Biomedical subjects

M Reboud-Ravaux

Publications and source records attributed to M Reboud-Ravaux.

At least 19 recordsLinked to original sources

PubMed2003

3-Bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate inhibits cancer cell invasion in vitro and tumour growth in vivo.

In search for new anticancer agents, we have evaluated the antiinvasive and antimigrative properties of recently developed synthetic coumarin derivatives among which two compounds revealed important activity: 3-chlorophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate and 3-bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate. Both drugs were able to inhibit cell invasion markedly in a Boyden chamber assay, the bromo derivative being more potent than the reference matrix metalloprotease (MMP) inhibitor GI 129471. In vivo, tumour growth was reduced when nude mice grafted with HT1080 or MDA-MB231 cells were treated i.p. 3 days week(-1) with the bromo coumarin derivative. These effects were not associated with the inhibition of urokinase, plasmin, MMP-2 or MMP-9. The mechanism of action of the drugs remains to be elucidated. However, these two coumarin derivatives may serve as new lead compounds of an original class of antitumour agents.

Animals↗

PubMed2002

RNA-acting antibiotics: in-vitro selection of RNA aptamers for the design of new bioactive molecules less susceptible to bacterial resistance.

During the last few years, antibiotic multiresistance has been increasing, not only in hospitals, but also, more worryingly, in general medicine. Different ways are being explored to bypass this problem. RNA-acting antibiotics such as aminosides (aminoglycosides) bind to bacterial RNA causing premature termination of proteins and mistranslation in bacteria. It is now possible to study the interactions of such antibiotics with their target by in-vitro selection of RNA molecules that recognize these antibiotics (RNA aptamers, SELEX method). The knowledge of the antibiotic-RNA interactions represents a promising way for the rational design of new bioactive compounds less susceptible to bacterial resistance.

Aminoglycosides↗

PubMed2001

beta-Amyloid protein aggregation: its implication in the physiopathology of Alzheimer's disease.

beta-Amyloid protein (A beta), a 39-42 residue peptide resulting from the proteolytic processing of a membrane-bound beta-amyloid precursor protein (APP), is one of the major components of the fibrillar deposits observed in Alzheimer patients. A beta fibril formation is a complex process which involves changes in A beta conformation and self-association to form cross-beta pleated sheets, protofibrils, and fibrils. Since the aggregation of soluble A beta peptide into fibrils is viewed as a critical event in the physiopathology of Alzheimer's disease (AD), preventing, altering, or reversing fibril formation may thus be of therapeutic value. This review will focus on the current state of knowledge of A beta fibril formation, with special emphasis on physiological and exogenous inhibitors which may have a therapeutic potential.

Alzheimer Disease↗

PubMed2001

[Elastase inhibitors].

The serine proteinase elastase is located in the azurophil granules of mature circulating polymorphonuclear neutrophils. This neutrophil elastase or NE is a potent non specific serine protease which plays a role as bactericidal agent and in the degradation of immune complexes by intraphagosomal processes. It promotes inflammation when the granule contents are secreted in the extracellular environment. In certain pathological circumstances, an imbalance between NE and its major plasmatic inhibitor alpha 1-PI (formerly, alpha 1-antitrypsin) leads to abnormal tissue destruction and disease development. Genetic or acquired alpha 1-PI deficiency is thought to be involved in the pathogenesis of pulmonary emphysema. A variety of degenerative and degradative disorders are also associated to uncontrolled proteolysis by NE (rheumatoid arthritis, glomerulonephritis, adult respiratory distress symptom, psoriasis, cancer). Numerous inhibitors of NE have been reported. Various molecules are currently undergoing clinical trials for emphysema and other pulmonary diseases.

Animals↗

PubMed2000

Coumarinic derivatives as mechanism-based inhibitors of alpha-chymotrypsin and human leukocyte elastase.

