A high-throughput screening-compatible homogeneous time-resolved fluorescence assay measuring the glycohydrolase activity of human poly(ADP-ribose) glycohydrolase

Anal Biochem. 2016 Jun 15:503:58-64. doi: 10.1016/j.ab.2016.03.016. Epub 2016 Mar 29.

Abstract

Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition.

Keywords: High-throughput screening; Homogeneous time-resolved fluorescence; Poly(ADP-ribose) glycohydrolase; Poly(ADP-ribose) polymerase; Small molecule inhibitors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Enzyme Inhibitors / pharmacology
  • Fluorescence*
  • Glycoside Hydrolases / analysis
  • Glycoside Hydrolases / antagonists & inhibitors
  • Glycoside Hydrolases / metabolism*
  • High-Throughput Screening Assays / methods*
  • Humans
  • Luminescent Measurements / methods*
  • Structure-Activity Relationship
  • Time Factors

Substances

  • Enzyme Inhibitors
  • Glycoside Hydrolases
  • poly ADP-ribose glycohydrolase