Sbustrate specificity of the elastase and the chymotrypsin-like enzyme of the human granulocyte.

Human granulocyte elastase (EC 3.4.21.11) differs from hog pancreatic elastase in its specificity for synthetic substrates. Although hydrolyzing peptide bonds adjacent to the carboxyl group of alanine, the granulocyte enzyme prefers valine at the cleaved bond, in contrast to the pancreatic enzyme which prefers alanine. Peptide bonds involving the carboxyl group of isoleucine can be hydrolyzed by the granulocyte enzyme but are not hydrolyzed to any significant extent extent by pancreatic elastase. This difference in specificty could explain the lower sensitivity of the granulocyte enzyme to inhibitors containing alanine analogs, such as the peptide chloromethyl ketones and elastatinal. The human granulocyte chymotrypsin-like enzyme differs from pancreatic chymotrypsin by being able to cleave substrates containing leucine in addition to those containing the aromatic amino acids.

Proteinase inhibitors. I. Inhibitors of elastase.

A series of peptides and depsipeptides containing 2-methylcarbazic acid (H-Mec-OH), the 2-aza analogue of alanine, was prepared and tested as inhibitors of pancreatic and human granulocyte elastases. A requirement for a minimum chain length as well as specific amino acid sequence was observed which correlates well with both substrate and inhibitor studies by others in this field. The most active inhibitors have the structure Ac-Ala-Ala-Pro-Mec-Lac-R. When Lac-R is an ester, only the pancreatic enzyme is inhibited. When Lac-R is an amide or hydrazide, then both enzymes are inhibited. The inhibitory activity is reversible; the inhibitors are not hydrolyzed by the enzyme and the inhibition is noncompetitive with synthetic substrates of similar structure, suggesting that binding at the sites adjacent to the carboyl group of the amino acid analogue, 2-methylcarbazic acid, is important for this inhibition. The data further demonstrate the differences between pancreatic and granulocyte elastases.

Orally active beta-lactam inhibitors of human leukocyte elastase. 2. Effect of C-4 substitution.

The effect of changing the C-4 substituent of 3,3-diethyl-1-[(benzylamino)carbonyl]-2-azetidinone on inhibition of HLE and in a model of HLE-induced lung damage in hamsters was explored. Substituents at this position do not appear to interact strongly with HLE with the most potent compounds having k(obs)/[I] = 6900 M-1 s-1. However, substituents at this position had a marked effect on in vivo activity. The greatest oral activity in the lung hemorrhage assay was achieved with C-4 aryl carboxylic acid ethers (60-85% inhibition at 30 mg/kg po). Based upon the established mechanism of inhibition by these compounds, the C-4 substituent would be released, and therefore, the pharmacological potential of these C-4 substituents was of considerable concern. Fortunately, compounds containing 4-hydroxybenzoic acid and 4-hydroxyphenylacetic acid ethers at C-4 were among the most active analogs. These phenolic acids are also found as urinary metabolites in healthy humans. Other heteroaryls at C-4 were also orally active in this model despite relatively modest enzyme activity.

Orally active beta-lactam inhibitors of human leukocyte elastase. 3. Stereospecific synthesis and structure-activity relationships for 3,3-dialkylazetidin-2-ones.

The stereospecific synthesis of several 4-[(4-carboxyphenyl)oxy]- 3,3-dialkyl-1-[[(1-phenylalkyl)-amino]carbonyl]azetidin-2-on es 3 is described in which the C-3 alkyl groups were varied from methyl to butyl as well as allyl, benzyl and methoxymethyl. The structure-activity relations for these compounds are discussed in terms of the hydrolytic stability of the beta-lactam ring, their in vitro inhibitory potency for human leukocyte elastase (HLE), and their in vivo oral efficacy in an HLE-mediated hamster lung hemorrhage assay. Further alkyl substitution on the benzylic urea moiety, especially in the R configuration, afforded enhanced HLE inhibition and in vivo efficacy. The stereochemical assignments for (3R,4S)-4-[(4-carboxyphenyl)oxy]-3-ethyl-3-methyl-1-[[((R)-1- phenylpropyl)amino]carbonyl]azetidin-2-one (42a) (kobs/[I] = 91,000 M-1 s-1) were confirmed with an X-ray structure determination, which was also utilized to develop an HLE inhibition model.

Inhibition of human leukocyte elastase. 4. Selection of a substituted cephalosporin (L-658,758) as a topical aerosol.

