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Accueil > Agenda > Les séminaires Jean Roche > Fonction des métalloprotéases à zinc matricelles (MMPs) : apport de la (...)

Fonction des métalloprotéases à zinc matricelles (MMPs) : (...)

Lundi 27 juin 2005, 11h, salle Lissitzky.


Biochimie. 2005 Mar-Apr ;87(3-4):393-402.

Future challenges facing the development of specific active-site-directed synthetic inhibitors of MMPs.

Cuniasse P, Devel L, Makaritis A, Beau F, Georgiadis D, Matziari M, Yiotakis A, Dive V.

CEA, Departement d’Ingenierie et d’Etudes des Proteines (DIEP), CE-Saclay, 91191 Gif/Yvette cedex, France.

Despite a deep knowledge on the 3D-structure of several catalytic domains of MMPs, the development of highly specific synthetic active-site-directed inhibitors of MMPs, able to differentiate the different members of this protease family, remains a strong challenge. Due to the flexible nature of MMP active-site, the development of specific MMP inhibitors will need to combine sophisticated theoretical and experimental approaches to decipher in each MMP the specific structural and dynamic features that can be exploited to obtain the desired selectivity.

2 : Int J Cancer. 2005 Feb 20 ;113(5):775-81.

Dosing and scheduling influence the antitumor efficacy of a phosphinic peptide inhibitor of matrix metalloproteinases.

Dive V, Andarawewa KL, Boulay A, Matziari M, Beau F, Guerin E, Rousseau B, Yiotakis A, Rio MC.

CEA, Departement d’Ingenierie et d’Etudes des Proteines, Gif/Yvette, France. vincent.dive

The in vivo disposition and antitumor efficacy of a newly developed phosphinic matrix metalloproteinase inhibitor (RXP03) were examined. RXP03 potently inhibits MMP-11, MMP-8 and MMP-13, but not MMP-1 and MMP-7. Twenty-four hours after i.p. injection into mice, most of the RXP03 was recovered intact in plasma, feces (biliary excretion) and tumor tissue. Pharmacokinetic parameters indicated that, after an i.p. dose of 100 microg/day, the plasma concentration of RXP03 over 24 hr remained higher than the Ki values determined for MMP-11, MMP-8 and MMP-13. Efficacy of RXP03 on the growth of primary tumors induced by s.c. injection of C(26) colon carcinoma cells in mice was observed to depend both on RXP03 doses and treatment schedules. Tumor volumes in mice treated for 18 days with 50, 100 and 150 microg/day of RXP03 were decreased compared with control tumor volumes, 100 microg/day being the most effective dose. Treatment at higher dose (600 microg/day) did not significantly reduce the tumor size as compared to control. Short treatments with RXP03 100 microg/day, 3 to 7 days after C(26) inoculation, were more effective on tumor growth than continuous treatment over 18 days. Strikingly, RXP03 treatment started 6 days after the C(26) injection and continued until day 18 led to stimulation of tumor growth, as compared to control. These paradoxical effects, depending on the RXP03 treatment schedule, underline the need to define carefully the spatiotemporal function of each MMP at various stages of tumor growth to achieve optimal therapeutic effects by MMP inhibitor treatment.

3 : Cell Mol Life Sci. 2004 Aug ;61(16):2010-9.

Phosphinic peptides as zinc metalloproteinase inhibitors.

Dive V, Georgiadis D, Matziari M, Makaritis A, Beau F, Cuniasse P, Yiotakis A.

Department d’Ingenierie et d’Etudes des Proteines (DIEP), CEA, CE-Saclay, 91191, Gif/Yvette Cedex, France. vincent.dive

Solid-phase synthesis of phosphinic peptides was introduced 10 years ago. A major application of this chemistry has been the development of potent synthetic inhibitors of zinc metalloproteases. Specific properties of the inhibitors produced in recent years are reviewed, supporting the notion that phosphinic pseudo-peptides are useful tools for studying the structural and functional biology of zinc proteases.

