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A Amour

Publications and source records attributed to A Amour.

11 recordsLinked to original sources
PubMed2004

General considerations for proteolytic cascades.

Proteases are involved in the regulation of a wide variety of essential physiological processes, often by participating in a highly orchestrated sequence of events termed a 'proteolytic cascade'. Four major proteolytic cascades with disease relevance are candidates for therapeutic intervention, namely caspase-mediated apoptosis, blood coagulation, the matrix metalloproteinase cascade and the complement cascade. Understanding the various steps involved in the functioning of a cascade is key to deciding possible points of intervention for the design of potential drug molecules. This brief review illustrates some of the common features of proteolytic cascades using the blood coagulation pathway as an example.

Blood Coagulation↗
PubMed2000

Localization of the death domain of tissue inhibitor of metalloproteinase-3 to the N terminus. Metalloproteinase inhibition is associated with proapoptotic activity.

The tissue inhibitors of metalloproteinases (TIMPs) are a family of four secreted inhibitors of matrix metalloproteinases (MMPs). Recently, additional functions have been attributed to the TIMPs, including cell growth and inhibition of angiogenesis. In particular, we demonstrated that TIMP-3 overexpression using gene transfer induces apoptosis in a variety of cell types and can inhibit vascular neointima formation in vivo. However, little is know about the mechanisms underlying TIMP-3-mediated apoptosis. Here, using both purified recombinant proteins and novel adenoviral vectors we demonstrate that the prodeath domain of TIMP-3 is located within the N-terminal three loops of TIMP-3. Although both wild type and N-terminal TIMP-3 proteins promoted apoptosis, a T-2/T-3 chimera, in which the N-terminal three loops of TIMP-3 are replaced by those of TIMP-2, failed to induce cell death. Furthermore, a point mutation at residue 1 of TIMP-3 totally abolished MMP-inhibitory activity of TIMP-3 and also failed to promote apoptosis. This study demonstrates, using multiple apoptosis assays, that the prodeath function of TIMP-3 is located within the N-terminal three loops and the presence of functional metalloproteinase-inhibitory activity is associated with the induction of apoptosis.

Animals↗
PubMed2000

The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3.

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.

ADAM Proteins↗
PubMed2000

Membrane type 4 matrix metalloproteinase (MMP17) has tumor necrosis factor-alpha convertase activity but does not activate pro-MMP2.

Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein. We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated. Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2. The pro-catalytic domain was expressed in Escherichia coli as insoluble inclusions and active enzyme recovered after refolding. Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3. The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin. mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor alpha (TNFalpha) cleavage site, a glutathione S-transferase-pro-TNFalpha fusion protein, and was found to shed pro-TNFalpha when co-transfected in COS-7 cells. MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNFalpha-converting activity suggests a role in inflammation.

ADAM Proteins↗
PubMed1998

Design and synthesis of hydrazinopeptides and their evaluation as human leukocyte elastase inhibitors.

The name hydrazinopeptide designates peptidic structures in which one of the native CONH links is replaced by a CONHNH (hydrazido) fragment. In this paper, we report the synthesis of such hydrazinohexapeptides (3-5) analogous to Z-Ala-Ala-Pro-Val-Ala-Ala-NHiPr (6), a substrate of human leukocyte elastase (HLE; EC 3.4.21.37), cleaved by this serine protease between the Val4 and Ala5 residues. In hydrazinopeptides 3-5, the Ala5, Val4, or Pro3 residue, respectively, of the model peptide, has been replaced by the corresponding alpha-L-hydrazino acid. In 3, the bond likely to be cleaved by HLE is the one involving the CONHNH link, while in 4 and 5, this link is normally shifted away by one or two amino acid units from the catalytic serine. On incubation with HLE, hydrazinopeptide 3 proved to be a substrate and was cleaved between Val4 and NHAla5, like peptide 6. In contrast, 4 and 5 proved to bind to HLE without being cleaved, featuring properties consistent with reversible competitive inhibition. General guidelines for the synthesis of hydrazinopeptides are also reported in this paper. These guidelines take into account the chemical specificity of hydrazino acids, while being fully compatible with the conventional peptide coupling techniques. The utilization of orthogonally bisprotected hydrazino acids 1 where the Nbeta and Nalpha atoms bear a Boc and a Bzl group, respectively, is recommended for the easy construction of such hydrazinopeptides.

Drug Design↗
PubMed1998

TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3.

TNF-alpha converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-alpha to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-alpha secretion is involved.

ADAM Proteins↗
PubMed1998

Stereoselective synthesis of peptidyl trifluoromethyl alcohols and ketones: inhibitory potency against human leucocyte elastase, cathepsin G, porcine pancreatic elastase and HIV-1 protease.

