Human leukocyte elastase

The design of peptidomimetic HLE inhibitors by researchers at Zeneca Pharmaceuticals23-2Z as based largely on information obtained from the X-ray crystal structures of peptidic inhibitors bound to HLE and the closely related enzyme, porcine pancreatic elastase (PPE). These two enzymes show relatively high structural homology, especially in the active site region. 

Inspection of the X-ray structures of the third domain of turkey ovomucoid inhibitor (TOMI, a 56-residue peptidic inhibitor) with HLE and the peptidic inhibitor Ace-Ala-Pro-Val-trifluoromethylketone, 1 [Xj =210 80 

Aqueous molecular dynamics (MD) - simulations of 3 complexed to HLE run with the program AMBER- revealed that the benzyl tail of the benzylcarbamate (Cbz) portion of the inhibitor remained quite exposed to the 

The reader will note that the of 101 10 nM obtained for 4 is significantly higher than that observed for other peptidomimetic derivatives from this series. It is important to remember, however, that as in many drug candidate design efforts, the focus of this design effort was the optimization of in vivo performance. In this case, the best in vivo performance was not obtained from the most active in vitro HLE inhibitor. 

Subsequent to these peptidomimetic design efforts, X-ray crystal structures were obtained of inhibitors from the 3-amino-pyridin-2-one and 5-amino-pyrimidin-6-one classes complexed to These experimental 

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Figure 15.6 Chromatogram of a plasma standard of human leukocyte elastase inhibitors obtained by using LC-LC. Adapted from Journal of Liquid Chromatography and Related Technologies, 19, R. A. Earley and L. R Tini, Versatile multidimensional chromatographic system for di ug discovery as exemplified by the analysis of a non-peptidic inhibitor of human leukocyte elastase , pp. 2527-2540, 1996, by courtesy of Marcel DekkeiTnc.

Irreversible inhibition is probably due to the alkylation of a histidine residue.43 Chymotrypsin is selectively inactivated with no or poor inhibition of human leukocyte elastase (HLE) with a major difference the inactivation of HLE is transient.42,43 The calculated intrinsic reactivity of the coumarin derivatives, using a model of a nucleophilic reaction between the ligand and the methanol-water pair, indicates that the inhibitor potency cannot be explained solely by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack 43 Studies on pyridyl esters of 6-(chloromethyl)-2-oxo-2//-1 -benzopyran-3-carboxylic acid (5 and 6, Fig. 11.5) and related structures having various substituents at the 6-position (7, Fig. 11.5) revealed that compounds 5 and 6 are powerful inhibitors of human leukocyte elastase and a-chymotrypsin thrombin is inhibited in some cases whereas trypsin is not inhibited.21... 

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)... 

TABLE 11.1 Inhibition of a-Chymotrypsin and Human Leukocyte Elastase by Phenolic Esters of 6-(Chloromethyl)-2-oxo-2H-l-Benzopyran-3-Carboxylic Acid42... 

TABLE 11.2 Inhibition of Human Leukocyte Elastase, a-Chymotrypsin and Thrombin by 5 -Chloropyrid-3 -yl Derivatives at pH 8.0 and 25°C21... 

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. 

Doucet, C. Pochet, L. Thierry, N. Pirotte, B. Delarge, J. Reboud-Ravaux, M. 6-Substituted 2-OXO-2H-1 -benzopyran-3-carboxyI ic acid as a core structure for specific inhibitors of human leukocyte elastase. J. Med. Chem. 1999, 42, 4161—4171. 

Pochet, L. Doucet, C. Dive, G. Wooters,J. Masereel, B. Reboud-Ravaux,M. Pirotte, B. Coumarinic derivatives as mechanism-based inhibitors of a-chymotrypsin and human leukocyte elastase. Bioorg. Med. Chem. 2000, 8, 1489-1501. 

Vergely, I. Laugaa, P. Reboud-Ravaux, M. Interaction of human leukocyte elastase with a /V-aryl azetidinone suicide substrate conformational analyses based on the mechanism of action of serine proteinases. j. Mol. Graphics 1996, 14, 158-167. 

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). 

Hantzsch four-component reaction hexafluoroisopropanol human immunodefficiency virus human leukocyte elastase hydroxymethylglutamate hexamethylphosphoric triamide highest occupied molecular orbital histrionicotoxin... 

In addition to the aforementioned allenic steroids, prostaglandins, amino acids and nucleoside analogs, a number of other functionalized allenes have been employed (albeit with limited success) in enzyme inhibition (Scheme 18.56) [154-159]. Thus, the 7-vinylidenecephalosporin 164 and related allenes did not show the expected activity as inhibitors of human leukocyte elastase, but a weak inhibition of porcine pancreas elastase [156], Similarly disappointing were the immunosuppressive activity of the allenic mycophenolic acid derivative 165 [157] and the inhibition of 12-lipoxygenase by the carboxylic acid 166 [158]. In contrast, the carboxyallenyl phosphate 167 turned out to be a potent inhibitor of phosphoenolpyruvate carboxylase and pyruvate kinase [159]. Hydrolysis of this allenic phosphate probably leads to 2-oxobut-3-enoate, which then undergoes an irreversible Michael addition with suitable nucleophilic side chains of the enzyme. 

The /3-lactam structure can also react with active-serine hydrolases other than PBPs and /3-lactamases. It has been shown that appropriately substituted cephalosporins (e.g., 5.18) are potent mechanism-based inactivators of human leukocyte elastase (HLE, EC 3.4.21.37), a serine endopeptidase involved in the pathogenesis of pulmonary emphysema and other connective tissue diseases [57-60]. Subsequent work has demonstrated that substituted /3-lactams such as 5.19 or 5.20 are more stable HLE inhibitors and have improved potencies [61-63]. 

M. Alpegiani, P. Bissolino, R. Corigli, S. Del Nero, E. Perrone, V. Rizzo, N. Sacchi, G. Cassinelli, G. Franceschi, A. Baici, Cephem Sulfones as Inactivators of Human Leukocyte Elastase. 5. 7a-Methoxy- and 7a-Chloro-l,l-dioxocephem 4-Ketones , J. Med. Chem. 1994, 37, 4003-4019. 

FR901277 - cyclic peptide that inhibits human leukocyte elastase... 

The chemistry of trifluoroacetaldehyde is more extensive. Inhibitors of Human Leukocyte Elastase [153-155] and antagonists of Leukotrienes D4 and E4 [156] were prepared recently using trifluoral chemistry. 

The intermolecular cycloaddition of lV-(azidomethyl)benzisothiazolone 124 with various electron-deficient acetylenes 125 generates potential inhibitors of human leukocyte elastases 1,2,3-triazoles 126 and their regioisomers 127 in 72-99% overall yields (equation 52)177. As the steric effect increases due to the increase size of R in 125,... 

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. 

Powers, J. C., et al. 1977. Specificity of porcine pancreatic elastase, human leukocyte elastase and cathepsin G. Inhibition with peptide chloromethyl ketones. Biochim Biophys Acta 485 156. 

Another system based on an SEC pre-column was employed by Earley and Tini for the analysis of a nonpeptidic inhibitor of human leukocyte elastase as part of a drug discovery project (13). The analytical column used here was octadecylsilane, although the authors commented that, depending on the particular application, any stationary phase could be used as the analytical column. In their application, a fraction of the effluent from the SEC column was transferred to the analytical column by the use of a C18 collection column that served to pre-concentrate and focus the analytes. They noted that frequent solvent flushing was required to avoid build-up of impurities on the collection column. A chromatogram obtained from a 50 p.L plasma standard injection is shown in Figure 15.6. This separation provides an excellent... 

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