Fitting of kinact and KI Values from Endpoint Pre-incubation IC50 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" IC50 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 kinact and KI values. Herein we report EPIC-Fit, a new method to determine kinact and KI values from global fitting of Endpoint Pre-incubation IC50 data that can be implemented using Microsoft Excel. Experimental characterization of a known tissue transglutaminase inhibitor, AA9, using EPIC-Fit provided kinact and KI values with strong correlations to the values determined by other, previously established methods of evaluation. This unprecedented method serves to finally include time-dependent pre-incubation endpoint assays in the medicinal chemist's toolbox for rigorous characterization of irreversible inhibitors.

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. By characterizing pathways and gene networks, we were able to define the effects of TG2 inhibitors (AA9, membrane-permeable, and NCEG2, impermeable) in relation to the roles of the enzyme in the intra- and extracellular space within the context of breast cancer. The deregulated genes revealed p53 and integrin signaling to be the common pathways with some genes showing opposite changes in expression. In MDA-MB-436, AA9 induced apoptosis, modulated cadherin, Wnt, gastrin and cholecystokinin receptors (CCKR) mediated signaling, with RHOB and GNG2 playing significant roles, and affected the Warburg effect by decreasing glycolytic enzymes. In MDA-MB-231 cells, AA9 strongly impacted HIF-mediated hypoxia, including AKT and mTOR pathway. These effects suggest an anti-tumor activity by blocking intracellular TG2 functions. Conversely, the use of NCEG2 stimulated the expression of ATP synthase and proteins involved in DNA replication, indicating a potential promotion of cell proliferation through inhibition of extracellular TG2. To effectively utilize these molecules as an anti-tumor strategy, an appropriate delivery system should be evaluated to target specific functions and avoid adverse effects. Additionally, considering combinations with other pathway modulators is crucial.

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., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.

Stabilizing transglutaminase 2 in the open conformation results in reactive astrocytes being more neurosupportive.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). Inflammatory conditions bring about a range of poorly understood, heterogeneous, reactive phenotypes in astrocytes. Finding ways to manipulate the phenotype of reactive astrocytes, and leveraging a pro-recovery phenotype, holds promise in treating CNS injury. Previous studies have shown that the protein transglutaminase 2 (TG2) plays a significant role in determining the phenotype of reactive astrocytes. Recently it has been demonstrated that ablation of TG2 from astrocytes improves injury outcomes both in vitro and in vivo. Excitingly, in an in vivo mouse model, pharmacological inhibition of TG2 with the irreversible inhibitor VA4 phenocopies the neurosupportive effects of TG2 deletion in astrocytes. The focus of this study was to provide insights into the mechanisms by which TG2 deletion or inhibition of TG2 with VA4 result in a more neurosupportive astrocytic phenotype. Using a neuron-astrocyte co-culture model of neurite outgrowth, we show that VA4 treatment improves the ability of astrocytes to support neurite outgrowth on an injury-relevant matrix, further validating the ability of VA4 to phenocopy astrocytic TG2 deletion. VA4 treatment of neurons alone had no effect on neurite outgrowth. VA4 covalently binds to active site residues of TG2 that are exposed in its open conformation and are critical for its enzymatic function, and prevents TG2 from taking on a closed conformation, which interferes with its protein scaffolding function. To begin to understand how pharmacologically altering TG2's conformation affects its ability to regulate reactive astrocyte phenotypes, we assayed the impact of VA4 on TG2's interaction with Zbtb7a, a transcription factor that we have previously identified as a TG2 interactor, and whose functional outputs are significantly regulated by TG2. The results of these studies demonstrated that VA4 significantly decreases the interaction of TG2 and Zbtb7a. Further, previous findings indicate that TG2 may act as an epigenetic regulator, through its nuclear protein-protein interactions, to modulate gene expression. Since both TG2 and Zbtb7a interact with members of the Sin3a chromatin repressor complex, we assayed the effect of TG2 deletion and VA4 treatment on histone acetylation and found significantly greater acetylation with TG2 deletion or inhibition with VA4. Overall, this work points toward a possible epigenetic mechanism by which genetic deletion or acute inhibition of TG2 leads to enhanced astrocytic support of neurons.

