• Androgen receptor and poly(ADP-ribose) glycohydrolase inhibition increases efficiency of androgen ablation in prostate cancer cells

      Zhang, M; Lai, Y; Vasquez, JL; James, Dominic I; Smith, Kate M; Waddell, Ian D; Ogilvie, Donald J; Liu, Y; Agoulnik, IU; Biochemistry Ph.D. Program, Florida International University, Miami, FL, (2020)
      There is mounting evidence of androgen receptor signaling inducing genome instability and changing DNA repair capacity in prostate cancer cells. Expression of genes associated with base excision repair (BER) is increased with prostate cancer progression and correlates with poor prognosis. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are key enzymes in BER that elongate and degrade PAR polymers on target proteins. While PARP inhibitors have been tested in clinical trials and are a promising therapy for prostate cancer patients with TMPRSS2-ERG fusions and mutations in DNA repair genes, PARG inhibitors have not been evaluated. We show that PARG is a direct androgen receptor (AR) target gene. AR is recruited to the PARG locus and induces PARG expression. Androgen ablation combined with PARG inhibition synergistically reduces BER capacity in independently derived LNCaP and LAPC4 prostate cancer cell lines. A combination of PARG inhibition with androgen ablation or with the DNA damaging drug, temozolomide, significantly reduces cellular proliferation and increases DNA damage. PARG inhibition alters AR transcriptional output without changing AR protein levels. Thus, AR and PARG are engaged in reciprocal regulation suggesting that the success of androgen ablation therapy can be enhanced by PARG inhibition in prostate cancer patients.
    • Anilinoquinazoline inhibitors of the RET kinase domain-elaboration of the 7-position.

      Jordan, Allan M; Begum, Habiba; Fairweather, Emma E; Fritzl, Samantha J R; Goldberg, Kristin M; Hopkins, Gemma V; Hamilton, Niall M; Lyons, Amanda J; March, H Nikki; Newton, Rebecca; et al. (2016-06-01)
      We have previously reported a series of anilinoquinazoline derivatives as potent and selective biochemical inhibitors of the RET kinase domain. However, these derivatives displayed diminished cellular potency. Herein we describe further optimisation of the series through modification of their physicochemical properties, delivering improvements in cell potency. However, whilst cellular selectivity against key targets could be maintained, combining cell potency and acceptable pharmacokinetics proved challenging.
    • An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.

      James, Dominic I; Durant, S; Eckersley, K; Fairweather, Emma E; Griffiths, Louise A; Hamilton, Nicola S; Kelly, Paul; O'Connor, M; Shea, K; Waddell, Ian D; et al. (2016)
      After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.
    • Cell-active small molecule inhibitors of the DNA-damage repair enzyme poly(ADP-ribose) glycohydrolase (PARG): discovery and optimization of orally bioavailable quinazolinedione sulphonamides.

      Waszkowycz, Bohdan; Smith, Kate M; McGonagle, Alison E; Jordan, Allan M; Acton, Ben; Fairweather, Emma E; Griffiths, Louise A; Hamilton, Niall M; Hamilton, Nicola S; Hitchin, James R; et al. (2018)
      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.
    • Development of a screening cascade to identify selective small molecule inhibitors of DNMT1

      Stowell, Alexandra I J; Thomson, Graeme J; Cockerill, Mark J; Burt, Charlotte; Fairweather, Emma E; Waddell, Ian D; Raoof, Ali; Jordan, Allan M; Ogilvie, Donald J; Pappalardi, Melissa; et al. (2018)
    • The discovery of 2-substituted phenol quinazolines as potent RET kinase inhibitors with improved KDR selectivity.

      Newton, Rebecca; Bowler, K; Burns, E; Chapman, Philip J; Fairweather, Emma E; Fritzl, Samantha J R; Goldberg, Kristin M; Hamilton, Niall M; Holt, Sarah V; Hopkins, Gemma V; et al. (2016-04-13)
      Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor using vandetanib 1 as the starting point for structure-based drug design. Phenolic anilinoquinazolines exemplified by 6 showed improved affinities towards RET but, unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte stability, but could also impart a significant gain in selectivity. This culminated in the identification of 36; a potent RET inhibitor with much improved selectivity against KDR.
    • Discovery, validation and targeting of novel synthetic lethal interactions in academic drug discovery.

      March, H Nikki; Chapman, Philip J; Blaikley, Elizabeth; Eberlein, Cath; Cockerill, Mark J; Hitchin, Samantha; Waddell, Ian D; Ogilvie, Donald J; Cancer Research UK Manchester Institute, Manchester, United Kingdom. (2017)
    • A fluorescence-based assay for the apurinic/apyrimidinic-site cleavage activity of human tyrosyl-DNA phosphodiesterase 1.

