Inhibition of the activation of multiple serine proteases with a cathepsin C inhibitor requires sustained exposure to prevent pro-enzyme processing

Cathepsin C is a cysteine protease required for the activation of several pro-inflammatory serine proteases and, as such, is of interest as a therapeutic target. In cathepsin C-deficient mice and humans, the N-terminal processing and activation of neutrophil elastase, cathepsin G, and proteinase-3 is abolished and is accompanied by a reduction of protein levels. Pharmacologically, the consequence of cathepsin C inhibition on the activation of these serine proteases has not been described, due to the lack of stable and non-toxic inhibitors and the absence of appropriate experimental cell systems. Using novel reversible peptide nitrile inhibitors of cathepsin C, and cell-based assays with U937 and EcoM-G cells, we determined the effects of pharmacological inhibition of cathepsin C on serine protease activity. We show that indirect and complete inhibition of neutrophil elastase, cathepsin G, and proteinase-3 is achievable in intact cells with selective and non-cytotoxic cathepsin C inhibitors, at concentrations approximately 10-fold higher than those required to inhibit purified cathepsin C. The concentration of inhibitor needed to block processing of these three serine proteases was similar, regardless of the cell system used. Importantly, cathepsin C inhibition must be sustained to maintain serine protease inhibition, because removal of the reversible inhibitors resulted in the activation of pro-enzymes in intact cells. These findings demonstrate that near complete inhibition of multiple serine proteases can be achieved with cathepsin C inhibitors and that cathepsin C inhibition represents a viable but challenging approach for the treatment of neutrophil-based inflammatory diseases.     

The inhibition of human neutrophil elastase and cathepsin G by peptidyl 1,2-dicarbonyl derivatives

Neutrophil elastase and cathepsin G are serine proteases that can damage connective tissue and trigger other pathological reactions. Compounds containing a peptide sequence to impart specificity and bearing an alpha-dicarbonyl unit (alpha-diketone or alpha-keto ester) at the carboxy terminus are potent inhibitors of the neutrophil serine proteases (human neutrophil elastase: R-Val-COCH3, Ki = 0.017 microM; R-Val-COOCH3, Ki = 0.002 microM; human neutrophil cathepsin G: R-Phe-COCH3, Ki = 0.8 microM; R-Phe-COOCH3, Ki = 0.44 microM; R = N-(4-[(4-chlorophenyl)sulfonylaminocarbonyl]phenylcarbonyl)+ ++ValylProlyl).     

Inhibition of human leukocyte elastase, cathepsin G, chymotrypsin A alpha, and porcine pancreatic elastase with substituted isobenzofuranones and benzopyrandiones

Several 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones, 3-(1-haloalkylidene)-1(3H)-isobenzofuranones, and 3-bromomethyl-1H-2-benzopyran-1-ones containing masked halo ketone functional groups were synthesized and tested as inhibitors of several serine proteases including human leukocyte (HL) elastase and cathepsin G. While many of the 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones were quite potent inhibitors of the enzymes tested, the alkylideneisobenzofuranones and benzopyran-1-ones inhibited poorly or not at all. The 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones decomposed rapidly upon addition to buffer to give the corresponding 3-alkyl-1H-2-benzopyran-1,4(3H)-diones. The pure benzopyran-1,4-diones were extremely potent inhibitors of HL elastase and chymotrypsin A alpha but did not inactivate porcine pancreatic elastase or cathepsin G. Enzymes inhibited by the isobenzofuranones and benzopyran-1,4-diones regained activity slowly upon standing or after dialysis (t1/2 = 5-16 h) and more rapidly in the presence of 0.5 M hydroxylamine, which indicated the presence of labile acyl moieties in the inhibited enzyme. These results are consistent with a scheme in which the active site serine of the protease reacts with the lactone carbonyl of these inhibitors to give a stable acyl enzyme and alkylation of another active site residue by the unmasked halo ketone functional group does not occur.     

