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

Abstract

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. KEYWORDS: plasma stability, computer-aided drug design, SAR, acrylamides, Celiac disease, ADME T issue transglutaminase 2 (TG2) is a multifunctional enzyme primarily known for its calcium-dependent cross-linking activity via isopeptide bond formation.1 Less well-studied activities of TG2 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 TG2 overactivity has been most closely associated with Celiac disease and Huntington's disease (HD), and 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. TG2 expression and transglutaminase activity have been shown to be increased in the brains of HD patients, 13 and in vitro and in vivo studies have implicated TG2 in HD pathophysiology. 14−17Several classes of TG2 inhibitors have been reported, most of which are irreversible in nature. 18 In addition, we have reported potent and selective covalent inhibitors bearing an acrylamide warhead as in 1 (Figure 1). 19 Compound 1 has TG2 IC 50 of 0.11 μM; however, in vitro metabolism profiling studies identified plasma stability as an issue cautioning against in vivo evaluation. Compound 1 was one of the examples showing better stability in plasma, having a half-life of 209 min in mouse plasma. Evidence supporting the acrylamide amide linkage as the site of metabolic instability was obtained by testing the plasma stability of aniline analogue 2 (Figure 1) and detecting its formation in plasma incubation of cyclopropylamide 1. Aniline 2 showed no indication of metabolism over a 24 h period in mouse plasma. The acrylamide warhead has been featured in several EGFR irreversible inhibitors that have progressed to clinical trials, 20 suggesting that this moiety is not especially susceptible to metabolism and enabling compounds to reach acceptable plasma exposures in vivo. Thus, we present here a structure−activity relationship (SAR) study of a new class of acrylamide-bearing TG2 inhibitors that show improved plasma stability.Our approach to stabilize the compounds to plasma exposure initially focused on replacing the problematic anilide with a more robust alkylamide. The compounds in Table 1 were prepared mainly to expand rapidly the SAR with respect to TG2 as this series of compounds still possessed ...

Strategies towards in vivo imaging of active transglutaminase type 2 using positron emission tomography

“…More potent and selective irreversible TG2 inhibitors carrying an acrylamide moiety have recently been reported ( 1 – 3 in Fig. 3 ) (Prime et al 2012a , b ; Wityak et al 2012 ). Conveniently, carbon-11 radiosynthesis of acryl amides has been reported using various strategies resulting in the carbon-11 atom at the acrylamide carbonyl position, rendering this class of Michael acceptor inhibitors amenable to radiolabelling (Eriksson et al 2007 ).…”

“…Other potent acrylamide inhibitors based on an aminopiperidine core have been reported. Apart from [ 11 C]CO aminocarbonylation, the strongest inhibitor within this class, compound 9q , potentially allows for radiolabelling at the methoxy position by straightforward [ 11 C]MeI methylation of the corresponding O-desmethyl precursor (Prime et al 2012b ). Recently, Badarau et al also reported on the development of potent TG2 inhibitors carrying the acrylamide moiety (Badarau et al 2015 ).…”

“…As PET only measures the distribution of radioactivity and not the chemical form, it is important to carefully study metabolism of a putative radioligand (Pike 2009 ). At present, the in vivo kinetics of existing TG2 inhibitors which are largely unknown and metabolism data, if available, is largely depending on in vitro plasma or hepatocyte stability data (Wityak et al 2012 ; Prime et al 2012a , b ; Badarau et al 2015 ). This limited availability of stability data hampers selection of high-potential compounds for translation to PET agents.…”

Transglutaminse 2 and EGGL, the Protein Cross-Link Formed by Transglutaminse 2, As Therapeutic Targets for Disabilities of Old Age

“…Other irreversible inhibitors that are not based on peptides have also been designed. 270,271 Of the other binding sites on TG2, only the GTP binding site has been targeted by an inhibitor, Tyrphostin 47, an epidermal growth factor receptor (EGFR) kinase inhibitor that binds to TG2 competitively with GTP. 272 Some kinaserelated inhibitors of TG2 have been found, but are competitive inhibitors of enzyme activity, 272 not (as far as is known) allosteric inhibitors acting through the GTP site.…”

An NMR-Guided Screening Method for Selective Fragment Docking and Synthesis of a Warhead Inhibitor

“…Acrylamide moieties have been used to functionalize lead compounds by several researchers to develop irreversible inhibitors for some classes of proteins [2,3,4,5]. This acrylamide “warhead” can form a covalent interaction via alkylation with conserved Cys residues in the active sites of these proteins.…”