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. These two discrete activities are tightly regulated by both environmental stimuli and redox reactions. Ubiquitously expressed in humans, TG2 has been implicated in numerous disease pathologies that require extensive investigation. The catalytic activity of TG2 can be monitored through various mechanisms, including hydrolysis, transamidation, or cleavage of isopeptide bonds. Activity assays are required to monitor the activity of this isozyme not only for studying its transamidation reaction but also for validation of therapeutics designed to abolish this activity. Herein, we present the design, synthesis, and evaluation of a new TG2 activity substrate based on a previously optimized inhibitor scaffold. The substrate APH7 exhibits excellent affinity, selectivity, and reactivity with TG2 (KM = 3.0 µM). Furthermore, its application also allowed the discovery of unique hysteresis at play within the catalytic activity and inhibition reactivity of TG2.

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 Nε(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. The pleiotropic nature of TG2 and multi-faceted activities have resulted in TG2 being implicated in numerous disease pathologies including celiac disease, fibrosis, and cancer. Targeted TG2 therapies have not been selective for subcellular localization, such that currently no tools exist to selectively target extracellular over intracellular TG2. Herein, we have designed novel TG2-selective inhibitors that are not only highly potent and irreversible, but also cell impermeable, targeting only extracellular TG2. We have also further derivatized the scaffold to develop probes that are intrinsically fluorescent or bear an alkyne handle, which target both intra- and extracellular TG2, in order to facilitate cellular labelling and pull-down assays. The fluorescent probes were internalized and imaged in cellulo, and provide the first implicit experimental evidence that by comparison with their cell-impermeable analogues, it is specifically intracellular TG2, and presumably its G-protein activity, that contributes to transglutaminase-associated cancer progression.

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 1 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. This metabolomics study provides evidence of a major effect of AA9 on MDA-MB-231 cells, impacting glutamate and aspartate metabolism, rather than on MCF-7 cells, characterised by choline and O-phosphocholine decrease. Interestingly, AA9 treatment induces myo-inositol alteration in both cell lines, indicating action on phosphatidylinositol metabolism, likely modulated by the G protein activity of TG2 on phospholipase C. Considering the metabolic deregulations that characterise various breast cancer subtypes, the existence of a metabolic pathway affected by AA9 further points to TG2 as a promising hot spot. The metabolomics approach provides a powerful tool to monitor the effectiveness of inhibitors and better understand the role of TG2 in cancer.

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 binding assay with high-performance magnetic nanobeads and structural dynamic analysis with native gel electrophoresis and size-exclusion chromatography-coupled multi-angle light scattering or small-angle X-ray scattering showed that ACR binds directly to TG2, induces oligomer formation of TG2, and inhibits the transamidase activity of cytoplasmic TG2 in HCC cells. The loss-of-function of TG2 suppressed the expression of stemness-related genes, spheroid proliferation and selectively induced cell death in an EpCAM+ liver CSC subpopulation in HCC cells. Proteome analysis revealed that TG2 inhibition suppressed the gene and protein expression of exostosin glycosyltransferase 1 (EXT1) and heparan sulfate biosynthesis in HCC cells. In contrast, high levels of ACR increased intracellular Ca2+ concentrations along with an increase in apoptotic cells, which probably contributed to the enhanced transamidase activity of nuclear TG2. This study demonstrates that ACR could act as a novel TG2 inhibitor; TG2-mediated EXT1 signaling is a promising therapeutic target in the prevention of HCC by disrupting liver CSCs.

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. In vitro enzyme inhibition assays identified three compounds as promising radiotracer candidates. Efficient automated radiosyntheses provided [11C]5b, [11C]5f, and [18F]5j and enabled pharmacokinetic characterization in atherosclerotic mice. The radiotracers displayed substantial differences in their distribution and excretion. Most favorably for vascular imaging, [18F]5j exhibited low uptake in metabolic organs with minimal retention of myocardial radioactivity, substantial renal clearance, and high metabolic stability in plasma. Ex vivo aortic autoradiography and competition studies revealed that [18F]5j specifically binds to MMP-13 within atherosclerotic plaques and localizes to lipid-rich regions. This study demonstrates the utility of the quinazoline-2-carboxamide scaffold for MMP-13 selective positron emission tomography (PET) radiotracer development and identifies [18F]5j for imaging atherosclerosis.

