Current Enzyme Inhibition - Volume 1, Issue 3, 2005

A review of methods based on enzyme inhibition in environmental monitoring is presented, which is mainly focussed on the detection of pollutants, such as pesticides, heavy metal ions and other toxic compounds. The sensing principles, design of the biosensor and an evaluation of biosensors structures (i.e. electrochemical, optical, piezoelectric and calorimetric) that have been developed for environmental monitoring are also emphasised. Finally, future prospects of the enzyme based biosensor are discussed, particularly in term of novel strategies required for application to the real samples.

Nitro compounds are used in various industries, including the pharmaceutical industry. These compounds have considerable importance for human health for two distinctly different reasons. Some nitro compounds are toxic environmental contaminants and pose a serious threat to human health; others are used as therapeutic agents. Nitro compounds are used as agents for the treatment of infectious diseases caused by Grampositive and Gram-negative bacteria, as antifungal and antiparasitic drugs, and as chemotherapeutic agents. To exert either beneficial or adverse biological effects, nitro compounds require enzymatic activation, either by nitroreduction alone or by ring oxidation followed by nitroreduction. The mechanism of drug action relies on the toxicity of the products generated by nitroreductases. Nitroreductases differ in the range of specificity and levels of activity with various nitro prodrugs. In this review, factors affecting nitroreductase activity, especially compounds that inhibit reduction, will be discussed.

Nitric oxide (NO) exerts its action in several physiological and pathological events. The great propensity for Cys(NO)-(de)nitrosylation represents a mechanism which modulates cysteine protease action. Cys(NO)-(de)nitrosylation is assisted by basic and acid residues, within the environment of the Cys catalytic residue. In particular, Cys-nitrosylation is catalyzed by amino acid residues which stabilize the reactive deprotonated form of the Cys Sγ atom. By contrast, CysNO-denitrosylation is assisted by amino acid residues which facilitate the protonation of the Cys Sγ atom with the concomitant NO release. Note that Cysnitrosylated residues may undergo oxidation giving rise to sulfenic, sulfinic or sulfonic acid and lead to the formation of disulfide bridges. These structural consensus rules apply not only to cysteine proteases, but represent a generally accepted mechanism for (macro)molecular Cys(NO)-(de)nitrosylation.

Diabetes mellitus rate is rising worldwide and is now recognized as the most common cause of endstage renal disease and blindness among adults, and a major risk factor for cardiovascular disease. Diabetics present more extensive atherosclerosis, and particularly, the type 2 diabetes mellitus patients are often obese, hypertensive, and dyslipidaemic subjects, sharing the criteria of the metabolic syndrome. Nowadays, the reninangiotensin system (RAS) can be considered a cascade of peptide hormones which play an important role in the development of several cellular and hemodynamic alterations among diabetics. Diabetes mellitus is now considered an inflammatory disease, since its origin in either type 1 or type 2, and there is growing evidence that insulin resistance, nephropathy, increased thrombotic risk, and endothelial dysfunction are all linked to RAS activation. In recent years, several studies showed the clear efficacy of angiotensin-converting enzyme inhibitors and angiotensin receptor blocking agents in reducing vascular complications and metabolic disorders. However, the precise role of the RAS blockade, including aldosterone inhibition, in modifying the morbidity and mortality associated with diabetes, is waiting for large, randomized and controlled clinical trials.

Ischemia/reperfusion injury is unavoidable during cardiopulmonary bypass surgery because the surgery is conducted during ischemic arrest of the heart. Animal studies have shown that the administration of angiotensin converting enzyme (ACE) inhibitors can protect against lethal arrhythmias, preserve ventricular function, and improve coronary reserve after ischemia/reperfusion. Two factors to consider when using ACE inhibitors are their temperature dependency (because cardiopulmonary bypass surgery is usually done under hypothermic conditions) and their direct effect on the bypass material. In this paper, we review studies of ACE inhibitors and discuss both their temperature dependency and their effect on bypass material, especially on the coating of the circuit tube and artificial lung. Finally, we explore the potential clinical applications of ACE inhibitors in cardiopulmonary bypass surgery, taking into consideration the findings of our own preliminary clinical study.