Synthetic inhibitors of human leukocyte elastase, Part 3. Peptides with alkyl groups at the N- or C-terminus. Non-toxic competitive inhibitors of human leukocyte elastase

A series of carboxy-alkylamidated and N-acetylated amino acids and peptides were synthesized and examined for their ability to inhibit human leukocyte elastase. The Boc-amino acid alkylamides were found to be potent specific and competitive inhibitors of this enzyme. They were found not to or only poorly inhibit several other serine proteinases such as bovine trypsin, alpha-chymotrypsin, porcine pancreatic elastase and human leukocyte cathepsin G at concentrations less than 10(-4) M. Specificity and maximum inhibition of human leukocyte elastase were achieved when the N-terminus of the amino acid was protected by a t-butyloxy-carbonyl (Boc) group, the oligopeptide fragment consisted of valine residues and when the alkyl chain was between 10 and 12 carbon atoms in length and attached to the C-terminus of the peptide fragment. Highest inhibition was obtained with the compound Boc-[Val]3-NH[CH2]11--CH3 (Ki = 0.21 microM). These specific inhibitors were also found to be non-toxic after oral administration to mice and rats (LD50 greater than 3.0 g/kg body weight).     

Various properties of cathepsin G and elastase from swine peripheral blood neutrophils

Using gel filtration through Sephadex G-100 and bioaffinity chromatography on contrical-Sepharose, cathepsin G and elastase were isolated from pig peripheral blood neutrophil granules and purified to homogeneity. Both enzymes hydrolyzed the total histone from calf thymus as well as synthetic substrates--tert-butoxy-L-alanine p-nitrophenyl ester (elastase) and benzoyltyrosine ethyl ester (cathepsin G). The use of natural and synthetic protease inhibitors showed that both enzymes were related to the group of serine proteases. The molecular mass of the cathepsin G subunit as determined by SDS polyacrylamide gel electrophoresis is 28-29 kD, that of elastase--30-31 kD. The pH optima for the hydrolysis of proteinaceous and synthetic substrates for cathepsin G and elastase are 8.0-8.5 and 7.0-7.5, respectively. The isoelectric points for elastase and cathepsin G are 9.7-10.0 and greater than 10, respectively; the temperature optima--30-40 degrees C and 50-60 degrees C, respectively. The amino acid composition of the two enzymes from pig granulocytes revealed a high content of arginine and was similar to that of human granulocytes.     

Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates

Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.     

Inactivation of human neutrophil elastase by 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamides

The interaction of a series of 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamides with neutrophil-derived serine proteases was investigated. The nature of the amino acid component, believed to be oriented toward the S' subsites, had a profound effect on enzyme selectivity. This series of compounds were found to be potent, time-dependent inhibitors of human neutrophil elastase (HNE) and were devoid of any inhibitory activity toward neutrophil proteinase 3 (PR 3) and cathepsin G (Cat G). The results of these studies demonstrate that exploitation of differences in the S' subsites of HNE and PR 3 can lead to highly selective inhibitors of HNE.     

Degradation of cartilage matrix proteoglycan by human neutrophils involves both elastase and cathepsin G

