Andreas Scheel

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.

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.

Receptor-Ligand Interactions Studied with Homogeneous Fluorescence-Based Assays Suitable for Miniaturized Screening

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and β2-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1,IL without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.

Ligand Binding to Transmembrane Receptors on Intact Cells or Membrane Vesicles Measured in a Homogeneous 1-Microliter Assay Format

We have developed homogeneous miniaturized assays to measure ligand binding to either intact cells or receptor-containing membrane fragments by analysis of particle brightness. As an example, the affinities and inhibition constants of fluorescently labeled interleukin-8 (IL-8) and a low-molecular-weight antagonist toward the receptors CXCR1 and CXCR2, which belong to the superfamily of G protein-coupled receptors (GPCRs), were determined. Although the results were generally comparable between the two approaches, the cell-based measurements revealed a more complex pattern of both ligand and inhibitor titration curves, pointing to the influence of intracellular regulatory events. Both the vesicle- and cell-based membrane receptor assays were successfully miniaturized to a total volume of 1,l without compromising their sensitivity, indicating that screening of transmembrane receptors in these formats is feasible. This is the first report of a cellular ligand-binding assay performed in such low volumes. The resulting savings in reagent could potentially enable the use of primary cells for future HTS/ultra-HTS efforts.

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.