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.

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.

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.

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.

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.

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.

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. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.