Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.23 (MMP)
 4,246 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Meprin is a zinc endopeptidase of the astacin family, which is expressed as a membrane-bound or secreted protein in mammalian epithelial cells, in intestinal leucocytes and in certain cancer cells. There are two types of meprin subunits, alpha and beta, which form disulphide-bonded homo- and hetero-oligomers. Here we report on the cleavage of matrix proteins by hmeprin (human meprin) alpha and beta homo-oligomers, and on the interactions of these enzymes with inhibitors. Despite their completely different cleavage specificities, both hmeprin alpha and beta are able to hydrolyse basement membrane components such as collagen IV, nidogen-1 and fibronectin. However, they are inactive against intact collagen I. Hence the matrix-cleaving activity of hmeprin resembles that of gelatinases rather than collagenases. Hmeprin is inhibited by hydroxamic acid derivatives such as batimastat, galardin and Pro-Leu-Gly-hydroxamate, by TAPI-0 (tumour necrosis factor alpha protease inhibitor-0) and TAPI-2, and by thiol-based compounds such as captopril. Therapeutic targets for these inhibitors are MMPs (matrix metalloproteases), TACE (tumour necrosis factor alpha-converting enzyme) and angiotensin-converting enzyme respectively. The most effective inhibitor of hmeprin alpha in the present study was the naturally occurring hydroxamate actinonin ( K(i)=20 nM). The marked variance in the cleavage specificities of hmeprin alpha and beta is reflected by their interaction with the TACE inhibitor Ro 32-7315, whose affinity for the beta subunit (IC50=1.6 mM) is weaker by three orders of magnitude than that for the alpha subunit ( K(i)=1.6 microM). MMP inhibitors such as the pyrimidine-2,4,6-trione derivative Ro 28-2653 that are more specific for gelatinases do not bind to hmeprin, presumably due to the subtle differences in the mode of zinc binding and active-site structure between the astacins and the MMPs.
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PMID:Human meprin alpha and beta homo-oligomers: cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors. 1459 49

Because membrane type-1 matrix metalloproteinase (MT1-MMP) is expressed specifically on the angiogenic endothelium as well as tumor cells, an agent possessing the ability to bind to this molecule might be useful as a tool for active targeting of tumor angiogenic vessels. Based on the sequences of peptide substrates of MT1-MMP, which had been determined by using a phage-displayed peptide library, we examined the binding ability of peptide-modified liposomes for endothelial cells and targeting ability for tumor tissues by positron emission tomography (PET). Liposomes modified with stearoyl-Gly-Pro-Leu-Pro-Leu-Arg (GPLPLR-Lip) showed high binding ability to human umbilical vein endothelial cells and accumulated in the tumor about 4-fold more than did the unmodified liposomes. Because we reported previously that liposomalized 5'-O-dipalmitoylphosphatidyl 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (DPP-CNDAC), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, strongly suppressed tumor growth when delivered in liposomes modified with another angiogenic homing peptide, we examined the antitumor activity of DPP-CNDAC entrapped in GPLPLR-Lip. DPP-CNDAC/GPLPLR-Lip showed significant tumor growth suppression compared to DPP-CNDAC/unmodified liposomes. These results suggest that DPP-CNDAC-liposomes modified with MT1-MMP-targeted peptide are useful for cancer anti-neovascular therapy (ANET), namely, tumor growth suppression by damage to angiogenic endothelial cells.
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PMID:Anti-neovascular therapy by liposomal drug targeted to membrane type-1 matrix metalloproteinase. 1463 19

