Transglutaminase-catalyzed reactions responsible for the pathogenesis of celiac disease and neurodegenerative diseases: from basic biochemistry to clinic

Transglutaminases (TGases) are enzymes which catalyze the cross linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate with the formation of an N-gamma-(epsilon-L-glutamyl)-L-lysine [GGEL] cross link (isopeptidic bond) and the concomitant release of ammonia. Such cross-linked proteins are often highly insoluble. The TGases are closely related enzymes and can also catalyze other important reactions for cell life. Recently, several findings concerning the relationships between the biochemical activities of the TGases and the basic molecular mechanisms responsible for some human diseases, have been reported. For example, some neurodegenerative diseases, such as Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), supranuclear palsy, etc., are characterized in part by aberrant cerebral TGase activity and by increased cross-linked proteins in affected brains. Our article describes the biochemistry and the physio-pathological roles of the TGase enzymes, with particular reference to human pathologies in which the molecular mechanism of disease can be due to biochemical activities of the tissue TGase enzyme (tTGase, type 2), such as in a very common human disease, Celiac Disease (CD), and also in certain neuropsychiatric disorders.     

Transglutaminases - possible drug targets in human diseases

Transglutaminases (TGases) belong to a family of closely related proteins that catalyze the cross linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate with the formation of an Nepsilon-(gamma-L-glutamyl)-L-lysine [GGEL] cross link and the concomitant release of ammonia. Such cross-linked proteins are often highly insoluble. Neurodegenerative diseases, such as Alzheimer disease (AD), Parkinson disease (PD), supranuclear palsy and Huntington disease (HD), are characterized in part by aberrant cerebral TGase activity and by increased cross-linked proteins in affected brain. In support of the hypothesis that TGases contribute to neurodegenerative disease, a recent study shows that knocking out TGase 2 in HD-transgenic mice results in increased lifespan. Moreover, recent studies show that cystamine, an in vitro TGase inhibitor, prolongs the lives of HD-transgenic mice. However, these findings are not definitive proof of TGase involvement in HD neuropathology. In neurodegenerative diseases, the brain is under oxidative stress and cystamine can theoretically be converted to the potent antioxidant cysteamine in vivo. Cystamine is also a caspase 3 inhibitor. In addition to neurodegenerative diseases, aberrant TGase activity is associated with celiac disease. Interestingly, a subset of celiac patients develops neurological disorders. This review focuses on the strategies that have been recently employed in the design of TGase inhibitors, and on the possible therapeutic benefits of selective TGase inhibitors to patients with neurodegenerative disorders or to patients with celiac disease.     

Transglutaminase inhibitors: a patent review

Introduction: Transglutaminases (TGases) are a class of enzymes that play multifunctional roles. Their protein-crosslinking activity has been linked to fibrosis and Huntington’s disease, their glutamine deamidation activity has been related to celiac disease and their GTP-binding activity has been implicated in cancer. All of these physiological disorders have prompted the development of inhibitors, which has accelerated dramatically over the past decade.

Areas covered: This review presents an overview of TGase inhibitors published in the patent literature, from the first compounds developed in the late 1980’s, to the current date. This article is focussed on the chemical structure of new inhibitors and their probable mechanism of action.

Expert opinion: Comparison of effective TGase inhibitors reveals common structural features that may guide future design. Many of these elements are embodied in the first TGase inhibitor to recently enter into clinical trials.     

Assay of transglutaminase activity by electrophoretic removal of the unreacted monodansyl cadaverine

Transglutaminases (TGases) catalyze the transfer of acyl groups between the γ-carboxyamide group of a glutamine residue and a primary amine. Rapid and precise determination of TGase activity is an important issue because improper function of TGases has been suggested to be associated with a variety of diseases. There have been tremendous efforts to develop the TGase assay methods to be more rapid, convenient and accurate. In the conventional assay method, fluorescence-tagged amine molecules such as monodansyl cadaverine (MDC) are coupled with casein by the action of transglutaminase. The removal step of unreacted MDC would require time-consuming work-up processes such as acid-precipitation and centrifugation. These processes would also interrupt the precise measurements of enzymatic activities. In this study, we have developed a new fluorometric assay methods to assay transglutaminase activity based on electrodialysis where the unreacted MDC is removed by electrophoresis. We have found the optimized condition to remove the unreacted MDC while preserving the β-casein protein. We also found the linear relationship between fluorescence intensity associated with β-casein and TGase can maintain in the range of 0–1.6 mU as well as 0–0.4 mU. The results show us as few as 0.1 mU of TGase could be detected by this method.     

Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase

Transglutaminase (TGase, E.C. 2.3.2.13) catalyzes acyl transfer reactions between the gamma-carboxamide groups of protein-bound glutamine (Gln) residues, which serve as acyl donors, and primary amines, resulting in the formation of new gamma-amides of glutamic acid and ammonia. By using an amino-derivative of poly(ethylene glycol) (PEG-NH(2)) as substrate for the enzymatic reaction with TGase it is possible to covalently bind the PEG polymer to proteins of pharmaceutical interest. In our laboratory, we have conducted experiments aimed to modify proteins of known structure using TGase and, surprisingly, we were able to obtain site-specific modification or PEGylation of protein-bound Gln residue(s) in the protein substrates. For example, in apomyoglobin (apoMb, myoglobin devoid of heme) only Gln91 was modified and in human growth hormone only Gln40 and Gln141, despite these proteins having many more Gln residues. Moreover, we noticed that these proteins suffered highly selective limited proteolysis phenomena at the same chain regions being attacked by TGase. We have analysed also the results of other published experiments of TGase-mediated modification or PEGylation of several proteins in terms of protein structure and dynamics, among them alpha-lactalbumin and interleukin-2, as well as disordered proteins. A noteworthy correlation was observed between chain regions of high temperature factor (B-factor) determined crystallographically and sites of TGase attack and limited proteolysis, thus emphasizing the role of chain mobility or local unfolding in dictating site-specific enzymatic modification. We propose that enhanced chain flexibility favors limited enzymatic reactions on polypeptide substrates by TGases and proteases, as well as by other enzymes involved in a number of site-specific post-translational modifications of proteins, such as phosphorylation and glycosylation. Therefore, it is possible to predict the site(s) of TGase-mediated modification and PEGylation of a therapeutic protein on the basis of its structure and dynamics and, consequently, the likely effects of modifications on the functional properties of the protein.     

Calpains as potential anti-cancer targets

Introduction: The intracellular signaling cysteine proteases, calpains (specifically the ubiquitous calpains 1 and 2), are involved in numerous physiological and pathological phenomena. Several works have highlighted the implication of calpains in processes crucial for cancer development and progression. For these reasons, calpains are considered by several authors as potential anti-cancer targets.

Areas covered: How calpains are implicated in cancer formation and development, how these enzymes are deregulated in cancer cells and how these proteases could be targeted by anti-cancer drugs. Studies published in the last 10 years are focused on.

Expert opinion: Targeting calpain activity with specific inhibitors could be a novel approach to limiting development of primary tumors and formation of metastases, by inhibiting tumor cell migration and invasion, which allows dissemination as well as tumor neovascularization, which in turn allows expansion. However, such drugs could interfere with anti-cancer treatments, as ubiquitous calpains play crucial roles in chemotherapy-induced apoptosis. For these reasons, drugs targeting calpains would have to be used selectively to avoid interference with other treatments and physiological processes. Further studies will be required concerning the other members of the calpain family and their potential implication in cancer development before considering treatments targeting their activity.     

Synthesis and structure-activity relationship of alpha-sulfonylhydroxamic acids as novel,orally active matrix metalloproteinase inhibitors for the treatment of osteoarthritis

The matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that play a key role in both physiological and pathological tissue degradation. These enzymes are strictly regulated by endogenous inhibitors such as tissue inhibitors of MMPs and alpha(2)-macroglobulins. Overexpression of these enzymes has been implicated in various pathological disorders such as arthritis, tumor metastasis, cardiovascular diseases, and multiple sclerosis. Developing effective small-molecule inhibitors to modulate MMP activity is one approach to treat these degenerative diseases. The present work focuses on the discovery and SAR of novel N-hydroxy-alpha-phenylsulfonylacetamide derivatives, which are potent, selective, and orally active MMP inhibitors.     

