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1.
Arterioscler Thromb Vasc Biol ; 31(3): 528-35, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21212406

ABSTRACT

OBJECTIVE: Matrix metalloproteinase (MMP)-12 has been implicated in plaque progression and instability and is also amenable to selective inhibition. In this study, we investigated the influence of a greater than 10-fold selective synthetic MMP-12 inhibitor on plaque progression in the apolipoprotein E knockout mouse model of atherosclerosis. METHODS AND RESULTS: A phosphinic peptide (RXP470.1) that is a potent, selective murine MMP-12 inhibitor significantly reduced atherosclerotic plaque cross-sectional area by approximately 50% at 4 different vascular sites in male and female apolipoprotein E knockout mice fed a Western diet. Furthermore, RXP470.1 treatment resulted in less complex plaques with increased smooth muscle cell:macrophage ratio, less macrophage apoptosis, increased cap thickness, smaller necrotic cores, and decreased incidence of calcification. Additional in vitro and in vivo findings indicate that attenuated monocyte/macrophage invasion and reduced macrophage apoptosis probably underlie the beneficial effects observed on atherosclerotic plaque progression with MMP-12 inhibitor treatment. CONCLUSIONS: Our data demonstrate that a selective MMP-12 inhibitor retards atherosclerosis development and results in a more fibrous plaque phenotype in mice. Our study provides proof of principle to motivate translational work on MMP-12 inhibitor therapy in humans.


Subject(s)
Apolipoproteins E/deficiency , Atherosclerosis/prevention & control , Matrix Metalloproteinase Inhibitors , Peptides/pharmacology , Protease Inhibitors/pharmacology , Animals , Apolipoproteins E/genetics , Apoptosis/drug effects , Atherosclerosis/enzymology , Atherosclerosis/genetics , Atherosclerosis/pathology , Body Weight , Calcinosis/enzymology , Calcinosis/prevention & control , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Fibrosis , Infusion Pumps, Implantable , Infusions, Subcutaneous , Lipids/blood , Macrophages/drug effects , Macrophages/enzymology , Macrophages/pathology , Male , Matrix Metalloproteinase 12/deficiency , Matrix Metalloproteinase 12/genetics , Mice , Mice, Knockout , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Necrosis , Peptides/administration & dosage , Phenotype , Protease Inhibitors/administration & dosage , Rabbits
2.
J Med Chem ; 53(15): 5597-606, 2010 Aug 12.
Article in English | MEDLINE | ID: mdl-20684601

ABSTRACT

Urease inhibitors can be considered as a tool to control the damaging effect of ureolytic bacteria infections in humans which occur commonly in the developed countries. Computer-aided optimization of the aminomethylphosphinate structures by modifying both their N- and P-termini led to the invention of a novel group of inhibitors of bacterial ureases. Introduction of P-hydroxymethyl group into the molecule resulted in considerable increase of the inhibitory activity against enzymes purified from Bacillus pasteurii and Proteus vulgaris as compared with their P-methyl counterparts described previously. The designed compounds represent a competitive reversible class of urease inhibitors. The most potent, N-methyl-aminomethyl-P-hydroxymethylphosphinic acid, displayed K(i) = 360 nM against P. vulgaris enzyme.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Phosphinic Acids/chemical synthesis , Urease/antagonists & inhibitors , Bacillus/enzymology , Bacterial Proteins/chemistry , Computer Simulation , Drug Stability , Hydrolysis , Models, Molecular , Phosphinic Acids/chemistry , Proteus vulgaris/enzymology , Structure-Activity Relationship , Urease/chemistry
3.
J Phys Chem B ; 114(1): 421-8, 2010 Jan 14.
Article in English | MEDLINE | ID: mdl-20014753