New fluorinated inhibitors have been designed to target two major proteases-human leucocyte elastase and HIV-1 protease. Two series of beta-peptidyl trifluoromethyl alcohols (TFMAs) Z-L-Val-NH-*CH(Y)*CH(OH)-CF3, where *C is the chiral centre, varied in the nature of the substituent Y, a phenylethyl [-(CH2)2-C6H5] or an isopropyl [-CH(CH3)2] group. These TFMAs were first synthesized as two pairs of the syn and anti diastereoisomers. The inhibitory effects of these mixtures were then assessed on three serine proteases chosen on the basis of the aromatic and aliphatic nature of the substituents-human leucocyte elastase (HLE), human cathepsin G (HCG) and porcine pancreatic elastase (PPE). In the presence of detectable inhibition, each epimer at C2 was separated to determine its inhibition constant (Ki) towards HLE, HCG and PPE. The stereoisomerically pure TFMAs were then oxidized into peptidyl trifluoromethyl ketones (TFMKs) for similar inhibition assays. The absolute configuration of the compounds remained unknown. One epimer at C2 of each syn and anti TFMA with the phenylethyl substituent behaved as a competitive inhibitor towards HLE and HCG with inhibition constants below the millimolar range, whereas their TFMK counterparts were non-inhibitors. In the second series, the two ketones inhibited both elastases with Ki values in the micromolar range, whereas only the syn TFMA was active towards HLE (Ki = 5.65 x 10(-4)M). The tested compounds also had structural properties compatible with recognition by HIV-1 protease. The inhibition of the enzyme was observed with TFMK only (IC50 = 15-200 microM). The phenylethyl substituent promoted inhibition by a factor of 10 (IC50 = 15 microM) compared with the isopropyl substituent (IC50 = 200 microM) leading to selective inhibition of HIV-1 protease. Isomerically pure TFMKs were more potent towards HLE than the alcohols from which they were obtained. However, an enantiomerically pure TFMA selectively inhibited HLE unlike its TFMK analogue which also inhibited PPE. This last result together with the selective inhibition of HIV-1 protease by TFMK with a phenylethyl substituent might be relevant to the design of specific HLE and HIV-1 inhibitors as therapeutic agents.

Alcohols↗
PubMed1996

Protection of rat lung from elastase-induced elastic fiber degradation in vitro and from emphysema in vivo by a trifluoroacetylpeptide anilide inhibitor.

Trifluoroacetylpeptide anilides are powerful reversible inhibitors of human neutrophil elastase (HNE), a serine protease implicated in the pathogenesis of pulmonary emphysema. The in vitro effectiveness of three inhibitors, CF3CO-Phe-Ala-NH-p-C6H4-CF3 (1), CF3CO-Val-Ala-NH-p-C6H4-CF3 (2) and CF3CO-Lys-Ala-NH-p-C6H4-CH(CH3)2 (3) was analyzed. The protection of lung tissue sections of rats from the degradation induced by HNE has been evaluated quantitatively by automated image analysis. Inhibitor 1 (22 microM), 2 (50 microM) or 3 (35 and 70 microM) significantly reduced the HNE-induced degradation of the elastin network by 75, 42, 54 and 44%, respectively. Inhibitor 3 was tested intratracheally on an experimental model of pulmonary emphysema. Rats that received the elastase inhibitor 1 h before instillation of HNE were significantly protected by 40% from experimental emphysema. Reduced protections were observed with the treatment by the inhibitor 1 or 4 h after challenge with the enzyme.

Anilides↗
PubMed1994

Synthesis and protease-catalyzed hydrolysis of a novel hydrazinopeptide.

In order to explore the potentiality of hydrazinopeptides as protease inhibitors, the resistance of the hydrazinopeptidic bond toward proteolysis was investigated. To this end, the novel hydrazinohexapeptide Z-Ala2-Pro-Val-hIle-Leu-OMe (1), where hIle represents hydrazinoisoleucine, was designed and synthesized together with the parent peptide Z-Ala2-Pro-Val-Ile-Leu-OMe (2). The interactions of 1 and 2 with human leukocyte elastase (HLE) and porcine pancreatic elastase (PPE) were analyzed comparatively. We observed that 1 behaved as a substrate for both elastases, without the formation of a stable acyl-enzyme as in the case of azapeptides. Compounds 1 and 2 were cleaved at the same site (-Val-parallel-NH-) with a slight delay of hydrolysis for 1 compared to 2 (kcat/KM for 1 vs. 2 decreased by a factor of 2.7 for the HLE-catalyzed hydrolysis at pH 8.0 and 25 degrees C). The presence of the hydrazinopeptide bond (-CONHNH-) in 1 reduced by a factor of 4.7 the apparent enzyme affinity without abolishing it. These results indicate that suitably designed hydrazinopeptides may represent interesting targets in the search for protease resisting pseudopeptides.

Amino Acid Sequence↗