Novel coumarinic derivatives were synthesized and tested for their inhibitory potency toward alpha-CT and HLE. Cycloalkyl esters and amides were found to be essentially inactive on both enzymes. On the opposite, aromatic esters strongly inactivated alpha-CT whereas HLE was less efficiently inhibited with dichlorophenyl ester derivatives (kinact/K(I) = 4000 M(-1) s(-1) for 36). Representative examples of amide, ester, thioester and ketone derivatives were prepared in order to evaluate the influence of the link between the coumarinic ring and the phenyl side chain. The irreversible inactivation of alpha-CT by 6-chloromethyl derivatives should be due to alkylation of a histidine residue as suggested by the amino acid analysis of the modified chymotrypsin. Conversely the inhibition of HLE was transient. Intrinsic reactivity of coumarins has been calculated using a model of a nucleophilic reaction between the ligand and the couple methanol-water. From this calculation, it appears that differences in the inhibitory potency expressed by these molecules cannot only be explained by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack.

Chymotrypsin↗

PubMed1999

6-Substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid as a core structure for specific inhibitors of human leukocyte elastase.

Pyridyl esters of 6-substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid were designed as mechanism-based inhibitors of human leukocyte elastase. Compounds of series 4 specifically inhibited this enzyme. Several of the tested compounds (series 2 and 3) acted as powerful time-dependent inhibitors of both human leukocyte elastase and alpha-chymotrypsin; some compounds of these series inhibited thrombin. Trypsin was not inhibited. A transient inactivation was observed for human leukocyte elastase (k(i)/K(I) = 107 000 M(-1). s(-1) for 4c) and thrombin (k(i)/K(I) = 7 200 M(-1).s(-1) for 3b) as demonstrated by spontaneous or hydroxylamine-accelerated reactivation, irrespective of the nature of the substituent at the 6-position. Conversely, alpha-chymotrypsin was irreversibly inhibited by 6-chloromethyl derivatives (k(i)/K(I) = 107 400 M(-1). s(-1) for 3b). The presence of a latent alkylating function at the 6-position (chloromethyl group) was required for leading to this inactivation. In the absence of such an alkylating function (series 4), human leukocyte elastase was specifically inhibited suggesting that this new series of human leukocyte elastase inhibitors may be of potential therapeutic interest in degradative and degenerative processes involving this enzyme.

Chromatography, High Pressure Liquid↗

PubMed1999

Design, synthesis, and evaluation of conformationally constrained tongs, new inhibitors of HIV-1 protease dimerization.

The active form of human immunodeficiency virus type 1 protease (HIV-1 PR) is a homodimeric structure in which two subunits are linked through a two-stranded antiparallel beta-sheet consisting of the N- and C-termini of each monomer. To inhibit the dimerization process or disrupt the dimeric interface leading to inactive enzyme, conformationally constrained "molecular tongs" have been designed and synthesized to interfere with one monomer end in a beta-sheet fashion. These molecules are based on two peptidic strands attached to an aromatic scaffold. Inhibitions (submicromolar range) were obtained with molecular tongs containing tripeptidic or tetrapeptidic arms attached to a pyridinediol- or naphthalenediol-based scaffold (Kid = 0.56-4.5 microM at pH 4.7 and 30 degrees C). Kinetic studies are in agreement with an interface inhibition mechanism.

Anti-HIV Agents↗

PubMed1999

Lipopeptides as dimerization inhibitors of HIV-1 protease.

In AIDS therapy, attempts have been made to inhibit the virus-encoded enzymes, e.g. HIV-1 protease, using active site-directed inhibitors. This approach is questionable, however, due to virus mutations and the high toxicity of the drugs. An alternative method to inhibit the dimeric HIV protease is the targeting of the interface region of the protease subunits in order to prevent subunit dimerization and enzyme activity. This approach should be less prone to inactivation by mutation. A list of improved 'dimerization inhibitors' of HIV-1 protease is presented. The main structural features are a short 'interface' peptide segment, including non-natural amino acids, and an aliphatic N-terminal blocking group. The high inhibitory power of some of the lipopeptides [e.g. palmitoyl-Tyr-Glu-Leu-OH, palmitoyl-Tyr-Glu-(L-thyronine)-OH, palmitoyl-Tyr-Glu-(L-biphenyl-alanine)-OH] with low nanomolar Ki values in the enzyme test suggests that mimetics with good bio-availability can be derived for AIDS therapy.