Calpains as potential anti-cancer targets

INTRODUCTION: The intracellular signaling cysteine proteases, calpains (specifically the ubiquitous calpains 1 and 2), are involved in numerous physiological and pathological phenomena. Several works have highlighted the implication of calpains in processes crucial for cancer development and progression. For these reasons, calpains are considered by several authors as potential anti-cancer targets. AREAS COVERED: How calpains are implicated in cancer formation and development, how these enzymes are deregulated in cancer cells and how these proteases could be targeted by anti-cancer drugs. Studies published in the last 10 years are focused on. EXPERT OPINION: Targeting calpain activity with specific inhibitors could be a novel approach to limiting development of primary tumors and formation of metastases, by inhibiting tumor cell migration and invasion, which allows dissemination as well as tumor neovascularization, which in turn allows expansion. However, such drugs could interfere with anti-cancer treatments, as ubiquitous calpains play crucial roles in chemotherapy-induced apoptosis. For these reasons, drugs targeting calpains would have to be used selectively to avoid interference with other treatments and physiological processes. Further studies will be required concerning the other members of the calpain family and their potential implication in cancer development before considering treatments targeting their activity.     

孕烷X受体在肾脏疾病中的研究进展

核受体是一类在机体内广泛分布的转录因子，在人体生理、病理过程中发挥重要作用。孕烷X受体（pregnane X receptor，PXR）为核受体家族成员之一，参与机体物质代谢，尤其在药物代谢中起关键作用。肾脏是药物代谢的主要器官之一，PXR参与肾脏药物代谢、转运等多种调节并参与多种肾脏疾病的病理生理过程。近年来多项研究关注PXR及其调控作用对肾脏及肾脏疾病的影响。本文将从PXR在肾脏与药物代谢和转运相关酶的相互作用以及PXR在肾脏疾病中的作用等方面进行综述。     

The pharmacological role of phosphatases (acid and alkaline phosphomonoesterases) in snake venoms related to release of purines - a multitoxin

Snake venom components, acting in concert in the prey, cause their immobilization and initiate digestion. To achieve this, several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as phosphatases (acid and alkaline phosphomonoesterases) are less studied and their pharmacological role in venoms is not clearly defined. Also, they show overlapping substrate specificities and have other common biochemical properties causing uncertainty about their identity in venoms. The near-ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It appears that these enzymes may play a central role in liberating purines (mainly adenosine) - a multitoxin and through the action of purines help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities as venom enzymes have been evolved to interfere in diverse physiological processes. This has not been verified by pharmacological studies using purified enzymes. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established.     

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in the respiratory tract: potential implications in asthma and other lung diseases

In healthy lung, Matrix Metalloproteinases (MMPs) and their physiological inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs), are produced in the respiratory tract by a panel of different structural cells. These activities are mandatory for many physiological processes including development, wound healing and cell trafficking. Deregulation of proteolytic-antiproteolytic network and inappropriate secretion of various MMPs by stimulated structural or inflammatory cells is thought to take part to pathophysiology of numerous lung diseases including asthma, chronic obstructive pulmonary disease (COPD), lung fibrosis and lung cancer. Cytokines and growth factors are involved in these inflammatory processes and some of those mediators interact directly with MMPs and TIMPs leading either to a regulation of their expression or changes in their biological activities by proteolytic cleavage. In turn, cytokines and growth factors modulate secretion of MMPs establishing a complex network of reciprocal interactions. Every MMP seem to play a rather specific role and some variations of their expression are observed in different lung diseases. The precise role of these enzymes and their inhibitors is now studied in depth as they could represent relevant therapeutic targets for many diseases. Indeed, MMP inhibition can lead either to a decrease of the intensity of a pathological process or, in the contrary for some of them, to an increase of disease severity. In this review, we focus on the role played by MMPs and TIMPs in asthma and we provide an overview of their potential roles in COPD, lung fibrosis and lung cancer, with a special emphasis on loops including MMPs and cytokines and growth factors relevant in these diseases.     

