Pharmacological Inhibition of Astrocytic Transglutaminase 2 Facilitates the Expression of a Neurosupportive Astrocyte Reactive Phenotype in Association with Increased Histone Acetylation.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury.

Diamino variants of piperazine-based tissue transglutaminase inhibitors.

Tissue transglutaminase (TG2) is a multifunctional protein that can catalyze the cross-linking between proteins, and function as a G-protein. TG2's unregulated behaviour has been associated with fibrosis, celiac disease and cancer metastasis. Recently, small molecule irreversible inhibitors have been designed, bearing an electrophilic warhead that can react with the catalytic cysteine, abolishing TG2's catalytic and G-protein capabilities.

A practical guide for the assay-dependent characterisation of irreversible inhibitors.

Irreversible targeted covalent inhibitors, in the past regarded as inappropriately reactive and toxic, have seen a recent resurgence in clinical interest. This paradigm shift is attributed to the exploitation of the two-step mechanism, in which a high affinity and selectivity (, low ) scaffold binds the target and only then does a pendant low intrinsic reactivity warhead react with the target (moderate ). This highlights the importance of evaluating inhibitors by deriving both their and values.

Enabling Unfavorable Hydroamination Reactions Using a Chemoselective N-O Bond Reduction.

Despite major advances, intramolecular alkene hydroamination reactions often face limitations. Herein, a redox-enabled process featuring oxidation of an amine to a hydroxylamine, a concerted hydroamination step, followed by catalytic reduction of -oxide is shown to be broadly applicable. Catalyst screening and optimization showed that a KOsO(OH)-pinacol complex rapidly and chemoselectively reduces the -oxide cycloadduct in the presence of hydroxylamine and dimethyl sulfoxide.

Structurally Minimalized and Druglike TGase2 Inhibitors Based on 7-Aminoquinoline-5,8-dione Scaffolds for the Treatment of Diabetic Retinopathy.

Diabetic retinopathy is a disease that can cause vision loss leading to blindness in people with diabetes. Improved methods to treat and prevent vision loss in diabetic patients are in high demand owing to limited current treatment procedures. Herein, we report a new class of transglutaminase 2 (TGase2) inhibitors for the treatment of diabetic retinopathy based on 7-aminoquinoline-5,8-dione derivatives.

Targeted Genome Mining Facilitates the Discovery of a Promiscuous, Hyperthermostable Amidase from Thermovenabulum Gondwanense with Notable Nylon-Degrading Capacity.

Plastics are ubiquitous in our ecosystems, and microplastic accumulation in the environment is an emerging global health concern. Since available recycling technologies are not economically competitive with primary plastic production, global use is expected to reach 1231 megatons by 2060, with 493 megatons leeching into the environment each year. To identify new nylon-recycling biotechnologies, targeted genome mining was used to identify thermostable enzymes capable of degrading polyamides.

Small molecule substrates for the rapid quantification of acyl transfer activity of nylon hydrolase NylC.

The widespread use of polyamides such as nylons has led to the accumulation of nylon waste, which is particularly resistant to decomposition due to the intrinsic stability of the amide bond. New methods are required for the true recycling of these waste materials by depolymerization. Enzymes that are capable of hydrolyzing polyamides have been proposed as biocatalysts that may be suitable for this application.

Fitting of and Values from Endpoint Pre-incubation IC Data.

Experiments comprising a "pre-incubation" phase, where enzyme is incubated with inhibitor prior to the addition of assay substrate, are commonly used to evaluate covalent inhibitors, often via discontinuous or "endpoint" IC assays. However, due to the lack of mathematical tools to describe its biphasic time-dependent nature, this experiment has thus far been unable to provide and values. Herein we report EPIC-Fit, a new method to determine and values from global fitting of Endpoint Pre-incubation IC data that can be implemented using Microsoft Excel.

Conformational Modulation of Tissue Transglutaminase via Active Site Thiol Alkylating Agents: Size Does Not Matter.

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g.

Pharmacological inhibition of astrocytic transglutaminase 2 facilitates the expression of a neurosupportive astrocyte reactive phenotype in association with increased histone acetylation.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury.

Transcriptomics Studies Reveal Functions of Transglutaminase 2 in Breast Cancer Cells Using Membrane Permeable and Impermeable Inhibitors.

Transglutaminase 2 (TG2) performs many functions both under physiological and pathological conditions. In cancer, its expression is associated with aggressiveness, propensity to epithelial-mesenchymal transition, and metastasis. Since TG2 performs key functions both outside and inside the cell, using inhibitors with different membrane permeability we analyzed the changes in the transcriptome induced in two triple-negative cell lines (MDA-MB-436 and MDA-MB-231) with aggressive features.

High-Affinity Fluorogenic Substrate for Tissue Transglutaminase Reveals Enzymatic Hysteresis.

Transglutaminases (TGases) are a family of calcium-dependent enzymes primarily known for their ability to cross-link proteins. Transglutaminase 2 (TG2) is one isozyme in this family whose role is multifaceted. TG2 can act not only as a typical transamidase through its catalytic core but also as a G-protein via its GTP binding site.

Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype.

Transglutaminase 2 (TG2) is a multifunctional enzyme primarily responsible for crosslinking proteins. Ubiquitously expressed in humans, TG2 can act either as a transamidase by crosslinking two substrates through formation of an N(ɣ-glutaminyl)lysine bond or as an intracellular G-protein. These discrete roles are tightly regulated by both allosteric and environmental stimuli and are associated with dramatic changes in the conformation of the enzyme.

Metabolic characterisation of transglutaminase 2 inhibitor effects in breast cancer cell lines.

