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Related Concept Videos

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Affinity Enrichment Chemoproteomics for Target Deconvolution and Selectivity Profiling.

Thilo Werner1, Michael Steidel1, H Christian Eberl1

  • 1Cellzome GmbH, GlaxoSmithKline, Heidelberg, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|May 5, 2021
PubMed
Summary

This study introduces a chemoproteomics method using functionalized compounds to identify drug targets and off-targets. This approach efficiently determines drug-target interactions, binding affinity, and selectivity for drug discovery.

Keywords:
Affinity enrichmentChemoproteomicsCompetition binding assaySelectivity profilingTandem mass tag

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Area of Science:

  • Chemical biology
  • Proteomics
  • Drug discovery

Background:

  • Understanding drug mechanisms requires characterizing interactions with both intended targets and off-targets.
  • Current methods for target identification can be complex and time-consuming.

Purpose of the Study:

  • To develop and validate a chemoproteomics workflow for efficient target identification and characterization.
  • To enable the determination of drug-target binding affinities and selectivity.

Main Methods:

  • Utilized functionalized analogs for affinity enrichment of target proteins from cell/tissue extracts.
  • Employed competition binding assays with varying drug concentrations.
  • Integrated TMT10 isobaric mass tags for multiplexed mass spectrometry analysis.
  • Performed rebinding experiments to assess matrix influence on binding equilibrium.

Main Results:

  • Successfully generated dose-response curves and determined half-binding concentrations.
  • Enabled efficient identification of drug targets and off-targets.
  • Determined apparent dissociation constants (Kd) and selectivity profiles.
  • Demonstrated the workflow's efficacy using the kinase inhibitor sunitinib.

Conclusions:

  • The described chemoproteomics approach provides an efficient means to characterize small molecule interactions with protein targets.
  • This method facilitates the comprehensive understanding of drug mechanisms of action and aids in selectivity profiling.
  • The workflow is valuable for advancing drug discovery and development processes.