Protease human leukocyte elastase

Full kinetic characterization for mechanism-based inhibition can be a challenge. Not only are there multiple rates to determine, but the mechanism of inhibition is often a combination of several different steps. The dividing line between alternate substrate inhibitors and the more eom-plex suicide inhibitors is often blurred, with some alternate substrates being virtually irreversible and some suicide substrates with high partition ratios and a significant alternate substrate element of inhibition. The following examples describe the characterization of an alternate substrate inhibitor and a suicide inhibitor of the serine protease human leukocyte elastase. 

Finally, coumarin derivatives may act as general inhibitors of serine proteases or as specific inhibitors of human leukocyte elastase, depending on the nature of the substituents, through two distinct mechanisms, suicide substrates (a-chymotrypsin)... 

The acyl-enzyme can eliminate the 4-chlorine atom to generate this reactive intermediate that can then react with a nearby nucleophile such as His57 to give an alkylated acyl-enzyme derivative in which the inhibitor moiety is bound to the enzyme by two covalent bonds (Scheme 11.5). Inhibition is irreversible.59 The mechanism has been confirmed by X-ray structural analysis of protease-isocoumarin complexes. There is a cross-link between the inhibitor and the Serl95 and His57 residues of PPE.60 Human leukocyte elastase is also very efficiently inactivated.61... 

Human leukocyte elastase is a protease that degrades elastin and other connective tissue components. It is implicated in the pathogenesis of pulmonary emphysema and other inflammatory diseases such as rheumatoid arthritis and cystic fibrosis. Porcine pancreatic elastase has often been used as a model for HLE. Both enzymes have a small primary binding site Si. 

This approach has been mainly applied to peptide-based inhibitors of proteases, where the inhibitory molecule is a peptide with a transition state isostere appended to it, and the cognate substrate is simply a peptide of the same amino acid sequence, but lacking the isostere functionality. Examples where good correlations between the free energy of inhibitor binding and the free energy of kcJKM have been found, include peptide-trifluoromethyl ketone inhibitors of human leukocyte elastase (Stein et al., 1987) and peptide-phosphonamidate inhibitors of the metalloprotease ther-molysin (Bartlett and Marlowe, 1983). 

Very recently, (3-lactam antibiotics have been shown to offer neuroprotection by increasing glutamate transporters expression via gene activation [15] in addition, the discoveries of new biologically active (3-lactams such as cholesterol acyl transferase inhibitors [16-18], thrombin inhibitors [19], human cytomegalovirus protease inhibitors [20], matrix-metallo protease inhibitors [21], inhibitors of human leukocyte elastase (HLE) [22, 23] and cysteine protease [24, 25], and apoptosis inductors [26, 27] have provided much needed motivation for continuous development of new (3-lactam systems. 

The reactive site of i-PI has a Ala-Ile-Pro-Met Ser-Ile-Pro-Pro sequence where the asterisk indicates the bond cleaved when i-PI pro-tase complexes are dissociated at high pH or by using nucleophiles. We have synthesized a number of peptides with the amino acid sequence at the ai-PI reactive site and have shown them to be perfectly adequate substrates for human leukocyte elastase. However, oxidation of the methionine residue of the substrates to a methionine sulfoxide residue (see Table III) almost completely destroys their reactivity toward human leukocyte elastase and other proteases (8,9). Oxidation of ai-PI itself destroys its ability to inhibit most proteases (10). 

Peptide chloromethyl ketone inhibitors have been developed for almost every serine protease that has been characterized adequately (30). For example, human leukocyte elastase, due to its involvement in emphysema, has been studied extensively with this class of inhibitor (32). The rate at which peptide chloromethyl ketones inhibit elastase is influenced by their interaction with the primary substrate binding site (Si) of the enzyme and by interactions at other subsites. The most effective chloromethyl ketone elastase inhibitor found thus far is MeO-Suc-Ala-Ala-Pro-ValCH2Cl (MeO-Suc- = CH3OCOCH2CH2CO-). This will not inhibit the other major leukocyte protease, cathepsin G (see Table VI). In contrast, Z-Gly-Leu-Phe-CH2C1 (Z = C6H5CH2OCO-) inhibits cathepsin G, but not elastase. Both enzymes can be inhibited with Ac-Ala-Ala-Pr o-V alCH2Cl. 

Sulfonyl Fluorides. Sulfonyl fluorides inhibit serine proteases by reacting with the active-site serine residue. Previously we investigated the rates of inhibition of human leukocyte elastase and cathepsin G by a variety of sulfonyl fluorides and found relatively little selectivity or reactivity (38). However, we have discovered recently that the introduction of fluoroacyl groups into the sulfonyl fluoride structure gives considerable reactivity and selectivity for elastase (39). 

Human leukocyte elastase (HLE) is a serine protease which is used in the lung to degrade necrotic tissue and invading bacteria. Under normal conditions, the activity of elastase is controlled by several endogenous inhibitors such as al-protease-inhibitor. If the balance between protease and inhibitor is shifted, then elastase also attacks healthy tissue, leading to emphysema. 

The isothiazol-3(2//)-one 1,1-dioxides 254-257 with stabilizing aryl substituents in the 2-, 4- and/or 5-position are potential inhibitors toward human leukocyte elastase (HLE) (03ZN(B)111, 05JEIMC341). HLE is a serine protease implicated in several inflammatory diseases and represents a major target for the development of low-molecular weight inhibitors. 

K. Nakajima, J. C. Powers, B. M. Ashe, and M. Zimmerman. Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the alpha 1-protease inhibitor reactive site. J. Biol. Chem. 254 4027 (1979). 

Human leukocyte elastase (HLE) is a serine protease produced and stored by PMNL and involved in the tissue destruction observed in many inflammatory diseases such as chronic arthritis. In fact, administration of exogenous elastase inhibitors could be a means of protecting tissues from proteolytic attack. 

E. C. Luccy, P. J. Stone. D. EL Ciccolclla, R. Bcuer, T. 0. Christensen, R. C. Thompson, and G- L. Snider. Recombinant human secretory leukocyte-protease inhibitor in vitro properties, and amelioration of human neutrophil elastase-induced emphysema and secretory cell metaplasia in the hamster. /. Lab. Clin. hied. 7/5 224... 

R. C. Thompson and K. Ohlsson. Isolation, properties and complete amino acid sequence of human leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc. Natl. Acad. Sci. USA 83 6692 (1986). 

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