Interaction of zona pellucida glycoproteins, sulphated carbohydrates and synthetic polymers with proacrosin, the putative egg-binding protein from mammalian spermatozoa.

Fertilization in mammals is a unique cell-cell recognition event that involves specific receptors on the surface of each gamete. Previous work has shown that proacrosin, a protein found within the acrosome of mammalian spermatozoa, binds non-enzymatically to zona pellucida glycoproteins (ZPGPs) that surround the egg and that this binding can be inhibited by sulphated polysaccharides such as fucoidan. The mechanism of this interaction has been investigated using 125I-ZPGPs and 125I-fucoidan as probes. Results show that it involves poly(sulphate) groups on zona glycoproteins that bind with high affinity (Kd = 1.2 to 5.0 x 10(-8)M) to complementary 'docking' sites on proacrosin. The spatial orientation of these sulphates, together with the tertiary structure of the target protein, determines the selectivity of polymer binding. Thus, dextran sulphate and poly(vinyl sulphate) are strong inhibitors of the above probes whereas dextran, chondroitin sulphates A and C and poly(vinyl phosphate) are ineffective. Proacrosin, therefore, has properties analogous to those described for 'bindin', the egg adhesion protein found within the acrosomal vesicle of sea urchin spermatozoa.     

The inhibition of acrosin by sterol sulphates

Four 3 beta-hydroxy-delta 5-steroid sulphates were found to be potent and specific inhibitors of the sperm acrosomal proteinase, acrosin. Two of these acrosin inhibitors, desmosteryl sulphate and cholesteryl sulphate, occur naturally in spermatozoa. Desmosteryl sulphate, an inhibitor of the in-vitro capacitation of hamster spermatozoa, has a Ki of 3.5 x 10(-6) M for the inhibition of acrosin. The mechanism of inhibition of sperm capacitation by sterol sulphates is probably due to their inhibition of acrosin.     

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.     

2,4,5-Triphenylisothiazol-3 (2H)-one 1,1-dioxides as inhibitors of human leukocyte elastase

A series of substituted 2,4,5-triphenylisothiazol-3(2H)-one 1,1-dioxides 9 was synthesized and investigated as inhibitors of human leukocyte elastase (HLE). All compounds were found to inhibit HLE in a time-dependent manner and most of them exhibited kobs/[I] values > 300M(-1)s(-1). The most potent 3-oxosultam of this series was 91 (kobs/[I] = 2440 M(-1)s(-1)). Kinetic investigations performed with 9g and different substrate concentrations did not allow to clearly distinguish between a competitive or noncompetitive mode of inhibition. A more complex interaction is supported by the failure of a linear dependency of kobs values on the inhibitor concentration.     

Interaction between leukocyte elastase and elastin: quantitative and catalytic analyses

Solubilization of elastin by human leukocyte elastase (HLE) cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. We now report quantitative measurements of the binding and catalytic interaction between HLE and elastin permitted by analogy to receptor-ligand systems. Our results indicated that a limited and relatively constant number of enzyme binding sites were available on elastin, and that new sites became accessible as catalysis proceeded. The activation energies and solvent deuterium isotope effects were similar for catalysis of elastin and a soluble peptide substrate by HLE, yet the turnover number for HLE digestion of elastin was 200-2000-fold lower than that of HLE acting on soluble peptide substrates. Analysis of the binding of HLE to elastin at 0 degrees C, in the absence of significant catalytic activity, demonstrated two classes of binding sites (Kd=9.3x10(-9) M and 2.5x10(-7) M). The higher affinity sites accounted for only 6% of the total HLE binding capacity, but essentially all of the catalytic activity, and dissociation of HLE from these sites was minimal. Our studies suggest that interaction of HLE with elastin in vivo may be very persistent and permit progressive solubilization of this structurally important extracellular matrix component.     

