Proteolytic Enzyme Inhibitors Research Articles

Potential protease inhibitors based on a functionalized cyclic sulfamide scaffold.

Exploratory studies related to the design and synthesis of functionalized cyclic sulfamides (I) as potential inhibitors of proteolytic enzymes were carried out. The structural motif and three diversity sites embodied in the scaffold render it amenable to combinatorial parallel synthesis and the facile generation of lead discovery prospecting libraries. The scaffold was readily assembled starting with (DL) serine methyl ester, and a series of compounds was generated and screened against human leukocyte elastase. Modification of the P(1) recognition element, believed to be accommodated at the primary specificity site (S(1) subsite) of the enzyme, yielded compounds that inhibited the enzyme by an apparent hyperbolic partial mixed-type inhibition.

On the possible mechanism of enhanced absorption of oral immobilized insulin preparations

The mechanism of the hypoglycemic effect of insulin immobilized in a polymer hydrogel modified by glycoprotein, an inhibitor of proteolytic enzymes, has been investigated. This mechanism involves the quick passage of hydrogel particles through the stomach; the inhibition of insulin proteolysis is due to the neutralization of proteolytic enzymes by glycoprotein and an increase in the rate of insulin diffusion from the hydrogel particles through the membrane of the small intestine. The latter is associated with a specific reaction between the polysaccharide part of glyco-protein, which is chemically bound with hydrogel, and lectin, contained in the mucosal membrane of the intestine.

Water-soluble polymers with low critical solution temperature (LCST) as carriers for protein drug delivery

The copolymers of N,N-diethylacrylamide and ovomucoid from duck egg white (an inhibitor of proteolytic enzymes) with low critical solution temperature (LCST) have been synthesized. The behaviour of these copolymers at the point of phase transition was investigated. It was shown that the relation between LCST and physiological activity for these copolymers is a function of their composition. The increase of ovomucoid content leads to the increase of LCST. When the content of ovomucoid rises above 0.2 mol%, LCST disappears. At the same time the physiological activity of obtained copolymers decrease with increasing of N,N-diethylacrylamide content.

Role of gene therapy in tissue engineering procedures in rheumatology: the use of animal models

Tissue engineering is not only the application of cells and scaffolds to generate a new tissue but should also bring into play biological principles to guide cellular behavior. A way to modify cellular behavior is genetic modification of the cells used for tissue engineering (gene therapy). In the field of rheumatic diseases, cellular modification by overexpressing anabolic factors, such as insulin-like growth factor-I or transforming growth factor β, or inhibitors of catabolic cytokines or proteolytic enzymes can protect tissues form further destruction and stimulate tissue repair. To test the effect of transgenes on tissue engineering adequate test systems have to be available. Initial testing can be done in simple in vitro systems. However, animal models are unavoidable to study the interaction between the environment and tissue engineering. Optimal models to study gene therapy in combination with tissue engineering in the field of rheumatology are not available at this moment. Arthritis models are mainly developed in small animals while high-quality tissue engineering experiments ask for a large animal model. Development of animal models that can be used for tissue engineering experiments and mimic end stage arthritic diseases is needed.

Urinary trypsin inhibitor improves peripheral microcirculation and bronchospasm associated with systemic anaphylaxis in rabbits in vivo.

Using intravital microscopy of the rabbit ear for quantitative studies of microvascular dynamics, we examined the impact of urinary trypsin inhibitor (UTI), a proteolytic enzyme inhibitor, on microvascular changes during immune complex-mediated anaphylaxis. A total of 50 rabbits, previously sensitized with horse serum, were anesthetized and mechanically ventilated with pentobarbital and isoflurane for the intravital microscopy. Rabbits were then challenged with intravenous injection of horse serum to induce systemic anaphylaxis. One minute after the challenge, each rabbit was randomly assigned to receive saline (group C), 50,000 units x kg(-1) of UTI (group U1), or 150,000 units x kg(-1) (group U2). There were no statistical differences between hemodynamic variables, including heart rate (HR), mean arterial pressure (MAP), and central venous pressure (CVP), among the survivors in each treatment group. Peak inspiratory pressure rose in all three groups but at a much higher rate in group C (P < 0.05). In contrast with the moderate effects of UTI on the above parameters, microscopic evaluation revealed a substantial difference among treatment groups: upon the initiation of anaphylaxis, the arteriole started to reduce in diameter, but UTI prevented vasoconstriction in the arteriole in a dose-dependent manner. Similar results were observed with blood flow velocity. Because flow rate was calculated as the product of blood flow velocity and vascular cross-sectional area proportional to the square of the vessel diameter, these results indicate that UTI preserves microvascular flow rate during anaphylaxis. Rabbit ear microcirculation is highly preserved in the UTI-treated groups during systemic anaphylactic shock.

