Synthesis of N-glyoxylyl peptides and their in vitro evaluation as HIV-1 protease inhibitors.

A series of novel synthetic peptides containing an N-terminal glyoxylyl function (CHOCO-) have been tested as inhibitors of HIV-1 protease. The N-glyoxylyl peptide CHOCO-Pro-Ile-Val-NH2, which fulfills the specificity requirements of the MA/CA protease cleavage site together with the criteria of transition state analogue of the catalyzed reaction, was found to be a moderate competitive inhibitor although favorable interactions were visualized between its hydrated form and the catalytic aspartates using molecular modeling. Increasing the length of the peptide sequence led to compounds acting only as substrates.

Synthesis and inhibition of human leucocyte elastase by functionalized N-aryl azetidin-2-ones: effect of different substituents on the aromatic ring.

N-aryl-3,3-difluoroazetidin-2-ones featured by a latent electrophilic methylene quinoniminium function have been synthesized and evaluated as inhibitors of human leucocyte elastase. To promote hydrophobic interactions with the enzyme, to increase the rates of beta-lactam ring opening and of benzylic group departure, or to induce hydrosolubility, these compounds incorporate on their aromatic ring either an alkyl moiety, a methoxy substituent or a carboxylic group. Some of these beta-lactams proved to be good inactivators of human leucocyte elastase.

Interaction of human leukocyte elastase with a N-aryl azetidinone suicide substrate: Conformational analyses based on the mechanism of action of serine proteinases.

The three-dimensional interaction of the enzyme-activated (suicide) inhibitor AA 231-1 [N-(2-chloromethyl)-3, 3-difluoro-azetidin-2-one] with human leukocyte elastase has been studied using computer graphics and molecular mechanics. Systematic conformational analyses and energy minimizations have been performed for the inhibitor AA 231-1 and its presumed complexes formed during the enzymatic process of inactivation, i.e., the Michaelis complex, the acyl-enzyme, and the inactivated enzyme with the covalently bound inhibitor. The beta-lactam ring characteristics of modeled AA 231-1 were in agreement with crystallographic data of related structures. Lowest energy conformations were found when the angle between the planes of the beta-lactam ring and that of its phenyl substituent was about -60 or 60 degrees. To study the interaction with the enzyme, the enzyme-inhibitor complexes were constructed by docking the inhibitor in the active site using enzyme coordinates from an X-ray crystallographic structure. The whole enzyme structure was used for conformational analyses and energy mechanics. Favorable conformations for the Michaelis complex have been obtained in which the carbonyl oxygen of the inhibitor was located in the oxyanion hole and the hydroxyl of Ser195 was in position to interact with the beta-lactam carbonyl carbon on the alpha face of AA 231-1. Simulations of the approach of the benzylic carbon by the nucleophilic amino acid His40 or His57 through an SN2 displacement on the halomethyl group of AA 231-1 were performed. The results agreed with the alkylation of the imidazole nitrogen N epsilon 2 of His57 leading to the inactivated enzyme (bis-adduct form).

[Synthetic inhibitors targeting serine and aspartic acid proteases]. / Inhibiteurs synthétiques à visée pharmacologique ciblant les protéases à sérine et à acides aspartiques.

The interaction of novel series of synthetic inhibitors with various serine proteases (leukocyte elastase, thrombin, cathepsin G, chymotrypsin, plasminogen activators and plasmin) and an aspartic protease (HIV-1 protease) were studied. Various aspects were analyzed: mechanism of action, structure-activity relationships, and in some cases, molecular modelling and biological evaluation. Functionalized cyclopeptides and N-aryl azetidin-2-ones behaved as suicide substrates acting specifically on trypsin-like proteases (thrombin or urokinase) and elastases, respectively. Novel hydrazinopeptides acted as reversible inhibitors of elastases. Coumarin derivatives inactivated very efficiently chymotrypsin-like proteases (k(inact)/K(I) = 760,000 M(-1) .s(-1)). Inhibitors of HIV-1 protease acting either as inactivators or dimerization inhibitors are under investigation. The inhibitors described above are useful for elucidating the biological roles of the target enzymes and constitute potential drugs.

Evaluation of the inhibitory activity on serine and aspartic proteases of 4-amino-4H-1,2,4-triazole and 5-aminothiazole derivatives structurally related to beta-lactam antibiotics.

