[Show abstract][Hide abstract] ABSTRACT: Crotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian reproductive tract proteins, and tumor necrosis factor α convertase, TACE, involved in the release of the inflammatory cytokine, TNFα. The structure of adamalysin II in noncovalent complex with two small-molecule right-hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X-ray diffraction data up to 2.6 and 2.8 Å resolution. The inhibitors bind to the -side of the proteinase, inserting between two protein segments, establishing a mixed parallel-antiparallel three-stranded β-sheet and coordinate the central zinc ion in a bidentate manner via their two C-terminal oxygen atoms. The proteinase-inhibitor complexes are described in detail and are compared with other known structures. An adamalysin-based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors.
[Show abstract][Hide abstract] ABSTRACT: The phosphotryptophan derivative l-Pro-l-Leu-l-(P)Trp(OH)(2) (2b) was reported as the first example of left-hand-sideLeft-hand-side inhibitors: inhibitors that bind in the unprime region of the enzyme active site, in reference to the convention of drawing the unprimed residues of a peptide substrate on the left side. [R.P. Beckett et al., Drug Discov. Today 1 (1996) 16-26]. The opposite applies to right-hand-side inhibitors. phosphonate inhibitor of MMP-8. Its uncommon mode of binding to MMP-8 was mainly ascribed to the presence of the proline residue in P(3). Ten new analogues of 2b were obtained by replacement of the aminoterminal l-Pro with aminoacid residues bearing small side chains. Most of the new analogues show an increase of affinity for MMP-2 and MMP-8, and different profiles of selectivity. Computer simulations were performed to explain the effects of substitutions on the preferred mode of binding. They reveal that most of the new analogues are probably accommodated in the right, rather than left-hand side of MMP-8 active site.
European Journal of Medicinal Chemistry 04/2005; 40(3):271-9. DOI:10.1016/j.ejmech.2004.10.013 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report a molecular dynamics simulation study of a zinc-protease--gelatinase A or MMP2--which is a major target for drug design, being involved in tumor metastasis and other degenerative diseases. Two structures have been employed as starting conditions, one based on the crystal of multi-domain proMMP2, the other consisting of the catalytic domain only. The overall fold of the two models is maintained over the 1260 ps trajectory, enabling us to analyze correlations of fluctuations among domains, and to observe the presence of correlations within the catalytic domain in the multi-domain enzyme only, hence due to the presence of hemopexin and fibronectin domains. In the multi-domain protein, two cavities are conserved over the trajectory, one of them pointing to a key region, a crevice surrounding the catalytic zinc. The other one is localized across the three domains of the MMP2 metalloproteinase. These areas are partially covered by the propeptide in the crystal structure of proMMP2. We propose a model of MMP2-collagen interaction that involves both identified cavities and takes into account the inter/intra domain cross-correlations.
[Show abstract][Hide abstract] ABSTRACT: The cleavage of bovine collagen I by neutrophil collagenase MMP-8 has been followed at pH 7.4, 37 degrees C. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990) Biochemistry 29, 10628-10634; Knäuper, V., Osthues, A., DeClerk, Y. A., Langley, K. A., Bläser, J., and Tschesche, H. (1993) Biochem. J. 291, 847-854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657-18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met(80) of the proenzyme (MetMMP-8) and the former Phe(79) of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much faster k(cat)/K(m), fully attributable to a lower K(m), suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227-233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.
[Show abstract][Hide abstract] ABSTRACT: We have studied the effect of small molecular weight inhibitors of snake venom metalloproteinases (SVMP) and matrix metalloproteinases (MMPs) on the induction and effector phase of the contact hypersensitivity reaction (CHR) in a mouse model. Identification of nonsteroid small molecules is very important for the development of new anti-inflammatory drugs. The compounds that we tested were synthetically modified tripeptides (peptidomimetic compounds) POL-257, POL-509, POL-443, POL-491, and POL-647, with structures based on natural occurring peptides in snake venom. A well-known hydroxamate-based inhibitor of the MMPs, Batimastat (BB-94), was also used. We have shown that these peptidomimetics possess in vitro inhibitory activity against the MMP-2 (gelatinase-A), MMP-9 (gelatinase-B), and MMP-3 (stromelysin). They also inhibit metalloproteinases purified from the venom of Crotalus adamanteus and C. atrox snakes, which are very similar to the so-called A Desintegrine, A Metalloproteinase (ADAMs) enzymes. When injected intraperitonealy before the topical application of the contact sensitizer (picryl chloride) or before the challenge, these compounds significantly inhibited the development of CHR. BB-94 at doses 0.4 and 4 mg/kg before the sensitization or before the challenge almost completely abrogated the reaction. POL-257 and POL-443 were among the most active peptidomimetics tested. They inhibited the inflammatory reaction up to 70-80%, when applied either immediately before sensitization or before challenge. POL-509, a methylated derivative of POL-257, inhibited the CHR to 40-50% when administered at either challenge or sensitization. However, when applied 24 h before the challenge, it completely abrogated the inflammatory reaction. The results show that these small molecular weight peptidomimetic compounds, as well as BB-94, are able to significantly inhibit the CHR. This finding opens possibilities for using metalloproteinase inhibitors in the treatment of allergic contact dermatitis and other inflammatory diseases.