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

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. TG2 and ERK1/2 bind to the CD44v6 C-terminal intracellular cytoplasmic domain to activate ERK1/2 and stimulate cell proliferation and invasion. This is the same region that binds to ERM proteins and ankyrin to activate CD44v6-dependent cell proliferation, invasion, and migration. We further show that treatment with hyaluronan (HA), the physiologic CD44v6 ligand, stimulates CD44v6 activity, as measured by ERK1/2 activation, but that this response is severely attenuated in TG2 or CD44v6 knockdown or knockout cells. Moreover, treatment with TG2 inhibitor reduces tumor growth and that is associated with reduced CD44v6 level and ERK1/2 activity, and reduced stemness and epithelial-mesenchymal transition (EMT). These changes are replicated in CD44v6 knockout cells. These findings suggest that a unique TG2/CD44v6/ERK1/2 complex leads to increased ERK1/2 activity to stimulate an aggressive cancer phenotype and stimulate tumor growth. These findings have important implications for cancer stem cell maintenance and suggest that cotargeting of TG2 and CD44v6 with specific inhibitors may be an effective anticancer treatment strategy. IMPLICATIONS: TG2 and CD44v6 are important procancer proteins. TG2 and ERK1/2 bind to the CD44v6 C-terminal domain to form a TG2/CD44v6/ERK1/2 complex that activates ERK1/2 to stimulate the cancer phenotype.

A kinetic study of thiol addition to N-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. In that context, we have studied the details of the mechanism of thiol addition to N-phenylchloroacetamide (NPC). A kinetic assay was developed to accurately monitor the reaction progress between NPC and a small library of thiols with varying pKa values. From these data, a Brønsted-type plot was constructed, from which a ßnucRS- value of 0.22 ± 0.07 was derived, indicative of a relatively early transition state with respect to attack by the thiolate. The halide leaving group was also varied, for the reaction with one thiol, providing rate constants consistent with a transition state that is early with respect to leaving group departure. The effects of temperature and ionic strength were also studied, and all data are consistent with an early transition state for a concerted SN2 mechanism of addition. Molecular modelling was also performed, and these calculations confirm the concerted transition state and relative reactivity of the haloacetamides. Finally, this study allows a detailed comparison of the reactivity and reaction mechanisms of the chloroacetamide group with the benchmark acrylamides used in many irreversible inhibitor drugs.

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. To combat these shortcomings, we designed, synthesised, and evaluated a library of 21 novel FXIIIa inhibitors. Electrophilic warheads, linker lengths, and hydrophobic units were varied on small molecule and peptidic scaffolds to optimize isozyme selectivity and potency. A previously reported FXIIIa inhibitor was then adapted for the design of a probe bearing a rhodamine B moiety, producing the innovative KM93 as the first known fluorescent probe designed to selectively label active FXIIIa with high efficiency (kinact/KI = 127,300 M-1 min-1) and 6.5-fold selectivity over TG2. The probe KM93 facilitated fluorescent microscopy studies within bone marrow macrophages, labelling FXIIIa with high efficiency and selectivity in cell culture. The structure-activity trends with these novel inhibitors and probes will help in the future study of the activity, inhibition, and localization of FXIIIa.

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. This study reveals a strong influence on the kinetic parameters k inact and K I with even subtle variation in warhead structure, suggesting that the warhead plays a significant role in not only reactivity, but also binding affinity, which consequently extends to isozyme selectivity. Warhead structure also influences in vivo stability, which we model by measuring intrinsic reactivity with glutathione, as well as stability in hepatocytes and in whole blood, giving insight into degradation pathways and relative therapeutic potential of different functional groups. This work provides fundamental structural and reactivity information highlighting the importance of strategic warhead design for the development of potent hTG2 inhibitors.