Amino Acid Sequence↗

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

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↗

PubMed1997

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.

Amino Acids↗

PubMed1996

Esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid as inhibitors of alpha-chymotrypsin: significance of the "aromatic" nature of the novel ester-type coumarin for strong inhibitory activity.

A series of esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid were synthesized and evaluated in vitro for their inhibitory activity toward bovine alpha-chymotrypsin and human leukocyte elastase. Both series behaved as time-dependent inhibitors of alpha-chymotrypsin, but ester-type coumarins were clearly more efficient than the corresponding amides in inactivating the serine proteinase. The best inactivations were observed with "aromatic" esters, in particular with meta-substituted phenyl esters such as m-chlorophenyl 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylate, which appears to be one of the most powerful inactivators of alpha-chymotrypsin yet reported (kinact/KI = 760,000 M-1 S-1 at pH 7.5 and 25 degrees C). Usually, the coumarin derivatives failed to inhibit significantly human leukocyte elastase. As a result, the reported series of aromatic coumarinic esters behaves as a new chemical family of selective alpha-chymotrypsin inhibitors.

Animals↗

PubMed1996

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).

Azetidines↗

PubMed1996

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.

Humans↗

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↗

PubMed1995

Role of monocyte/macrophage derived matrix-metalloproteinases (gelatinases) in prolonged skin inflammation.

Neutral metalloproteinase activities in dermal extracellular space have been studied in several models of prolonged cutaneous inflammation in guinea pigs, by the following techniques: lysis of type I 14C-collagen fibrils, electrophoretic analysis of types I or IV collagen hydrolytic fragments and zymography. For 2-3 weeks, in parallel to mononuclear cell infiltration, collagenase activity was increased 3-4-fold. Constitutive gelatinases (67 and 72 kDa) augmented and larger molecular species emerged (92, 110 and 185 kDa), all of neutral metalloproteinase type. Guinea pig peritoneal monocytes/macrophages cultured with appropriate stimulation released large gelatinases in a similar set (92, 110, 210 kDa). These were purified from culture media by gelatin affinity and used in vivo as follows: (a) direct injection of monocyte/macrophage gelatinases; (b) injection of collagen I fragments (M(r) < 10,000) split off by gelatinases from preincubated (pH 5 and 38.5 degrees C) collagen I. In both instances, a mononuclear cell invasion of dermis occurred, indistinguishable from prolonged inflammation. These analogies suggest that monocyte/macrophage-derived metalloproteinases have an early and basic participation in the mechanism of prolonged inflammation.

Animals↗

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↗

PubMed1993

New mechanism-based inactivators of trypsin-like proteinases. Selective inactivation of urokinase by functionalized cyclopeptides incorporating a sulfoniomethyl-substituted m-aminobenzoic acid residue.

In order to obtain selective suicide substrates of trypsin-like proteases including plasminogen activators, plasmin, and thrombin, a series of cyclopeptides cyclo[Arg or Lys-aB(CH2X)-Gly4], in which a substituted o- or m-aminobenzoyl group constitutes a latent electrophile, have been prepared. Treatment of the corresponding phenyl ethers cyclo[P1-aB(CH2OC6H5)-Gly4] with HBr/HOAc or R1R2S/TFA gives the bromides (X = Br) or the sulfonium salts (X = +SR1R2 with R1 = R2 = Me or R1 = Me and R2 = C6H5), respectively. These water-soluble cyclopeptides behave as time-dependent inhibitors of bovine trypsin and human urokinase (u-PA) but have no effect on tissue plasminogen activator (t-PA) and no or poor effect on plasmin and thrombin. The compounds containing a m-aminobenzoic acid residue are more efficient inactivators than their anthranilic analogues. The kinetic criteria expected for a suicide inhibition are met. A mechanism of inhibition involving the formation of a quinonimmonium methide intermediate is proposed. The activity of the inhibitors is very sensitive to the nature of the X benzylic substituent. An increased efficiency for the inactivation of human urokinase is observed with the sulfonium salts. The selectivity of the inactivation of u-PA compared to t-PA could be of therapeutical significance in controlling cell proliferation and invasion.

Aminobenzoates↗