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Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
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Target Identification Using Cell Permeable and Cleavable Chloroalkane Derivatized Small Molecules.

Jacqui L Mendez-Johnson1, Danette L Daniels1, Marjeta Urh1

  • 1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711, USA.

Methods in Molecular Biology (Clifton, N.J.)
|August 16, 2017
PubMed
Summary

Identifying protein targets for small molecules is challenging. This study introduces a chloroalkane (CA) capture handle for efficient isolation and validation of small molecule-interacting proteins.

Keywords:
Chemical cleavageChemoproteomicsChloroalkaneDerivatized small moleculeHaloTagMass spectrometryPalladium catalystPhenotypic screeningSmall moleculeTarget identification

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Area of Science:

  • Chemical Biology
  • Proteomics
  • Drug Discovery

Background:

  • Phenotypic screening identifies bioactive small molecules, but their protein targets are often unknown.
  • Identifying interacting proteins is crucial for understanding small molecule mechanisms of action.
  • Current methods for target identification can be complex and inefficient.

Purpose of the Study:

  • To develop a novel chemical approach for efficient isolation and identification of small molecule-interacting proteins.
  • To enable validation of small molecule potency and cell permeability before target capture.
  • To facilitate the capture of weak or low-abundance protein interactors.

Main Methods:

  • Small molecules are derivatized with a chloroalkane (CA) moiety capture handle.
  • Derivatized small molecules retain cell permeability and potency, allowing phenotypic validation.
  • CA-derivatized molecules covalently bind to HaloTag-coated magnetic beads for protein capture.
  • Elution of bound proteins is achieved via chemical cleavage, competitive elution, or SDS.

Main Results:

  • The CA moiety capture handle effectively isolates small molecule-interacting proteins.
  • Derivatization does not significantly affect small molecule cell permeability or potency.
  • The method allows for target identification in live cells.
  • Robust capture of weak or low-abundance interactors is achieved.

Conclusions:

  • This chloroalkane-based approach provides a robust and efficient method for small molecule target identification.
  • The technique facilitates functional insight into small molecule activity by revealing interacting proteins.
  • This method enhances the drug discovery process by enabling rapid target validation.