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. TG2 is expressed ubiquitously in humans and is localized both intracellularly and extracellularly. Targeted TG2 therapies have been developed but have faced numerous hurdles including decreased efficacy in vivo. Our latest efforts in inhibitor optimization involve the modification of a previous lead compound's scaffold by insertion of various amino acid residues into the peptidomimetic backbone, and derivatization of the N-terminus with substituted phenylacetic acids, resulting in 28 novel irreversible inhibitors. These inhibitors were evaluated for their ability to inhibit TG2 in vitro and their pharmacokinetic properties, and the most promising candidate 35 (k inact/K I = 760 × 103 M-1 min-1) was tested in a cancer stem cell model. Although these inhibitors display exceptional potency versus TG2, with k inact/K I ratios nearly ten-fold higher than their parent compound, their pharmacokinetic properties and cellular activity limit their therapeutic potential. However, they do serve as a scaffold for the development of potent research tools.

A mechanistic study of thiol addition to N-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. In this work we have focussed on the reaction of N-acryloylpiperidine (AcrPip), which represents a motif found in many targeted covalent inhibitor drugs. Using a precise HPLC-based assay, we measured the second order rate constants for the reaction of AcrPip with a panel of thiols possessing different pKa values. This allowed construction of a Brønsted-type plot that reveals the relative insensitivity of the reaction to the nucleophilicity of the thiolate. By studying temperature effects, we were able to construct an Eyring plot from which the enthalpy and entropy of activation were calculated. Ionic strength and solvent kinetic isotope effects were also studied, informing on charge dispersal and proton transfer in the transition state. DFT calculations were also performed, providing information on the potential structure of the activated complex. Taken together, these data strongly support one cohesive addition mechanism that is the microscopic reverse of the E1cb elimination, and highly relevant to the intrinsic thiol selectivity of AcrPip inhibitors and their subsequent design.

A mechanistic study of thiol addition to N-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. The electrophilic warhead most commonly used in this way is the acrylamide functional group. Although the acrylamide group is well known for its ability to undergo thiol-addition reactions, very few studies have been conducted to elucidate the detailed mechanism of this reaction, which inspired us to conduct a thorough kinetic investigation. First, we developed a robust kinetic assay to accurately monitor reaction progress between N-phenylacrylamide (NPA) and a small library of alkyl thiols having widely varying pKa values. This allowed us to construct a Brønsted-type plot for the thiol addition reaction, revealing a ßnucRS- value of 0.07 ± 0.04. We also studied the solvent kinetic isotope effects (SKIEs), pH dependence, and temperature dependence of the reaction, which showed that reaction has a relatively large negative ΔS‡, and a small ΔH‡. Computational studies provided a structure for the transition state that is consistent with the experimental data. All of these data are consistent with rate-limiting nucleophilic attack, followed by rapid protonation of the enolate, corresponding to the microscopic reverse of the E1revcb elimination mechanism.

Structure-activity relationships of hydrophobic alkyl acrylamides as tissue transglutaminase inhibitors.

Tissue transglutaminase (TG2) is a multifunctional protein that plays biological roles based on its ability to catalyse protein cross-linking and to function as a non-canonical G-protein known as Ghα. The non-regulated activity of TG2 has been implicated in fibrosis, celiac disease and the survival of cancer stem cells, underpinning the therapeutic potential of cell permeable small molecule inhibitors of TG2. In the current study, we designed a small library of inhibitors to explore the importance of a terminal hydrophobic moiety, as well as the length of the tether to the irreversible acrylamide warhead. Subsequent kinetic evaluation using an in vitro activity assay provided values for the k inact and K I parameters for each of these irreversible inhibitors. The resulting structure-activity relationship (SAR) clearly indicated the affinity conferred by dansyl and adamantyl moieties, as well as the efficiency provided by the shortest warhead tether. We also provide the first direct evidence of the capability of these inhibitors to suppress the GTP binding ability of TG2, at least partially. However, it is intriguing to note that the SAR trends observed herein are opposite to those predicted by molecular modelling - namely that longer tether groups should improve binding affinity by allowing for deeper insertion of the hydrophobic moiety into a hydrophobic pocket on the enzyme. This discrepancy leads us to question whether the existing crystallographic structures of TG2 are appropriate for docking non-peptidic inhibitors. In the absence of a more relevant crystallographic structure, the data from rigorous kinetic studies, such as those provided herein, are critically important for the development of future small molecule TG2 inhibitors.

