Structures of the human poly (ADP-ribose) glycohydrolase catalytic domain confirm catalytic mechanism and explain inhibition by ADP-HPD derivatives

PLoS One. 2012;7(12):e50889. doi: 10.1371/journal.pone.0050889. Epub 2012 Dec 10.

Abstract

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5'-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain*
  • Computational Biology
  • Glycoside Hydrolases / chemistry*
  • Humans
  • Protein Conformation
  • Structure-Activity Relationship

Substances

  • Glycoside Hydrolases
  • poly ADP-ribose glycohydrolase

Associated data

  • PDB/4A0D
  • PDB/4B1G

Grant support

This work was carried out at and financially supported by AstraZeneca. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.