Human leukocyte elastase (HLE) is a serine protease which has been implicated as a causative agent in several pulmonary diseases. The continued modification of our previously reported cephalosporin-based HLE inhibitors has led to the identification of a series of C-2 amides with potent, topical activity in an in vivo hamster lung hemorrhage model. While the most potent in vitro HLE inhibition had previously been obtained with lipophilic ester derivatives, it was found that the less active, but more polar and stable, amide derivatives were much more effective in vivo. The development of the structure--activity relations for optimization of these activities is discussed. These results led to the selection of 3-(acetoxymethyl)-2-[(2(S)-carboxypyrrolidino)carbonyl]-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene, 5,5-dioxide (3, L-658,758) as a selective, potent, time-dependent HLE inhibitor suitable for formulation as a topical aerosol drug for possible clinical use.

Orally active beta-lactam inhibitors of human leukocyte elastase-1. Activity of 3,3-diethyl-2-azetidinones.

A thorough analysis of the mechanism of inhibition of human leukocyte elastase (HLE) by a monocyclic beta-lactam and the mechanism of beta-lactam hydrolysis led to the preparation of potent and highly stable inhibitors of HLE. This work led to the identification of 4-[(4-carboxyphenyl)-oxy]-3,3-diethyl-1- [[(phenylmethyl)amino]carbonyl]-2-azetidinone (2) as the first orally active inhibitor of human leukocyte elastase (HLE). Analogs of 2 with different substituents on the urea N were synthesized and evaluated for their activity in vitro against HLE as well as in vivo in a hamster lung hemorrhage model. Compounds with a methyl or a methoxy group in the para position of the benzene ring were very potent in both assays. The results are discussed on the basis of the proposed model for the binding of this class of inhibitors to HLE and a possible mechanism of inhibition is presented.

Inhibition of human leukocyte elastase. 1. Inhibition by C-7-substituted cephalosporin tert-butyl esters.

Time-dependent inhibitors of the enzyme human leukocyte elastase have been developed based on the cephem nucleus. A series of cephalosporin tert-butyl esters has been examined, and the activity of these compounds has been found to be very sensitive to C-7 substituents, with small, alpha-oriented, electron-withdrawing groups showing greatest activity. Additionally, the oxidation state of the sulfur atom has been found to play a role in potency, with sulfones showing considerably greater activity than the corresponding sulfides or beta-sulfoxides. The alpha-sulfoxides were inactive.

Inhibition of human leukocyte elastase. 2. Inhibition by substituted cephalosporin esters and amides.

A variety of 7 alpha-methoxycephalosporin ester and amide sulfones were prepared and tested to determine the structure-activity relations for inhibition of human leukocyte elastase (HLE), a serine protease which has been implicated in several degenerative lung and tissue diseases. The most potent IC50 values were obtained with neutral, lipophilic derivatives, with the esters being more active than the amides. However, the best time-dependent inhibition in this series was observed with the p- and m-carboxybenzyl esters 7b and 7c. These results are discussed in terms of the proposed mechanism of inhibition as well as a molecular modeling study using the recently solved X-ray crystal structure of HLE.

Inhibition of human leukocyte elastase. 3. Synthesis and activity of 3'-substituted cephalosporins.

Several 3'-substituted cephalosporin sulfones were synthesized from 3-(hydroxymethyl)cephalosporin, which was prepared by Ti(OiPr)4 hydrolysis of the corresponding acetate. A method was also developed to prepare a 3-vinylcephalosporin. Some of these compound were found to be potent time-dependent inhibitors of human leukocyte elastase (HLE). The HLE inhibitory activity was correlated with sigma 1 and it was concluded that the potency was determined by the electron-withdrawing ability as well as the size of the substituent. A mechanism for inhibition of HLE by cephalosporin sulfones is proposed.

Inhibition of elastase and other serine proteases by heterocyclic acylating agents.

The N-acylsaccharins and N-acylbenzoisothiazolinones form a new class of acylating inhibitors of the serine proteases with a broad spectrum of activity. However, they are unique in that they are able to differentiate between various serine proteases because of the differential stability of the presumptive acyl-enzyme formed. Furoyl saccharin was the best studied among this class of inhibitors. We report evidence that the amide bond in the heterocyclic ring of this compound is cleaved by porcine pancreatic and human leukocyte elastases and chymotrypsin, forming acyl-enzymes. Radioisotope studies indicate that the saccharin portion of furoyl saccharin is attached to these enzymes in approximately a 1:1 molar ratio with enzyme, blocking the active site serine. The acylelastases thus prepared are unusually stable to hydrolysis, with kdeacyl values at neutral pH of 2.3 x 10(-6) s-1 for porcine pancreatic elastase and 1.4 x 10(-6) s-1 for human leukocyte elastase. Trypsin appears to be inhibited by a different mechanism. These data suggest a new approach to the design of specific synthetic protease inhibitors.

