Chaperone mediated detection of small molecule target binding in cells

Nat Commun. 2020 Jan 23;11(1):465. doi: 10.1038/s41467-019-14033-0.

Abstract

The ability to quantitatively measure a small molecule's interactions with its protein target(s) is crucial for both mechanistic studies of signaling pathways and in drug discovery. However, current methods to achieve this have specific requirements that can limit their application or interpretation. Here we describe a complementary target-engagement method, HIPStA (Heat Shock Protein Inhibition Protein Stability Assay), a high-throughput method to assess small molecule binding to endogenous, unmodified target protein(s) in cells. The methodology relies on the change in protein turnover when chaperones, such as HSP90, are inhibited and the stabilization effect that drug-target binding has on this change. We use HIPStA to measure drug binding to three different classes of drug targets (receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases), via quantitative fluorescence imaging. We further demonstrate its utility by pairing the method with quantitative mass spectrometry to identify previously unknown targets of a receptor tyrosine kinase inhibitor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzoquinones / pharmacology
  • Bridged Bicyclo Compounds, Heterocyclic / metabolism
  • Bridged Bicyclo Compounds, Heterocyclic / pharmacology
  • Cell Line, Tumor
  • Estrogen Receptor alpha / metabolism
  • Fluorescent Antibody Technique
  • HSP90 Heat-Shock Proteins / antagonists & inhibitors
  • HSP90 Heat-Shock Proteins / metabolism*
  • High-Throughput Screening Assays / methods*
  • Humans
  • Hydroxybutyrates / metabolism
  • Hydroxybutyrates / pharmacology
  • Lactams, Macrocyclic / pharmacology
  • Mass Spectrometry
  • Molecular Chaperones / antagonists & inhibitors
  • Molecular Chaperones / metabolism*
  • Nuclear Proteins / metabolism
  • Protein Stability / drug effects
  • Proteome / analysis
  • Proto-Oncogene Proteins c-raf / metabolism
  • Receptor, ErbB-2 / metabolism
  • Small Molecule Libraries / metabolism*

Substances

  • Benzoquinones
  • Bridged Bicyclo Compounds, Heterocyclic
  • ESR1 protein, human
  • Estrogen Receptor alpha
  • HSP90 Heat-Shock Proteins
  • Hydroxybutyrates
  • Lactams, Macrocyclic
  • Molecular Chaperones
  • Nuclear Proteins
  • Proteome
  • Small Molecule Libraries
  • TSSC3 protein
  • tanespimycin
  • N-(2-(4-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)-5H-pyrrolo(3,2-d)pyrimidin-5-yl)ethyl)-3-hydroxy-3-methylbutanamide
  • ERBB2 protein, human
  • Receptor, ErbB-2
  • Proto-Oncogene Proteins c-raf
  • Raf1 protein, human