Molecular analysis of antimalarial therapies : from drug development to mode of action
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...
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| Format: | Thesis |
| Language: | English |
| Published: |
2019
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| Online Access: | https://hdl.handle.net/10356/105861 http://hdl.handle.net/10220/47871 |
| Summary: | 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. |
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