Selective inhibition of human leukocyte elastase and bovine alpha-chymotrypsin by novel heterocycles.

A number of N-arylbenzisothiazolinone 1,1-dioxides have been synthesized and examined for inhibitory activity against human leukocyte and porcine pancreatic elastase (EC 3.4.21.11), bovine alpha-chymotrypsin (EC 2.4.21.1), human leukocyte cathepsin G (EC 3.4.21.20), and bovine trypsin (EC 3.4.21.4). They are potent, selective, competitive inhibitors of human leukocyte elastase and chymotrypsin. The inhibitory capacity of these compounds is directly related to the electron-withdrawing capability of the aryl substituents. When sufficiently activated, the amide bond in the heterocyclic ring can be cleaved by the enzyme, resulting in inhibition which is highly specific. The most potent inhibitor of hummotrypsin. The inhibitory capacity of these compounds is directly related to the electron-withdrawing capability of the aryl substituents. When sufficiently activated, the amide bond in the heterocyclic ring can be cleaved by the enzyme, resulting in inhibition which is highly specific. The most potent inhibitor of human leukocyte elastase, the 2,4-dinitrophenyl derivative, has a Ki of 2.16 microM with elastase and 0.77 microM with chymotrypsin. This study demonstrates that it is possible to design specificity into non-peptide, low molecular weight serine protease inhibitors, which may have considerable pharmacologic potential.

Study of elastolytic activity Propionibacterium acnes and Staphylococcus epidermis in acne vulgaris and in normal skin.

Histopathological sections of anetoderma-like scars from 10 patients with acne vulgaris showed a selective absence of elastic fibers around pilosebaceous follicles. This finding is similar to the histologic changes of "perifollicular elastolysis" reported by Varadi. Bacteria isolated by anaerobic and aerobic cultures of swabs of the skin surface and pus of these 10 patients, 12 others with active acne vulgaris and 8 normal subjects were studied with particular attention to Staphylococcus epidermis and Propionibacterium acnes. These organisms were analysed for production of an elastolytic enzyme which might play a role in the observed selective loss of elastic fibers. No elastolytic activity was produced by S. epidermidis or P. acnes isolated from any of these individuals. Thus, we cannot attribute the perifollicular loss of elastic fibers in acne scarring to an elastase produced by organisms. The observed absence of elastic fibers might result from tissue necrosis produced by leukocytes during the inflammatory phase, followed by collagenous scar formation without regeneration of elastic fibers.

PMN elastases: a comparison of the specificity of human isozymes and the enzyme from other species toward substrates and inhibitors.

The human elastases isolated from polymorphonuclear neutrophils (PMN) and purulent sputum displayed identical kinetic constants toward substrates and inhibitors. The elastases from the two sources yield identical N-terminal sequences and were recognized by antiserum prepared against human sputum elastase (HSE) isozyme-4 (I-4). The data support the proposal put forth by Twumasi and Liener (1977, J. Biol. Chem. 252, 1917-1926) that the human elastase from sputum is of PMN origin. PMN elastases from other species displayed kinetic constants toward both substrates and inhibitors significantly different from the human enzyme. Therefore, extrapolation of inhibitor profiles from these elastases to the human source should be avoided. Four groups of isozymes were resolved from HSE by FPLC. Only the most basic isozyme (I-4) was obtained as a single species. The isozymes displayed identical macroscopic kinetic constants toward several substrates and two classes of inhibitors. The similar partition ratios observed with a cephalosporin-derived inhibitor suggest that the microscopic rate constants are also identical. The data support the proposal suggested by Baugh and Travis (1976, Biochemistry 15, 836-841) that HLE isozymes differ only in carbohydrate content. Whatever the source of human PMN elastase heterogeneity, it does not result in heterogeneous catalytic properties. In addition, a new protein was identified in elastase preparations derived from human sputum. This protein displayed homology to serine proteases and properties suggesting that it is identical to azurocidin.