ABSTRACT

Structure-activity relationship studies, regarding the influence of side chains of phosphinic pseudotripeptidic inhibitors on matrix metalloproteinases (MMPs), provided potent and selective inhibitors for this family of structurally and functionally related proteases. Among them, phosphinic pseudopeptide CbzPhepsi[P(O)(OH)CH(2)] phenylpropyl TrpNH(2), known as RXP03, has been extensively used for in vivo and in vitro studies so far. The large quantities of RXP03 required for in vivo studies, as well as the necessity for diastereoisomeric purity, motivated us to further explore and develop an efficient synthetic methodology, which allows separation of the four diastereoisomers of RXP03 based on the astonishing observed differences in solubility of the four isomers in various solvents. This fact prompted us to examine theoretically the conformational differences of these four isomers via computer simulations in the solvents used experimentally. Given the fact that the four examined diastereoisomeric forms of the phosphinic peptides exhibit different behavior in terms of potency and selectivity profiles toward zinc-metalloproteases, this theoretical study provides valuable information on the conformation of phosphinic inhibitors and therefore improves the design and synthesis of active structures. The differences in solubility of RXP03 diastereoisomers in the used solvents were examined in terms of intra- and intermolecular structure. It is found that the different solubility of the RRS and RSS diastereoisomers in EtOH is a result of the different number of hydrogen bonds formed by each isomer with EtOH molecules. In the case of SRS and SSS in Et(2)O, their different solubility might be attributed to the different intramolecular hydrogen bonds formed on these diastereoisomers.


Subject(s)
Enzyme Inhibitors/chemistry , Matrix Metalloproteinase Inhibitors , Oligopeptides/chemical synthesis , Phosphinic Acids/chemistry , Solvents/chemistry , Enzyme Inhibitors/pharmacology , Hydrogen Bonding , Matrix Metalloproteinases/metabolism , Molecular Dynamics Simulation , Oligopeptides/chemistry , Stereoisomerism , Structure-Activity Relationship
4.
J Med Chem ; 53(1): 208-20, 2010 Jan 14.
Article in English | MEDLINE | ID: mdl-19899765

ABSTRACT

A new series of phosphinic inhibitors able to interact with both angiotensin-converting enzyme (ACE) C-domain and endothelin-converting enzyme-1 (ECE-1), while sparing neprilysin (NEP), has been developed. The most potent and selective inhibitor in this series (compound 8(F2)) displays K(i) values of 0.65 nM, 150 nM, 14 nM and 6.7 microM toward somatic ACE C-domain, ACE N-domain, ECE-1, and NEP, respectively. Remarkably, in this series, the inhibitor's ability to discriminate between ECE-1 and NEP was observed to depend on the stereochemistry of the residue present in the inhibitor's P(1)' position. After iv administration, compound 8(F2) (10 mg/kg) lowered mean arterial blood pressure by 24 +/- 2 mmHg in spontaneously hypertensive rats, as compared with controls. Mixed ACE/ECE-1 inhibitor may lead to a new generation of vasopeptide inhibitors that should reduce the levels of angiotensin-II and endothelin-1, without interfering with bradykinin cleavage.


Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Metalloendopeptidases/antagonists & inhibitors , Oligopeptides/pharmacology , Peptidyl-Dipeptidase A/metabolism , Angiotensin-Converting Enzyme Inhibitors/chemical synthesis , Angiotensin-Converting Enzyme Inhibitors/chemistry , Animals , Blood Pressure/drug effects , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Endothelin-Converting Enzymes , Matrix Metalloproteinase Inhibitors , Molecular Conformation , Neprilysin/antagonists & inhibitors , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Rats , Rats, Inbred SHR , Stereoisomerism , Structure-Activity Relationship
5.
Curr Pharm Des ; 15(31): 3606-21, 2009.
Article in English | MEDLINE | ID: mdl-19925415