Levels of alpha1-antitrypsin in plasma and risk of spontaneous cervical artery dissections: a case-control study.

Abnormalities of dermal connective tissue have been detected in patients with spontaneous cervical artery dissections (sCAD), suggesting an underlying structural defect of the arterial wall. Alpha1-antitrypsin (A1-AT) is a circulating serine proteinase inhibitor of proteolytic enzymes that helps to maintain the integrity of elastic and collagen fibers. To test the hypothesis that moderate deficiency of A1-AT may be a risk factor for sCAD, 22 cases with sCAD and 113 controls were included in the study. Patients with sCAD had significantly mean lower levels of A1-AT compared with controls (116.0+/-24.9 versus 141.1+/-31.7 mg/dL; P<0.01). Low levels of A1-AT (<90 mg/dL) were more frequently observed in patients with sCAD compared with controls (27.3% versus 2.7%; P<0.001). A positive correlation between age and plasma levels of A1-AT was found (r=0.22; P<0.01). A1-AT levels were not affected by sex or vascular risk factors, including smoking habit. On multivariate analysis, A1-AT <90 mg/dL was associated with sCAD independently of age, sex, or vascular risk factors (odds ratio, 17.7; 95% confidence interval, 2.9 to 105.6). Low plasma levels of A1-AT may be a risk factor for sCAD.

ChemInform Abstract: Vinyl Sulfonium as Novel Proteolytic Enzyme Inhibitor.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Vinyl sulfonium as novel proteolytic enzyme inhibitor

Vinyl sulfoniums were synthesized from vinyl sulfides by methylation, and inhibited the proteolytic enzyme papain. Inhibition studies suggest a mechanism by which the vinyl sulfonium inhibitor covalently and irreversibly modifies the enzyme.

Optimization of cytokine stability in stored amniotic fluid

Objective: Many studies use stored amniotic fluid samples to assay cytokines and other proteins for outcome-based research; however, there is little information on the optimal methods of storage. The objective of our study was to evaluate cytokine stability in amniotic fluid stored at different temperatures both with and without a proteolytic enzyme inhibitor. Study Design: Patients undergoing midtrimester genetic amniocentesis for routine indications gave consent for the study. After the sample was centrifuged, the acellular portion of the sample was mixed to homogeneity and aliquoted in 0.5-mL increments and stored for 1 year at 4°C, –20°C, and –80°C with and without the protease inhibitor aprotinin. Enzyme-linked immunoassays for angiogenin, interleukin-6, and vascular endothelial growth factor were performed simultaneously on each aliquot. Results: Thirty samples were assayed for each storage condition. Results were calculated as the percentage of its own sister aliquot stored at -80°C without aprotinin. In all samples, there was a significant relation between storage temperature and cytokine levels, with the lowest levels found at 4°C and the highest at –80°C (angiogenin, P =.004; interleukin-6, P <.001; vascular endothelial growth factor, P =.02). The addition of aprotinin improved stability only for angiogenin at all temperatures (all P <.05). Conclusions: Degradation of cytokines occurs when amniotic fluid samples are stored for prolonged periods at temperatures greater than –80°C. The addition of a protease inhibitor helps stem the degradation of some cytokines. (Am J Obstet Gynecol 2001;185:459-62.)