Cytochrome P450 in the central nervous system as a therapeutic target in neurodegenerative diseases

Cytochromes P450 (CYPs) constitute a family of enzymes that can be found in the endoplasmic reticulum (ER), mitochondria or the cell surface of the cells. CYPs are characterized by carrying out the oxidation of organic compounds and they are mainly recognized as mediators of the biotransformation of xenobiotics to polar hydrophilic metabolites that can be eliminated from the organism. However, these enzymes play a key role in many other physiological processes, being involved in diverse indispensable metabolic pathways since they metabolize many endogenous substrates. Various CYP isoforms are expressed in the brain, and it is believed that this could be in part due to the particular function of brain CYPs. In the brain, CYPs are involved in the cholesterol turnover, the biosynthesis of dopamine, serotonin, morphine, hormones, and protective lipid mediators (epoxyeicosatrienoic acids), in addition to their already recognized role in xenobiotics detoxification and psychotropic drug metabolism. Increasing evidence suggests that this group of enzymes is fundamental for the normal functioning and maintenance of brain homeostasis. This review is focused on highlighting the importance of CYP-mediated endogenous metabolism in the central nervous system (CNS) and its relationship with recent findings regarding CYP involvement in neurodegenerative diseases. Some therapeutic approaches focused on CYP regulation are also discussed.     

Therapeutic perspectives of inhibitors of endocannabinoid degradation

The study of the cannabinoids can be established in the middle sixties with the elucidation of the structure of the active principle of Cannabis sativa plant, the delta9-tetrahydrocannabinol. However, the existence of an endogenous cannabinoid system (ECS) has not been unequivocally accepted until recently. The last two decades have witnessed an impressive advance in the knowledge about cannabinoids, their chemistry, the enzymes involved in their metabolism, and their physiological and pathological roles. In particular, we have made progress in modifying the activity of the ECS with selective compounds, validating the ECS as a new therapeutic target. Endocannabinoids play a role in physiological and pathological processes, and their levels are affected in several disorders. Therefore, it should be possible to ameliorate these pathologies by correcting their altered levels. This review focuses on the current therapeutic opportunities of endocannabinoid-directed drugs, and pays special attention to the therapeutic possibilities underlying the inhibition of the endocannabinoid inactivation. The strategy of manipulating the ECS might open new avenues in the development of therapeutic approaches for a number of disorders, both central and peripheral, that lack as yet effective treatments.     

低氧诱导因子-1的病理生理及相关药物研究进展

低氧诱导因子-1（hypoxia-inducible factors, HIF-1）是介导哺乳动物和人体细胞低氧适应性反应的主要核转录因子,是专一调节氧稳态的关键物质。 HIF-1对胚胎的正常发育,软骨及骨的形成等多种生理过程起保护及促进作用,也与肿瘤、糖尿病及其并发症等多种缺血低氧性疾病密切相关。 HIF-1在这些疾病中的分子机制已成为目前的研究热点,笔者就HIF-1的结构特征、调控机制、生物学效用及在药物研发等方面进行综述。     

Matrix metalloproteinases in asthma and COPD

Asthma and chronic obstructive pulmonary disease (COPD) are both highly prevalent, chronic inflammatory lung diseases that lead to significant morbidity and mortality. Matrix metalloproteinases (MMPs) are extracellular matrix degrading enzymes that play a critical role in normal development and physiological tissue remodeling and repair. In addition, they play an important role in the regulation of the kinetics and function of inflammatory cells. There is increasing evidence that MMPs are involved in the pathogenesis of both asthma and COPD, and several MMPs are possible therapeutic targets in these common chronic airway diseases.     

Enzymatic procedure for site-specific pegylation of proteins

We have developed a novel methodology for site-specific pegylation of proteins by use of transglutaminase (TGase). In this methodology, alkylamine derivatives of poly(ethyleneglycol) (PEG) could be site-specifically incorporated into intact or chimeric proteins without decreasing the bioactivities. The incorporation site of the TGase-catalyzed modification is limited to the substrate Gln residues for TGases. The high homogeneity of the constructed conjugates and the ability to design conjugates with suitable incorporation sites will improve the applicability of PEG-protein conjugates for clinical use.     

New perspectives for the development of selective metabotropic glutamate receptor ligands.

