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Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR
Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
International Union of Crystallography
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489230/ https://www.ncbi.nlm.nih.gov/pubmed/34605430 http://dx.doi.org/10.1107/S2059798321009037 |
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author | Luchinat, Enrico Barbieri, Letizia Cremonini, Matteo Pennestri, Matteo Nocentini, Alessio Supuran, Claudiu T. Banci, Lucia |
author_facet | Luchinat, Enrico Barbieri, Letizia Cremonini, Matteo Pennestri, Matteo Nocentini, Alessio Supuran, Claudiu T. Banci, Lucia |
author_sort | Luchinat, Enrico |
collection | PubMed |
description | Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained in vitro, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding. |
format | Online Article Text |
id | pubmed-8489230 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | International Union of Crystallography |
record_format | MEDLINE/PubMed |
spelling | pubmed-84892302021-10-18 Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR Luchinat, Enrico Barbieri, Letizia Cremonini, Matteo Pennestri, Matteo Nocentini, Alessio Supuran, Claudiu T. Banci, Lucia Acta Crystallogr D Struct Biol Ccp4 Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained in vitro, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding. International Union of Crystallography 2021-09-27 /pmc/articles/PMC8489230/ /pubmed/34605430 http://dx.doi.org/10.1107/S2059798321009037 Text en © Enrico Luchinat et al. 2021 https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. |
spellingShingle | Ccp4 Luchinat, Enrico Barbieri, Letizia Cremonini, Matteo Pennestri, Matteo Nocentini, Alessio Supuran, Claudiu T. Banci, Lucia Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR |
title | Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR |
title_full | Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR |
title_fullStr | Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR |
title_full_unstemmed | Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR |
title_short | Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR |
title_sort | determination of intracellular protein–ligand binding affinity by competition binding in-cell nmr |
topic | Ccp4 |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489230/ https://www.ncbi.nlm.nih.gov/pubmed/34605430 http://dx.doi.org/10.1107/S2059798321009037 |
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