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Determination of intracellular protein-ligand binding affinity by competition binding in-cell NMR.

Authors
  • Luchinat, Enrico1
  • Barbieri, Letizia1
  • Cremonini, Matteo1
  • Pennestri, Matteo2
  • Nocentini, Alessio3
  • Supuran, Claudiu T3
  • Banci, Lucia1
  • 1 CERM - Magnetic Resonance Center, Università degli Studi di Firenze, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy. , (Italy)
  • 2 Pharmaceutical Business Unit, Bruker UK Limited, Banner Lane, Coventry CV4 9GH, United Kingdom. , (United Kingdom)
  • 3 Dipartimento Neurofarba, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Italy. , (Italy)
Type
Published Article
Journal
Acta crystallographica. Section D, Structural biology
Publication Date
Oct 01, 2021
Volume
77
Issue
Pt 10
Pages
1270–1281
Identifiers
DOI: 10.1107/S2059798321009037
PMID: 34605430
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

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. open access.

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