Trypsin inhibition, calcium and zinc ion binding of starch– g-poly(acrylic acid) copolymers and starch/poly(acrylic acid) mixtures for peroral peptide drug delivery

Newly synthesised starch– g-poly(acrylic acid) copolymers and starch/poly(acrylic acid) mixtures were evaluated for their in vitro inhibition potency towards the proteolytic enzyme trypsin. Their Ca 2+ and Zn 2+ binding capacity was measured. Carbopol ® 934P was used as reference polymer. Starch– g-poly(acrylic acid) copolymers were prepared by chemical grafting and 60Co irradiation, the starch/poly(acrylic acid) mixtures by freeze–drying. The influence of preparation method, the ratio starch:acrylic acid, the neutralisation degree and for the freeze–dried polymers the influence of heat treatment after freeze–drying was investigated. All freeze–dried polymers showed a higher inhibition factor (IF) than the chemically grafted and 60Co irradiated starches, which all showed significantly lower IF than Carbopol ® 934P. The heat treated freeze–dried polymer Amioca ®/poly(acrylic acid) (1:1) showed a significantly higher IF than the reference polymer (Mann–Whitney test, p<0.05). The Ca 2+ and Zn 2+ binding capacity of all chemically grafted starches was much lower than for Carbopol ® 934P. Only the 60Co irradiated starches and freeze–dried polymers with ratio 1:3 approached the binding capacity of the reference polymer. The freeze–dried polymers showed the highest proteolytic enzyme inhibition potency. Freeze–drying and 60Co irradiation could result in the highest ion binding capacity. This combination of proteolytic enzyme inhibition activity and ion binding capacity makes these polymers hopeful excipients for successful oral peptide delivery.

Conformational Selection of Inhibitors and Substrates by Proteolytic Enzymes: Implications for Drug Design and Polypeptide Processing

Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease.

Angiogenesis: new targets for the development of anticancer chemotherapies.

Angiogenesis is the process by which new blood vessels are formed from preexisting microvasculature. To ensure an adequate blood supply, tumor cells release angiogenic factors that are capable of promoting nearby blood vessels to extend vascular branches to the tumor. In addition, larger tumors have been shown to release angiogeneic inhibitory factors that prevent blood vessels from sending branches to smaller, more distant tumors that compete for oxygen and nutrients. Angiogenesis is a complex multistep biochemical process, and offers several potential molecular targets for non-cytotoxic anticancer therapies. Strategies for exploiting tumor angiogenesis for novel cancer drug discovery include: (i) inhibition of proteolytic enzymes that breakdown the extracellular matrix surrounding existing capillaries; (ii) inhibition of endothelial cell migration; (iii) inhibition of endothelial cell proliferation; (iv) enhancement of tumor endothelial cell apoptosis. There is also a host of miscellaneous agents that inhibit angiogenesis for which the specific mechanisms are not clear. Several methods have been developed for measuring antiangiogenic activity both in vitro and in vivo. Although there has been intensive research efforts focused at the phenomena of angiogenesis, as well as the search for antiangiogenic agents for more than two decades, many questions remain unanswered with regard to the overall biochemical mechanisms of the angiogenesis process and the potential therapeutic utility of angiogenic inhibitors. Nevertheless potent angiogenic inhibitors capable of blocking tumor growth have been discovered, and appear to have potential for development into novel anticancer therapeutics. However there are still hurdles to be overcome before these inhibitors become mainstream therapies.

Biological activity and pharmacological properties of anticoagulant complex (Hirudin, plasma kallikrein inhibitor, prostaglandin) fromHirudo Medicinalis

Antiprocoagulant complex isolated from lyophilized medicinal leeches exerted pronounced antithrombotic, thrombolytic, and hypotensive effects in experimental animals after intravenous injection and showed antithrombotic activity after oral administration in combination with hirudone, the source of hyaluronidase and inhibitors of digestive proteolytic enzymes. The antiprocoagulant complex can be used as a specific medicinal preparation.