Structure-activity relationships of N-terminal variants of peptidomimetic tissue transglutaminase inhibitors.

Tissue transglutaminase (TG2) is a multifunctional protein that catalyses protein crosslinking in the extracellular matrix, and functions as an intracellular G-protein. While both activities have been associated with human diseases, its role as a G-protein has been linked to cancer stem cell survival and maintenance of a metastatic phenotype. Recently we have shown that targeted covalent inhibitors (TCIs) can react selectively with the enzyme active site of TG2, to allosterically abolish its ability to bind GTP. In the present work, we focused on the variation of the N-terminal group of these peptidomimetic inhibitors, in order to enhance efficiency, while reducing log P and the number of rotatable bonds. This approach led to the synthesis and evaluation of 41 novel inhibitors, some of which had greatly improved efficiency and affinity for TG2 (e.g. TCI 72: KI = 1.0 µM, kinact/KI = 4.4 × 105 M-1 min-1). Molecular modelling provided a hypothetical binding mode for these TCIs. The most efficient inhibitors were evaluated further and shown to have excellent isozyme selectivity, to block GTP binding, and to have improved pharmacokinetic properties, as expected. Their biological activity was also confirmed, in a cellular invasion assay, although with less potency than expected.

Dysregulation of Transglutaminase type 2 through GATA3 defines aggressiveness and Doxorubicin sensitivity in breast cancer.

The role of transglutaminase type 2 in cell physiology is related to protein transamidation and signal transduction (affecting extracellular, intracellular and nuclear processes) aided by the expression of truncated isoforms and of two lncRNAs with regulatory functions. In breast cancer TG2 is associated with disease progression supporting motility, epithelial-mesenchymal transition, invasion and drug resistance. The aim of his work is to clarify these issues by emphasizing the interconnections among TGM2 variants and transcription factors associated with an aggressive phenotype, in which the truncated TGH isoform correlates with malignancy. TGM2 transcripts are upregulated by several drugs in MCF-7, but only Doxorubicin is effective in MDA-MB-231 cells. These differences reflect the expression of GATA3, as demonstrated by silencing, suggesting a link between this transcription factor and gene dysregulation. Of note, NC9, an irreversible inhibitor of enzymatic TG2 activities, emerges to control NF-ĸB and apoptosis in breast cancer cell lines, showing potential for combination therapies with Doxorubicin.

Sulforaphane covalently interacts with the transglutaminase 2 cancer maintenance protein to alter its structure and suppress its activity.

Type 2 transglutaminase (TG2) functions as an important cancer cell survival protein in a range of cancers including epidermal squamous cell carcinoma. TG2 exists in open and closed conformations each of which has a distinct and mutually exclusive activity. The closed conformation has GTP-binding/GTPase activity while the open conformation functions as a transamidase to catalyze protein-protein crosslinking. GTP-binding/GTPase activity is required for TG2 maintenance of the aggressive cancer phenotype. Thus, identifying agents that convert TG2 from the closed to the open GTP-binding/GTPase inactive conformation is an important cancer prevention/treatment strategy. Sulforaphane (SFN) is an important diet-derived cancer prevention agent that is known to possess a reactive isothiocyanate group and has potent anticancer activity. Using a biotin-tagged SFN analog (Biotin-ITC) and kinetic analysis we show that SFN covalently and irreversibly binds to recombinant TG2 to inhibit transamidase activity and shift TG2 to an open/extended conformation, leading to a partial inhibition of GTP binding. We also show that incubation of cancer cells or cancer cell extract with Biotin-ITC results in formation of a TG2/Biotin-ITC complex and that SFN treatment of cancer cells inhibits TG2 transamidase activity and shifts TG2 to an open/extended conformation. These findings identify TG2 as a direct SFN anticancer target in epidermal squamous cell carcinoma.