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Human neutrophil elastase (HNE), a main actor in the development of chronic obstructive pulmonary diseases, has been recently involved in non-small cell lung cancer progression. It can act at several levels (i) intracellularly, cleaving for instance the adaptor molecule insulin receptor substrate-1 (IRS-1) (ii) at the cell surface, hydrolyzing receptors as CD40 (iii) in the extracellular space, generating elastin fragments i.e. morphoelastokines which potently stimulate cancer cell invasiveness and angiogenesis. Since decades, researchers identified natural compounds and/or synthesized agents which antagonize HNE activity that will be described in this review article. Some of these compounds might be of value as therapeutic agents in lung cancer. However, it is now widely accepted that lung tumor invasion and metastasis involve proteolytic cascades. Accordingly, we will here mainly focus our attention to natural substances able to display a dual inhibitory capacity (i.e. lipids and derivatives, phenolics) towards HNE and matrix metalloproteinases (MMPs), particularly MMP-2. To that purpose, we recently synthesize substances named "LipoGalardin" (Moroy G. et al., Biochem. Pharmacol., 2011, 81(5), 626-635) exhibiting such inhibitory bifunctionality. At last, we will propose an original synthetic scheme for designing a potent biheaded HNE/MMP-2 inhibitor.

Molecular modeling was undertaken at aims to analyze the interactions between oleic acid and human leukocyte elastase (HLE), plasmin and matrix metalloproteinase-2 (MMP-2), involved in the inhibitory capacity of fatty acid towards those proteases. The carboxylic acid group of the fatty acid was found to form a salt bridge with Arg(217) of HLE while unsaturation interacted with Phe(192) and Val(216) at the S(3) subsite, and alkyl end group occupied S(1) subsite. In keeping with the main contribution of kringle 5 domain in plasmin-oleic acid interaction [Huet E et al. Biochem Pharmacol 2004;67(4):643-54], docking computations revealed that the long alkyl chain of fatty acid inserted within an hydrophobic groove of this domain with the carboxylate forming a salt bridge with Arg(512). Finally, blind docking revealed that oleic acid could occupy both S'(1) subsite and Fn(II)(3) domain of MMP-2. Several residues involved in Fn(II)(3)/oleic acid interaction were similarly implicated in binding of this domain to collagen. Oleic acid was covalently linked to galardin (at P'(2) position): OL-GAL (CONHOH) or to its carboxylic acid counterpart: OL-GAL (COOH), with the idea to obtain potent MMP inhibitors able to also interfere with elastase and plasmin activity. OL-GALs were found less potent MMP inhibitors as compared to galardin and no selectivity for MMP-2 or MMP-9 could be demonstrated. Docking computations indicated that contrary to oleic acid, OL-GAL binds only to MMP-2 active site and surprisingly, hydroxamic acid was unable to chelate Zn, but instead forms a salt bridge with the N-terminal Tyr(110). Interestingly, oleic acid and particularly OL-GALs proved to potently inhibit MMP-13. OL-GAL was found as potent as galardin (K(i) equal to 1.8nM for OL-GAL and 1.45nM for GAL) and selectivity for that MMP was attained (2-3 log orders of difference in inhibitory potency as compared to other MMPs). Molecular modeling studies indicated that oleic acid could be accommodated within S'(1) pocket of MMP-13 with carboxylic acid chelating Zn ion. OL-GAL also occupied such pocket but hydroxamic acid did not interact with Zn but instead was located at 2.8Å from Tyr(176). Since these derivatives retained, as their oleic acid original counterpart, the capacity to inhibit the amidolytic activity of HLE and plasmin as well as to decrease HLE- and plasmin-mediated pro MMP-3 activation, they might be of therapeutic value to control proteolytic cascades in chronic inflammatory disorders., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Hydrazide derivatives of Ilomastat, carrying either aryl groups or distinct alkyl and arylsulfonyl moieties were synthesized and evaluated for their MMP inhibitory activity. Potent and selective MMP-9 inhibition (IC(50)=3 nM) was observed for compound 3m (arylsulfonyl group: 4-(4-Br-C6H4)-C6H4-SO(2)-). Interaction with the S2 enzyme subsite is mainly responsible for the inhibitory properties of this derivative as confirmed by molecular docking computation.