High-Speed Detection of the Two Common α1-Antitrypsin Deficiency Alleles Pi*Z and Pi*S by Real-Time Fluorescence PCR and Melting Curves

Protease inhibitor 1 (α1-antitrypsin; AT) is the main serum inhibitor of proteolytic enzymes. In AT deficiency, enzymes such as neutrophil elastase can damage the lung tissues, leading to pulmonary emphysema. More than 90 different alleles have been identified to date for the protease inhibitor 1 gene (PI) . The three most important variants are type M (90% of population), type S (PiS) , and type Z (PiZ) , of which types S and Z are two of the more common abnormal variants. Homozygotes of type Z have a considerable reduction in the serum AT concentration and may develop pulmonary emphysema or hepatic cirrhosis. SZ-heterozygotes are less severely affected (1)(2)(3). The allele frequencies of the most common alleles, PiS and PiZ , are 0.02–0.04 or 0.01–0.02, respectively, with 0.2% of the general population being homozygous for either the PiZ or PiS allele. Typically, different phenotypes have been characterized by isoelectric focusing. PCR-based technologies are now being used widely for the identification of the mutations underlying the PiZ allele (Glu342Lys, GAG to AAG) and the PiS allele (Glu264Val, GAA to GTA), thus simplifying mutation detection (4). Although these methods give rise to unequivocal results, they are time-consuming and require optimization of the PCR reaction to eliminate nonspecific PCR products that would disturb the genetic analysis. Recently, a new detection methodology based on hybridization of amplicon-specific oligonucleotides with adjacent fluorophores capable of fluorescence resonance energy transfer was introduced and was used in a new high-speed thermal cycler (LightCyclerTM; Roche Diagnostics) that uses glass capillaries and hot air for heating (5). This technology enables the real-time detection of the specific PCR product followed by detection of the mutation by identification of the melting behavior of one of the two hybridization oligonucleotides (6). To this end, one …

Complexation graft copolymer networks: swelling properties, calcium binding and proteolytic enzyme inhibition.

Graft copolymer networks of poly(methacrylic acid-g-ethylene glycol) were prepared by free radical solution UV-polymerization of methacrylic acid (MAA) and poly(ethylene glycol) monomethacrylate. Dynamic swelling studies indicated that complexation/decomplexation processes occurred due to hydrogen bonding between the carboxylic groups of the poly(methacrylic acid) (PMAA) and the ether groups of poly(ethylene glycol) (PEG). The effects of copolymer composition, graft chain molecular weight, environmental pH and ion content on network structure and gel behavior were studied. The largest change in swelling ratio and mesh size of the gel structure was observed in gels containing the highest content of PEG and the longest molecular weight PEG grafts. Complexation was greatest in hydrogels containing the longest PEG grafts and equimolar amounts of MAA and PEG. The swelling was much less pronounced in the presence of calcium chloride compared to sodium chloride which could be attributed to the complexation of calcium of the carboxylic groups in the polymer. The copolymers showed significant but less binding of calcium compared to poly(acrylates) like Carbopol 934P and polycarbophil. The P(MAA-g-EG) copolymers inhibited trypsin but to a lesser extent than the known protease inhibitors Carbopol 934P and polycarbophil. Results suggest that P(MAA-g-EG) copolymers are good drug delivery carrier candidates due to their pH-sensitive and controllable swelling behavior. Additionally, they possess some protease inhibition effect along with their bioadhesive properties which make them promising carriers for peptides or proteins.

Chronic allograft vasculopathy: new strategies for drug development

Chronic rejection reflects cumulative trauma to the allograft regardless of its origin. The main histologic feature in chronic rejection in all allografts is proliferative allograft vasculopathy. According to the current paradigm, chronic rejection can be prevented by more intensive and selective immunosuppression. Experimental retransplantation to donor strain suggests that elimination of histoincompatibility does not prevent progression of the disorder after the initial stimulus has been sufficiently strong or long-lasting. In order to design additional sites for intervention after this point of no return, attention has focused on the regulation of the synthesis and/or action of smooth muscle cell growth factors. In several preclinical models, the blocking of growth factor synthesis, blocking of the binding of the growth factors to their receptors, and/or growth factor signaling downstream of the receptor, have successfully been used in the inhibition of the intimal response and allograft vasculopathy. Inhibition of proteolytic enzymes, necessary for smooth muscle cell locomotion, may provide additional sites of intervention. However, on most occasions, the inhibitory effect is incomplete only 30% to 50% of maximal. The future will demonstrate whether several approaches must be applied concomitantly, or whether genes regulating several of these ligands and their receptors simultaneously can be identified and exploited.