Transglutaminase 2 (TG2), which mediates post-translational modifications of multiple intracellular enzymes, is involved in the pathogenesis and progression of cancer. We used H-NMR metabolomics to study the effects of AA9, a novel TG2 inhibitor, on two breast cancer cell lines with distinct phenotypes, MCF-7 and MDA-MB-231. AA9 can promote apoptosis in both cell lines, but it is particularly effective in MD-MB-231, inhibiting transamidation reactions and decreasing cell migration and invasiveness.

Targeting transglutaminase 2 mediated exostosin glycosyltransferase 1 signaling in liver cancer stem cells with acyclic retinoid.

Transglutaminase 2 (TG2) is a multifunctional protein that promotes or suppresses tumorigenesis, depending on intracellular location and conformational structure. Acyclic retinoid (ACR) is an orally administered vitamin A derivative that prevents hepatocellular carcinoma (HCC) recurrence by targeting liver cancer stem cells (CSCs). In this study, we examined the subcellular location-dependent effects of ACR on TG2 activity at a structural level and characterized the functional role of TG2 and its downstream molecular mechanism in the selective depletion of liver CSCs.

A kinetic study of thiol addition to -phenylchloroacetamide.

Irreversible enzyme inhibitors bind covalently to their target and permanently limit its function. The redox-sensitive thiol group on the side chain of cysteine (Cys) residues is often the nucleophilic group that is targeted for reaction with the electrophilic warhead of irreversible inhibitors. While the acrylamide group is the warhead applied most frequently currently in the design of inhibitors with therapeutic potential, the chloroacetamide group offers a comparable reactivity profile.

Transglutaminase 2 Binds to the CD44v6 Cytoplasmic Domain to Stimulate CD44v6/ERK1/2 Signaling and Maintain an Aggressive Cancer Phenotype.

Unlabelled: Transglutaminase 2 (TG2) is a key cancer cell survival protein in many cancer types. As such, efforts are underway to characterize the mechanism of TG2 action. In this study, we report that TG2 stimulates CD44v6 activity to enhance cancer cell survival via a mechanism that involves formation of a TG2/CD44v6/ERK1/2 complex that activates ERK1/2 signaling to drive an aggressive cancer phenotype.

Quinazoline-2-Carboxamides as Selective PET Radiotracers for Matrix Metalloproteinase-13 Imaging in Atherosclerosis.

Matrix metalloproteinase-13 (MMP-13) plays a critical role in the progression of unstable atherosclerosis. A series of highly potent and selective MMP-13 inhibitors were synthesized around a quinazoline-2-carboxamide scaffold to facilitate radiolabeling with fluorine-18 or carbon-11 positron-emitting nuclides and visualization of atherosclerotic plaques. enzyme inhibition assays identified three compounds as promising radiotracer candidates.

Novel irreversible peptidic inhibitors of transglutaminase 2.

Transglutaminase 2 (TG2), also referred to as tissue transglutaminase, plays crucial roles in both protein crosslinking and cell signalling. It is capable of both catalysing transamidation and acting as a G-protein, these activities being conformation-dependent, mutually exclusive, and tightly regulated. The dysregulation of both activities has been implicated in numerous pathologies.

The war on hTG2: warhead optimization in small molecule human tissue transglutaminase inhibitors.

Human tissue transglutaminase (hTG2) is a multifunctional enzyme with protein cross-linking and G-protein activity, both of which have been implicated in the progression of diseases such as fibrosis and cancer stem cell propagation when dysregulated, prompting the development of small molecule targeted covalent inhibitors (TCIs) possessing a crucial electrophilic 'warhead'. In recent years there have been significant advances in the library of warheads available for the design of TCIs; however, the exploration of warhead functionality in hTG2 inhibitors has remained relatively stagnant. Herein, we describe a structure-activity relationship study entailing rational design and synthesis for systematic variation of the warhead on a previously reported small molecule inhibitor scaffold, and rigorous kinetic evaluation of inhibitory efficiency, selectivity, and pharmacokinetic stability.

Peptidic Inhibitors and a Fluorescent Probe for the Selective Inhibition and Labelling of Factor XIIIa Transglutaminase.

Factor XIIIa (FXIIIa) is a transglutaminase of major therapeutic interest for the development of anticoagulants due to its essential role in the blood coagulation cascade. While numerous FXIIIa inhibitors have been reported, they failed to reach clinical evaluation due to their lack of metabolic stability and low selectivity over transglutaminase 2 (TG2). Furthermore, the chemical tools available for the study of FXIIIa activity and localization are extremely limited.

A mechanistic study of thiol addition to -acryloylpiperidine.

Nucleophilic cysteine (Cys) residues are present in many enzyme active sites and represent the target of many different irreversible enzyme inhibitors. Given its fine balance between aqueous stability and thiolate reactivity, the acrylamide group is a particularly popular warhead pharmacophore among inhibitors designed for biological and therapeutic application. The acrylamide group is well known to undergo thiol addition, but the precise mechanism of this addition reaction has not been studied in as much detail.

A Multidisciplinary Approach Establishes a Link between Transglutaminase 2 and the Kv10.1 Voltage-Dependent K Channel in Breast Cancer.

Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K channels using the specific inhibitors 4-aminopyridine and astemizole.

A mechanistic study of thiol addition to -phenylacrylamide.

Cysteine (Cys) residues contain a redox-sensitive thiol and are commonly found in enzyme active sites. In recent years, the presence of a reactive thiolate group on a protein has been exploited in the development of irreversible enzyme inhibitors as therapeutic agents. Many targeted covalent inhibitors (TCIs) are designed to covalently react with a specific Cys residue on a target protein active site, irreversibly modifying the target and inhibiting its normal function.