ABSTRACT

The fusion of therapeutics and diagnostic medicine in an effort to provide individualized pharmacotherapy frequently requires the manipulation of drugs that target different enzymes and receptors. To this end, and as a strategy to increase the efficiency of drug development pipelines, new chemical entities are often developed that interact with more than one target. Angiotensin-converting enzyme (ACE), its homologue ACE2, neutral endopeptidase (NEP) and endothelin-converting enzyme (ECE-1) are metallopeptidases that are involved in the metabolism of biologically active peptides that impact on the regulation of the cardiovascular system. The benefit of the ACE/NEP; NEP/ECE and ACE/NEP/ECE dual and triple inhibitors is not only their possible increased efficacy with respect to blood pressure control, but also their other activities, such as antiproliferative, anti-fibrotic and anti-inflammatory, mediated by angiotensin II and atrial natriuretic peptide. Over the last few years a number of three-dimensional structures of these metallopeptidases have advanced our understanding of the mode of interaction between various ligands and their target binding sites. This information is invaluable in the rational design of new and improved drugs. Here we review the structural basis for the design of single and multiple metallopeptidase inhibitors for the treatment of cardiovascular disease. Moreover, we present recent advances in the development of ACE/ECE-1 inhibitors that are likely to have high potency and improved side effect profiles.


Subject(s)
Cardiovascular Diseases/drug therapy , Enzyme Inhibitors/pharmacology , Metalloendopeptidases/antagonists & inhibitors , Animals , Binding Sites , Cardiovascular Diseases/physiopathology , Drug Delivery Systems , Drug Design , Enzyme Inhibitors/adverse effects , Humans , Ligands , Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Protein Conformation , Zinc/metabolism
6.
Org Lett ; 11(20): 4696-9, 2009 Oct 15.
Article in English | MEDLINE | ID: mdl-19775105

ABSTRACT

An unprecedented coupling of a P-C and a C-C bond-forming event in a practical operation was developed to access medicinally relevant phosphinic structures. The strategy relies on an Ireland-Claisen rearrangement triggered by the phospha-Michael addition of silyl phosphonites to allyl acrylates. This protocol was extended to a more versatile three-component variant that utilizes phosphinic acids, acryloyl chlorides, and allylic alcohols as starting materials.


Subject(s)
Medicine , Phosphinic Acids/chemistry , Acrylates/chemistry , Carbon/chemistry , Phosphinic Acids/pharmacology , Phosphorus/chemistry
7.
J Proteome Res ; 8(5): 2484-94, 2009 May.
Article in English | MEDLINE | ID: mdl-19271733

ABSTRACT

Various attempts to detect matrix metalloproteinase (MMP) active forms from complex proteomes, based on the use of specific photoactivatable affinity probes, have up to now failed. To overcome this failure, an affinity approach has been evaluated as an alternative to the photoaffinity one. For this purpose, two probes were synthesized to interact specifically with the active site of MMPs and allow isolation of MMP/probe complexes on magnetic beads through a biotin linker. Using phosphinic peptide chemistry, we prepared an affinity probe displaying picomolar potency toward several MMPs, and a related photoaffinity probe incorporating a photoactivatable azido group exhibiting subnanomolar affinity toward these targets. By a combination of silver-staining detection and MALDI peptide mass fingerprints, a systematic comparison was made of both strategies in terms of hMMP-12 and hMMP-8 recovery and identification when present in mixtures of different complexity. The results obtained show that the affinity protocol is superior to the photoaffinity strategy in terms of quantity of captured MMPs and number of MMP tryptic fragments detected in MALDI-MS. The specificity and efficiency of the affinity capture protocol developed in this study allowed easy, fast, and unambiguous detection by MALDI-MS of three hMMPs (2, 8, and 12), from a single affinity capture experiment, when added (10-36 ng of MMPs) to a tumor extract (10 microg). Thus, the tools and approaches reported should enable us to progress in the detection of endogenous active forms of MMPs in complex proteomes, an important objective with many diagnostic applications.


