DNA damage repair enzymes are promising targets in the development of new therapeutic agents for a wide range of cancers and potentially other diseases. The enzyme poly(ADP-ribose) glycohydrolase (PARG) plays a pivotal role in the regulation of DNA repair mechanisms; however, the lack of potent drug-like inhibitors for use in cellular and in vivo models has limited the investigation of its potential as a novel therapeutic target. Using the crystal structure of human PARG in complex with the weakly active and cytotoxic anthraquinone 8a, novel quinazolinedione sulfonamides PARG inhibitors have been identified by means of structure-based virtual screening and library design. 1-Oxetan-3-ylmethyl derivatives 33d and 35d were selected for preliminary investigations in vivo. X-ray crystal structures help rationalize the observed structure−activity relationships of these novel inhibitors.
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 (PARPs). PARG depletion, using RNAi, results in several effects such as PAR chain persistence, progression of single- to double-strand DNA lesions and NAD+ depletion. Given these findings, inhibition of PARG with a small molecule agent offers a potential opportunity to interfere with DNA repair mechanisms and induce cell death in those cells with increased susceptibility to DNA damage, such as tumour cells. Previous efforts to develop small molecule inhibitors of PARG activity have generally been hampered by poor physicochemical properties, off-target pharmacology and a lack of cell permeability, leading some to suggest that PARG may be undruggable. In contrast, we have now developed a series of first-in-class PARG inhibitors which display drug-like properties and attractive pharmacokinetic parameters. These compounds have proved to be useful biological tool compounds. Moreover, displaying selective activity in both biochemical and, more importantly, cellular assays of PARG function, these derivatives have allowed an exploration of the phenotypes resulting from reversible, pharmacological PARG inhibition in both in vitro cell panels and in vivo models. Furthermore, our initial bioinformatic analysis suggests that deficiency of a known tumour suppressor confers sensitivity to PARG inhibition, suggesting patient populations that will potentially benefit from PARGi therapies. Citation Format: Bohdan Waszkowycz, Dominic James, Ben Acton, Emma Fairweather, Sam Fritzl, Niall Hamilton, Nicola Hamilton, Sarah Holt, James Hitchen, Colin Hutton, Stuart Jones, Allan Jordan, Alison McGonagle, Daniel Mould, Helen Small, Kate Smith, Alexandra Stowell, Ian D. Waddell, Donald Ogilvie. First-in-class inhibitors of the putatively undruggable DNA repair target Poly(ADP-ribose) glycohydrolase (PARG). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C39.
In recent years, many proteins involved in DNA repair, such as ATR, ATM and PARP, have received considerable attention as potential points of therapeutic intervention in cancer. Indeed, these efforts have recently delivered several agents into clinical evaluation or FDA regulatory approval. However, the DNA repair protein poly(ADP ribose) glycohydrolase (PARG), which plays an equally critical role in DNA single stand break repair, to successful drug discovery efforts. Through our innovative collaboration with AstraZeneca, we have discovered a novel PARG-binding pharmacophore and have employed this information to discover drug-like chemotypes, facilitating the development of potent and selective inhibitors. This poster will describe our emerging results in this area, where a novel benzimidazolone sulphonamide scaffold has been shown potently to inhibit PARG in both biochemical and cellular assays with potencies of 40 nM and 60 nM respectively. Moreover, these agents display pharmacology consistent with the anticipated mode of action, appropriate drug-like properties and are selective against PARP1 and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of this scaffold will be described, alongside the recent biological results obtained. Ultimately, this work has helped deliver tool compounds which may help to elucidate the true pharmacology and roles of PARG in cancer and other disease settings. Citation Format: Allan Jordan, Ben Acton, Nicola Hamilton, James Hitchin, Colin Hutton, Dominic James, Cliff Jones, Stuart Jones, Alison McGonagle, Helen Small, Kate Smith, Alex Stowell, Julie Tucker, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. Benzimidazolone sulphonamides - potent, selective and drug-like inhibitors of poly(ADP Ribose) Glycohydrolase (PARG). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3715.