International Immunopharmacology 05/2002; 2(5):699-710. DOI:10.1016/S1567-5769(02)00005-X · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metalloproteases are metalloenzymes secreted in the extracellular fluid and involved in inflammatory pathologies or events, such as extracellular degradation. A Zn(2+) metal, present in the active site, is involved in the catalytic mechanism, and it is generally coordinated with histidyl and/or cysteinyl residues of the protein moiety. In this study we have investigated the effect of both pH (between pH 4.8 and 9.0) and temperature (between 15 degrees C and 37 degrees C) on the enzymatic functional properties of the neutrophil interstitial collagenase (MMP-8), gelatinases A (MMP-2) and B (MMP-9), using the same synthetic substrate, namely MCA-Pro-Leu-Gly approximately Leu-DPA-Ala-Arg-NH(2). A global analysis of the observed proton-linked behavior for k(cat)/K(m), k(cat), and K(m) indicates that in order to have a fully consistent description of the enzymatic action of these metalloproteases we have to imply at least three protonating groups, with differing features for the three enzymes investigated, which are involved in the modulation of substrate interaction and catalysis by the enzyme. This is the first investigation of this type on recombinant collagenases and gelatinases of human origin. The functional behavior, although qualitatively similar, displays significant differences with respect to what was previously observed for stromelysin and porcine collagenase and gelatinase (Stack, M. S., and R. D. Gray. 1990. Arch. Biochem. Biophys. 281:257-263; Harrison, R. K., B. Chang, L. Niedzwiecki, and R. L. Stein. 1992. Biochemistry. 31:10757-10762). The functional characterization of these enzymes can have some relevant physiological significance, since it may be related to the marked changes in the environmental pH that collagenase and gelatinases may experience in vivo, moving from the intracellular environment to the extracellular matrix.
[Show abstract][Hide abstract] ABSTRACT: The enzymatic processing of bovine collagen I by neutrophil collagenase (MMP-8) has been monitored at 37 degrees C, envisaging the occurrence of multiple intermediate steps, following the initial cleavage, which leads to the formation of (1/4) and (3/4) fragments. Further, the first cleavage event has been investigated at 37 degrees C as a function of pH, and catalytic parameters have been obtained through a global analysis of steady-state kinetic data, such as to get an overall consistent picture of k(cat)/K(m), k(cat), and K(m). These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimental conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK(a) values for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymatic activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natural substrates.
[Show abstract][Hide abstract] ABSTRACT: In the last few years, Indol-3-pyruvic acid (IPA) has been subjected to 5 pilot clinical trials in volunteers and a phase II study on patients affected by anxiety, with or without sleep problems. Overall, results indicated a very good safety profile, relief from anxiety and a better quality of sleep in patients treated with IPA. Moreover, the drug showed no withdrawal signs, but positive effects on mood, improving feelings of relaxation, calmness and happiness. The mechanism of action of IPA, depending on increased turnover of some indoles in the CNS, appears clearly distinct from that of benzodiazepines, suggesting that the drug might be used in the treatment of symptoms of mild anxiety and stress experienced by busy and anxious people.
Advances in Experimental Medicine and Biology 02/1999; 467:227-32. DOI:10.1007/978-1-4615-4709-9_29 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The search of reprolysin inhibitors offers the possibility of intervention against both matrixins and ADAMs. Here we report the crystal structure of the complex between adamalysin II, a member of the reprolysin family, and a phosphonate inhibitor modeled on an endogenous venom tripeptide. The inhibitor occupies the primed region of the cleavage site adopting a retro-binding mode. The phosphonate group ligates the zinc ion in an asymmetric bidentate mode and the adjacent Trp indole system partly fills the primary specificity subsite S1'. An adamalysin-based model of tumor necrosis factor-alpha-converting enzyme (TACE) reveals a smaller S1' pocket for this enzyme.
[Show abstract][Hide abstract] ABSTRACT: Venom-induced necrosis is a common local debilitating sequela of bites by many vipers, frequently resulting in severe permanent scarring and deformity. Antivenoms are not effective under these circumstances unless administered within a few minutes of the bite; this is unlikely to occur in the rural tropics where most victims take a long time to reach medical care. We have shown that two venom zinc metalloproteinases (jararhagin from Bothrops jararaca venom and a metalloproteinase from Echis pyramidum leakeyi venom) successfully cleaved the recombinant glutathione-S-transferase-tumor necrosis factor-alpha fusion protein (GST-TNF-alpha) substrate to form biologically active TNF-alpha which was shown to be neutralized by ovine TNF-alpha Fab antibodies. This resulted in a reduction of venom-induced necrosis in mice when injected intravenously or intradermally both before and after intradermal injections of E.p.leakeyi venom. A peptidomimetic (POL 647) was also found to inhibit the Echis metalloproteinase, thus preventing the processing of the TNF precursor; this was shown using a TNF-alpha-sensitive cell culture assay and electrophoresis. These observations demonstrate the possible importance of TNF-alpha in the development of the resulting necrotic lesion and leads to the hypothesis that increased levels of venom metalloproteinases following snake bite release active TNF-alpha. This cytokine may contribute to the local necrosis and also induce the production of endogenous matrix metalloproteinases, which in turn generate a positive feedback mechanism resulting in continued cleavage of pro-TNF-alpha. The results indicate that inhibition or neutralization of endogenous TNF-alpha appears to result in a significant reduction in venom-induced necrosis. This could help to explain the clinical observations that treatment of local necrosis following snake bite by antivenom is only minimally successful.
European Journal of Immunology 09/1996; 26(9):2000-5. DOI:10.1002/eji.1830260905 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress is believed to be involved in the pathogenesis of many important degenerative diseases, especially in the Central Nervous System, where oxygen is utilized in very high amount, and scavengers of toxic substances might be insufficient when neurons are over-stimulated or ageing processes accumulate peroxide lipids (Halliwell, 1992).
Advances in Experimental Medicine and Biology 02/1996; 398:291-8. DOI:10.1007/978-1-4613-0381-7_46 · 1.96 Impact Factor