High-throughput screening of histone deacetylases and determination of kinetic parameters using fluorogenic assays

STAR Protoc. 2021 Feb 3;2(1):100313. doi: 10.1016/j.xpro.2021.100313. eCollection 2021 Mar 19.

Abstract

Histone deacetylases (HDACs) are ubiquitous enzymes that cleave post-translational ε-N-acyllysine modifications. The continued identification of diverse acyl modifications at lysine residues in proteins has resulted in discovery of new insight into the biological roles of these enzymes. Here, we describe a fluorogenic high-throughput screening protocol to identify deacylase activities. We describe the careful optimization of continuous, coupled enzyme assays, which provide efficient determination of kinetic parameters. These techniques can facilitate inhibitor assay design and provide fundamental understanding of HDAC biochemistry. For complete details on the use and execution of this protocol, please refer to Moreno-Yruela et al. (2018).

Keywords: Molecular/chemical probes; Protein biochemistry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods
  • Enzyme Assays / methods*
  • Fluorescent Dyes / analysis
  • Fluorescent Dyes / chemistry
  • High-Throughput Screening Assays / methods*
  • Histone Deacetylase Inhibitors / pharmacokinetics
  • Histone Deacetylase Inhibitors / pharmacology
  • Histone Deacetylases / chemistry*
  • Histone Deacetylases / metabolism
  • Kinetics
  • Lysine / chemistry
  • Protein Processing, Post-Translational / drug effects
  • Substrate Specificity

Substances

  • Fluorescent Dyes
  • Histone Deacetylase Inhibitors
  • Histone Deacetylases
  • Lysine