Identifying the proteins to which small-molecule probes and drugs bind in cells

Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4617-22. doi: 10.1073/pnas.0900191106. Epub 2009 Mar 2.

Abstract

Most small-molecule probes and drugs alter cell circuitry by interacting with 1 or more proteins. A complete understanding of the interacting proteins and their associated protein complexes, whether the compounds are discovered by cell-based phenotypic or target-based screens, is extremely rare. Such a capability is expected to be highly illuminating--providing strong clues to the mechanisms used by small-molecules to achieve their recognized actions and suggesting potential unrecognized actions. We describe a powerful method combining quantitative proteomics (SILAC) with affinity enrichment to provide unbiased, robust and comprehensive identification of the proteins that bind to small-molecule probes and drugs. The method is scalable and general, requiring little optimization across different compound classes, and has already had a transformative effect on our studies of small-molecule probes. Here, we describe in full detail the application of the method to identify targets of kinase inhibitors and immunophilin binders.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Carbazoles / metabolism
  • HeLa Cells
  • Humans
  • Immunophilins / chemistry
  • Immunophilins / metabolism
  • Indole Alkaloids / metabolism
  • Isotope Labeling
  • Ligands
  • Microspheres
  • Microtubule-Associated Proteins / metabolism
  • Molecular Probes / metabolism*
  • Pharmaceutical Preparations / metabolism*
  • Protein Kinase Inhibitors / metabolism
  • Protein Kinases / metabolism
  • Proteins / metabolism*
  • Proteomics
  • Solubility

Substances

  • Carbazoles
  • Indole Alkaloids
  • Ligands
  • Microtubule-Associated Proteins
  • Molecular Probes
  • Pharmaceutical Preparations
  • Protein Kinase Inhibitors
  • Proteins
  • staurosporine aglycone
  • Protein Kinases
  • Immunophilins