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

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• 作者：Alexandra I.J. Stowell^a ; ^{alex.stowell@cruk.manchester.ac.uk" class="auth_mail" title="E-mail the corresponding author} ; Dominic I. James^a ; Ian D. Waddell^a ; Neil Bennett^b ; Caroline Truman^b ; Ian M. Hardern^b ; Donald J. Ogilvie^a
• 关键词：Poly(ADP-ribose) glycohydrolase ; Poly(ADP-ribose) polymerase ; Homogeneous time-resolved fluorescence ; Small molecule inhibitors ; High-throughput screening
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：15 June 2016
• 年：2016
• 卷：503
• 期：Complete
• 页码：58-64
• 全文大小：1004 K

文摘

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.