Subject(s)
Matrix Metalloproteinases/metabolism , Proteome/metabolism , Proteomics/methods , Affinity Labels/chemistry , Amino Acid Sequence , Animals , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Humans , Matrix Metalloproteinase 12/chemistry , Matrix Metalloproteinase 12/metabolism , Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 8/chemistry , Matrix Metalloproteinase 8/metabolism , Matrix Metalloproteinases/chemistry , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Molecular Structure , Photoaffinity Labels/chemistry , Proteome/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
8.
J Org Chem ; 73(21): 8591-3, 2008 Nov 07.
Article in English | MEDLINE | ID: mdl-18826326

ABSTRACT

The synthesis of the phosphinic analogue of thyrotropin releasing hormone (TRH) GlpPsi[P(O)(OH)]HisProNH2, where the scissile peptide bond of TRH has been replaced by the hydrolytically stable phosphinic bond, has been achieved by a multistep synthetic strategy, providing thus one of the most potent synthetic inhibitors of pyroglutamyl peptidase II (PPII) reported to date (170 nM). The key synthetic step, an Ugi-type condensation reaction, produced directly the suitably protected for solid-phase peptide synthesis pseudodipeptidic block FmocGlu(OMe)Psi[P(O)(OH)]His(Tr)OH. Formation of the pyroglutamic ring was performed on solid phase, providing thus a general method for synthesizing pyroglutamyl phosphinic peptides on solid phase. Using this strategy, the phosphinic analogue of TRH has been synthesized for the first time.


Subject(s)
Aminopeptidases/antagonists & inhibitors , Phosphinic Acids/chemistry , Pyrrolidonecarboxylic Acid/analogs & derivatives , Thyrotropin-Releasing Hormone/analogs & derivatives , Protease Inhibitors/chemical synthesis , Pyrrolidonecarboxylic Acid/antagonists & inhibitors , Thyrotropin-Releasing Hormone/chemical synthesis
9.
Bioorg Med Chem ; 16(19): 8781-94, 2008 Oct 01.
Article in English | MEDLINE | ID: mdl-18790648

ABSTRACT

Zn-metalloproteinases are an important class of hydrolytic enzymes that are characterized by the presence of a catalytic zinc(II) atom in their active center which is fundamental for proteolytic activity. Metzincins, a superfamily of Zn-metalloproteinases with many structural and functional commonalities among its members, are responsible for the fine tuning of key physiological functions in mammals and the deregulation of their activity is directly connected to numerous inflammatory and degenerative diseases such as arthritis or cancer. Development of small-molecule exogenous inhibitors of metzincins able to re-establish normal proteolytic activity in pathological conditions has been a field of intense research effort for many years but applications in the clinic were not always successful. One of the main reasons for this failure is the uncontrolled action of these inhibitors on target as well as anti-target metzincin family members. Current medicinal efforts have been shifted to the discovery of target-specific inhibitors that will help to improve our understanding of metzincins biological function and provide the basis for the development of safer pharmaceutical agents. This review focuses on the cases of certain medicinally important metzincins [matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), ADAMs with thrombospondin motifs (ADAMTSs), and procollagen C-proteinase (PCP)] and summarizes the latest advances on the discovery of inhibitors of these enzymes that display improved selectivity profiles.


Subject(s)
Chelating Agents/chemistry , Enzyme Inhibitors/pharmacology , Matrix Metalloproteinase Inhibitors , Zinc/chemistry , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/chemistry , ADAM Proteins/metabolism , ADAM17 Protein , Animals , Bone Morphogenetic Protein 1/antagonists & inhibitors , Bone Morphogenetic Protein 1/chemistry , Bone Morphogenetic Protein 1/metabolism , Catalysis , Catalytic Domain , Extracellular Matrix Proteins/antagonists & inhibitors , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Humans , Matrix Metalloproteinases/chemistry , Matrix Metalloproteinases/metabolism , Structure-Activity Relationship , Thrombospondins/antagonists & inhibitors , Thrombospondins/chemistry , Thrombospondins/metabolism
10.
J Biol Chem ; 283(45): 31058-67, 2008 Nov 07.
Article in English | MEDLINE | ID: mdl-18775985