Utilization of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold in the design of potent inhibitors of serine proteases: SAR studies using carboxylates

A series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1 dioxide scaffolds has been synthesized and the inhibitory profile of these compounds toward human leukocyte elastase (HLE), cathepsin G (Cat G) and proteinase 3 (PR 3) was then determined. Most of the compounds were found to be potent, time-dependent inhibitors of elastase, with some of the compounds exhibiting k(inact)/K1 values as high as 4,928,300 M(-1) s(-1). The inhibitory potency of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide platform was found to be influenced by both the pKa and the inherent structure of the leaving group. Proper selection of the primary specificity group (R(I)) was found to lead to selective inhibition of HLE over Cat G, however, those compounds that inhibited HLE also inhibited PR 3, albeit less efficiently. The predictable mode of binding of these compounds suggests that, among closely-related serine proteases, highly selective inhibitors of a particular serine protease can be fashioned by exploiting subtle differences in their S' subsites. This study has also demonstrated that the degradative action of elastase on elastin can be abrogated in the presence of inhibitor 17.     

Synthesis and in-vitro evaluation of novel low molecular weight thiocarbamates as inhibitors of human leukocyte elastase

A series of novel low molecular weight thiocarbamate esters (1e-6e) were synthesized and evaluated as inhibitors of human leukocyte elastase (HLE). The thiocarbamate esters studied consist of a substituted primary or secondary aliphatic or aromatic amine and a 1-phenyl-1H-tetrazole-5-thiol (Table I). The HLE catalyzed hydrolysis of N-methoxysuccinyl- L-Ala-L-Ala-L-Pro-L-Val-p-nitroanilide substrate was utilized as the measure of inhibition. N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (1e) exhibited the highest inhibitory activity (k(obs) /[I] = 2.1 x 10(5) M(-1). min(-1) ) and N-allyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (2e) (K(obs) /[I] = 6.1 x 10(4) M(-1). min(-1) ) exhibited the second highest inhibitory activity of all the thiocarbamates. The aromatic N-phenyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (4e) showed the lowest inhibitory activity (K(obs) /[I] = 1.9 x 10(2) M(-1). min(-1) ) among the N-monosubstituted derivatives, similar to that of N-ethyl-N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (5e) (K(obs) /[I] = 1.8 x 10(2) M(-1).min(-1) ). The N-isopropyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (3e) (K(obs) /[I] = 3.3 x 10(3) M(-1).min(-1) ) was about 10 fold more active than (4e) and N, N-diisopropyl, 1-phenyl-1H-tetrazole- 5-thiocarbamate (6e) showed no inhibitory activity against HLE. In the present work less than 3% of HLE specific activity was regained after 24 hours incubation with each of the tested N-monosubstituted thiocarbamates (1e-4e). The time-dependent inhibition of HLE by the thiocarbamate compounds (1e-5e) seems to involve the interaction and possible chemical modification of one enzyme residue. Straight chain nonpolar aliphatic substituents on the nitrogen of the thiocarbamate functionality may be essential for high inhibitory activity. As the degree of substitution (branching) on the nitrogen of the thiocarbamate functionality increases the inhibitory activity of the compounds decreases. The time-dependent inhibition of HLE and the slow deacylation rates by the N-monosubstituted thiocarbamates are consistent with irreversible inhibition.     

Structure-activity studies of fluoroketone inhibitors of alpha-lytic protease and human leukocyte elastase