The metabotropic glutamate receptors are GTP-binding-protein (G-protein) coupled receptors that play important roles in regulating the activity of many synapses in the central nervous system. As such, these receptors are involved in a wide number of physiological and pathological processes. Within the last few years, new potent and selective agonists and antagonists as well as radioligands acting on these receptors have been developed. Molecular modeling studies revealed the structural features of the glutamate binding site, and will be useful for the design of more selective and potent ligands. More interestingly, recent data revealed new regulatory sites on the receptor protein, able either to decrease or potentiate the action of the endogenous ligand. No doubt that in the near future a multitude of new tools to modulate the activity of these receptors will be discovered, enabling the identification of the possible therapeutic applications for these new neuroactive molecules.     

Acute actions and novel targets of matrix metalloproteinases in the heart and vasculature

Matrix metalloproteinases (MMPs) have been shown to play significant roles in a number of physiological as well as pathological processes. Best known to proteolyse components of the extracellular matrix, MMPs have recently been discovered to also target a growing list of proteins apart from these, both inside and outside the cell. MMPs have also been traditionally thought of as enzymes involved in chronic processes such as angiogenesis, remodelling and atherosclerosis on a days-week time-scale. However they are now understood to also act acutely in response to oxidative stress on a minutes time-scale on non-extracellular matrix substrates. This review focuses on the acute actions and both extracellular and intracellular targets of two prominent MMP family members, MMP-2 and -9, in cardiovascular diseases including ischaemia/reperfusion injury, inflammatory heart disease, septic shock and pre-eclampsia. Also discussed are various ways of regulating MMP activity, including post-translational mechanisms, the endogenous tissue inhibitors of metalloproteinases and pharmacological inhibitors. A comprehensive understanding of MMP biology is necessary for the development of novel pharmacological therapies to combat the impact of cardiovascular disease.     

Progress in the development of selective nitric oxide synthase (NOS) inhibitors.

Nitric oxide (NO), a molecular messenger synthesized by nitric oxide synthase (NOS) from L-arginine and molecular oxygen, is involved in a number of physiological and pathological processes in mammalians. Three structurally distinct isoforms of NOS have been identified: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). Although NO mediates several physiological functions, overproduction of NO by nNOS has been reported in a number of clinical disorders including acute (stroke) and chronic (schizophrenia, Alzheimer s, Parkinson s and AIDS dementia) neurodegenerative diseases, convulsions and pain; overproduction of NO by iNOS has been implicated in various pathological processes including septic shock, tissue damage following inflammation and rheumatoid arthritis. On the contrary, NO produced by eNOS has only physiological roles such as maintaining physiological vascular tone. Accordingly, selective inhibition of nNOS or iNOS vs eNOS may provide a novel therapeutic approach to various diseases; in addition selective inhibitors may represent useful tools for investigating other biological functions of NO. For these reasons, after the identification of N-methyl-L-arginine (L-NMA) as the first inhibitor of NO biosynthesis, design of selective NOS inhibitors has received much attention. In this article the recent developments of new molecules endowed with inhibitory properties against the various isoforms of NOS are reviewed. Major focus is placed on structure-activity-selectivity relationships especially concerning compounds belonging to the non-amino acid-based inhibitors.     

Fyn kinase in brain diseases and cancer: the search for inhibitors

Fyn is a non-receptor tyrosine kinase belonging to the Src family kinases. It has been shown to play important roles in neuronal functions, including myelination and oligodendrocytes formation, and in inflammatory processes. It has also demonstrated its involvement in signaling pathways that lead to severe brain pathologies, such as Alzheimer's and Parkinson's diseases. Moreover, Fyn is upregulated in some malignancies. Experimental studies demonstrated that Fyn inhibition could be useful in the disruption of metabolic processes involved in cancer neurodegenerative diseases. Unfortunately, no specific Fyn inhibitor has been discovered till today, active compounds on other members of Src family or on different tyrosine kinases have also been reported. However, multitargeted inhibitors might be endowed with therapeutic potential. Indeed, as increasingly reported, also a not completely selective inhibitor of a specific protein could be therapeutically useful, affecting a number of cell pathways involved especially in cancer development. In this review, we report some examples of small molecule tyrosine kinase inhibitors for which data on Fyn inhibition, both in enzymatic and in cell assays, have been reported, with the aim of giving information as starting point for the researchers working in this field.