The macrodomain protein poly(ADP ribose) glycohydrolase (PARG) has been shown to be a critical component in the repair of single stand DNA breaks and counteracts the function of the ARTD family of poly(ADP ribose) polymerases, commonly known as the PARPs. As PARG exists as a single protein, it presents an attractive target for therapeutic intervention in cancer cells with enhanced dependence upon DNA repair. Inhibitors of this enzyme have proved difficult to discover and develop. Moreover, intact cell-active tool compounds which have the propensity to be used as robust chemical probes to understand PARG pharmacology, are absent from the literature. This poster will describe our work in this emerging area, optimising a series of drug-like quinazolinedione derivatives to deliver molecules with the correct physicochemical and biochemical properties to function as in vitro cell probe compounds. These unprecedented agents display potent on-target biochemical (5 nM) and cell (10 nM) activity with a significant window to acute 3-day cytotoxicity. Moreover, these agents are selective against PARP family members and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of the scaffold will be described, alongside the outline pharmacology demonstrating on-target, selective inhibition of PARG in cells. Such tool compounds will be of value in revealing the detailed mechanisms of action of PARG in DNA repair and other PAR chain-mediated cellular processes, with the ultimate goal of delivering novel and clinically relevant therapeutic agents. Citation Format: Kate Smith, Ben Acton, Dominic James, Cliff Jones, Stuart Jones, Allan Jordan, Nicola Hamilton, Alison McGonagle, Daniel Mould, Helen Small, Alex Stowell, Julie Tucker, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. Optimisation of quinazolinedione sulphonamides as novel inhibitors of poly(ADP Ribose) glycohydrolase (PARG). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3714.
Background: DNA single strand breaks (SSBs) are the most common type of damage occurring in cells. Poly (ADP ribose) polymerase (PARP) binds to SSBs and auto-ribosylates itself using NAD+ as a substrate. PARG is the only enzyme known to efficiently catalyse the hydrolysis of O-glycosidic linkages of ADP-ribose polymers and exists (unlike PARP) as a single gene. We have developed novel inhibitors of PARG and here we describe our efforts to understand their sensitivity against a range of cell lines Methods: A robust and detailed screening cascade for small molecule inhibition of PARG has been developed. Active compounds are tested in cells for PAR chain persistence and for cytotoxicity using a 3-day HeLa assay. In addition, suitable compounds were then evaluated for their physico-chemical properties and their in vivo PK profiles determined. Compounds with the desired PK properties are subsequently profiled in tumour bearing mice to determine their pharmacodynamic effect before progressing to efficacy studies. Results: We have developed nM PARG inhibitors that are highly selective against ARH3 and PARP1and these derivatives show potent activity in cells. We have designed a breast cancer cell panel against which our compounds have been tested. Our initial bioinformatics analysis suggests that deficiency of a known tumour suppressor confers sensitivity to PARG inhibition. Conclusions: We have developed two drug-like series of PARG inhibitors that block the breakdown of PAR chains in cells after exogenous DNA damage by methylating agents. These tool compounds are potent, selective and have pharmacokinetic and pharmacodynamics properties that have allowed us to explore their anti-tumour potential. The discovery of a tumour suppressor profile indicating cell line sensitivity will aid the about identification of patient populations that will potentially benefit from PARGi therapies. Citation Format: Ian D. Waddell, Dominic James, Kate Smith, Sarah Holt, Ben Acton, Emma Fairweather, Niall Hamilton, Nicola Hamilton, James Hitchen, Colin Huttom, Allan Jordan, Alison McGonagle, Helen Small, Alex Stowell, Bohdan Waszkowycz, Donald Ogilvie. PARG inhibition: development of novel compounds and a biomarker strategy to determine cell line sensitivity in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3656. doi:10.1158/1538-7445.AM2015-3656
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