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The recently discovered enzyme tyrosyl-DNA phosphodiesterase 2 (TDP2) has been implicated in the topoisomerase-mediated repair of DNA damage. In the clinical setting, it has been hypothesized that TDP2 may mediate drug resistance to topoisomerase II (topo II) inhibition by etoposide. Therefore, selective pharmacological inhibition of TDP2 is proposed as a novel approach to overcome intrinsic or acquired resistance to topo II-targeted drug therapy. Following a high-throughput screening (HTS) campaign, toxoflavins and deazaflavins were identified as the first reported sub-micromolar and selective inhibitors of this enzyme. Toxoflavin derivatives appeared to exhibit a clear structure-activity relationship (SAR) for TDP2 enzymatic inhibition. However, we observed a key redox liability of this series, and this, alongside early in vitro drug metabolism and pharmacokinetics (DMPK) issues, precluded further exploration. The deazaflavins were developed from a singleton HTS hit. This series showed distinct SAR and did not display redox activity; however low cell permeability proved to be a challenge.

Novel derivatives of the steroid DHEA 1, a known uncompetitive inhibitor of G6PD, were designed, synthesized, and tested for their ability to inhibit this dehydrogenase enzyme. Several compounds with approximately 10-fold improved potency in an enzyme assay were identified, and this improved activity translated to efficacy in a cellular assay. The SAR for steroid inhibition of G6PD has been substantially developed; the 3β-alcohol can be replaced with 3β-H-bond donors such as sulfamide, sulfonamide, urea, and carbamate. Improved potency was achieved by replacing the androstane nucleus with a pregnane nucleus, provided a ketone at C-20 is present. For pregnan-20-ones incorporation of a 21-hydroxyl group is often beneficial. The novel compounds generally have good physicochemical properties and satisfactory in vitro DMPK parameters. These derivatives may be useful for examining the role of G6PD inhibition in cells and will assist the future design of more potent steroid inhibitors with potential therapeutic utility.

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.

RET (REarranged during Transfection) is a receptor tyrosine kinase, which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activation of RET is a mechanism of oncogenesis in medullary thyroid carcinomas where both germline and sporadic activating somatic mutations are prevalent. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been opportunistically identified through selectivity profiling of compounds initially designed to target other tyrosine kinases. Vandetanib and cabozantinib, both multi-kinase inhibitors with RET activity, are approved for use in medullary thyroid carcinoma, but additional pharmacological activities, most notably inhibition of vascular endothelial growth factor - VEGFR2 (KDR), lead to dose-limiting toxicity. The recent identification of RET fusions present in ~1% of lung adenocarcinoma patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. In an earlier publication [Newton et al, 2016; 1] we reported the discovery of a series of 2-substituted phenol quinazolines as potent and selective RET kinase inhibitors. Here we describe the development of the robust screening cascade which allowed the identification and advancement of this chemical series. Furthermore we have profiled a panel of RET-active clinical compounds both to validate the cascade and to confirm that none display a RET-selective target profile.

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.

Background:RET is a receptor tyrosine kinase (RTK) and forms part of a macromolecular receptor complex containing dimerised RET receptor, two co-receptors and a bound ligand. Signalling networks downstream of RET play an important role in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activating mutations in RET (e.g. C634W and M918T) are known drivers in medullary thyroid carcinomas (MTC). More recently, oncogenic RET fusions (e.g. CCDC6-RET and KIF5B-RET) have been identified in 1-2% of lung adenocarcinoma patients. We are currently developing novel, selective inhibitors of RET, and at the same time, investigating a number of biomarker approaches for the stratification of RET fusion-positive lung cancer patients who might benefit from such therapy. Methods: We have undertaken collaborative studies using established techniques including immunohistochemistry (IHC) and FISH (DNA break apart and RNA). In addition, we have investigated hybrid capture DNA sequencing of both biopsy material and circulating tumour DNA in the blood. Here we, compare and contrast the benefits of each biomarker assay evaluated and consider how these approaches could be translated for use in Phase I clinical trials at The Christie. Conclusion: Our data supports the successful implementation of predictive biomarkers to identify patients who might benefit from treatment with selective RET inhibitors. Acknowledgements:This work was funded by Cancer Research UK (Grant numbers C480/A1141 and C5759/A17098). Citation Format: Mandy Watson, Helen Small, Phil Chapman, Gemma Hopkins, Habiba Begum, Ian D. Waddell, Garry Ashton, Caron Abbey, Jade Harris, Mahmood Ayub, Sumitra Mohan, Dominic Rothwell, Ged Brady, Caroline Dive, Allan Jordan, Donald Ogilvie. RET inhibition: Development of novel compounds and a personalized medicine strategy in lung adenocarcinoma. [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 A176.

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

RET (REarranged during Transfection) is a receptor tyrosine kinase (TK), which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activating mutations in RET (C634W and M918T) have been identified in both familial and sporadic forms of medullary thyroid carcinoma (MTC) and correlate with aggressive disease progression, validating RET as a classical oncogene. Furthermore the recent identification of RET fusions (CCDC6-RET and KIF5B-RET) present in ∼1% of lung adenocarcinoma (LAD) patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been identified opportunistically through selectivity profiling of compounds initially designed to target other TKs. Such “secondary RET inhibitors” include the clinical agents Vandetanib and Cabozantinib, both approved for use in MTC, but additional pharmacological activities (most notably inhibition of KDR) lead to dose-limiting toxicity. Using a robust screening cascade developed in house, we have measured RET and KDR inhibitory activity in vitro and in relevant cell line models to assess compound potency and selectivity. Anti-proliferative activity and off-target toxicity of these agents have also been measured. Although these competitor compounds displayed reasonable RET potency in cellular assays and this translated into anti-proliferative effects in our MTC and LAD disease models, as expected none met our target candidate criteria, clearly highlighting the need for therapeutic agents with improved selectivity. Guided by structure-based drug design, we have identified and optimised a novel series of potent and selective inhibitors of the RET kinase domain. These agents met our stringent criteria for enzyme and cell selectivity and, whilst potent in a RET-driven cell line, display little overt toxicity in a matched non-RET driven cell line. Herein, we describe the chemical optimisation of these agents and, using structural information, rationalise their improved selectivity. Citation Format: Roger J. Butlin, Rebecca Newton, Mandy Watson, Gemma Hopkins, Ben Acton, Kate Bowler, Samantha Fritzl, Kristin Goldberg, Niall Hamilton, Sarah Holt, Stuart Jones, Allan Jordan, Nikki March, Daniel Mould, Helen Small, Alexandra Stowell, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. The identification and structure-guided optimisation of potent and selective inhibitors of oncogenes in medullary thyroid carcinoma and lung adenocarcinoma. [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 778. doi:10.1158/1538-7445.AM2015-778