Current Enzyme Inhibition - Volume 2, Issue 1, 2006

It is known that peroxidase oxidation of nitrite to toxic products is one of the basic mechanisms of its toxicity. Therefore, the ratio between activities of hydrogen peroxide metabolized enzymes and the impact of nitrite on these enzymes is an important factor determining its toxicity. By use of calorimetric method developed by us that makes possible to control H2O2 destroying kinetics and distinguish different pathways of its destruction, we established that nitrite even in micromolar concentrations significantly decreases the ability of catalase to destroy hydrogen peroxide in the presence of chloride, bromide and thiocyanate, but not without them. Also it was established that this decrease is the result of reversible inhibition of the enzyme without any change in the essence of H2O2 destroying pathway. Mechanisms of the inhibition are discussed. Heme-containing peroxidases, such as horseradish peroxidase, lactoperoxidase and methemoglobin, manifested multiple higher stability towards nitrite inhibition than catalase does independently without halides and thiocyanate. Therefore, the presence of Cl-, Br- and SCN- in nitrite, catalase- and peroxidasecontaining systems should lead to increase the peroxidase pathway of H2O2 destroying and, consequently, the peroxidase oxidation of nitrite to a toxic product (probably NO2 •). This finding was demonstrated in the example of nitrite-induced hemoglobin oxidation. Low anion concentration in intracellular media and also the presence of effective nitrite competitors for peroxidases appear to be the most important mechanism of cell protection from nitrite toxicity.

Phospholamban (PLB) is a major target of β-adrenergic signaling, whose phosphorylation results in enhanced rates of relaxation in the heart. Prior to phosphorylation, PLB functions to reduce the calcium sensitivity of the Ca-ATPase, resulting in slower rates of calcium resequestration into the sarcoplasmic reticulum after each contractile event. Recent structures indicate that the inhibitory interaction between PLB and the Ca-ATPase requires PLB to assume an extended structure, where the transmembrane and cytosolic portions of PLB undergo specific binding interactions with distant sites on the Ca-ATPase. In the extended conformation, PLB binding to the Ca-ATPase functions to inhibit the Ca-ATPase through a reduction in the rates of catalytically important motions involving the nucleotide binding domain. Phosphorylation of PLB at either Ser16 or Thr17 releases the inhibitory interaction between PLB and the Ca-ATPase. These sites of phosphorylation are within a hinge region in PLB that separates the highly structured transmembrane and cytosolic portions that associate with the Ca-ATPase. The helical content of the hinge region increases following the phosphorylation of PLB, which induces a shortening of the maximal dimensions of PLB and a release of the inhibitory interaction with the Ca-ATPase. Following phosphorylation, PLB remains associated with the Ca-ATPase in a more compact form that has no inhibitory capability. Thus, the conformational switch involving PLB regulation of the Ca-ATPase relies upon a physical mechanism, whereby the phosphorylationdependent stabilization of the structure of PLB functions to destabilize the inhibitory interaction between PLB and the Ca-ATPase. Upon hydrolysis of the phosphoester linkages by endogenous phosphatases, PLB is poised to reassume the inhibited state through re-association with inhibitory sites on the nucleotide binding domain of the Ca-ATPase.

Endo-(1,4)-β-D-xylanases of plant and microbial origin play an important role in the degradation of arabinoxylan from plant cell wall. To date, two distinct types of xylanase inhibitors, the TAXI (Triticum aestivum xylanase inhibitor) and XIP (Xylanase inhibitor protein) types have been identified in cereals (rye, barley, maize, rice, durum and bread wheat). TAXI inhibits fungal and bacterial xylanases from glycoside hydrolase (GH) family 11 (GH11) whereas XIP inhibitors display species selectivity for both GH10 and GH11 xylanases. The evolution and biological role of the xylanase inhibitors are discussed in the light of the features which have recently become available by resolution of the crystal structures of the inhibitors isolated from wheat, XIP-I and TAXI-I, free and in complex with target xylanases.

The Na/K ATPase, or Na/K pump, is an enzyme converting chemical energy of ATP hydrolysis into the energy of electrochemical sodium and potassium gradient that is used to maintain cellular volume, pH and Ca2+ levels. Apart from maintaining the cellular Na+ and K+ concentrations, Na/K ATPase is involved in signal transduction including activation of mitogenic and proliferative signalling cascades. Changes in the Na/K ATPase activity have an impact on cellular metabolic and redox state, since it controls mitochondrial reactive oxygen species (ROS) levels and ATP utilisation rates. Na/K ATPase is known to be oxygen- and redoxsensitive. Alterations in the Na/K pump activity in response to hypoxia-reoxygenation or shifts in cellular and environmental redox state play an important role in pathological and adaptive responses of cells to hypoxia or ischemia. Despite intensive studies, the mechanisms involved in redox- and oxygen-induced regulation of the Na/K ATPase remain largely unknown. The present review focuses on the possible targets of action of oxygen, oxidants and reductants on the Na/K ATPase and mechanisms of oxygen- and redox-sensitivity of the enzyme at the molecular and cellular levels. Finally, we also consider factors involved in systemic responses to hypoxia that influence activity of the transporter as well as potential physiological and pathological outcome of such changes.