ABSTRACT

Mass spectroscopy, microsequencing, and site-directed mutagenesis studies have been performed to identify in human matrix metalloelastase (hMMP-12) residues covalently modified by a photoaffinity probe, previously shown to be able to covalently label specifically the active site of matrix metalloproteinases (MMPs). Results obtained led us to conclude that photoactivation of this probe in complex with hMMP-12 affects a single residue in human MMP-12, Lys(241), through covalent modification of its side chain epsilon NH(2) group. Because x-ray and NMR studies of hMMP-12 indicate that Lys(241) side chain is highly flexible, our data reveal the existence of particular Lys(241) side-chain conformation in which the epsilon NH(2) group points toward the photolabile group of the probe, an event explaining the high levels of cross-linking yield between hMMP-12 and the probe. Lys(241) is not conserved in MMPs, thus differences in cross-linking yields observed with this probe between MMP members may be linked to the residue variability observed at position 241 in this family.


Subject(s)
Lysine/chemistry , Matrix Metalloproteinase 12/chemistry , Photoaffinity Labels/chemistry , Catalytic Domain/physiology , Humans , Lysine/genetics , Mass Spectrometry , Matrix Metalloproteinase 12/genetics , Mutagenesis, Site-Directed
11.
J Med Chem ; 51(18): 5736-44, 2008 Sep 25.
Article in English | MEDLINE | ID: mdl-18717581

ABSTRACT

A new group of organophosphorus inhibitors of urease, P-methyl phosphinic acids was discovered by using the structure based inhibitor design approach. Several derivatives of the lead compound, aminomethyl(P-methyl)phosphinic acid, were synthesized successfully. Their potency was evaluated in vitro against urease from Bacillus pasteurii and Proteus vulgaris. The studied compounds constitute a group of competitive, reversible inhibitors of bacterial ureases. Obtained thiophosphinic analogues of the most effective structures exhibited kinetic characteristics of potent, slow binding urease inhibitors, with Ki = 170 nM (against B. pasteurii enzyme) for the most active N-( N'-benzyloxycarbonylglycyl)aminomethyl(P-methyl)phosphinothioic acid.


Subject(s)
Bacillus/enzymology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/pharmacology , Proteus vulgaris/enzymology , Urease/antagonists & inhibitors , Computer-Aided Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Organophosphorus Compounds/chemical synthesis , Spectrometry, Mass, Electrospray Ionization
12.
Peptides ; 29(11): 1953-64, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18703099

ABSTRACT

Ecto-peptidases modulate the action of peptides in the extracellular space. The relationship between peptide receptor and ecto-peptidase localization, and the physiological role of peptidases is poorly understood. Current evidence suggests that pyroglutamyl peptidase II (PPII) inactivates neuronally released thyrotropin-releasing hormone (TRH). The impact of PPII localization in the anterior pituitary on the endocrine activities of TRH is unknown. We have studied whether PPII influences TRH signaling in anterior pituitary cells in primary culture. In situ hybridization (ISH) experiments showed that PPII mRNA was expressed only in 5-6% of cells. ISH for PPII mRNA combined with immunocytochemistry for prolactin, beta-thyrotropin, or growth hormone, showed that 66% of PPII mRNA expressing cells are lactotrophs, 34% somatotrophs while none are thyrotrophs. PPII activity was reduced using a specific phosphorothioate antisense oligodeoxynucleotide or inhibitors. Compared with mock or scrambled oligodeoxynucleotide-treated controls, knock-down of PPII expression by antisense targeting increased TRH-induced release of prolactin, but not of thyrotropin. Similar data were obtained with either a transition-state or a tight binding inhibitor. These results demonstrate that PPII expression in lactotrophs coincides with its ability to control prolactin release. It may play a specialized role in TRH signaling in the anterior pituitary. Anterior pituitary ecto-peptidases may fulfill unique functions associated with their restricted cell-specific expression.


