Stephen Martin Courtney

A Profiling Platform for the Characterization of Transglutaminase 2 (TG2) Inhibitors

Huntington’s disease (HD) is associated with increased expression levels and activity of tissue transglutaminase (TG2), an enzyme primarily known for its cross-linking of proteins. To validate TG2 as a therapeutic target for HD in transgenic models and for eventual clinical development, a selective and brain-permeable inhibitor is required. Here, a comprehensive profiling platform of biochemical and cellular assays is presented which has been established to evaluate the potency, cellular efficacy, subtype selectivity and the mechanism-of-action of known and novel TG2 inhibitors. Several classes of inhibitors have been characterized including: the commonly used pseudo-substrate inhibitors, cystamine and putrescine (which are generally nonspecific for TG2 and therefore not practical for drug development), the various peptidic inhibitors that target the active site cysteine residue (which display excellent selectivity but in general have poor cellular activity), and the allosteric reversible small-molecule hydrazides (which show poor selectivity and a lack of cellular activity and could not be improved despite considerable medicinal chemistry efforts). In addition, a set of inhibitors identified from a collection of pharmacologically active compounds was found to be unselective for TG2. Moreover, inhibition at the guanosine triphosphate binding site has been examined, but apart from guanine nucleotides, no such inhibitors have been identified. In addition, the promising pharmacological profile of a TG2 inhibitor is presented which is currently in lead optimization to be developed as a tool compound.

Phthalazinone Pyrazoles as Potent, Selective, and Orally Bioavailable Inhibitors of Aurora-A Kinase

The inhibition of Aurora kinases in order to arrest mitosis and subsequently inhibit tumor growth via apoptosis of proliferating cells has generated significant discussion within the literature. We report a novel class of Aurora kinase inhibitors based upon a phthalazinone pyrazole scaffold. The development of the phthalazinone template resulted in a potent Aurora-A selective series of compounds (typically >1000-fold selectivity over Aurora-B) that display good pharmacological profiles with significantly improved oral bioavailability compared to the well studied Aurora inhibitor VX-680.

Discovery and Structure–Activity Relationship of Potent and Selective Covalent Inhibitors of Transglutaminase 2 for Huntington’s Disease

Tissue transglutaminase 2 (TG2) is a multifunctional protein primarily known for its calcium-dependent enzymatic protein cross-linking activity via isopeptide bond formation between glutamine and lysine residues. TG2 overexpression and activity have been found to be associated with Huntingtons disease (HD); specifically, TG2 is up-regulated in the brains of HD patients and in animal models of the disease. Interestingly, genetic deletion of TG2 in two different HD mouse models, R6/1 and R6/2, results in improved phenotypes including a reduction in neuronal death and prolonged survival. Starting with phenylacrylamide screening hit 7d, we describe the SAR of this series leading to potent and selective TG2 inhibitors. The suitability of the compounds as in vitro tools to elucidate the biology of TG2 was demonstrated through mode of inhibition studies, characterization of druglike properties, and inhibition profiles in a cell lysate assay.

Development of a Series of Aryl Pyrimidine Kynurenine Monooxygenase Inhibitors as Potential Therapeutic Agents for the Treatment of Huntington's Disease

We report on the development of a series of pyrimidine carboxylic acids that are potent and selective inhibitors of kynurenine monooxygenase and competitive for kynurenine. We describe the SAR for this novel series and report on their inhibition of KMO activity in biochemical and cellular assays and their selectivity against other kynurenine pathway enzymes. We describe the optimization process that led to the identification of a program lead compound with a suitable ADME/PK profile for therapeutic development. We demonstrate that systemic inhibition of KMO in vivo with this lead compound provides pharmacodynamic evidence for modulation of kynurenine pathway metabolites both in the periphery and in the central nervous system.

Exploiting differences in caspase-2 and -3 S(2) subsites for selectivity: Structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors.

Several caspases have been implicated in the pathogenesis of Huntingtons disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P(2) residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P(2) elements for their predicted ability to clash sterically with a residue of the caspase-3 S(2) pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and ∼200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD.

SAR Development of Lysine-Based Irreversible Inhibitors of Transglutaminase 2 for Huntington's Disease

We report a series of irreversible transglutaminase 2 inhibitors starting from a known lysine dipeptide bearing an acrylamide warhead. We established new SARs resulting in compounds demonstrating improved potency and better physical and calculated properties. Transglutaminase selectivity profiling and in vitro ADME properties of selected compounds are also reported.

Irreversible 4-Aminopiperidine Transglutaminase 2 Inhibitors for Huntington's Disease.

A new series of potent TG2 inhibitors are reported that employ a 4-aminopiperidine core bearing an acrylamide warhead. We establish the structure-activity relationship of this new series and report on the transglutaminase selectivity and in vitro ADME properties of selected compounds. We demonstrate that the compounds do not conjugate glutathione in an in vitro setting and have superior plasma stability over our previous series.

Modulation of the Kynurenine Pathway for the Potential Treatment of Neurodegenerative Diseases

Modulation of tryptophan metabolism and in particular the kynurenine pathway is of considerable interest in the discovery of potential new treatments for neurodegenerative diseases. A number of small molecule inhibitors of the kynurenine metabolic pathway enzymes have been identified over recent years; a summary of these and their utility has been reviewed in this chapter. In particular, inhibitors of kynurenine monooxygenase represent an opportunity to develop a therapy for Huntington’s disease; progress in the optimization of small molecule inhibitors of this enzyme is also described.