Proteolytic enzyme inhibitors

KROGDAHL A, HOLM H (1981) Soybean proteinase inhibitors and human proteolytic enzymes selective inactivation of inhibitors by treatment with human gastric juice. /M/fr. Ill 2045-51. 

Plasminogen activator inhibitors have been shown to be present in a large variety of different cells and tissues. These inhibitors are thought to play an important role in regulating tissue fibrinolysis. One of these inhibitors has been purified from cultured bovine aortic epithelial cells. This inhibitor has been shown to be a serine protease inhibitor and inhibits the function of two proteolytic enzymes urokinase and tissue plasminogen activator, both of which cleave and activate plasminogen. The mechanism by which this inhibitor functions is very similar to that described above with a-l-PI. Thus, the inhibitor forms a binary complex with the proteolytic enzyme and thereby inhibits its activity. Again in a situation comparable to that with a-l-PI, it was found that when the purified bovine aortic epithelial inhibitor was exposed to Al-chlorosuccinimide,... 

Volume 222. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A Mammalian Blood Coagulation Factors and Inhibitors)... 

In this laboratory, we also include the metal ion chelators EDTA (ethylene diamine tetraacetic acid binds, e.g., Mg2 1 -ions) and EGTA (ethylene glycol-bis(2-aminoethyl)-Al,iV,iV/,iV/,-tetraacetic acid binds, e.g., Ca2+-ions) in our lysis buffers. These agents help prevent phosphatase action (by the metal ion-dependent phosphatase PP2C, which is not inhibited by microcystin-LR), metal (Ca2+) dependent proteinases, and protein kinases, which require divalent cations such as Mg2 1 (and, in some cases, also Ca2+). We also use a mix of proteinase inhibitors that inhibit a broad range of proteolytic enzymes, including serine and cysteine proteinases. 

An additional approach to IL-1 down-regulation could entail development of inhibitors of the proteolytic enzymes that release the active interleukin from its inactive precursor. Moreover, such inhibitors could probably be taken orally and, thus, would be suitable to treat chronic inflammation (the alternatives outlined above would be administered parenterally). 

Involvement of several proteolytic enzymes, secretases, is probably crucial for this process but other hypotheses, including, for example, cholinergic transmission or accumulation of metal ions, have also been considered. Future perspectives in this area concern the search for novel pharmaceuticals that cross the blood-brain barrier, without side effects (e.g., the dyskinesias of L-Dopa), or potent and selective inhibitors of improper cleavage of amyloid protein, or even stem cell therapy to restore neuronal cells. 

A recent study, however, has shown that aminopeptidase activity is present on the surface of porcine buccal mucosa, and that various aminopeptidase inhibitors, including amastatin and sodium deoxycholate, reduce the mucosal surface degradation of the aminopeptidase substrate, leucine-enkephalin [149], Since the peptidases are present on the surface of the buccal mucosa, they may act as a significant barrier to the permeability of compounds which are substrates for the enzyme. In addition to proteolytic enzymes, there exist some esterases, oxidases, and reductases originating from buccal epithelial cells, as well as phosphatases and carbohydrases present in saliva [154], all of which may potentially be involved in the metabolism of topically applied compounds. 

The failure in increasing residence time of mucoadhesive systems in the human intestinal tract has led scientists to the evaluation of multifunctional mucoadhesive polymers. Research in the area of mucoadhesive drug delivery systems has shed light on other properties of some of the mucoadhesive polymers. One important class of mucoadhesive polymers, poly(acrylic acid) derivatives, has been identified as potent inhibitors of proteolytic enzymes [72-74]. The interaction between various types of mucoadhesive polymers and epithelial cells has a direct influence on the permeability of mucosal epithelia by means of changing the gating properties of the tight jrmctions. More than being only adhesives, some mucoadhesive polymers can therefore be considered as a novel class of multifunctional macromolecules with a number of desirable properties for their use as delivery adjuvants [72,75]. 