The granule proteases of human neutrophils are thought to be responsible for the connective tissue destruction associated with certain inflammatory diseases. Using a model system for the degradation of a macromolecular connective tissue substrate, purified neutrophil elastase and cathepsin G were both individually able to degrade cartilage matrix proteoglycan and this degradation was blocked by the appropriate specific inhibitors. Neutrophil granule lysate also produced cartilage matrix degradation but little inhibition of degradation occurred when either elastase or cathepsin G inhibitor was used alone. However, a combination of elastase and cathepsin G inhibitors each at 100 microM or each at 10 microM blocked cartilage matrix degradation by 89% +/- 1 and 65% +/- 9 (mean +/- SEM, n = 3), respectively. The magnitude of the cartilage degradation mediated by neutrophil lysate, and its sensitivity to specific inhibitors, was reproduced using purified elastase and cathepsin G at the concentrations at which they are present in neutrophil lysate. Human neutrophils stimulated with opsonized zymosan degraded cartilage matrix in a dose-dependent manner in the presence of serum antiproteases. Supernatants from stimulated neutrophils cultured in the presence of serum did not degrade cartilage matrix, indicating that neutrophil mediated degradation in the presence of serum was confined to the protected subjacent region between the inflammatory cell and the substratum. A combination of elastase and cathepsin G inhibitors each at 500 microM or each at 100 microM blocked subjacent cartilage matrix degradation by stimulated human neutrophils by 91% +/- 3 and 54% +/- 8 (mean +/- SEM, n = 5), respectively, whereas either the elastase or cathepsin G inhibitor alone was much less effective. These studies demonstrate that neutrophil-mediated cartilage matrix degradation is produced primarily by elastase and cathepsin G. Furthermore, these results support the hypothesis that inflammatory neutrophils form zones of close contact with substratum that exclude serum antiproteases and that this subjacent degradation of cartilage matrix by stimulated neutrophils can be blocked by a combination of synthetic elastase and cathepsin G inhibitors.     

Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids

The kinetic parameters for peptide boronic acids in their interaction with alpha-lytic protease were determined and found to be similar to those of other serine proteases [Kettner, C., & Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114]. alpha-Lytic protease hydrolyzes substrates with either alanine or valine in the P1 site and has a preference for substrate with a P1 alanine. The most effective inhibitors are tri- and tetrapeptide analogues that have a -boroVal-OH residue in the P1 site. At pH 7.5, MeOSuc-Ala-Ala-Pro-boroVal-OH has a Ki of 6.4 nM and Boc-Ala-Pro-boroVal-OH has a Ki of 0.35 nM. Ac-boroVal-OH and Ac-Pro-boroVal-OH are 220,000- and 500-fold less effective, respectively, than the tetrapeptide analogue. The kinetic properties of the tri- and tetrapeptide analogues are consistent with the mechanism for slow-binding inhibition, E + I in equilibrium EI in equilibrium EI*, while the less effective inhibitors are simple competitive inhibitors. MeO-Suc-Ala-Ala-Pro-boroAla-OH is a simple competitive inhibitor with a Ki of 67 nM at pH 7.5. Other peptide boronic acids, which are analogues of nonsubstrates, are less effective than substrate analogues but still are effective competitive inhibitors. For example, MeOSuc-Ala-Ala-Pro-boroPhe-OH has a Ki of 0.54 microM although substrates with a phenylalanine in the P1 position are not hydrolyzed. Binding for boronic acid analogues of both substrate and nonsubstrate analogues is pH dependent with higher affinity near pH 7.5. Similar binding properties have been observed for pancreatic elastase. Both enzymes have almost identical requirements for an extended peptide inhibitor sequence in order to exhibit highly effective binding and slow-binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.     

Novel secretory vesicle serpins,endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.     

