Jeffrey W. Keillor

Cinnamoyl inhibitors of tissue transglutaminase.

Transglutaminases (TGases) catalyze the intermolecular cross-linking of certain proteins and tissue TGases (TG2) are involved in diverse biological processes. Unregulated, high TGase activities have been implicated in several physiological disorders, but few reversible inhibitors of TG2 have been reported. Herein, we report the synthesis of a series of novel trans-cinammoyl derivatives, discovered to be potent inhibitors of guinea pig liver transglutaminase. The most effective inhibitors evaluated can be sorted into two subclasses: substituted cinnamoyl benzotriazolyl amides and the 3-(substituted cinnamoyl)pyridines, referred to more commonly as azachalcones. Kinetic evaluation of both of these subclasses revealed that they display reversible inhibition and are competitive with acyl donor TGase substrates at IC50 values as low as 18 microM. An analysis of structure-activity relationships within these series of inhibitors permitted the identification of potentially important binding interactions. Further testing of some of the most potent inhibitors demonstrated their selectivity for TG2 and their potential for further development.

Gamma-glutamyl transpeptidase substrate specificity and catalytic mechanism.

The enzyme gamma-glutamyltranspeptidase (GGT) is critical to cellular detoxification and leukotriene biosynthesis processes, as well as amino acid transport in kidneys. GGT has also been implicated in many important physiological disorders, including Parkinsons disease and inhibition of apoptosis. It binds glutathione as donor substrate and initially forms a gamma-glutamyl enzyme that can then react with a water molecule or an acceptor substrate (usually an amino acid or a dipeptide) to form glutamate or a product containing a new gamma-glutamyl isopeptide bond, respectively, thus regenerating the free enzyme. Given the importance of GGT in human physiology, we have undertaken studies of its substrate specificity and catalytic mechanism. In the course of these studies, we have developed methods for the indirect evaluation of donor substrate affinity and stereospecificity and applied others for the measurement of steady state and pre-steady state kinetics and linear free-energy relationships. These methods and the pertinent results obtained with them are presented herein.

Reversible and Competitive Cinnamoyl Triazole Inhibitors of Tissue Transglutaminase

A series of 15 cinnamoyl triazole derivatives was prepared by Cu(I)‐catalyzed azide/alkyne [3+2]‐cycloaddition reactions and examined as inhibitors of guinea‐pig liver transglutaminase. Several compounds exhibited activity as reversible inhibitors that were competitive with acyl donor transglutaminase substrates. For example, triazole 4d has a Ki value of 174 nm and represents one of the most potent reversible transglutaminase inhibitors reported to date.

Synthesis of dipeptide-bound epoxides and α,β-unsaturated amides as potential irreversible transglutaminase inhibitors

Abstract Herein we report the synthesis of 24 novel peptides as potential irreversible inactivators of transglutaminase (TGase). These peptides were designed to resemble Cbz- l -Gln-Gly, known to be a good TGase substrate, and to include either α,β-unsaturated amide groups or the corresponding epoxide groups. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect.

Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrix secretion and deposition by affecting microtubule dynamics.

Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to ‘block –and-track’ enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.

Synthesis and evaluation of novel dipeptide-bound 1,2,4-Thiadiazoles as irreversible inhibitors of guinea pig liver transglutaminase

Herein we report the synthesis and evaluation of 14 novel peptides as potential irreversible inactivators of guinea pig liver transglutaminase (TGase). These peptides were designed to resemble Cbz-L-Gln-Gly, known to be a good TGase substrate, and to include a 1,2,4-thiadiazole group. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 0.330 to 0.89 microM(-1) min(-1).

Inhibitors of tissue transglutaminase

Tissue transglutaminase (TG2) catalyzes the cross-linking of proteins by the formation of isopeptide bonds between glutamine (Gln) and lysine (Lys) side chains. Although TG2 is essential for the stabilization of the extracellular matrix, its unregulated activity has been implicated in celiac disease, fibrosis, and cancer metastasis, among other disorders. Given the importance and range of TG2-related pathologies, recent work has focused on the development of potent and selective inhibitors against TG2. In this review, we present the latest and most noteworthy irreversible and reversible inhibitors of TG2, and offer perspectives for the design of future inhibitors, in the hope that lead compounds with therapeutic potential may soon be discovered.