Several metals have been shown to be carcinogenic to humans and/or experimental animals. The mechanisms of their carcinogenicity are not completely understood, even if DNA repair inhibition seems to be the most probable. Current evidence suggests that DNA repair systems are very sensitive target for nickel (II), cadmium (II), cobalt (II) and arsenic (III) leading to a diminished removal of endogenous and exogenously induced DNA damage, which increase the risk of tumor formation. Metal compounds inhibit DNA repair probably by oxidative damage and direct interactions of metal-ions with reparative enzyme. Metals carcinogenic potential depends from their solubility and oxidation state. Oxidative DNA damage by metal compounds is due to ROS generation by Fenton-type reactions and inactivation of anti-oxidative enzymes. DNA repair inhibition seems to depend on the ability of metal ions to compete with magnesium ions or to displace zinc ions in zinc finger structures of DNA repair enzymes with consequent their inactivation. The DNA repair inhibition has been shown for low non-cytotoxic metal concentrations. DNA repair systems inhibited by metal compounds include essentially base-excision repair (BER) including Formamidopyrimidinglycosilase (Fpg)-oxidized base excision repair and nucleotide excision repair (NER). Several studies on metal inhibition activity use prevalently the comet test, a rapid and sensitive technique, to evaluate direct and oxidative DNA damage and its repair in human cells. Comet test consents to study the influence of metals on the different steps involved in DNA repair, evaluating the recovery of DNA damage induced by exposure to different genotoxic agents in absence or presence of metal compounds. This technique and its modifications can clarify the mechanisms of inhibitory processes of enzymes involved in carcinogenesis and could be a useful tool in the development of new anticancer drugs.

Somatostatin (SRIF) is a cyclic peptide widely distributed throughout the body with important physiological effects (mostly inhibitory) on several organ systems. SRIF may act as a neurohormone, neurotransmitter, neuromodulator or as a local factor, and exhibits potent antiproliferative activity. SRIF effects have formed the basis for the clinical use of SRIF analogues in the treatment of endocrine tumours, acromegaly and gastrointestinal disorders. Several data suggest that SRIF may also be a therapeutic target in a number of different diseases. The binding of SRIF to its five G-protein coupled receptors leads to modulation of multiple transduction pathways, including adenylyl cyclase, guanylyl cyclase, phospholipase C, K+ and Ca2+ channels, phospholipase A2, nitric oxide, Na+/H+ exchanger, protein phosphatases and MAP kinases. The diversity of the transduction pathways reflects the pleiotropic actions of SRIF. However, our current understanding depicts a rather complicated picture and conflicting results have also been reported. Data are mostly based on in vitro experiments, and parallels with the real in vivo conditions are not so obvious. Due to the clinical relevance of the SRIF system, the elucidation of the intracellular role of endogenous SRIF receptors may offer new therapeutic perspectives. These will enable development of specific pharmacological signalling modulators which can be incorporated into the therapeutic arsenal. The present review represents a detailed and exhaustive summary which covers the latest advances in the transduction pathways of SRIF receptors.

Caspases are a family of aspartate-specific cysteine proteinases that play a critical role in the execution phase of apoptosis. They are normally present in cells in a proenzyme form that requires limited proteolysis for enzymatic activity. The processing of caspases needs the reduction of a cysteine residue as well as other cysteine residues around the catalytic site for enzymatic activity and dimerization via sulfhydryl groups. Therefore, both processing and activity are inhibited in the presence of an excess of an oxidant such as hydrogen peroxide or nitric oxide and thiol-oxidizing agents, and enhanced in the presence of an anti-oxidant such as dithiothreitol or glutathione. On the other hand, reactive oxygen species (ROS) released from mitochondria decrease the concentration of cellular glutathione during apoptosis. Under physiological conditions, ROS are needed for survival. The redox valance decides the fate of cells, survival or death involving apoptosis and necrosis. In this review, we discussed the relationship between the redox control of caspase activity and cell death.

Very little is known about the numerous causes and molecular mechanisms of aneuploidy in any cell type. However, recent data suggest that alterations in proteasome activity may be one of several potential mechanisms that predispose cells to faulty chromosome segregation. Proteasomes represent multicatalytic 26S proteases consisting of a 20S central catalytic core bordered by two 19 S components that hydrolyze the Cterminal peptide bonds to acidic, basic, and hydrophobic amino acid residues of unwanted proteins in eukaryotic cells. Key cell-cycle regulatory proteins such as cyclins, cyclin-dependent kinase inhibitors, and anaphase-inhibitory proteins that have been ubiquinated by the anaphase promoting complex are subject to degradation by proteasomes. An association between proteasomes and chromosome segregation exists because proteasomes are responsible for proteolyzing subunits of the securin-cohesin protein complex which helps bind homologues together prior to meiotic anaphase I and sister chromatids during meiosis II and mitosis. This association suggests that if cohesins are not timely removed from chromosomes at anaphase onset, the probability of abnormal chromosome segregation will be increased. Recent data obtained from fission yeast and rodent cells exposed to proteasome inhibitors have revealed that faulty proteasome activity can lead to aberrant chromosome segregation. Certain peptide aldehydes that function as transition-state analogues can readily enter cells and inhibit 26S proteasome activity. The ability to alter the function of the proteasome/ubiquitin pathway in regulating cell-cycle control, chromosome segregation, and apoptosis provides a venue for addressing aneuploidy and underlies the current interest in the use of proteasome inhibitors as chemotherapeutic agents.

