Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington’s disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

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. (Journal of Biomolecular Screening 2010:478-487)

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 Huntington's 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.

ABSTRACT: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.KEYWORDS: plasma stability, polar surface area, acrylamides, celiac disease, in vitro ADME T issue transglutaminase 2 (TG2) is a multifunctional enzyme primarily known for its calcium-dependent protein cross-linking activity. 1 Less well-studied functions include simple amidase, GTPase, ATPase, and protein disulfide isomerase activities. 2−4 TG2 has been characterized in at least three forms, including open, 5 closed, 6 and an open-inactive form. 7 Genetic deletion of TG2 in mice suggests a role for TG2 activity in mitochondrial energy function, 8 and its overactivity has been most closely associated with celiac disease and Huntington's disease (HD). In addition, there is growing support for roles in inflammation and cancer. 9−12 HD is an autosomal dominant, progressive, neurodegenerative disease that is characterized clinically by motor, cognitive, and behavioral deficits. 13 TG2 expression and transglutaminase activity have been shown to be increased in the brains of HD patients, 14 and in vitro and in vivo models have implicated TG2 in HD pathophysiology, 15−18 although more recent contradictory animal data have appeared. 19 The subject of irreversible inhibitors of TG2 has been recently reviewed. 20 Our studies have focused on irreversible inhibitors bearing an acrylamide warhead, 21,22 during which we became interested in dipeptides A and B 23 (Figure 1) as leads due to their attractive potency and specificity for TG2. In a prior report, 21 we established that an excellent correlation exists for several transglutaminase isoforms between the IC 50 values using a 30 min compound incubation and the irreversible inhibition constants, k inact /K i . With this correlation in hand, we relied on the IC 50 values to guide our medicinal chemistry effort. We began by benchmarking dipeptide A, resulting in the selectivity profile illustrated in Figure 1. We also resynthesized and tested several compounds from the Marrano paper, 23 the results of which are found in the Supporting Information. To summarize, the results confirmed that dipeptide A was one of the most potent TG2 inhibitors from this report. As shown in Figure 1, ADME profiling studies on this compound indicated good solubility, low permeability potentially accompanied by efflux, and rapid metabolism in mouse liver microsomes (mLM). Our goal was to identify a tool molecule from this series for in vivo proof of concept studies in HD, where brain is the target organ. Therefore, we focused on increasing potency, improving the absorption profile, and increasing microsomal stability. Lowering the polar surface area (PSA), the number of hydrogen bond donors, and the number of rotatable bon...

The point mutation serine 120 to glycine in the human nucleoside diphosphate kinase A has been identified in several aggressive neuroblastomas (Chang, C. L., Zhu, X. X., Thoraval, D. H., Ungar, D., Rawwas, J., Hora, N., Strahler, J. R., Hanash, S. M. & Radany, E. (1994) Nature 370, 335-336). We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A. The mutant enzyme had enzymatic and structural properties similar to the wild-type enzyme, whereas its stability to denaturation by heat and urea was markedly reduced. More importantly, upon renaturation of the urea-denatured mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule. It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered. The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift. The hydrodynamic size was between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse urea gradient displayed no renaturation curve, and the protein showed the tendency to aggregate at the lowest urea concentrations. The existence of a molten globule folding intermediates resulting from an altered folding in the mutated protein might be related to the aggressiveness of neuroblastomas.