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Updated: Jul 5, 2025

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor
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Ligand-Based Competition Binding by Real-Time 19F NMR in Human Cells.

Enrico Luchinat1,2, Letizia Barbieri2, Ben Davis3

  • 1Dipartimento di Scienze e Tecnologie Agro-Alimentari, Alma Mater Studiorum─Università di Bologna, Piazza Goidanich 60, Cesena 47521, Italy.

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|January 12, 2024
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Summary

This study introduces real-time in-cell 19F NMR for measuring drug-target binding affinities within living human cells. This method overcomes limitations of traditional NMR, aiding in the design of more effective drugs.

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Area of Science:

  • Biochemistry
  • Chemical Biology
  • Drug Discovery

Background:

  • Effective drug development necessitates understanding bioavailability and binding efficacy within the cellular environment.
  • In-cell nuclear magnetic resonance (NMR) spectroscopy enables direct investigation of ligand-target interactions in living cells.
  • Observing NMR-invisible targets or proton (1H) NMR signals in cellular backgrounds presents significant challenges.

Purpose of the Study:

  • To develop a novel real-time in-cell 19F NMR approach for measuring ligand binding affinities.
  • To overcome limitations of traditional in-cell NMR for observing ligand-target interactions.
  • To facilitate early-stage drug design by assessing compound efficacy in a native cellular context.

Main Methods:

  • Utilized real-time in-cell 19F NMR spectroscopy.
  • Employed a competition binding assay with a fluorinated reference compound.
  • Investigated the binding of various compounds to Heat Shock Protein 90 alpha (Hsp90α) in human cells.

Main Results:

  • Successfully measured ligand binding affinities directly within living human cells using 19F in-cell NMR.
  • Demonstrated the feasibility of observing fluorinated ligands binding to intracellular targets.
  • Quantified the binding interactions of a set of compounds with Hsp90α.

Conclusions:

  • The developed real-time in-cell 19F NMR method is a powerful tool for drug discovery.
  • This approach can overcome challenges associated with NMR-invisible targets and cellular background noise.
  • The methodology holds potential for application to other pharmacologically relevant targets, accelerating the design of improved therapeutics.