4 : Curr Organ Chem. 2004 Aug ;8(12):1135-1158

Phosphinic peptides : Synthetic approaches and biochemical evaluation as Zn-metalloprotease inhibitors

Yiotakis, A Georgiadis, D Matziari, M Makaritis, A Dive, V

Over the course of the last decades. phosphinic peptides have emerged as an extremely important class of Zn-metalloprotease inhibitors. The intense interest in these compounds in the field of medicinal chemistry reflects a conjunction of synthetic advances and ideal physicochemical and biochemical properties. Among the latter, the optimal tetrahedral structure of phosphinic acid moiety, which perfectly fills the requirements of transition state theory, clearly defines the mechanistical frame of their function as metalloprotease inhibitors (transition state analogues, TSA). In particular. phosphinic peptides are peptidic isosters containing a chemically stable phosphinic acid moiety which mimics the transition state tetrahedral geometry of a scissile peptide bond during enzymatic hydrolysis. Moreover, the ionic interactions of phosphinic oxyanion with catalytic Zn2+ combined with the substrate-like primary interactions of pseudopeptidic backbone with enzyme’s active site, can aid the rationalization of the design and development of potent and selective Zn-protease inhibitors. In these review, we aim to resume the major achievements in this field over the last decades. In the first part, the fundamental principles of phosphinopeptidic chemistry, as these have been established by the pioneers of the field, as well as the synthesis of first-generation inhibitors are presented. In the second part, accounting as a turning point the introduction of SPPS and combinatorial practices in the field, a comprehensive description of all the recent synthetic novelties as well as the outstanding applications of phosphinopeptidic derivatives in enzymatic, biochemical and crystallographic studies is attempted.

5 : Biochemistry. 2004 Jun 29 ;43(25):8048-54.

Structural determinants of RXPA380, a potent and highly selective inhibitor of the angiotensin-converting enzyme C-domain.

Georgiadis D, Cuniasse P, Cotton J, Yiotakis A, Dive V.

Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece.

RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhibitor of the C-domain of somatic angiotensin-converting enzyme (ACE), able to differentiate the two active sites of somatic ACE by a selectivity factor of more than 3 orders of magnitude. The contribution of each RXPA380 residue toward this remarkable selectivity was evaluated by studying several analogues of RXPA380. This analysis revealed that both pseudo-proline and tryptophan residues in the P(1)’ and P(2)’ positions of RXPA380 play a critical role in the selectivity of this inhibitor for the C-domain. This selectivity is not due to a preference of the C-domain for inhibitors bearing pseudo-proline and tryptophan residues, but rather reflects the poor accommodation of these inhibitor residues by the N-domain. A model of RXPA380 in complex with the ACE C-domain, based on the crystal structure of germinal ACE, highlights residues that may contribute to RXPA380 selectivity. >From this model, striking differences between the N- and C-domains of ACE are observed for residues defining the S(2)’ pocket. Of the twelve residues that surround the tryptophan side chain of RXPA380 in the C-domain, five are different in the N-domain. These differences in the S(2)’ composition between the N- and C-domains are suggested to contribute to RXPA380 selectivity. The structural insights provided by this study should enhance understanding of the factors controlling the selectivity of the two domains of somatic ACE and allow the design of new selective ACE inhibitors.

6 : J Med Chem. 2004 Jan 15 ;47(2):325-36.

Evaluation of P1’-diversified phosphinic peptides leads to the development of highly selective inhibitors of MMP-11.

Matziari M, Beau F, Cuniasse P, Dive V, Yiotakis A.

Department of Chemistry, Laboratory of Organic Chemistry, University of Athens, Panepistimiopolis Zografou 15771, Athens, Greece.

Phosphinic peptides were previously reported to be potent inhibitors of several matrixins (MMPs). To identify more selective inhibitors of MMP-11, a matrixin overexpressed in breast cancer, a series of phosphinic pseudopeptides bearing a variety of P(1)’-side chains has been synthesized, by parallel diversification of a phosphinic template. The potencies of these compounds were evaluated against a set of seven MMPs (MMP-2, MMP-7, MMP-8, MMP-9, MMP-11, MMP-13, and MMP-14). The chemical strategy applied led to the identification of several phosphinic inhibitors displaying high selectivity toward MMP-11. One of the most selective inhibitors of MMP-11 in this series, compound 22, exhibits a K(i) value of 0.23 microM toward MMP-11, while its potency toward the other MMPs tested is 2 orders of magnitude lower. This remarkable selectivity may rely on interactions of the P(1)’-side chain atoms of these inhibitors with residues located at the entrance of the S(1)’-cavity of MMP-11. The design of inhibitors able to interact with residues located at the entrance of MMPs’ S(1)’-cavity might represent an alternative strategy to identify selective inhibitors that will fully differentiate one MMP among the others.

7 : Circ Res. 2003 Jul 25 ;93(2):148-54. Epub 2003 Jun 12. Comment in : Circ Res. 2003 Jul 25 ;93(2):91-3.

Roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin I and bradykinin : insights from selective inhibitors.