Twenty new derivatives of 4-amino-4H-1,2,4-triazole and 5-aminothiazole have been examined for their inhibitory potential towards serine and aspartic proteases. Upon prolonged incubation with enzyme, the phenylacetylaminothiazolium salts exhibit progressive, time-dependent inhibition of chymotrypsin according to a first-order process. The formation of a tetrahedral transition state-like complex by attack of the active-site serine at the C2-position of the pseudobase form of the thiazolium may be responsible for the observed effect. Triazolium salts appeared to be simple competitive inhibitors of this enzyme, effective in the mM range concentration. Poor inhibitions of trypsin and pepsin were also obtained in the triazolium series. In spite of their structural analogy with beta-lactams, the selected derivatives failed to inhibit human leucocyte elastase.

Protection of vascular basement membrane and microcirculation from elastase-induced damage with a fluorinated beta-lactam derivative.

N-(2-chloromethylphenyl) 3,3-difluoroazetidin-2-one (AA 231-1), a specific suicide-type inhibitor of elastase which is known to suppress the lysis of chromogenic oligopeptides, elastin and elastic fibers, is effective also in preventing the degradation of the vascular basement membrane. The degradation of porcine glomerular basement membrane by purified human leukocyte elastase (HLE), was reduced in proportion of inhibitor dose (8.3 microM for 50% inhibition). It is noteworthy that there was no reduction of the inhibitory effect when the addition of AA 231-1 was delayed for 1 h after the addition of the enzyme to the substrate. In the guinea pig, reduction of the dermal microhemorrhage due to HLE was related to the dose of inhibitor and to its preincubation time with HLE before intradermal injection. The inflammatory hemorrhage associated with the Arthus skin reaction was moderately depressed by AA 231-1 in situ. A part of the vascular permeability induced by HLE also responded to the inhibitor. In spite of the tissular diffusion and the time-dependence parameters which restrict responsiveness of elastase to AA 231-1 in vivo this biochemical compound should be helpful in the study and possibly the cure of vascular injury related to elastase.

Prevention of some types of inflammatory damage using AA 231-1, a fluorinated beta-lactam.

The leukocyte elastase inhibitory activity of AA 231-1, a suicide substrate, was investigated in the presence of elastin, a natural substrate of elastase, and its efficiency to reduce the degradation of basement membrane and haemorrhage induced by elastase was analysed. Elastin only moderately decreased the inhibitory efficiency of AA 231-1. The digestion by human leukocyte elastase (HLE) of glomerular basement membrane prepared from pig kidney was prevented in the presence of AA 231-1. Intradermal microvascular haemorrhage was also significantly inhibited by AA 231-1. These results suggest that AA 231-1 may be a valuable candidate as an anti-inflammatory agent.

Functionalized N-aryl azetidinones as novel mechanism-based inhibitors of neutrophil elastase.

A functionalized N-aryl azetidinone has been shown to inactivate human leukocyte elastase (HLE) and porcine pancreatic elastase (PPE) by an enzyme-mediated process. The inactivation is characterized by the following kinetic constants at pH 8.0 and 37 degrees C: kinact = 0.035 s-1, KI = 1.2 x 10(-4) M for HLE, 0.08 s-1 and 2.7 x 10(-4) M for PPE, respectively. Two parent molecules devoid of the latent leaving group failed to inactivate HLE and PPE and behaved as substrates of these enzymes. A suicide mechanism is postulated involving the formation of an acyl-enzyme and the simultaneous unmasking of a latent quinonimmonium methide ion which irreversibly reacts with an active site nucleophile. Moreover, the inhibitor is still effective at inhibiting elastase preabsorbed onto elastin.

Acyloxybenzyl halides, inhibitors of elastases.

3-Benzyl-6-chloromethyl-3,4-dihydrocoumarin inhibits human leucocyte elastase (HLE) and porcine pancreatic elastase (PPE) through a mechanism-based process characterized by the following apparent enzyme-inhibitor dissociation constants, Ki, and limiting inactivation rate constants k2: 200 microM (HLE), 69 microM (PPE) and 5.10(-2) s-1 (HLE), 17.7.10(-2) s-1 (PPE) at pH 8.0, 37 degrees C. Bis(4-acyloxyphenyl)methane derivatives with a benzylic halogen as potential leaving group have also been synthesized and studied. They transiently inactivate PPE and HLE through the formation of an acyl-enzyme.