      Thomson, Graeme J; Hamilton, Nicola S; Hopkins, Gemma V; Waddell, Ian D; Watson, Amanda J; Ogilvie, Donald J; Cancer Research UK Drug Discovery Unit, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK. Electronic address: gthomson@picr.man.ac.uk. (2013-05-18)
      Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of phosphodiester bonds between the DNA 3'-phosphate and tyrosine residues and plays a major role in the repair of stalled topoisomerase I-DNA covalent complexes. Given this role, Tdp1 is of interest as a potential target for anticancer therapy. Inhibiting Tdp1 in combination with clinically used Top1 inhibitors may potentiate the effects of the latter and help to overcome some of the chemoresistance issues currently observed. In addition, Tdp1 can function during DNA repair to remove a variety of other 3' adducts from DNA such as phosphoglycolates and abasic or apurinic/apyrimidinic (AP) sites. Here we describe a new mix-and-read homogeneous fluorogenic assay for the measurement of the AP-site cleavage activity of Tdp1 that is compatible with high-throughput screening. The application of such an assay will open up further avenues for the discovery of novel Tdp1 inhibitors.
    • Fluoromethylcyclopropylamine derivatives as potential in vivo toxicophores - a cautionary disclosure

      Acton, Ben; Small, Helen F; Smith, Kate M; McGonagle, Alison E; Stowell, Alexandra I J; James, Dominic I; Hamilton, Niall M; Hamilton, Nicola S; Hitchin, James R; Hutton, Colin P; et al. (2019)
      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.
    • Generation of assays and antibodies to facilitate the study of human 5'-tyrosyl DNA phosphodiesterase.

      Thomson, Graeme J; Watson, Amanda J; Caldecott, K; Denneny, Olive; Depledge, Paul; Hamilton, Nicola S; Hopkins, Gemma V; Jordan, Allan M; Morrow, Christopher J; Raoof, Ali; et al. (2013-02-12)
      Topoisomerases regulate DNA topology by the transient cleavage and re-ligation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5' DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5'-tyrosyl DNA phosphodiesterase which repairs this topoisomerase-mediated DNA damage, thus playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in an increased susceptibility and sensitivity to Topo II-induced DNA double strand breaks, thereby revealing Tdp2 as a potentially attractive anti-cancer target. No drug-like inhibitors of Tdp2 have been identified to date and assays suitable for high throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogenous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. Additionally, a new and specific Tdp2 antibody is described. Together these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.
    • An HTS-compatible HTRF assay measuring the glycohydrolase activity of human PARG.

      Stowell, Alexandra I J; James, Dominic I; Waddell, Ian D; Bennett, N; Truman, C; Hardern, I; Ogilvie, Donald J; Cancer Research UK Manchester Institute Drug Discovery Unit, University of Manchester, Manchester, (2016-03-29)
      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.
    • Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation

      Joshi SR; Kitagawa A, Jacob C; Hashimoto R; Dhagia V; Ramesh A; Zheng C; Zhang H; Jordan, Allan M; Waddell, Ian D; Leopold J; et al. (2020)
    • Identification of selective inhibitors of RET and comparison with current clinical candidates through development and validation of a robust screening cascade.

      Watson, Amanda J; Hopkins, Gemma V; Hitchin, Samantha; Begum, Habiba; Jones, Stuart; Jordan, Allan M; Holt, Sarah V; March, H Nikki; Newton, Rebecca; Small, Helen F; et al. (2016)
      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.
    • In vitro and in vivo induction of fetal hemoglobin with a reversible and selective DNMT1 inhibitor

      Gilmartin, AG; Groy, A; Gore, ER; Atkins, C; Long, ER, 3rd; Montoute, MN; Wu, Z; Halsey, W; McNulty, DE; Ennulat, D; et al. (2020)
      Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2'-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo. Keywords: DNA methyltransferase; Fetal Hemoglobin; Hemoglobinopathies; Molecular Pharmacology; Sickle Cell Disease.
    • IncucyteDRC: An R package for the dose response analysis of live cell imaging data.