We have synthesized a series of peptidyl fluoroketones that reversibly inhibit the serine proteases human leukocyte elastase (HLE) and alpha-lytic protease (alpha-LP). Ac-ambo-AlaCF3 (1) inhibits HLE and alpha-LP with Ki's of 2.4 and 15 mM, respectively. The effects of structural variations on this parent compound on Ki and the kinetics of inhibition were studied. The acetyl group was replaced by the tripeptide Z-L-Ala-L-Ala-L-Pro to yield the tetrapeptide trifluoroketone (TFK) Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF3 (2). This extension reduced Ki 3500-fold for HLE and 3000-fold for alpha-LP. Removal of a fluorine atom from a TFK decreases Ki about 15- to 30-fold with both enzymes. Replacement of one fluorine atom of 2 by a residue (-CH2-CH2-COLeuOMe) (6) which can interact with the S'1 and S'2 subsites decreased Ki 30-fold for HLE and 150-fold for alpha-LP compared to Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF2H (3). The Ki of 6 for HLE is approximately equal to that of trifluoroketone 2. For alpha-LP Ki of 6 is 10-fold lower than that for the trifluoroketone 2. Inhibitors with Ki values less than 10(-7) M exhibit slow binding kinetics. By analogy to cholinesterases and chymotrypsin, it is likely that these enzymes combine with the keto form of the inhibitor to form the enzyme-inhibitor complex. Therefore, kon and Ki were corrected for the ketone concentration. The corrected kon values for the slow binding inhibitors are in most cases less than diffusion controlled, ranging between 8.2 X 10(4) and 4.68 X 10(6) M-1 s-1. An exception is Z-L-Ala-L-Ala-L-Pro-ambo-ValCF3 (8) where kon = 9 X 10(7) M-1 s-1, which is nearly diffusion controlled.     

Synthesis and in vitro evaluation of pseudosaccharinamine derivatives as potential elastase inhibitors

Pseudosaccharinamine derivatives were evaluated for elastase inhibitory activity. Ester derivatives of pseudosaccharinamine displayed reversible and high inhibition of human leukocyte elastase (HLE) as compared to porcine pancreatic elastase (PPE). Cyanomethyl (2S,3S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylpentanoate was found to inhibit HLE at Ki=0.8 microM.     

A new class of inhibitors of human leucocyte elastase

Studies of the inhibition of elastases at a molecular level have resulted in the identification of protected dipeptides which are reversible and highly specific inhibitors of human leucocyte elastase (HLE). These have been further developed by increasing their hydrophilicity and potency to give a new family of elastase inhibitors, typically N alpha-(1-adamantanesulphonyl)-N epsilon-(4-carboxybenzoyl)-L-lysyl-L-alanyl-L-valinal. These compounds are active in pharmacological models designed to detect compounds of potential therapeutic value in the treatment of emphysema.     

Initiation of altered heparan sulphate on beta-D-xyloside in rat hepatocytes

Inhibition of protein synthesis by cycloheximide 10(-3)M reduced the incorporation of [35S]sulphate into heparan sulphate to about 5% of untreated hepatocytes. Addition of rho-nitrophenyl beta-D-xyloside could partially revert this inhibitory effect. The sulphated material isolated from the cell layer or secretions of hepatocytes grown in presence of cycloheximide and rho-nitrophenyl beta-D-xyloside were shown to be mostly free heparan sulphate chains not bound to core protein. Covalent association of beta-xylosides to the heparan sulphates was demonstrated for heparan sulphate synthetized in the presence of [35S]sulphate, cycloheximide and the fluorogenic 4-methylumbelliferyl beta-D-xyloside. Beta-Xylosides served as an initiator of heparan sulphate chain synthesis in rat hepatocytes only in the absence of protein synthesis. Heparan sulphates primed on artificial beta-xylosides are slightly smaller in molecular size and are more sulphated than chains linked to core protein.     

Cleavage of CD14 on human gingival fibroblasts cocultured with activated neutrophils is mediated by human leukocyte elastase resulting in down-regulation of lipopolysaccharide-induced IL-8 production