Subject(s)
Aminopeptidases/physiology , Pituitary Gland, Anterior/enzymology , Prolactin/metabolism , Pyrrolidonecarboxylic Acid/analogs & derivatives , Thyrotropin-Releasing Hormone/physiology , Animals , Cells, Cultured , Female , In Situ Hybridization , RNA, Messenger/metabolism , Rats , Rats, Wistar
13.
Mol Aspects Med ; 29(5): 329-38, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18657570

ABSTRACT

The involvement of many zinc metalloproteinases belonging to the metzincin family with a variety of pathological states raises the possibility of therapeutic intervention using synthetic inhibitors with appropriate selectivity. Knowledge of the catalytic domain 3D-structures for various members of the metzincin family has been successfully exploited by chemists to develop potent synthetic inhibitors. However, despite intense efforts, very few highly selective inhibitors of metzincins have been discovered up to now. A survey of the literature suggests that the over-exploitation of the hydroxamate function as a zinc-binding group to develop inhibitors might be responsible for this situation. The use of alternative zinc-binding groups has led to more selective inhibitors, but the most encouraging results have been obtained for MMP-13 with compounds that do not incorporate zinc-binding groups in their structure. This new family of inhibitors exploits the presence of a deep S(1)(') cavity in the protease active site, a specific trait shared by many members of the metzincin family. However, to be successfully transposed to the metzincin members, this strategy will not only be able to exploit the structural detail of these S(1)(') cavities, but probably also subtle difference in their dynamics.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Zinc/metabolism , Animals , Catalytic Domain , Enzyme Inhibitors/chemical synthesis , Metalloendopeptidases/genetics , Models, Molecular , Molecular Structure , Protein Conformation
14.
J Med Chem ; 51(7): 2216-26, 2008 Apr 10.
Article in English | MEDLINE | ID: mdl-18324760

ABSTRACT

Angiotensin-converting enzyme 2 (ACE2), a recently identified human homologue of angiotensin-converting enzyme, is a zinc metallocarboxypeptidase which may play a unique role in cardiovascular and renal function. Here we report the discovery of potent and selective inhibitors of ACE2, which have been identified by evaluating a series of phosphinic di- and tripeptides of the general formula: Z-Xaa(PO 2-CH 2)YaaOH and Ac-Zaa-Xaa(PO 2-CH 2)YaaOH. The most potent inhibitor in this series is a tripeptide that displays a K i value of 0.4 nM toward ACE2 and is 3 orders of magnitude less potent toward carboxypeptidase A. Phosphinic tripeptides exhibit high potency exclusively when the Xaa position is occupied by a pseudoproline. A model of interaction between one inhibitor of this series and ACE2 suggests that the critical role played by a proline in inhibitors, but also for substrates hydrolysis, may rely on the presence of Tyr (510) in the ACE2 active site.


Subject(s)
Angiotensin-Converting Enzyme Inhibitors/chemical synthesis , Oligopeptides/chemical synthesis , Oligopeptides/pharmacology , Peptidyl-Dipeptidase A/drug effects , Phosphinic Acids/chemistry , Angiotensin-Converting Enzyme 2 , Angiotensin-Converting Enzyme Inhibitors/chemistry , Binding Sites , Crystallography, X-Ray , Drug Design , Humans , Models, Molecular , Molecular Structure , Oligopeptides/chemistry , Stereoisomerism , Structure-Activity Relationship
15.
Hepatology ; 47(1): 177-85, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18008367

ABSTRACT

UNLABELLED: Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia-warm reperfusion (CI-WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after 2 conditions of CI (4 degrees C for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI-WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, whereas gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10 microM) was added to the University of Wisconsin solution, and livers were preserved for 42 hours (4 degrees C), then reperfused for 1 hour in Krebs solution (37 degrees C), containing 20% erythrocytes. Liver viability parameters were recorded, and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2%-60% of liver biopsies; mean, 26% +/- 9%) to isolated necrotic hepatocytes as found in treated livers (0.2%-12%; mean, 3% +/- 2%) (P < 0.05). CONCLUSION: These data demonstrate that MMPs, by altering the ECM, play a major role in liver CI-WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions.


