Mechanism of inhibition of human leucocyte elastase by beta-lactams. 3. Use of electrospray ionization mass spectrometry and two-dimensional NMR techniques to identify beta-lactam-derived E-I complexes.

A combination of NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) was used to probe the identity of beta-lactam-derived complexes with serine proteases. The carbon and proton NMR chemical shifts of the human leucocyte elastase (HLE)-inhibitor complex derived from [4-13C]-L-680,833, [S-(R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-oxo-4- azetidinyl)oxy]benzeneacetic acid, were consistent with an sp3 hybridized carbon. The ESI-MS spectrum of the L-680,833-derived HLE-I complex indicated an increase of 333 Da over the mass of the free enzyme. The data are consistent with acylation of the active site serine, loss of p-hydroxybenzeneacetic acid, and formation of a carbinolamine at the carbon deriving from C-4 of the lactam ring. The complexes produced from HLE and the diastereomers of L-680,833 display identical masses. Since the 4R-isomers produce more stable complexes [Green et al. (1995) Biochemistry 34, 14331-14343], these data suggest that these complexes differ in their stereochemistry or conformation. The structural model of the HLE-I complexes derived from the diastereomers predicts that the hydroxyl of the carbinolamine derives from a structurally observed water molecule yielding S-stereochemistry in all cases. In this model, the 4S- and 4R-diastereomers produce complexes that differ by the location of the side chain of a phenylalanine residue. The mass of HLE was increased by that of L-684,481, (R)-1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-azetidino ne, which lacks a leaving group at C-4 in the complex derived from this compound. L-691,886, [S-(R*,S*)]-4-[(1-(((1-(4-ethoxyphenyl)butyl)amino)carbonyl)- 3,3-diethyl-4-oxo-2-azetidinyl)-oxy]benzeneacetic acid, produces two complexes of different mass that reactivate with different rates. The mass of the less stable complex is consistent with the acyl-enzyme of 2,2-ethyl-3-oxopropanoic acid while the mass of the more stable complex is analogous to the carbinolamine observed during L-680,833 inactivation. Porcine pancreatic elastase (PPE) produces a complex with a mass consistent with replacement of the C-4 leaving group by water to produce a carbinolamine from L-684,248, [S-(R*,S*)]-4-[(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-dimethy l - 2-oxo-4-azetidinyl)oxy]benzoic acid. The C-4 diastereomer, L-684,249, produces two PPE-I complexes with different masses. One of these complexes has a mass identical to the mass of the complex derived from L-684,248 while the mass of the other complex indicates the presence of the entire inhibitor molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

An inhibitor of leukocyte elastase prevents immune complex-mediated hemorrhage in the rat lung.

The typical reverse passive Arthus reaction (RPA) was attained in rats by the instillation of a rabbit antiovalbumin serum into the lungs and intravenous injection of ovalbumin. Instillation of antiserum alone caused accumulation of polymorphonuclear leukocytes (PMN) and increased vascular permeability, but did not cause hemorrhage. However, when an intravenous injection of ovalbumin was also given, the vascular permeability of the lungs increased dramatically and PMN, as well as hemoglobin, were measurable in the lung lavage fluids by 4 hr after initiation of the reaction. Various proteinase inhibitors were instilled into the lungs after the initial stages of the RPA had developed, specifically to investigate their effect on the development of the hemorrhage, which we chose to monitor as an indicator of severe vascular damage. A cephalosporin-based beta-lactam, L-658,758, which is a time-dependent inhibitor of human and rat PMN elastase, effectively prevented the lung hemorrhage associated with the RPA reaction (ED50 = 2 x 55 micrograms doses/animal when instilled at 1.5 and 2.5 hr after initiating the RPA). The PMN elastase inhibitor, methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone, also inhibited hemorrhage in this model. Compounds of the same chemical class as these elastase inhibitors, but having no activity against PMN elastase in vitro, did not affect the hemorrhage associated with the RPA. Several specific inhibitors of proteinases other than PMN elastase (e.g., pepstatin and methoxysuccinyl-prolyl-glycyl-alanyl-lysine-chloromethylketone) were found to have little effect on the hemorrhage associated with the RPA reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical, biochemical, pharmacokinetic, and biological properties of L-680,833: a potent, orally active monocyclic beta-lactam inhibitor of human polymorphonuclear leukocyte elastase.

A series of potent and highly selective time-dependent monocyclic beta-lactam inhibitors of human polymorphonuclear leukocyte elastase (PMNE, EC 3.4.21.37) is described. The intrinsic potency of these compounds, as exemplified by L-680,833 (k(inactivation)/K(i) of 622,000 M-1.s-1), is reflected at the cellular level where it inhibits generation of the specific N-terminal cleavage product A alpha-(1-21) from the A alpha chain of fibrinogen by enzyme released from isolated polymorphonuclear leukocytes stimulated with fMet-Leu-Phe with an IC50 of 0.06 microM. The inhibitory activity of L-680,833 is also apparent in whole blood stimulated with A23187, where it inhibits formation of A alpha-(1-21) and PMNE-alpha 1-proteinase inhibitor complex formation with IC50 values of 9 microM. Pharmacokinetic studies indicate that after oral dosing L-680,833 is bioavailable in rats and rhesus monkeys. This oral bioavailability is reflected by the inhibition (i) of tissue damage elicited in hamster lungs by intratracheal instillation of human PMNE and (ii) enzyme released from human PMN stimulated after their transfer into the pleural cavity of mice. The properties of L-680,833 allow it to effectively supplement the activity of natural inhibitors of PMNE in vivo, suggesting that this type of low-molecular-weight synthetic inhibitor could have therapeutic value in diseases where PMNE damages tissue.

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.

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. 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.

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)

Crystallographic study of a beta-lactam inhibitor complex with elastase at 1.84 A resolution.

The continuing discovery and development of beta-lactams as antibiotics has had an unparalleled impact on the overall health and well-being of society. Recently, appropriately substituted cephalosporins were shown to be potent inhibitors of elastase, suggesting a novel therapeutic role for the beta-lactams in the control of emphysema and other degenerative diseases. We have now solved and partially refined at atomic resolution the structure of a complex of porcine pancreatic elastase with the time-dependent irreversible inhibitor 3-acetoxymethyl-7-alpha-chloro-3-cephem-4-carboxylate-1,1-dioxide tert-butyl ester (I), the most potent of the beta-lactam elastase inhibitors yet reported. (Porcine pancreatic elastase is a close relative of the desired drug target, human polymorphonuclear leukocyte elastase.) A mechanism of action is presented, based on the structure and on biochemical evidence (T.-Y.L. et al., in preparation), which clarifies the operational similarities and differences between beta-lactam elastase inhibitors and antibiotics. Features of the reaction include the expulsion of a leaving group at the cephalosporin 3' position and the formation of two covalent bonds with the active site of porcine pancreatic elastase at residues Ser 195 and His 57.

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.