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Graphical abstract, Fluorination of metabolic hotspots in a molecule is a common medicinal chemistry strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.

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.

A series of reversible inhibitors of lysine specific demethylase 1 (LSD1) with a 5-hydroxypyrazole scaffold have been developed from compound 7, which was identified from the patent literature. Surface plasmon resonance (SPR) and biochemical analysis showed it to be a reversible LSD1 inhibitor with an IC50 value of 0.23 µM. Optimisation of this compound by rational design afforded compounds with Kd values of

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.

Background: Constitutive activation of RET kinase activity following mutation or rearrangement can lead to the development of cancers such as medullary thyroid carcinoma and lung adenocarcinoma. The currently approved therapeutics for these diseases are significantly compromised due to dose-limiting toxicities associated with off-target activity vs KDR (VEGFR2) and lack of potency vs anticipated secondary resistance (e.g., gatekeeper) mutations. Consequently there is considerable interest in the development of highly selective inhibitors targeting diverse RET alterations including the putative resistance mutation, V804M. Methods: We have established a robust screening cascade complemented by structure-enabled drug design and effective medicinal chemistry. Biochemical activity vs RET, KDR, and RETV804M protein was assessed using a HTRF assay. Cellular activity was quantified in BaF3 cells dependent on activity of RET, KDR, or RETV804M for proliferation. Tumor growth inhibition and supporting PK/PD studies were carried out in a number of disease-relevant models including a KIF5B-RET lung cancer patient-derived xenograft (PDX) model, a medullary thyroid carcinoma (MZ-CRC-1) xenograft model, and a lung cancer control (Calu-6) xenograft model. Results: Using this optimized, robust platform, we have identified a number of selective compounds offering a range of interesting biochemical and cellular profiles, targeting either, or both, RET and the gatekeeper mutant, RETV804M. We believe certain examples of these compounds offer the first cell-active RETV804M-selective derivatives. More importantly perhaps, we have also delivered a highly selective preclinical candidate compound demonstrating potency vs both RET fusion and RETV804M. This compound is well tolerated in vivo after oral dosing at up to 80mg/kg bid and, in a KIF5B-RET lung cancer PDX model, demonstrates efficacy at much lower doses: 50% tumor regression at 20mg/kg bid and 92% tumor growth inhibition at 10mg/kg bid. Importantly, this agent shows no efficacy in the (non-RET driven) Calu-6 xenograft model, demonstrating selective inhibition of the RET kinase domain. Conclusions: We believe that the identification of well-tolerated, selective RET inhibitors with potent activity against diverse RET alterations (including the anticipated resistance mutation, V804M) offers a clear therapeutic advantage over the present clinically approved compounds. Our most advanced compound fulfills all of these challenging criteria and has now entered preclinical development. Citation Format: Mandy Watson, Rebecca Newton, Ben Acton, Helen Small, Habiba Begum, Samantha Hitchin, Paul Kelly, Donald Ogilvie, Ian Waddell, Allan Jordan. Delivery of a potent, selective, and efficacious RET inhibitor for the treatment of RET-driven lung adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A123.

Background: The aim of this CRUK-MI Drug Discovery project is to deliver a RET-selective inhibitor for the treatment of cancers with RET activating mutations, which include 1-2% of lung adenocarcinomas and medullary thyroid cancers (MTC). Recent data supports the hypothesis that the efficacy of vandetanib and cabozantinib, clinically approved multi-kinase inhibitors, is limited by toxicities associated with potent activity against KDR. Therefore, a RET-selective inhibitor would represent a best-in-class agent for the treatment of these cancers. Methods: We have established a robust screening cascade to develop a potent, selective RET inhibitor and developed several in vivo models to evaluate compound PKPD and antitumor efficacy. Tumor growth inhibition and PKPD studies were carried out in BaF3 mouse allograft models overexpressing KIF5B-RET or RETV804M and other disease relevant models, including an MTC xenograft (MZ-CRC-1), a KIF5B-RET lung cancer patient derived xenograft (PDX) model (CTG-0838, Champions Oncology) and a lung cancer control xenograft (Calu-6). Results: Two orally bioavailable compounds displaying nanomolar RET potency and >10 fold selectivity over KDR in cellular assays were selected from the lead series and further evaluated in our in vivo PD and efficacy models. Both compounds demonstrated efficacy in the BaF3 KIF5B-RET driven model (71% and 103% tumor growth inhibition (TGI), respectively), accompanied by reduced levels of pRET in the tumor tissue. Following further lead optimisation; a compound displaying an improved DMPK profile and additional nanomolar potency versus the gatekeeper mutation (RETV804M) was identified and accelerated through our DMPK/in vivo cascade. We consider this additional activity versus RETV804M beneficial since mutations at the gatekeeper residue in other tyrosine kinases (e.g. EGFR) have been shown to mediate acquired drug resistance in the clinic. This compound demonstrated significant TGI of 58% and 82% respectively in the BaF3 KIF5B-RET and BaF3 RETV804M allograft models. Moreover, tumor growth in the lung cancer PDX model was strongly inhibited (95% TGI) and tumor regression induced in the MTC xenograft model (109% TGI). As expected, this potent and selective RET inhibitor was not active in the Calu-6 model, which is sensitive to KDR inhibition, whereas vandetanib, a potent KDR inhibitor, significantly inhibited tumor growth (84% TGI). Additional in vitro and in vivo DMPK analyses further support the nomination of this compound as a preclinical candidate. Conclusions: The identification of selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of the currently available clinical compounds. We have made considerable progress towards this goal and show here the compelling data supporting our nomination of a preclinical development compound. Citation Format: Mandy Watson, Helen Small, Ben Acton, Habiba Begum, Samantha Hitchin, Allan Jordan, Paul Kelly, Rebecca Newton, Ian Waddell, Gina Paris, Donald Ogilvie. A potent and selective RET inhibitor with efficacy in RET-driven mouse models of medullary thyroid carcinoma and lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2092. doi:10.1158/1538-7445.AM2017-2092

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.

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