Phosphorus plays a fundamental role in cell since it is a part of many biomolecules and biological metabolites which include phospholipids, nucleic acids, proteins, polysaccharides or nucleotide cofactors. Phosphorus is most commonly found under its highest oxidized state as in orthophosphate or phosphate esters. However, there are also examples of naturally occurring molecules bearing phosphorus atoms at lower oxidized state that contain one carbon to phosphorus (P-C) bond. These compounds, so-called phosphonates, are much more resistant to chemical and enzymatic hydrolysis, thermal decomposition and photolysis than phosphates. The first example of a natural phosphonate, 2-aminoethylphosphonic acid (2-AEP), was reported in 1959 and shown to be a constituent of lipids, proteins and polysaccharides. Since the beginning of the 20th century, with the discovery of the Arbuzov reaction, synthetic phosphonates have been developed which have applications as therapeutic agents (e.g. antibiotics, anti-viral, or trypanocidal drugs) as well as insecticides and herbicides. Recently, phosphonate derivatives have been extensively used as enzyme inhibitors due to their specific structural and electronic properties. First, since they differ from phosphates by the single substitution of one oxygen atom with a carbon atom, phosphonates have been widely used as isosteric mimics of phosphates in the design of analogues of enzyme substrates or cofactors. They offer the advantage of a much greater stability to hydrolysis and resistance to proteases than phosphates and therefore can be used to mimic highly chemically unstable phosphates like carboxy-phosphates. Such a strategy was shown successful for generating competitive inhibitors of glycolytic enzymes (triose/hexose phosphate analogues), phosphatases or viral DNA polymerases (phosphonate nucleotides), with applications as chemotherapeutic agents against trypanosomiasis, hepatitis B or human immunodeficiency virus. Moreover, introduction of one or two fluorine atoms on the α-methylene group was also used to provide a better mimic of phosphate geometric and electronic properties by lowering the pKa of the phosphonate and increasing its P-Cα-Cβ dihedral angle. Phosphonic acids have also been extensively studied as enzyme inhibitors mimicking tetrahedral transition states. They proved to be potent competitive inhibitors of peptidases as mimics of the tetrahedral gem-diolate transition state of peptide bond hydrolysis, and of lipases, as mimics of configuration and charge distribution of the first transition state in triglyceride hydrolysis. This chapter summarizes the most recent studies on phosphonate-based enzyme inhibitors with special emphasis on isosteric analogues of phosphorylated substrates and mimics of tetrahedral transition states for peptide bond hydrolysis.

Obesity has reached epidemic proportions worldwide and is now a major healthcare problem. Likewise, there are a number of cardiovascular risk factors and metabolic factors associated with obesity and this clustering contributes to the disease known as metabolic syndrome or syndrome X. Metabolic syndrome over a number of years can cause end organ damage resulting in morbidity and mortality. Metabolic syndrome and obesity are major contributing factors to the increase in nephropathy and end stage renal disease. Interestingly, an imbalance between cyclooxygenase-2 (COX-2) and cytochrome P450 (CYP450) enzymes in the kidney may contribute to the nephropathy associated with metabolic syndrome. Recent studies have demonstrated that COX-2 inhibition decreases renal cytokine levels and glomerular injury in obese rats. Therefore, COX-2 and CYP450 metabolites are therapeutic targets for the treatment of renal disease related to metabolic syndrome.

Diabetes mellitus type 2 is a metabolic disease associated with chronic hyperglycaemia, which leads to a wide range of complications, including microvascular and macrovascular alterations, retinopathy, nephropathy and renal disease or peripheral neuropathy. Several intracellular pathways have been shown to be associated to type 2 diabetes mellitus, ranging from an altered insulin receptor-associated signalling to an abnormal intracellular calcium homeostasis or disturbances in Na+ handling. A number of diabetes-associated complications have been reported to be associated with hyperactivity of certain protein tyrosine kinases, such as those cytosolic kinases of the Src family, involved in the altered intracellular calcium mobilisation and platelet-derived cardiovascular problems and in glomerular injury, or the JAK family of tyrosine kinases involved in hyperglycaemia-induced renal failure. There has been a considerable effort in several laboratories to identify suitable targets for the design of drugs against this disease. The development of tyrosine kinase inhibitors suitable for medical purposes might represent a significant advance in the therapy of complications associated to type 2 diabetes mellitus.

Histone deacetylase (HDAC) inhibitors are now widely recognized as powerful anticancer agents. Unlike conventional chemotherapeutics, they not only inhibit tumor growth and survival but also reinitiate differentiation processes. Surprisingly, little attention has been paid so far to their capacity as differentiating agents for primary non-malignant cells. Dedifferentiation, however, is a common pernicious event occurring in almost all primary epithelial cell cultures and limits their use in fundamental and applied research. In this review, we elaborate on the innovative concept of HDAC inhibition as a key tool in the development of functional long-term cultures of primary hepatocytes. The need for long-term hepatocyte-based in vitro models is high and underscored by the fact that (sub)-chronic liver toxicity testing still consumes an important number of animals and by recent changes in EU legislation (e.g. chemicals policy REACH and cosmetics Directive 2003/15/EC). In addition, many cell-based therapies would benefit from the availability of highly differentiated primary hepatocytes as well. We believe that the HDAC inhibition approach could be applicable to create stable, differentiated culture systems of other primary cell types as well. In addition, HDAC inhibitors, in combination with tissue-specific growth factors, can be used to generate tissue-specific cell types from stem cells.