Dominic I. James

Mitochondrial Dynamics and Apoptosis: A Painful Separation

Reporting in Molecular Cell, Sheridan et al. (2008) and Breckenridge et al. (2008) show that mitochondrial fragmentation is not required to induce cell death. Meanwhile, Yamaguchi et al. show that proapoptotic Bcl-2 family members promote cytochrome c mobilization through Opa1-mediated cristae remodeling. Therefore, the connection between mitochondrial structure and apoptosis is more complex than previously imagined.

Novel Steroid Inhibitors of Glucose 6-Phosphate Dehydrogenase

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.

Loss of ATF2 Function Leads to Cranial Motoneuron Degeneration during Embryonic Mouse Development

The AP-1 family transcription factor ATF2 is essential for development and tissue maintenance in mammals. In particular, ATF2 is highly expressed and activated in the brain and previous studies using mouse knockouts have confirmed its requirement in the cerebellum as well as in vestibular sense organs. Here we present the analysis of the requirement for ATF2 in CNS development in mouse embryos, specifically in the brainstem. We discovered that neuron-specific inactivation of ATF2 leads to significant loss of motoneurons of the hypoglossal, abducens and facial nuclei. While the generation of ATF2 mutant motoneurons appears normal during early development, they undergo caspase-dependent and independent cell death during later embryonic and foetal stages. The loss of these motoneurons correlates with increased levels of stress activated MAP kinases, JNK and p38, as well as aberrant accumulation of phosphorylated neurofilament proteins, NF-H and NF-M, known substrates for these kinases. This, together with other neuropathological phenotypes, including aberrant vacuolisation and lipid accumulation, indicates that deficiency in ATF2 leads to neurodegeneration of subsets of somatic and visceral motoneurons of the brainstem. It also confirms that ATF2 has a critical role in limiting the activities of stress kinases JNK and p38 which are potent inducers of cell death in the CNS.

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

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.

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

Highlights • A synthetic lethal screen for Poly(ADP-ribose)glycohydrolase (PARG) is presented.• SiRNA and the PARG inhibitors Gallotannin and PDD00017273 are used.• PARG is synthetically lethal with BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1.• PARG inhibition induces DNA damage, stalled replication forks and homologous recombination.• The data support the validity of PARG as a target for therapy.

An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.

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.

Selective Loss of PARG Restores PARylation and Counteracts PARP Inhibitor-Mediated Synthetic Lethality

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically.

Abstract 2745: Pharmacological characterisation of cell active inhibitors of Poly(ADP-ribose) glycohydrolase (PARG)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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. Total PARG deficiency leads to cell death whilst PARG depletion, using RNAi, leads to pleiotropic effects such as PAR chain persistence, progression of single- to double-strand DNA lesions and NAD+ depletion. Whilst efforts to develop small molecule inhibitors of PARG activity have generally been hampered by poor physiochemical properties, off-target pharmacology and a lack of cell permeability, we have now developed a series of PARG inhibitors which have proved to be useful biological tool compounds. 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. Citation Format: Dominic James, Allan Jordan, Nicola Hamilton, Alison McGonagle, Kate Smith, Alexandra Stowell, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. Pharmacological characterisation of cell active inhibitors of Poly(ADP-ribose) glycohydrolase (PARG). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2745. doi:10.1158/1538-7445.AM2014-2745

Radiosensitization with an inhibitor of poly(ADP-ribose) glycohydrolase: A comparison with the PARP1/2/3 inhibitor olaparib

Graphical abstract

Studying Catabolism of Protein ADP-Ribosylation.

Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD+) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.