Matrix metalloproteinases (MMPs) and the related tumor necrosis factor converting enzyme (TACE) are involved in tissue remodeling, cell migration, and processing of signaling molecules, such as cytokines and adhesion molecules. Fluorescence-quenched peptide substrates have been widely used to quantitate the actual enzymatic activity of MMPs. However, the various MMPs have very different specific activities toward these substrates. This restricts their value for the determination of composite proteolytic activity of mixtures of metalloproteinases in biological fluids. The N-terminal elongation of the most widely used MMP substrate (FS-1) with a Lys to the sequence Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH(2) (FS-6) yields a fluorogenic peptide with improved substrate properties. As compared to FS-1, the specificity constant (kcat/Km) of FS-6 for collagenases (MMP-1, MMP-8, MMP-13) and MT1-MMP (MMP-14) is increased two- to ninefold and threefold, respectively, while those for gelatinases and matrilysin remain equally high. Using high-performance liquid chromatography-fluorescence detection, MMP activity can be quantitated in the picomolar range. FS-6 shows up to twofold higher specificity constants (kcat/Km of 0.8x10(6)M(-1)s(-1)) for TACE, as compared to standard substrates Mca-PLAQAV-Dpa-RSSSAR-NH(2) and Dabcyl-LAQAVRSSSAR-EDANS. FS-6 is fully water soluble and thus allows measurement of metalloproteinase activity in tissue culture conditions, e.g., on the surface of viable cells in situ.
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PMID:Characterization of Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, a fluorogenic substrate with increased specificity constants for collagenases and tumor necrosis factor converting enzyme. 1511 93

A variety of therapeutic strategies in oncology are focused on the inhibition of tumor-induced angiogenesis. Thus, there is a keen interest in methods which allow non-invasive monitoring of molecular targets involved in angiogenesis which would support information for planning and controlling corresponding therapies. Moreover, such techniques would provide an insight into the formation of new sprouting blood vessels, the involved processes and regulatory mechanisms in patients. At the moment, development of radiotracer based techniques is mainly concentrated on three different targets which include peptidic and non-peptidic alpha v beta 3-integrin binding antagonists, matrix metalloproteinase inhibitors and single chain anti-fibronectin antibody fragments. Development of radiolabeled MMP inhibitors is based on either the decapeptide Cys-Thr-Thr-His-Trp-Gly-Phe-Thr-Leu-Cys resulting from a phage display library or small molecular weight compounds. The in vitro data for these tracers are very promising. However, more detailed in vivo data are necessary to evaluate the potency of MMP-inhibitors for in-vivo imaging. The radiolabelled anti-ED-B single chain antibody fragment scFv L-19 shows selective accumulation in the tumor vasculature in a murine tumour model. In a first patient study a selective localisation of the (123)I-labeled tracer in lesions of different tumours was found. On the basis of the lead structure cyclo(-Arg-Gly-Asp-dPhe-Val) a variety of different radiolabeled RGD-peptides has been developed for the non-invasive determination of the alpha v beta 3 expression. These developments include peptides labeled with minimum structural alteration, peptide carbohydrate conjugates, peptidomimetics based on the RGD-structure as well as heterodimeric, homodimeric and homotetrameric ligand systems. Many of the tracers show high alpha v beta 3-affinity and selectivity in vitro and receptor selective tumour accumulation with high image contrast in different murine tumour models. Further studies have to demonstrate that this approach can be translated to clinical settings allowing visualisation of alpha v beta 3-positive tumours and alpha v beta 3 expression during tumour-induced angiogenesis in patients.
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PMID:Radiolabeled tracers for imaging of tumor angiogenesis and evaluation of anti-angiogenic therapies. 1513 68

The in vitro inhibitory activity of the rice Bowman-Birk inhibitor (rBBI) or soybean Bowman-Birk inhibitor (sBBI) against trypsin-catalyzed activation of pro-matrix metalloproteinase 1 or 9 (pro-MMP-1 or pro-MMP-9), respectively, was investigated using electrophoresis with silver staining, heparin-enhanced zymography, biotinylated gelatin, Biotrak assay, and fluorescence quenched substrate hydrolysis. rBBI at concentrations of 0.08-0.352 mg/mL dose-dependently inhibited the in vitro activation of 45 microg/mL pro-MMP-1 by trypsin. Heparin-enhanced zymography analysis of pro-MMP-1, trypsin-activated MMP-1, and a mixture of pro-MMP-1-trypsin-rBBI showed clear zones associated with trypsin-activated MMP-1 and the absence of clear zones in lanes containing pro-MMP-1 or a mixture of pro-MMP-1, trypsin, and rBBI. The results of the Biotrak assay also indicated that rBBI dose-dependently suppressed the activation of pro-MMP-1 by trypsin. sBBI dose-dependently inhibited the activation of 100 microg/mL of pro-MMP-9 by trypsin. Biotinylated gelatin assays demonstrated that pro-MMP-9 or pro-MMP-9 in the presence of trypsin and BBI did not hydrolyze gelatin, whereas p-aminophenylmercury acetate (APMA)-activated MMP-9 and trypsin-activated MMP-9 caused significant hydrolysis of gelatin. Quenched fluorescence substrate hydrolysis for total MMP activity showed that pro-MMP-1 or pro-MMP-9 did not hydrolyze the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; active MMP-1 or MMP-9 hydrolyzed the substrate, but lower substrate hydrolysis was obtained when pro-MMP-1 or pro-MMP-9 was incubated with trypsin in the presence of increasing concentrations of rBBI. The results are discussed in light of the role of MMP-1 and MMP-9 in the process of angiogenesis and the potential of rBBI or sBBI as a functional food ingredient.
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PMID:In vitro inhibition of the activation of Pro-matrix Metalloproteinase 1 (Pro-MMP-1) and Pro-matrix metalloproteinase 9 (Pro-MMP-9) by rice and soybean Bowman-Birk inhibitors. 1526 7