Tissue transglutaminase acylation: Proposed role of conserved active site Tyr and Trp residues revealed by molecular modeling of peptide substrate binding

Transglutaminases (TGases) catalyze the cross‐linking of peptides and proteins by the formation of γ‐glutamyl‐ε‐lysyl bonds. Given the implication of tissue TGase in various physiological disorders, development of specific tissue TGase inhibitors is of current interest. To aid in the design of peptide‐based inhibitors, a better understanding of the mode of binding of model peptide substrates to the enzyme is required. Using a combined kinetic/molecular modeling approach, we have generated a model for the binding of small acyl‐donor peptide substrates to tissue TGase from red sea bream. Kinetic analysis of various N‐terminally derivatized Gln‐Xaa peptides has demonstrated that many CBz‐Gln‐Xaa peptides are typical in vitro substrates with KM values between 1.9 mM and 9.4 mM, whereas Boc‐Gln‐Gly is not a substrate, demonstrating the importance of the CBz group for recognition. Our binding model of CBz‐Gln‐Gly on tissue TGase has allowed us to propose the following steps in the acylation of tissue TGase. First, the active site is opened by displacement of conserved W329. Second, the substrate Gln side chain enters the active site and is stabilized by hydrophobic interaction with conserved residue W236. Third, a hydrogen bond network is formed between the substrate Gln side chain and conserved residues Y515 and the acid‐base catalyst H332 that helps to orient and activate the γ‐carboxamide group for nucleophilic attack by the catalytic sulphur atom. Finally, an H‐bond with Y515 stabilizes the oxyanion formed during the reaction.

Microbial transglutaminase displays broad acyl-acceptor substrate specificity

The great importance of amide bonds in industrial synthesis has encouraged the search for efficient catalysts of amide bond formation. Microbial transglutaminase (MTG) is heavily utilized in crosslinking proteins in the food and textile industries, where the side chain of a glutamine reacts with the side chain of a lysine, forming a secondary amide bond. Long alkylamines carrying diverse chemical entities can substitute for lysine as acyl-acceptor substrates, to link molecules of interest onto peptides or proteins. Here, we explore short and chemically varied acyl-acceptor substrates, to better understand the nature of nonnatural substrates that are tolerated by MTG, with the aim of diversifying biocatalytic applications of MTG. We show, for the first time, that very short-chain alkyl-based amino acids such as glycine can serve as acceptor substrates. The esterified α-amino acids Thr, Ser, Cys, and Trp—but not Ile—also showed reactivity. Extending the search to nonnatural compounds, a ring near the amine group—particularly if aromatic—was beneficial for reactivity, although ring substituents reduced reactivity. Overall, amines attached to a less hindered carbon increased reactivity. Importantly, very small amines carrying either the electron-rich azide or the alkyne groups required for click chemistry were highly reactive as acyl-acceptor substrates, providing a robust route to minimally modified, “clickable” peptides. These results demonstrate that MTG is tolerant to a variety of chemically varied natural and nonnatural acyl-acceptor substrates, which broadens the scope for modification of Gln-containing peptides and proteins.

Using FLIM-FRET to Measure Conformational Changes of Transglutaminase Type 2 in Live Cells

Transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family, capable of mediating a transamidation reaction between a variety of protein substrates. TG2 also has a unique role as a G-protein with GTPase activity. In response to GDP/GTP binding and increases in intracellular calcium levels, TG2 can undergo a large conformational change that reciprocally modulates the enzymatic activities of TG2. We have generated a TG2 biosensor that allows for quantitative assessment of TG2 conformational changes in live cells using Förster resonance energy transfer (FRET), as measured by fluorescence lifetime imaging microscopy (FLIM). Quantifying FRET efficiency with this biosensor provides a robust assay to quickly measure the effects of cell stress, changes in calcium levels, point mutations and chemical inhibitors on the conformation and localization of TG2 in living cells. The TG2 FRET biosensor was validated using established TG2 conformational point mutants, as well as cell stress events known to elevate intracellular calcium levels. We demonstrate in live cells that inhibitors of TG2 transamidation activity can differentially influence the conformation of the enzyme. The irreversible inhibitor of TG2, NC9, forces the enzyme into an open conformation, whereas the reversible inhibitor CP4d traps TG2 in the closed conformation. Thus, this biosensor provides new mechanistic insights into the action of two TG2 inhibitors and defines two new classes based on ability to alter TG2 conformation in addition to inhibiting transamidation activity. Future applications of this biosensor could be to discover small molecules that specifically alter TG2 conformation to affect GDP/GTP or calcium binding.