Activated polymorphonuclear leukocytes (PMNs) release various types of proteases and express them on the cell surface. The proteases play important roles in PMN-mediated events. In the present study, flow cytometric analysis revealed that CD14 expression on human gingival fibroblasts (HGF) was markedly reduced by PMA-activated PMNs in a coculture system. We found that this reduction was caused by both secreted and cell surface proteases produced by activated PMNs. A protease responsible for the reduction was found to be human leukocyte elastase (HLE) secreted from the activated PMNs by use of various protease inhibitors, although HLE was only partially involved in CD14 reduction caused by cell-bound molecule(s) on fixed PMNs. Analysis with purified HLE revealed a time- and dose-dependent reduction of CD14 on HGF, and complete reduction was observed by 20 microg/ml HLE treatment for 30-60 min, but the other molecules such as CD26, CD59, CD157, and MHC class I on HGF were only slightly reduced. This reduction of CD14 resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD14 on fixed HGF and also on purified cell membranes. As a result of CD14 proteolysis, IL-8 production by HGF was suppressed when triggered by 10 ng/ml LPS, but not by IL-1alpha, indicating that HLE inhibited a CD14-dependent cell activation. These findings suggested that activated PMNs have a potential negative feedback mechanism for HGF function at the inflammatory site, particularly in periodontal tissues.     

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.     

Elafin and its precursor trappin-2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase

Elafin and its precursor trappin-2 (also called pre-elafin) are potent protein inhibitors of neutrophil serine proteases such as leukocyte elastase and proteinase 3. Trappin-2 has unique conserved sequence motifs rich in Gln and Lys residues. These motifs are substrates for transglutaminases that may enable trappin-2 to be cross-linked to extracellular matrix proteins, thus anchoring the inhibitor at its site of action. We have used Western blotting and ELISA-based assays to demonstrate that both elafin and trappin-2 can be conjugated to various extracellular matrix proteins in vitro by a type 2 transglutaminase. Cross-linked elafin and trappin-2 still inhibited their target proteases. Surface plasmon resonance studies allowed the determination of the kinetic constants governing the interaction of fibronectin-bound elafin and trappin-2 with neutrophil elastase and proteinase 3. Both inhibitors were potent inhibitors when cross-linked to fibronectin by transglutamination, with equilibrium dissociation constants K(i) for their interaction with target proteases of 0.3 nM (elastase-elafin), 20 nM (proteinase 3-elafin), 0.3 nM (elastase-trappin-2), and 12 nM (proteinase 3-trappin-2). The conjugated inhibitors reacted more slowly with their target enzymes than did the soluble inhibitors, perhaps due to their immobilization, with association rate constants of 2-7 x 10(5) M(-)(1) s(-)(1) for elastase and 1-4 x 10(4) M(-)(1) s(-)(1) for proteinase 3. We believe this is the first demonstration that transglutaminase-mediated cross-linking of serine protease inhibitors to proteins preserves their inhibitory capacities.     

Effect of sodium oleate on the hydrolysis of human plasma fibronectin by proteinases

Oleic acid binds in a saturable fashion to human plasma fibronectin (FN). Analysis of the binding indicated the presence of a high affinity binding site with nKa approximately equal to 10 uM-1. Furthermore, it was found that binding of sodium oleate to FN modulated its susceptibility to degradation by various proteinases. FN saturated with sodium oleate was hydrolysed at a higher rate by trypsin, cathepsin D, thermolysin and pancreatic elastase than native FN. In contrast, sodium oleate inhibits the activity of two human granulocyte proteinases, human leucocyte elastase (HLE) and cathepsin G on either FN or on their respective specific synthetic substrates (at concentrations ranging from 10(-6) mM to 10 mM). Cathepsin G inhibition was non-competitive and gave a Ki in the 10 uM range similar to the previously reported inhibitory constant of oleic acid toward HLE.     

Inhibition of human serine proteases by substituted 2-azetidinones.

trans-4-Ethoxycarbonyl-3-ethyl-1-(4-nitrophenyl-sulfonyl)-azetidin -3-one described by Firestone et al. (1990, Tetrahedron 46, 2255) as an inhibitor of human leucocyte elastase (HLE) displayed potent, time-dependent inhibition of both HLE and human cathepsin G (Cat-G). The cis-isomer was 7- and 180-fold less active, respectively. The mechanism likely involves opening of the beta-lactam ring by the active site serine to form an acyl-enzyme intermediate(s). This intermediate partitions with ratios of 4:1 between turnover of the inhibitor and formation of relatively stable enzyme-inhibitor complexes from both enzymes. The final HLE-inhibitor complex reactivated with a half-life of 48 h at 25 degrees C and was 16-fold more stable than the Cat-G-inhibitor complex. The stability of the acyl-enzymes supports a "double hit" chemical mechanism involving both serine acylation and alkylation of the histidine. These observations suggest that beta-lactams may be developed as a class of serine protease inhibitors.     