Interaction of heparin cofactor II with neutrophil elastase and cathepsin G

We investigated the interaction of the human plasma proteinase inhibitor heparin cofactor II (HC) with human neutrophil elastase and cathepsin G in order to examine 1) proteinase inhibition by HC, 2) inactivation of HC, and 3) the effect of glycosaminoglycans on inhibition and inactivation. We found that HC inhibited cathepsin G, but not elastase, with a rate constant of 6.0 x 10(6) M-1 min-1. Inhibition was stable, with a dissociation rate constant of 1.0 x 10(-3) min-1. Heparin and dermatan sulfate diminished inhibition slightly. Both neutrophil elastase and cathepsin G at catalytic concentrations destroyed the thrombin inhibition activity of HC. Inactivation was accompanied by a dramatic increase in heat stability, as occurs with other serine proteinase inhibitors. Proteolysis of HC (Mr 66,000) produced a species (Mr 58,000) that retained thrombin inhibition activity, and an inactive species of Mr 48,000. Amino acid sequence analysis led to the conclusion that both neutrophil elastase and cathepsin G cleave HC at Ile66, which does not affect HC activity, and at Val439, near the reactive site Leu444, which inactivates HC. Since cathepsin G is inhibited by HC and also inactivates HC, we conclude that cathepsin G participates in both reactions simultaneously so that small amounts of cathepsin G can inactivate a molar excess of HC. High concentrations of heparin and dermatan sulfate accelerated inactivation of HC by neutrophil proteinases, with heparin having a greater effect. Heparin and dermatan sulfate appeared to alter the pattern, and not just the rate, of proteolysis of HC. We conclude that while HC is an effective inhibitor of cathepsin G, it can be proteolyzed by neutrophil proteinases to generate first an active inhibitor and then an inactive molecule. This two-step mechanism might be important in the generation of chemotactic activity from the amino-terminal region of HC.     

Cathepsin G, a neutrophil-derived serine protease, increases susceptibility of macrophages to acute human immunodeficiency virus type 1 infection

Neutrophils dominate acute inflammatory responses that generally evolve into chronic inflammatory reactions mediated by monocyte/macrophages and lymphocytes. The latter cell types also serve as major targets for human immunodeficiency virus type 1 (HIV-1). In this study we have investigated the role of neutrophil products, particularly cathepsin G, in HIV infection. Cathepsin G induced chemotaxis and production of proinflammatory cytokines by macrophages but not CD4(+) T cells. Pretreatment with cathepsin G markedly increased susceptibility of macrophages but not CD4(+) T cells to acute HIV-1 infection. When macrophages were exposed to pertussis toxin prior to cathepsin G treatment, the cathepsin G-mediated effect was almost abrogated, suggesting that enhancement of HIV-1 replication by cathepsin G requires Gi protein-mediated signal transduction. Although prolonged exposure to cathepsin G suppressed HIV infection of macrophages, serine protease inhibitors, which are exuded from the bloodstream later during inflammatory processes, neutralized the inhibitory effect. Neutrophil extracts or supernatants from neutrophil cultures, which contain cathepsin G, had effects similar to purified cathepsin G. Thus, cathepsin G, and possibly other neutrophil-derived serine proteases, may have multiple activities in HIV-1 infection of macrophages, including chemoattraction of monocyte/macrophages (HIV-1 targets) to inflamed tissue, activation of target cells, and increase in their susceptibility to acute HIV-1 infection.     

Neutrophil elastase up-regulates cathepsin B and matrix metalloprotease-2 expression

Neutrophil elastase (NE) activity is increased in many diseases. Other families of proteases, including cathepsins and matrix metalloproteases (MMPs), are also present at elevated levels in similar disease conditions. We postulated that NE could induce expression of cathepsins and MMPs in human macrophages. NE exposure resulted in macrophages, producing significantly greater amounts of cathepsin B and latent and active MMP-2. Cathepsin B and MMP-2 activities were decreased in Pseudomonas-infected NE knockout mice compared with wild-type littermates. We also demonstrate that NE can activate NF-kappaB in macrophages, and inhibition of NF-kappaB resulted in a reduction of NE-induced cathepsin B and MMP-2. Also, inhibition of TLR-4 or transfection of macrophages with dominant-negative IL-1R-associated kinase-1 resulted in a reduction of NE-induced cathepsin B and MMP-2. This study describes for the first time a novel hierarchy among proteases whereby a serine protease up-regulates expression of MMPs and cathepsins. This has important implications for therapeutic intervention in protease-mediated diseases.     

Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates

The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. The whole procedure, including neutrophil purification and kinetic measurements, can be done in 4-5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.     

Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions

Polymorphonuclear neutrophils form a primary line of defense against bacterial infections using complementary oxidative and non-oxidative pathways to destroy phagocytized pathogens. The three serine proteases elastase, proteinase 3 and cathepsin G, are major components of the neutrophil primary granules that participate in the non-oxidative pathway of intracellular pathogen destruction. Neutrophil activation and degranulation results in the release of these proteases into the extracellular medium as proteolytically active enzymes, part of them remaining exposed at the cell surface. Extracellular neutrophil serine proteases also help kill bacteria and are involved in the degradation of extracellular matrix components during acute and chronic inflammation. But they are also important as specific regulators of the immune response, controlling cellular signaling through the processing of chemokines, modulating the cytokine network, and activating specific cell surface receptors. Neutrophil serine proteases are also involved in the pathogenicity of a variety of human diseases. This review focuses on the structural and functional properties of these proteases that may explain their specific biological roles, and facilitate their use as molecular targets for new therapeutic strategies.     

Interaction of peptide boronic acids with elastase: circular dichroism studies

Boronic acid derivatives of good peptide substrates of the serine proteases cause slow-binding inhibition, manifested as biphasic binding (Kettner and Shenvi: J. Biol Chem. 259:15106-15114, 1984). These inhibitors are thought to act as reaction-intermediate analogs. Three peptide boronic acids--Ac-Pro-boro-Val-OH, DNS-Ala-Pro-boro-Val-OH, and Ac-Ala-Ala-Pro-boro-Val-OH--were chosen for far-ultraviolet circular dichroism (CD) studies in order to determine whether the second phase involves a conformational change of pancreatic elastase. The dipeptide is a simple competitive inhibitor (Ki = 0.27 microM) and the latter are slow-binding inhibitors (Ki = 16.4 and 0.25 nM, respectively). Spectral deconvolution and correction for the formation of antiparallel beta-sheet by the peptide inhibitor itself indicate that there is no significant change in the secondary structure of the enzyme in either the initial or final inhibitor complex. A kinetic experiment confirmed that the slow-binding step was not associated with a CD spectral change, and that therefore a protein conformational change was not responsible for the slow binding.     

Evidence for a crucial role of neutrophil-derived serine proteases in the inactivation of interleukin-6 at sites of inflammation

The bioactivity of interleukin-6 (IL-6) was found to be dramatically reduced in fluids from sites of inflammation. Here, we provide evidence that the neutrophil-derived serine proteases elastase, proteinase 3 and cathepsin G are mainly involved in its degradation and subsequent inactivation. The initially hydrolyzed peptide bonds were detected to be Val(11)-Ala(12) and Leu(19)-Thr(20) (elastase), Phe(78)-Asn(79) (cathepsin G) and Ala(145)-Ser(146) (proteinase 3). The soluble IL-6 receptor elicits a protective effect against the IL-6 inactivation by cathepsin G only. The inactivation of IL-6 by neutrophil-derived serine proteases might act as a feedback mechanism terminating the IL-6-induced activation of neutrophils.     

Phosphonic pseudopeptides as human neutrophil elastase inhibitors--a combinatorial approach

Here we present a simple and rapid method for the construction of phosphonic peptide mimetic inhibitor libraries-products of Ugi and Passerini multicomponent condensations-leading to the selection of new biologically active phosphonic pseudopeptides. As the starting isonitriles, 1-isocyanoalkylphosphonate diaryl ester derivatives were applied. The structure of the synthesized inhibitors was designed to target human neutrophil elastase, a serine protease whose uncontrolled activity may lead to development of several pathophysiological states such as rheumatoid arthritis, cystic fibrosis or tumor growth and invasion. After screening the inhibitory activity of our constructed libraries, the most active compounds were synthesized as single molecules. One of the obtained inhibitors, Cbz-Met-O-Met-Val(P)(OC(6)H(4)-p-Cl)(2), displayed apparent second-order inhibition value at 40,105M(-1)s(-1) as the diastereomers mixture. Inhibition potency and selectivity of action toward other serine proteases as well as the results of initial in vitro experiments regarding inhibitors influence on cancer cell proliferation are presented.     

Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.