Subject(s)
Cold Ischemia/adverse effects , Liver Diseases/enzymology , Matrix Metalloproteinases/metabolism , Phosphinic Acids/therapeutic use , Reperfusion Injury/enzymology , Tissue Inhibitor of Metalloproteinases/metabolism , Tryptophan/analogs & derivatives , Animals , Apoptosis/drug effects , Endothelial Cells/drug effects , Enzyme Inhibitors/therapeutic use , Fibronectins/metabolism , Gene Expression , Hepatocytes/drug effects , Liver Diseases/metabolism , Male , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/genetics , Necrosis/prevention & control , Phosphinic Acids/pharmacology , Rats , Rats, Wistar , Reperfusion Injury/metabolism , Reperfusion Injury/prevention & control , Time Factors , Tissue Inhibitor of Metalloproteinases/genetics , Tissue Survival/drug effects , Tryptophan/pharmacology , Tryptophan/therapeutic use
16.
J Org Chem ; 72(19): 7222-8, 2007 Sep 14.
Article in English | MEDLINE | ID: mdl-17715974

ABSTRACT

The development of a novel 3-center 2-component annulation reaction between alpha,omega-carbamoylaldehydes and suitably monoalkylated phosphinic acids is reported. Depending on the starting alpha,omega-carbamoylaldehyde, diverse phosphinic scaffolds varying in the size of their rigidity element, the nature and stereochemistry of substituents, and the participation of heteroatoms in the azacyclic ring system can be obtained in one synthetic step and in high yield. In addition, this methodology allows the synthesis of Fmoc-protected constrained aminophosphinic acids that can be easily converted to suitable pseudodipeptide building blocks compatible with the requirements of peptide synthesis on the solid phase. Finally, the careful choice of both substituents and protecting groups can provide functionally diverse, orthogonally protected constrained scaffolds for extended derivatization of the target phosphinic peptidomimetic structures.


Subject(s)
Peptides/chemistry , Phosphinic Acids/chemistry , Aldehydes/chemistry , Amino Acids/chemistry , Cyclization , Fluorenes/chemistry , Peptides/chemical synthesis , Phosphinic Acids/chemical synthesis , Pipecolic Acids/chemical synthesis , Pipecolic Acids/chemistry
17.
J Mol Biol ; 371(4): 989-1006, 2007 Aug 24.
Article in English | MEDLINE | ID: mdl-17599356

ABSTRACT

Human matrix metalloproteinase 9 (MMP-9), also called gelatinase B, is particularly involved in inflammatory processes, bone remodelling and wound healing, but is also implicated in pathological processes such as rheumatoid arthritis, atherosclerosis, tumour growth, and metastasis. We have prepared the inactive E402Q mutant of the truncated catalytic domain of human MMP-9 and co-crystallized it with active site-directed synthetic inhibitors of different binding types. Here, we present the X-ray structures of five MMP-9 complexes with gelatinase-specific, tight binding inhibitors: a phosphinic acid (AM-409), a pyrimidine-2,4,6-trione (RO-206-0222), two carboxylate (An-1 and MJ-24), and a trifluoromethyl hydroxamic acid inhibitor (MS-560). These compounds bind by making a compromise between optimal coordination of the catalytic zinc, favourable hydrogen bond formation in the active-site cleft, and accommodation of their large hydrophobic P1' groups in the slightly flexible S1' cavity, which exhibits distinct rotational conformations of the Pro421 carbonyl group in each complex. In all these structures, the side-chain of Arg424 located at the bottom of the S1' cavity is not defined in the electron density beyond C(gamma), indicating its mobility. However, we suggest that the mobile Arg424 side-chain partially blocks the S1' cavity, which might explain the weaker binding of most inhibitors with a long P1' side-chain for MMP-9 compared with the closely related MMP-2 (gelatinase A), which exhibits a short threonine side-chain at the equivalent position. These novel structural details should facilitate the design of more selective MMP-9 inhibitors.