Improved bioavailability of orally delivered peptides and polypeptides in teleost fish

Summary A gonadoliberin analogue (GnRHa) whose biological role is the stimulation of gonadotropin (GTH II) secretion, has been encapsulated together with intestinal absorption enhancers and proteolytic enzyme inhibitors. The capsules have been coated to prevent their solubilization at acid pH in the stomach. After oral delivery of the capsules by forced-feeding, high GnRHa intestinal absorption and high bioavailability in the blood results. GTH II secretion was stimulated in a dose-dependent manner by the orally delivered GnRHa in three species having different digestive tract structures and different feeding habits: the rainbow trout, the common carp and the African catfish. In addition, the oral delivery of GnRHa also results in the induction of ovulation in all three species at doses similar to those usually used to induce spawning in these species. There was no competition of nutrient given together with the microcapsules on GnRHa absorption. From these results, it becomes possible to develop special diets containing microencapsulated bioactive peptides for use in therapy or to modify nonvital functions in fish. In the present case, an orally-induced ovulation treatment will be developed.

Chronic rejection: risk factors, regulation, and possible sites of therapeutic intervention

Clinical chronic rejection corresponds histologically to proliferative allograft vasculopathy. This disorder reflects the cumulative trauma to the allograft regardless of its origin. As a consequence of trauma, inflammation is generated, leading to endothelial and smooth muscle cell activation, smooth muscle replication and migration, and graft arterial intimal thickening. According to the current paradigm, chronic rejection can be avoided by more intensive and selective immunosuppression and by avoiding the known predisposing factors. Retransplantation experiments in experimental animals to donor or F1-strain suggest, however, that elimination of histoincompatibility does not prevent the progression of the disorder once it has been initiated. To design additional sites of intervention after the "point-of-no-return," attention has been focused on the regulation of the synthesis of growth factors, binding of the growth factors to the receptors, and to signalling downstream of the receptor. Also, inhibition of proteolytic enzymes, necessary for the locomotion of the smooth muscle cells, is a possible target. On most occasions, however, the inhibitory effect is at the most 30 to 50% of the maximal, and only the future will show whether several of these approaches must be applied concomitantly or whether genes regulating several of these processes simultaneously can be identified and exploited.

A possible association of alpha1-antitrypsin deficiency with the periodontal condition in adults.

Alpha1-antitrypsin (alpha1AT) is a serum inhibitor of several neutrophil-derived proteolytic enzymes, the serine proteinases. In certain individuals, a deficiency of this enzyme inhibitor is present, whereby only 10 to 20% of the normal concentration is present in the circulation. The purpose of this study was to evaluate the influence of this alpha1AT deficiency on the periodontal condition in a case control study. 10 patients aged 20-50 years with a mean age of 32 and showing a deficiency of alpha1AT were randomly selected from a national data bank, which contains persons known to be affected with the alpha1AT deficiency state. Control subjects were matched for age, gender and socio-economic status. Full periodontal charting on all subjects was performed on 6 sites per tooth, including probing attachment level, probing depth, bleeding on probing and a plaque score. Analysis of the data revealed no differences with regard to attachment loss between the two groups. The deficiency group showed 9.3% pockets > or =5 mm and the matched control group 5.9%. When these results were covariated with plaque scores, the deficiency group appeared to have a higher number of sites with probing depths > or =5 mm.

Bioremediation: Utilization of Aircraft Paint Waste as a Nutrient Source by Pseudomonas SP. VM1

Abstract The standard organic coatings applied to Navy aircraft consist of an epoxy primer and a polyurethane topcoat. The old coatings are routinely stripped in order for new layers to be applied. The stripping process involves removing the coating by sandblasting the aircraft with small plastic pellets. Although unpublished, we have demonstrated an ability of Pseudomonas sp. VM1 to biodegrade the waste products associated with the sandblasting process. A primary objective of this investigation was to determine the growth kinetics associated with the utilization of paint waste products as a sole carbon source for Pseudomonas. Another objective was to ascertain the nature of morphologic changes that occur among the particles of paint during biodegradation. In addition, the study was designed to determine whether phenylmethylsulfonyl fluoride (PMSF), a recognized inhibitor of proteolytic enzymes, is capable of modifying the biodegradative process of paint waste by Pseudomonas.