Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay
Mechanisms of action (MoAs) have been elusive for most antimalarial drugs in clinical use. Decreasing responsiveness to antimalarial treatments stresses the need for a better resolved understanding of their MoAs and associated resistance mechanisms. In the present work, we implemented the cellular t...
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sg-ntu-dr.10356-1524772021-08-18T02:06:36Z Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay Dziekan, Jerzy Michal Yu, Han Chen, Dan Dai, Lingyun Wirjanata, Grennady Larsson, Andreas Prabhu, Nayana Sobota, Radoslaw Mikolaj Bozdech, Zbynek Nordlund, Pär School of Biological Sciences Institute of Molecular and Cell Biology, A*STAR Science::Biological sciences Biological Assay Mefloquine Molecular Mechanisms of action (MoAs) have been elusive for most antimalarial drugs in clinical use. Decreasing responsiveness to antimalarial treatments stresses the need for a better resolved understanding of their MoAs and associated resistance mechanisms. In the present work, we implemented the cellular thermal shift assay coupled with mass spectrometry (MS-CETSA) for drug target identification in Plasmodium falciparum, the main causative agent of human malaria. We validated the efficacy of this approach for pyrimethamine, a folic acid antagonist, and E64d, a broad-spectrum cysteine proteinase inhibitor. Subsequently, we applied MS-CETSA to quinine and mefloquine, two important antimalarial drugs with poorly characterized MoAs. Combining studies in the P. falciparum parasite lysate and intact infected red blood cells, we found P. falciparum purine nucleoside phosphorylase (PfPNP) as a common binding target for these two quinoline drugs. Biophysical and structural studies with a recombinant protein further established that both compounds bind within the enzyme's active site. Quinine binds to PfPNP at low nanomolar affinity, suggesting a substantial contribution to its therapeutic effect. Overall, we demonstrated that implementation of MS-CETSA for P. falciparum constitutes a promising strategy to elucidate the MoAs of existing and candidate antimalarial drugs. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University National Medical Research Council (NMRC) This work was supported by NMRC MS-CETSA platform grant MOH/IAFCAT2/004/2015 to Z.B., P.N., A.L., and R.M.S.; Singapore Ministry of Education, Tier 2, MOE2015-T2-2-108 grant to Z.B.; Young Investigator Grant (YIG2015 A-STAR) to R.M.S.; Startup grant from NTU to P.N.; and grants from the Swedish Research Council and the Knut och Alice Wallenberg Foundation to P.N. 2021-08-18T02:06:36Z 2021-08-18T02:06:36Z 2019 Journal Article Dziekan, J. M., Yu, H., Chen, D., Dai, L., Wirjanata, G., Larsson, A., Prabhu, N., Sobota, R. M., Bozdech, Z. & Nordlund, P. (2019). Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay. Science Translational Medicine, 11(473), eaau3174-. https://dx.doi.org/10.1126/scitranslmed.aau3174 1946-6234 https://hdl.handle.net/10356/152477 10.1126/scitranslmed.aau3174 30602534 2-s2.0-85059497666 473 11 eaau3174 en MOH/IAFCAT2/004/2015 MOE2015-T2-2-108 YIG2015 A-STAR Science Translational Medicine © 2019 The Authors. Some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. |
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Science::Biological sciences Biological Assay Mefloquine Molecular Dziekan, Jerzy Michal Yu, Han Chen, Dan Dai, Lingyun Wirjanata, Grennady Larsson, Andreas Prabhu, Nayana Sobota, Radoslaw Mikolaj Bozdech, Zbynek Nordlund, Pär Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay |
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Mechanisms of action (MoAs) have been elusive for most antimalarial drugs in clinical use. Decreasing responsiveness to antimalarial treatments stresses the need for a better resolved understanding of their MoAs and associated resistance mechanisms. In the present work, we implemented the cellular thermal shift assay coupled with mass spectrometry (MS-CETSA) for drug target identification in Plasmodium falciparum, the main causative agent of human malaria. We validated the efficacy of this approach for pyrimethamine, a folic acid antagonist, and E64d, a broad-spectrum cysteine proteinase inhibitor. Subsequently, we applied MS-CETSA to quinine and mefloquine, two important antimalarial drugs with poorly characterized MoAs. Combining studies in the P. falciparum parasite lysate and intact infected red blood cells, we found P. falciparum purine nucleoside phosphorylase (PfPNP) as a common binding target for these two quinoline drugs. Biophysical and structural studies with a recombinant protein further established that both compounds bind within the enzyme's active site. Quinine binds to PfPNP at low nanomolar affinity, suggesting a substantial contribution to its therapeutic effect. Overall, we demonstrated that implementation of MS-CETSA for P. falciparum constitutes a promising strategy to elucidate the MoAs of existing and candidate antimalarial drugs. |
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School of Biological Sciences |
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School of Biological Sciences Dziekan, Jerzy Michal Yu, Han Chen, Dan Dai, Lingyun Wirjanata, Grennady Larsson, Andreas Prabhu, Nayana Sobota, Radoslaw Mikolaj Bozdech, Zbynek Nordlund, Pär |
format |
Article |
author |
Dziekan, Jerzy Michal Yu, Han Chen, Dan Dai, Lingyun Wirjanata, Grennady Larsson, Andreas Prabhu, Nayana Sobota, Radoslaw Mikolaj Bozdech, Zbynek Nordlund, Pär |
author_sort |
Dziekan, Jerzy Michal |
title |
Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay |
title_short |
Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay |
title_full |
Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay |
title_fullStr |
Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay |
title_full_unstemmed |
Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay |
title_sort |
identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay |
publishDate |
2021 |
url |
https://hdl.handle.net/10356/152477 |
_version_ |
1709685316789469184 |