan, Jerzy Michal
• Other Authors: Roderick Wayland Bates
• Format: Theses and Dissertations
• Language: English
• Published: 2019
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/105861; http://hdl.handle.net/10220/47871
• Institution: Nanyang Technological University

Mechanisms of action (MoA) have been elusive for most antimalarial drugs in clinical use. Decreasing responsiveness to antimalarial treatments stresses the need of a more resolved understanding of their MoA and associated resistance mechanisms. In the present work we have implemented the Cellular Thermal Shift Assay (CETSA) for drug target deconvolution in Plasmodium falciparum. We used the CETSA assay to characterise the thermal unfolding patterns of the P. falciparum proteome. Subsequently, we validated the efficacy of CETSA for antimalarial drugtarget identification using pyrimethamine, a drug with well-known MoA relying on inhibition of folic acid synthesis pathway and E64d, a broad-spectrum cysteine proteinase inhibitor. As a next step, we applied CETSA to quinine and mefloquine, two important anti-malarial drugs with poorly characterised MoA. Combining studies in parasite lysate and intact P. falciparum-infected red blood cells, we discovered P. falciparum Purine Nucleoside Phosphorylase (PfPNP) as a common putative target for these two quinoline drugs. The interactions were confirmed using biophysical and activity studies on recombinant proteins and crystal structures revealed binding of the two compounds in the enzyme’s active site. Our results suggest that PfPNP inhibition is likely contributing to the therapeutic effect of aryl aminoquinolines. Additionally, we coupled the CETSA assay with an in-house-developed natural products drug discovery pipeline from Traditional Chinese Medicine to demonstrate assay’s efficacy for drug discovery efforts. We isolated and identified skullcapflavone-II and wogonin, two previously uncharacterised molecules with antimalarial properties from Scutellaria baicalensis plant extract and used CETSA to determine the candidate molecular targets for skullcapflavone-II, the more potent of the two substances. This work demonstrates that implementation of CETSA for P. falciparum constitutes a new promising strategy to establish MoA for existing and candidate antimalarial drugs.