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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...

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Autores principales: Luchinat, Enrico, Barbieri, Letizia, Cremonini, Matteo, Pennestri, Matteo, Nocentini, Alessio, Supuran, Claudiu T., Banci, Lucia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2021
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.
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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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