As shown in several screenings, cyanobacteria (blue-green algae) are sources of very interesting metabolites, many of which possess significant biological activities [1]. Cyanopeptides, as a rule, metabolites of a peptidic nature, show in addition to hepato- and neurotoxic properties, a broad spectrum of biological activities, including antitumour [2], immunosuppressive [3] and antimicrobial effects [4], as well as angiotensin-converting enzyme inhibitory action [5] and cardioactive effects [6]. Many of the isolated, non-toxic compounds inhibit serine proteases, which play a central role in the human organism. Trypsin-like serine proteases (e.g. thrombin, plasmin, factor Xa, t-PA, or tryptase) are the leading factors in blood coagulation, fibrinolysis, kinin-kallikrein and complement systems as well as in inflammatory processes. Both the kinin-kallikrein and complement systems are thought to be closely related to inflammation and immune reactions. Failures of one or more of these enzymes may cause a state of imbalance between protease and antiprotease (endogenous protease inhibitors) and may lead to an excess of proteolytic activity and to the development of diseases such as thrombosis, heart failures, further thromboembolic events, asthma, multiple sclerosis and pancreatitis. The discovery and development of oral inhibitors of the above mentioned enzymes therefore presents a notable measure for improving the treatment of these disorders and remains a challenge for each medicinal chemist. This paper reviews the low-molecular weight, serine protease inhibitory cyanopeptides published over the last decade and reports on actual efforts and developments in establishing structure-activity relationships concerning the inhibition of trypsin-like serine proteases.

Lipoxygenases (LOs) constitute a family of dioxygenases containing one non-heme iron atom per molecule, which oscillates between Fe2+ (inactive enzyme) and Fe3+ (active form) during the catalytic cycle. They catalyze the oxygenation of polyunsaturated fatty acids containing a (1Z, 4Z)-penta-1,4-diene system to the corresponding hydroxyperoxy derivatives. Arachidonic acid, which contains four double bonds in its configuration, is metabolised via lipoxygenases to a number of products with the hydroperoxy group in different positions. The LOs family includes several members which have been identified according the recommendations of the nomenclature committee of the international union of biochemistry and molecular biology on the nomenclature and classification of the enzymes by inserting oxygen into the 5-, 8-, 9-, 11-, 12- and 15- positions of fatty acids with varying stereoconfiguration (S or R). Four of these (5-LOs, 8-LOs, 12- LOs, 15-LOs) have been discovered in mammals to date. Although the cyclooxygenases could be considered specialized in the arachidonate pathway, the detailed mechanism of the LO reaction still remains controversial. It has been found that LOs are implicated in several processes such as cell differentiation, inflammation, carcinogenesis. Development of drugs that interfere with the formation or effects of these metabolites would be important for the treatment of various diseases like asthma, psoriasis, ulcerative colitis, rheumatoid arthritis, atherosclerosis, cancer and blood vessel disorders. Asthma consists the only pathological case in which improvement has been shown by LO inhibitors. The most widely studied isoform of the enzyme is 5-LO, which is involved in the biosynthesis of potent inflammatory mediators. Accordingly research efforts have been directed towards the development of drugs that interfere with the formation of leukotrienes. Zileuton (Zyflo) is the only 5-LO inhibitor on the market in the USA for the treatment of asthma.

Diabetic retinopathy and diabetic macular edema (DME) remain important causes of visual loss worldwide despite the adoption of intensive glycemic control for the management of diabetes. Several interacting and mutually perpetuating abnormal biochemical systems, such as non-enzymatic glycation, protein kinase C (PKC) β activation, the polyol pathway, and oxidative stress, may be activated by sustained hyperglycemia in patients with diabetes. These abnormal systems may in turn influence several vasoactive factors, which are probably instrumental in the development of functional and morphological changes in the retina. Among the known vasoactive factors, vascular endothelial growth factor (VEGF), angiotensin II, and interleukin-6, are thought to be important in mediating the functional and structural alterations that occur in diabetic retinopathy and DME. Complex and interacting regulatory mechanisms, as well as enzyme inhibition, which are included advanced glycation end product inhibitor, PKC β inhibitor, aldose reductase inhibitor, antioxidant, angiotensin converting enzyme inhibitor, angiotensin II type 1 receptor blocker, and anti-VEGF, may modulate the ability of these molecules to produce biologically significant effects. Better understanding of these factors and their interactions should be of assistance in the development of new therapies for the treatment of diabetic retinopathy and DME.