6-Acylamino-2-1[(ethylsulfonyl)oxy]-1H-isoindole-1,3-diones mechanism-based inhibitors of human leukocyte elastase and cathepsin G: effect of chirality in the 6-acylamino substituent on inhibitory potency and selectivity

Inhibition of human leukocyte elastase(HLE) by a series of 6-acylamino-2-[(ethylsulfonyl)oxy)]-1H-isoindole-1,3-diones was determined and compared to their inhibition of ChT, PPE, and Cat G. The best inhibitor of the series was 6-((1'S)-camphanyl)amino-2-[(ethylsulfonyl) oxy]-1H-isoindole-1,3-dione 5b, with a k(obs)/[I] = 11,000 M(-1) s(-1). This study revealed that HLE shows a preference for the S stereochemistry and tolerates hydrophobic substituents in the Sn' binding sites. Molecular modeling of non-covalent HLE-inhibitor complexes was used as a tool to investigate our binding model. Buffer stability assays reveal that these compounds are susceptible to hydrolysis at physiological pH.     

Irreversible inhibition of serine proteases by peptide derivatives of (alpha-aminoalkyl)phosphonate diphenyl esters

Peptidyl derivatives of diphenyl (alpha-aminoalkyl)phosphonates have been synthesized and are effective and specific inhibitors of serine proteases at low concentrations. Z-PheP(OPh)2 irreversibly reacts with chymotrypsin (kobsd/[I] = 1200 M-1 s-1) and does not react with two elastases. The best inhibitor for most chymotrypsin-like enzymes including bovine chymotrypsin, cathepsin G, and rat mast cell protease II is the tripeptide Suc-Val-Pro-PheP(OPh)2 which corresponds to the sequence of an excellent p-nitroanilide substrate for several chymases. The valine derivative Z-ValP(OPh)2 is specific for elastases and reacts with human leukocyte elastase (HLE, 280 M-1 s-1) but not with chymotrypsin. The tripeptide Boc-Val-Pro-ValP(OPh)2, which has a sequence found in a good trifluoromethyl ketone inhibitor of HLE, is the best inhibitor for HLE (kobsd/[I] = 27,000 M-1 s-1) and porcine pancreatic elastase (PPE, kobsd/[I] = 11,000 M-1 s-1). The rates of inactivation of chymotrypsin by MeO-Suc-Ala-Ala-Pro-PheP(OPh)2 and PPE and HLE by MeO-Suc-Ala-Ala-Pro-ValP(OPh)2 were decreased 2-5-fold in the presence of the corresponding substrate, which demonstrates active site involvement. Only one of two diastereomers of Suc-Val-Pro-PheP(OPh)2 reacts with chymotrypsin (146,000 M-1 s-1), and the enzyme-inhibitor complex had one broad signal at 25.98 ppm in the 31P NMR spectrum corresponding to the Ser-195 phosphonate ester. Phosphonylated serine proteases are extremely stable since the half-time for reactivation was greater than 48 h for the inhibited elastases and 7.5-26 h for chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin enhances the rate of binding of fibronectin to collagen.

125I-labelled fibronectin is shown to bind to both native and denatured collagen immobilized on Sephadex beads in reactions that exhibit different kinetics. The rates of both reactions were enhanced by the presence of heparin or highly sulphated dextran sulphate but not by other glycosaminoglycans or dextran sulphates having low sulphate contents.     

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.