LueBen, H.L., Verhoef, J.C., Borchard, G., Lehr, C.-M., De Boer, A.G., and Junginger, H.E., Mucoadhesive polymers in peroral peptide drug delivery. II. Carbomer and polycarbophil are potent inhibitors of the intestinal proteolytic enzyme trypsin, Pharm. Res., 12 1293-1298 (1995). 

A compound that reduces the activity of an enzyme is known as an inhibitor. Inhibitors are usually small molecules but some are peptides or proteins. For example, there are a number of proteolytic enzymes in the blood that have serine in their active site. If the activities of these enzymes are too high, they can cause problems. Consequently, inhibitor proteins, known as serine proteinase inhibitors (serpins), are present in blood indeed, about 10% of all the plasma proteins are serpins (Box 3.4). 

To prevent self-digestion, the pancreas releases most proteolytic enzymes into the duodenum in an inactive form as proenzymes (zymogens). Additional protection from the effects of premature activation of pancreatic proteinases is provided by proteinase inhibitors in the pancreatic tissue, which inactivate active enzymes by complex formation (right). 

It is a naturally occurring proteolytic enzyme inhibitor acting on plasmin and kallikrein. 

Glucagon is extensively degraded in the liver and kidney as well as in plasma and at its tissue receptor sites. Because of its rapid inactivation by plasma, chilling of the collecting tubes and addition of inhibitors of proteolytic enzymes are necessary when samples of blood are collected for immunoassay of circulating glucagon. Its half-life in plasma is between 3 and 6 minutes, which is similar to that of insulin. 

Polymers may also inhibit proteolytic enzymes or be used to augment the activity of proteolytic inhibitors (Table 11.6). The administration of small molecule protease inhibitors in conjunction with the peptide, however, has been examined with some success (Fujii et al. 1985) however, the ability of these molecules to protect the protein has been hampered by the fact that they tend to cause systemic side effects (Yagi et al. 1980 McCaffrey and Jamieson 1993 Plumpton et al. 1994). Many protease inhibitors have been specific for the proteases in the stomach and intestine, but some of these factors are not specific and some control over absorption of the... 

The discovery of yet other nonhydrolyzable amide bond isosteres has particularly impacted the design of protease inhibitors, and these include hydroxymethylene or FfCF OH)], 12 hydroxyethylene or T fCF OFQCFy and T fCFkCHiOH)], 13 and 14, respectively dihydroxyethylene or ( [ )], 15, hydroxyethylamine or 4 [CH(0H)CH2N], 16, dihydroxyethylene 17 and C2-symmetric hydroxymethylene 18. In the specific case of aspartyl protease inhibitor design (see below) such backbone modifications have been extremely effective, as they may represent transition state mimics or bioisosteres of the hypothetical tetrahedral intermediate (e.g., xF[C(OH)2NH] for this class of proteolytic enzymes. 

Antitrypsin Blood and other body fluids contain a protein, ai -antitrypsin (ai-AT, currently also called ai-aritiproteinase), that inhibits a number of proteolytic enzymes (also called proteases or proteinases) that hydrolyze and destroy proteins. [Note The inhibitor was originally named oti-antitrypsin because it inhibits the activity of trypsin (a proteolytic enzyme synthesized as trypsinogen... 

A classic text, directed at the food sciences, covering the fundamental principles of enzymology. Chapters covering enzyme purification, pH effects, temperature effects, enzyme inhibitors, and the proteolytic enzymes are particularly relevant to this unit. 

Irreversible inhibitors often provide clues to the nature of the active site. Enzymes that are inhibited by iodo-acetamide, for example, frequently have a cysteine in the active site, and the cysteinyl sulfhydryl group often plays an essential role in the catalytic mechanism (fig. 7.18). An example is glyceraldehyde 3-phosphate dehydrogenase, in which the catalytic mechanism begins with a reaction of the cysteine with the aldehyde substrate (see fig. 12.21). As we discuss in chapter 8, trypsin and many related proteolytic enzymes are inhibited irreversibly by diisopropyl-fluorophosphate (fig. 7.18), which reacts with a critical serine residue in the active site. 

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