title: A high-throughput screening-compatible homogeneous time-resolved fluorescence assay measuring the glycohydrolase activity of human poly(ADP-ribose) glycohydrolase.
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.
authors.affiliation: Cancer Research UK Manchester Institute Drug Discovery Unit, University of Manchester, Manchester M20 4BX, UK. Electronic address: alex.stowell@cruk.manchester.ac.uk.
authors.forename: Alexandra I J
authors.initials: AI
authors.lastname: Stowell
authors.affiliation: Cancer Research UK Manchester Institute Drug Discovery Unit, University of Manchester, Manchester M20 4BX, UK.
authors.forename: Dominic I
authors.initials: DI
authors.lastname: James
authors.affiliation: Cancer Research UK Manchester Institute Drug Discovery Unit, University of Manchester, Manchester M20 4BX, UK.
authors.forename: Ian D
authors.initials: ID
authors.lastname: Waddell
authors.affiliation: Discovery Sciences, AstraZeneca, Alderley Park, Cheshire SK10 4TG, UK.
authors.forename: Neil
authors.initials: N
authors.lastname: Bennett
authors.affiliation: Discovery Sciences, AstraZeneca, Alderley Park, Cheshire SK10 4TG, UK.
authors.forename: Caroline
authors.initials: C
authors.lastname: Truman
authors.affiliation: Discovery Sciences, AstraZeneca, Alderley Park, Cheshire SK10 4TG, UK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Hardern
authors.affiliation: Cancer Research UK Manchester Institute Drug Discovery Unit, University of Manchester, Manchester M20 4BX, UK.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Ogilvie
mesh_terms: D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D006026:Glycoside Hydrolases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase
keywords: High-throughput screening; Homogeneous time-resolved fluorescence; Poly(ADP-ribose) glycohydrolase; Poly(ADP-ribose) polymerase; Small molecule inhibitors