Structural analogues of Ilomastat (Galardin), containing unsaturation(s) and chain extension carrying bulky phenyl group or alkyl moieties at P'1 were synthesized and purified by centrifugal partition chromatography. They were analyzed for their inhibitory capacity towards MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14, main endopeptidases involved in tumour progression. Presence of unsaturation(s) decreased the inhibitory potency of compounds but, in turn increased their selectivity for gelatinases. 2b and 2d derivatives with a phenyl group inhibited preferentially MMP-9 with IC50 equal to 45 and 38 nM, respectively, but also display activity against MMP-2 (IC50 equal to 280 and 120 nM, respectively). Molecular docking computations confirmed affinity of these substances for both gelatinases. With aims to obtain a specific gelatinase A (MMP-2) inhibitor, P'1 of Ilomastat was modified to carry one unsaturation coupled to an alkyl chain with pentylidene group. Docking studies indicated that MMP-2, but not MMP-9, could accommodate such substitution; indeed 2a proved to inhibit MMP-2 (IC50=123 nM), while displaying no inhibitory capacity towards MMP-9.

We previously demonstrated that the NC1[alpha3(IV)185-191] CNYYSNS peptide inhibited in vivo tumor progression. The YSNS motif formed a beta turn crucial for biological activity. The aim of the present study was to design a YSNSG cyclopeptide with a constrained beta turn on the YSNS residues more stable than CNYYSNS. By nuclear magnetic resonance and molecular modeling, we demonstrated that the YSNSG cyclopeptide actually adopted the expected beta-turn conformation. It promoted melanoma cell adhesion and prevented their adhesion to the native peptide. It inhibited in vitro cell proliferation and migration through Matrigel by downregulating proteolytic cascades. Moreover, intraperitoneal administration of the YSNSG cyclopeptide inhibited melanoma progression far more efficiently than the native peptide. The increased solubility and stability at low pH of the YSNSG cyclopeptide suggest this peptide as a potent antitumor therapeutic agent.

We report an experimental and theoretical study on type VIII beta-turn using a designed peptide of sequence GDNP. CD and NMR studies reveal that this peptide exists in equilibrium between type VIII beta-turn and extended conformations. Extensive MD simulations give a description of the free energy landscape of the peptide in which we retrieve the same two main conformations suggested by the experiments. The free energy difference between the two conformational states is very small and the transition between them occurs within a few kT at 300 K on a nanosecond timescale. The equilibrium is mainly driven by entropic contribution, which favors extended conformations over beta-turns. This confirms other theoretical studies showing that beta-turns are marginally stable in water solution because of the larger entropy of the extended state unless some stabilizing interactions exist. Our observations may be extended to any type of beta-turn and have important consequences for protein folding. A comparison of our MD and CD results also suggests a possible type VIII beta-turn CD signature indicated by one main band at 200 nm, close to that of random coil, and a fairly large shoulder at 220 nm. Last, our results clearly show that the XXXP motif can only fold into a type VIII beta-turn, which is consistent with its fairly strong propensity for this type of turn. This important finding may help for peptide design and is in line with recent studies on bioactive elastin peptides.

Mutations in the gene for fibrillin-1 cause Marfan syndrome (MFS), a common hereditary disorder of connective tissue. Recent findings suggest that proteolysis, increased matrix metalloproteinase activity, and fragmentation of fibrillin-rich microfibrils in tissues of persons with MFS contribute to the complex pathogenesis of this disorder. In this study we show that a fibrillin-1 fragment containing a EGFEPG sequence that conforms to a putative GxxPG elastin-binding protein (EBP) consensus sequence upregulates the expression and production of matrix metalloproteinase (MMP)-1 by up to ninefold in a cell culture system. A mutation of the GxxPG consensus sequence site abrogated the effects. This is the first demonstration of such an effect for ligands other than elastin fragments. Molecular dynamics analysis of oligopeptides with the wildtype and mutant sequence support our biochemical results by predicting significant alterations of structural characteristics such as the potential for forming a type VIII beta-turn that are thought to be important for binding to the EBP. These results suggest that fibrillin-1 fragments may regulate MMP-1 expression, and that the dysregulation of MMPs related to fragmentation of fibrillin might contribute to the development of MFS. Our Gene Ontology (GO) analysis of the human proteome shows that proteins with multiple GxxPG motifs are highly enriched for GO terms related to the extracellular matrix. Matrix proteins with multiple GxxPG sites include fibrillin-1, -2, and -3, elastin, fibronectin, laminin, and several tenascins and collagens. Some of these proteins have been associated with disorders involving alterations in MMP regulation, and the results of the present study suggest a potential mechanism for these observations.