Georgiadis D, Beau F, Czarny B, Cotton J, Yiotakis A, Dive V.

Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Zografou, Athens, Greece.

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. The in vivo contribution of each active site to the release of angiotensin II (Ang II) and the inactivation of bradykinin (BK) is still unknown. To gain insights into the functional roles of these two active sites, the in vitro and in vivo effects of compounds able to selectively inhibit only one active site of ACE were determined, using radiolabeled Ang I or BK, as physiological substrates of ACE. In vitro studies indicated that a full inhibition of the Ang I and BK cleavage requires a blockade of the two ACE active sites. In contrast, in vivo experiments in mice demonstrated that the selective inhibition of either the N-domain or the C-domain of ACE by these inhibitors prevents the conversion of Ang I to Ang II, while BK protection requires the inhibition of the two ACE active sites. Thus, in vivo, the cleavage of Ang I and BK by ACE appears to obey to different mechanisms. Remarkably, in vivo the conversion of Ang I seems to involve the two active sites of ACE, free of inhibitor. Based on these findings, it might be suggested that the gene duplication of ACE in vertebrates may represent a means for regulating the cleavage of Ang I differently from that of BK.

8 : Am J Physiol Heart Circ Physiol. 2003 Jun ;284(6) :H1978-84. Epub 2003 Feb 27.

Endopeptidases and 24.16 in endothelial cells : potential role in vasoactive peptide metabolism.

Norman MU, Reeve SB, Dive V, Smith AI, Lew RA.

Baker Heart Research Institute, Melbourne, Victoria, Australia 8008.

The closely related metalloendopeptidases EC (EP24.15 ; thimet oligopeptidase) and 24.16 (EP24.16 ; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells ; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.

9 : J Neurochem. 2003 May ;85(4):969-77.

The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme.

Hayashi MA, Murbach AF, Ianzer D, Portaro FC, Prezoto BC, Fernandes BL, Silveira PF, Silva CA, Pires RS, Britto LR, Dive V, Camargo AC.

Center for Applied Toxinology—CAT/CEPID, Instituto Butantan, Avenue.Vital Brazil 15600, Sao Paulo, SP 05530-900, Brazil.

The bradykinin-potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin-converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin-converting enzyme in blood pressure regulation. In the present study, seven bradykinin-potentiating peptides were identified within the C-type natriuretic peptide precursor cloned from snake brain. The bradykinin-potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin-converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin-potentiating peptides, one of which displays high specificity toward the N-domain active site of the somatic angiotensin-converting enzyme. In situ hybridization studies revealed the presence of the bradykinin-potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin-potentiating peptides, their presence within the neuroendocrine regulator C-type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.

10 : J Cell Biochem. 2003 Apr 15 ;88(6):1188-201.

Vascular smooth muscle cells efficiently activate a new proteinase cascade involving plasminogen and fibronectin.

Houard X, Monnot C, Dive V, Corvol P, Pagano M. 

INSERM Unite 36, College de France, 11 place Marcelin Berthelot, 75231 Paris Cedex 05, France.

The plasminogen/plasmin system is involved in vascular wall remodeling after injury, through extracellular matrix (ECM) degradation and proteinase activation. Vascular smooth muscle cells (VSMCs) synthesize various components of the plasminogen/plasmin system. We investigated the conversion of plasminogen into plasmin in primary cultured rat VSMCs. VSMCs efficiently converted exogenous plasminogen into plasmin in a time- and dose-dependent manner. We measured plasmin activity by monitoring the hydrolysis of Tosyl-G-P-R-Mca, a fluorogenic substrate of plasmin. Cell-mediated plasmin activation was associated with the degradation of ECM, as revealed by fibronectin proteolysis. Plasmin also activated a proteinase able to hydrolyze Mca-P-L-G-L-Dpa-A-R-NH(2), a fluorogenic substrate of matrix metalloproteinases (MMPs). However, this proteinase was not inhibited by an MMP inhibitor. Furthermore, this proteinase displayed similar biochemical and pharmacological properties to fibronectin-proteinase, a recently identified zinc-dependent metalloproteinase located in the gelatin-binding domain of fibronectin. These results show that VSMCs convert exogenous plasminogen into plasmin in their pericellular environment. By hydrolyzing matrix protein plasmin activates a latent metalloproteinase that differs from MMP, fibronectin-proteinase. This metalloproteinase may participate to vascular wall remodeling, in concert with other proteinases. Copyright 2003 Wiley-Liss, Inc.

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