      Chapman, Philip J; James, Dominic I; Watson, Amanda J; Hopkins, Gemma V; Waddell, Ian D; Ogilvie, Donald J; Drug Discovery Unit, Cancer Research UK Manchester Institute, Manchester, UK (2016)
      We present IncucyteDRC, an R package for the analysis of data from live cell imaging cell proliferation experiments carried out on the Essen Biosciences IncuCyte ZOOM instrument. The package provides a simple workflow for summarising data into a form that can be used to calculate dose response curves and EC50 values for small molecule inhibitors. Data from different cell lines, or cell lines grown under different conditions, can be normalised as to their doubling time. A simple graphical web interface, implemented using shiny, is provided for the benefit of non-R users. The software is potentially useful to any research group studying the impact of small molecule inhibitors on cell proliferation using the IncuCyte ZOOM.
    • Inhibition of G6PD activity attenuates right ventricle pressure and hypertrophy elicited by VEGFR inhibitor + hypoxia

      Kitagawa, A.; Jacob, C.; Jordan, Allan M; Waddell, Ian D; McMurtry, I. F.; Gupte, S. A.; Pharmacology, New York Medical College, United States (2021)
      Pulmonary hypertension (PH) is a disease of hyperplasia of pulmonary vascular cells. The pentose phosphate pathway (PPP) - a fundamental glucose metabolism pathway - is vital for cell growth. Because treatment for PH is inadequate, our goal was to determine whether inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, prevents maladaptive gene expression that promotes smooth muscle cell (SMC) growth, reduces pulmonary artery remodeling, and normalizes hemodynamics in experimental models of PH. PH was induced in mice by exposure to 10% oxygen (Hx) or weekly injection of vascular endothelial growth factor receptor blocker (Sugen5416 (SU); 20 mg.kg-1) during exposure to hypoxia (Hx+SU). A novel G6PD inhibitor (PDD4091;1.5 mg kg-1) was injected daily during exposure to Hx. We measured right ventricle (RV) pressure and left ventricle (LV) pressure-volume relationships, and gene expression in lungs of normoxic, Hx, and Hx+SU, and G6PD inhibitor-treated, mice. RV systolic and end-diastolic pressures were higher in Hx and Hx+SU than normoxic-control mice. Hx and Hx+SU decreased expression of epigenetic modifiers (writers and erasers), increased hypomethylation of the DNA, and induced aberrant gene expression in lungs. G6PD inhibition decreased maladaptive expression of genes and SMC growth, reduced pulmonary vascular remodeling, decreased RV pressures, and increased cardiac index, compared to untreated PH groups. Pharmacologic inhibition of G6PD activity, by normalizing activity of epigenetic modifiers and DNA methylation, efficaciously reduces RV pressure overload in Hx and Hx+SU mice, pre-clinical models of PH, appears to be a safe pharmacotherapeutic strategy. Significance Statement The results of this study demonstrated inhibition of a metabolic enzyme efficaciously reduces pulmonary hypertension. For first time, we show that a novel inhibitor of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the fundamental pentose phosphate pathway, modulates DNA methylation and alleviates pulmonary artery remodeling and dilates pulmonary artery to reduce pulmonary hypertension.
    • Inhibition of SMARCA2: a novel target for SMARCA4-deficient lung adenocarcinoma

      Chapman, Philip J; March, H Nikki; Thomson, Graeme J; Fairweather, Emma E; Fritzl, Samantha J R; Hitchin, James R; Hamilton, Nicola S; Jordan, Allan M; Waddell, Ian D; Ogilvie, Donald J; et al. (2016)
    • Mechanism of APTX nicked DNA sensing and pleiotropic inactivation in neurodegenerative disease.

      Tumbale, P; Schellenberg, M; Mueller, G; Fairweather, Emma E; Watson, Mandy; Little, J; Krahn, J; Waddell, Ian D; London, R; Williams, R; et al. (2018-07-13)
      The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks. The APTX RNA-DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder ataxia with oculomotor ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. Here, we report X-ray structures of APTX engaging nicked RNA-DNA substrates that provide direct evidence for a wedge-pivot-cut strategy for 5'-AMP resolution shared with the alternate 5'-AMP processing enzymes POLβ and FEN1. Our results uncover a DNA-induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. Further, based on comprehensive biochemical, X-ray and solution NMR results, we define a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and a dominant AOA1 variant unexpectedly allosterically modulates APTX active site conformations.
    • Novel steroid inhibitors of glucose 6-phosphate dehydrogenase.

      Hamilton, Niall M; Dawson, Martin J; Fairweather, Emma E; Hamilton, Nicola S; Hitchin, James R; James, Dominic I; Jones, Stuart D; Jordan, Allan M; Lyons, Amanda J; Small, Helen F; et al. (2012-05-10)
      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.
    • PARG inhibition: development of novel compounds and a biomarker strategy to determine cell line sensitivity in breast cancer

      Waddell, Ian D; James, Dominic I; Smith, Kate M; Holt, Sarah V; Acton, Ben; Fairweather, Emma E; Hamilton, Niall M; Hamilton, Nicola S; Hitchin, James R; Huttom, Colin; et al. (2015)