Cephalosporin antibiotics can be modified to inhibit human leukocyte elastase.

Several laboratories, including our own have reported the synthesis and activity of certain low relative molecular mass inhibitors of mammalian serine proteases, especially human leukocyte elastase (HLE, EC 3.4.21.37), an enzyme whose degradative activity on lung elastin has been implicated as a major causative factor in the induction of pulmonary emphysema, and which is present in the azurophil granules of human polymorphonuclear leukocytes (PMN). Normally, these granules fuse with phagosomes containing engulfed foreign material (such as bacteria), and HLE, in combination with other lysosomal enzymes, catabolizes the particles. Under certain pathological conditions, however, PMN become attached to host protein (elastin fibres, basement membrane, connective tissue, immune complexes), and in response to this adherence, the granules may fuse with the PMN outer membrane and release their contents, including HLE, directly onto the tissue. Besides emphysema, HLE may also contribute to the pathogenesis of disease states such as adult respiratory distress syndrome, and its potential involvement in rheumatoid arthritis makes HLE inhibitors of considerable interest. It is known that cephalosporin antibiotics (for example, cephalothin (compound I, Table 2)) are acylating inhibitors of bacterial serine proteases which help synthesize the cell wall by performing a transpeptidation reaction on a peptidyl substrate bearing a D-Ala-D-Ala terminus. We now report that neutral cephalosporins (that is, compounds not bearing a free carboxyl at position C-4) can be modified to become potent time-dependent inhibitors of HLE.

Mechanism of inhibition of human leukocyte elastase by two cephalosporin derivatives.

The cephalosporin derivatives L 658758 [1-[[3-(acetoxymethyl)-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-en-2-yl]carbonyl]proline S,S-dioxide] and L 659286 [1-[[7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo- 1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-aza-(6R)-bicyclo[4.2.0]-o ct-2-en-2-yl]carbonyl]pyrrolidine S,S-dioxide] are mechanism based inhibitors of human leukocyte elastase (HLE). The mechanism involves initial formation of a Michaelis complex followed by acylation of the active site serine. The group on the 3'-methylene is liberated during the course of these reactions, followed by partitioning of an intermediate between hydrolysis to regenerate active enzyme and further modification to produce a stable HLE-inhibitor complex. The partition ratio of 2.0 obtained for the reaction with L 658758 approaches that of an optimal inhibitor. These compounds are functionally irreversible inhibitors as the recovery of activity after inactivation is slow. The half-lives at 37 degrees C of the L 658758 and L 659286 derived HLE-I complexes were 9 and 6.5 h, respectively. The complexes produced by both inhibitors are similar chemically since the thermodynamic parameters for activation to regenerate active enzyme are essentially identical. The free energy of activation for this process is dominated primarily by the enthalpy term. The stability of the final complexes likely arises from Michael addition on the active site histidine to the 3'-methylene.

A comparison of alpha 1-proteinase inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone and specific beta-lactam inhibitors in an acute model of human polymorphonuclear leukocyte elastase-induced lung hemorrhage in the hamster.

A pharmacokinetic model is described for testing of polymorphonuclear leukocyte (PMN) elastase inhibitors administered by intratracheal or aerosol dosing of hamsters. Acute lung injury, measured as hemorrhage occurring within hours after intratracheal instillation of human PMN elastase, correlated directly with the amount of active enzyme instilled. Hemorrhage began within minutes of elastase instillation, was maximal within 1 h, and remained constant for up to 5 h subsequently. Therefore, inhibition of hemorrhage was used as an assay of the effectiveness of various PMN elastase inhibitors given by the intratracheal route. Lung hemorrhage could also be induced by intratracheal instillation of other elastolytic enzymes, such as thermolysin, and inhibition of hemorrhage was seen only with inhibitors active against the type of elastase used. Methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone (MeOSuc-AAPV-CMK), as well as alpha 1-proteinase inhibitor (alpha 1PI) but not tosyl-lysine-chloromethylketone (tosyl-lysine-CMK), inhibited the hemorrhage caused by human PMN elastase, but the specific inhibitors of this enzyme had no effect on thermolysin-induced lung hemorrhage. The duration of activity of these compounds as elastase inhibitors in this model correlated directly with the extent of their persistence in lung lavage fluid as determined by HPLC analysis of compound recovered by bronchoalveolar lavage. (ABSTRACT TRUNCATED AT 250 WORDS)