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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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2019
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| Online Access: | https://hdl.handle.net/10356/105861 http://hdl.handle.net/10220/47871 |
| _version_ | 1811681473637384192 |
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| author | Dziekan, Jerzy Michal |
| author2 | Roderick Wayland Bates |
| author_facet | Roderick Wayland Bates Dziekan, Jerzy Michal |
| author_sort | Dziekan, Jerzy Michal |
| collection | NTU |
| description | 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. |
| first_indexed | 2024-10-01T03:41:30Z |
| format | Thesis |
| id | ntu-10356/105861 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T03:41:30Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | ntu-10356/1058612023-02-28T18:35:29Z Molecular analysis of antimalarial therapies : from drug development to mode of action Dziekan, Jerzy Michal Roderick Wayland Bates Zbynek Bozdech School of Biological Sciences DRNTU::Science::Biological sciences::Molecular biology 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. Doctor of Philosophy 2019-03-20T14:21:36Z 2019-12-06T21:59:29Z 2019-03-20T14:21:36Z 2019-12-06T21:59:29Z 2018 Thesis Dziekan, J. M. (2018). Molecular analysis of antimalarial therapies : from drug development to mode of action. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/105861 http://hdl.handle.net/10220/47871 10.32657/10220/47871 en 204 p. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences::Molecular biology Dziekan, Jerzy Michal Molecular analysis of antimalarial therapies : from drug development to mode of action |
| title | Molecular analysis of antimalarial therapies : from drug development to mode of action |
| title_full | Molecular analysis of antimalarial therapies : from drug development to mode of action |
| title_fullStr | Molecular analysis of antimalarial therapies : from drug development to mode of action |
| title_full_unstemmed | Molecular analysis of antimalarial therapies : from drug development to mode of action |
| title_short | Molecular analysis of antimalarial therapies : from drug development to mode of action |
| title_sort | molecular analysis of antimalarial therapies from drug development to mode of action |
| topic | DRNTU::Science::Biological sciences::Molecular biology |
| url | https://hdl.handle.net/10356/105861 http://hdl.handle.net/10220/47871 |
| work_keys_str_mv | AT dziekanjerzymichal molecularanalysisofantimalarialtherapiesfromdrugdevelopmenttomodeofaction |