Subject(s)
Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase 9/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Amino Acid Sequence , Arginine/genetics , Arginine/metabolism , Barbiturates/chemistry , Barbiturates/pharmacology , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacology , Conserved Sequence , Crystallography, X-Ray , Heterocyclic Compounds, 1-Ring/chemistry , Heterocyclic Compounds, 1-Ring/pharmacology , Humans , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Matrix Metalloproteinase 9/genetics , Models, Molecular , Molecular Sequence Data , Phosphorous Acids/chemistry , Phosphorous Acids/pharmacology , Protein Binding , Protein Folding , Protein Structure, Tertiary , Sequence Alignment , Sulfones/chemistry , Sulfones/pharmacology
18.
Bioorg Med Chem ; 15(9): 3187-200, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17337192

ABSTRACT

A novel, general, and versatile method of diversification of the P1' position in phosphinic pseudodipeptides, presumable inhibitors of proteolytic enzymes, was elaborated. The procedure was based on parallel derivatization of the amino group in the suitably protected phosphinate building blocks with appropriate alkyl and aryl halides. This synthetic strategy represents an original approach to phosphinic dipeptide chemistry. Its usefulness was confirmed by obtaining a series of P1' modified phosphinic dipeptides, inhibitors of cytosolic leucine aminopeptidase, through computer-aided design basing on the structure of homophenylalanyl-phenylalanine analogue (hPheP[CH(2)]Phe) bound in the enzyme active site as a lead structure. In this approach novel interactions between inhibitor P1' fragment and the S1' region of the enzyme, particularly hydrogen bonding involving Asn330 and Asp332 enzyme residues, were predicted. The details of the design, synthesis, and activity evaluation toward cytosolic leucine aminopeptidase and aminopeptidase N are discussed. Although the potency of the lead compound has not been improved, marked selectivity of the synthesized inhibitors toward both studied enzymes was observed.


Subject(s)
Dipeptides/chemical synthesis , Leucyl Aminopeptidase/chemistry , Phosphinic Acids/chemical synthesis , Binding Sites , Dipeptides/chemistry , Hydrogen Bonding , Ligands , Models, Molecular , Molecular Structure , Phosphinic Acids/chemistry , Stereoisomerism , Structure-Activity Relationship
20.
Med Res Rev ; 27(4): 528-52, 2007 Jul.
Article in English | MEDLINE | ID: mdl-16710861

ABSTRACT

Matrix metalloproteinase (MMP)-11, or Stromelysin 3, is a particular member of MMP family, a group of zinc-dependent endopeptidases involved in matrix degradation and tissue remodeling. Despite intense efforts since its first characterization 15 years ago, its role and target substrates in different diseases remain largely unknown. While mice with MMP-11 deficiency display no particular phenotype, analysis of different tumorigenesis models with these mice lead to the conclusion that MMP-11 promotes tumor development. In contrast with other MMPs, MMP-11 is unable to degrade any major extracellular matrix component and unlike most of other MMPs that are secreted as inactive proenzymes and activated extracellularly, MMP-11 is secreted under active form. MMP-11 may thus play a unique role in tissue remodeling processes, including those associated with tumor progression. Although MMP-11 and other MMPs have been considered as promising targets to combat cancer, a first series of clinical trials using broad-spectrum MMP inhibitors have not led to significant therapeutic benefits. These disappointing results highlight the need for better understanding of the exact role played by each MMP during the different stages of tumor progression. Among the different strategies to fill this gap, highly specific MMP inhibitors would be of great value. This review provides an update on the selectivity profile of phosphinic MMP-11 synthetic inhibitors developed and discusses the opportunities and limitations to identify inhibitors able to fully discriminate MMP-11 from the other MMPs.


Subject(s)
Matrix Metalloproteinase Inhibitors , Protease Inhibitors/pharmacology , Animals , Humans , Matrix Metalloproteinase 11/chemistry , Matrix Metalloproteinase 11/metabolism , Models, Molecular , Neoplasms/physiopathology , Oligopeptides/chemistry , Phosphinic Acids/pharmacology
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