We have developed a method that predicts both the presence and the type of beta-turns, using a straightforward approach based on propensities and multiple alignments. The propensities were calculated classically, but the way to use them for prediction was completely new: starting from a tetrapeptide sequence on which one wants to evaluate the presence of a beta-turn, the propensity for a given residue is modified by taking into account all the residues present in the multiple alignment at this position. The evaluation of a score is then done by weighting these propensities by the use of Position-specific score matrices generated by PSI-BLAST. The introduction of secondary structure information predicted by PSIPRED or SSPRO2 as well as taking into account the flanking residues around the tetrapeptide improved the accuracy greatly. This latter evaluated on a database of 426 reference proteins (previously used on other studies) by a sevenfold crossvalidation gave very good results with a Matthews Correlation Coefficient (MCC) of 0.42 and an overall prediction accuracy of 74.8%; this places our method among the best ones. A jackknife test was also done, which gave results within the same range. This shows that it is possible to reach neural networks accuracy with considerably less computional cost and complexity. Furthermore, propensities remain excellent descriptors of amino acid tendencies to belong to beta-turns, which can be useful for peptide or protein engineering and design. For beta-turn type prediction, we reached the best accuracy ever published in terms of MCC (except for the irregular type IV) in the range of 0.25-0.30 for types I, II, and I' and 0.13-0.15 for types VIII, II', and IV. To our knowledge, our method is the only one available on the Web that predicts types I' and II'. The accuracy evaluated on two larger databases of 547 and 823 proteins was not improved significantly. All of this was implemented into a Web server called COUDES (French acronym for: Chercher Ou Une Deviation Existe Surement), which is available at the following URL: http://bioserv.rpbs.jussieu.fr/Coudes/index.html within the new bioinformatics platform RPBS.

Elastin macromolecular assembly is a highly complex mechanism involving many steps including coacervation, cross-linking, and probably other (not known) phenomena. In past studies, it has been proposed that the C-terminal part of tropoelastin is also involved in this process and may play a key role in tropoelastin interactions with other proteins of the final elastic fibres scaffold. Presented here are the results of the biophysical studies (biospectroscopy, bioinformatics) of the C-terminal domain of tropoelastin. We report the detailed structures adopted by the oxidized (native) and reduced forms of the free synthetic peptide with sequence encoded by exon 36 of human tropoelastin (GGACLGKACGRKRK) and propose a dynamical interpretation of which structures may be involved in interactions with other extra-cellular matrix proteins. We also suggest that these structures may be retrieved in other proteins sharing a consensus sequence; however no definitive conclusion can be drawn here on a possible structure-function relationship.

The elastic properties of elastin have essentially been discussed in terms of dominant entropic components, with questions still remaining about whether the basic mechanism is compatible with the classical theory of rubber elasticity. A better understanding of the structure-function relationships in terms of the protein's elastic properties remains an important goal in elastin science. Recently, we succeeded in the exon-by-exon synthesis of all polypeptide sequences encoded by the so-called hydrophobic exons and almost all of the cross-linking exons of human tropoelastin. Among these, the peptide encoded by exon 5 (PGGLAGAGLGA) has been extensively studied by classical spectroscopic methods, such as CD and NMR spectroscopy, and by molecular dynamics simulations. The results obtained clearly evidenced a large flexibility of the polypeptide chain, which oscillates between rather extended conformations, such as PPII, and folded ones, such as beta turns. At the supramolecular level, we obtained evidence by TEM that shows that the peptide encoded by exon 5 is able to self-assemble in fibrillar structures, a result indicating that the "information" for self-assembly is also contained within a small domain of tropoelastin.