A FRET-based assay for screening SIRT5 specific modulators

Bioorg Med Chem Lett. 2015 Apr 15;25(8):1671-1674. doi: 10.1016/j.bmcl.2015.03.018. Epub 2015 Mar 14.

Abstract

A fluorogenic assay for SIRT5 has been developed to screen their small molecule modulators based on the recent discovery that SIRT5 is a demalonylase and desuccinylase. However, this assay uses a fluorogenic peptide containing 7-amino-4-methylcoumarin (AMC), which becomes the cause of false positive hits from the screening. To overcome this, we have developed an alternative method called a FRET-based assay, which will be reliable and useful for screening SIRT5 modulators in a high-throughput format since no AMC group present in this assay.

Keywords: Deacetylation; Desuccinylation; FRET assay; Fluorogenic assay; Sirtuin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Coumarins / chemistry
  • Enzyme Assays
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / metabolism
  • Fluorescence Resonance Energy Transfer*
  • Fluorescent Dyes / chemistry*
  • Kinetics
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / metabolism
  • Sirtuins / antagonists & inhibitors
  • Sirtuins / metabolism*
  • Substrate Specificity
  • p-Dimethylaminoazobenzene / analogs & derivatives
  • p-Dimethylaminoazobenzene / chemistry

Substances

  • Coumarins
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Peptides
  • 4-(4-dimethylaminophenylazo)benzoic acid
  • p-Dimethylaminoazobenzene
  • Sirtuins
  • 7-amino-4-methylcoumarin