Processing of the laminin-5 (Ln-5) gamma 2 chain by membrane-type-1 matrix metalloproteinases (MT1-MMP) promotes migration and invasion of epithelial and tumor cells. We previously demonstrated that MT1-MMP cleaves the rat gamma 2 chain at two sites, producing two major C-terminal fragments of 100 (gamma 2') and 80 (gamma 2 x) kDa and releasing a 30-kDa fragment containing epidermal growth factor (EGF)-like motifs (domain III (DIII) fragment). The DIII fragment bound the EGF receptor (EGF-R) and stimulated cell scattering and migration. However, it is not yet clear whether human Ln-5 is processed in a similar fashion to rat Ln-5 because one of the two MT1-MMP cleavage sites present in rat gamma 2 is not found in human gamma 2. To identify the exact cleavage site for MT1-MMP in human Ln-5, we purified both the whole molecule as well as a monomeric form of human gamma 2 that is frequently expressed by malignant tumor cells. Like rat Ln-5, both the monomer of gamma 2, as well as the gamma 2 derived from intact Ln-5, were cleaved by MT1-MMP in vitro, generating C-terminal gamma 2' (100 kDa) and gamma 2 x (85 kDa) fragments and releasing DIII fragments (25 and 27k Da). In addition to the conserved first cleavage site used to generate gamma 2', two adjacent cleavage sites (Gly(559)-Asp(560) and Gly(579)-Ser(580)) were found that could generate the gamma 2 x and DIII fragments. Two of the three EGF-like motifs present in the rat DIII fragment are present in the 27-kDa human fragment, and like the rat DIII, this fragment can promote breast carcinoma cell migration by engaging the EGF-R. These results suggest that MT1-MMP processing of Ln-5 in human tumors may stimulate the EGF-R, resulting in increased tumor cell scattering and migration that could possibly increase their metastatic potential.
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PMID:Membrane-type matrix metalloproteinase-1 (MT1-MMP) is a processing enzyme for human laminin gamma 2 chain. 1552 52

Enzyme prodrug monotherapy takes advantage of the selectivity and specificity of enzymes that are differentially active in the immediate environment of tumor cells. Matrix metalloproteinases-2 and -9 (MMP-2 and -9, respectively) are cell-surface Zn-dependent endoproteases associated with diverse processes throughout tumor formation and progression. These enzymes have demonstrated high ratios of tumor- to nontumor-associated activity and may represent candidates for antitumor prodrug activation. Our MMP targeting strategy was to prepare and evaluate two classes of enzyme prodrugs, peptides and sequence-similar peptidomimetics, and determine which would be substrates for the enzymes and thus suitable for further in vitro and in vivo evaluation. We selected representatives of three structurally and mechanistically distinct classes of compounds for delivery, doxorubicin, several auristatins (novel synthetic members of the dolastatin class of tubulin polymerization inhibitors), and CBI-TMI (a duocarmycin class minor groove binder). The drugs were acylated on available amines with the broadly recognized MMP substrate P3-P1' sequence acetyl L-prolyl-L-leucyl-glycyl-L-leucine, or with a peptidomimetic analogue. From a panel of four peptides and four peptidomimetics, two compounds, both peptides, were found to be substrates, with specific activities in the range of 1-20 nmol min(-1) mg(-1). For MMP-9, complete conversion took place in 4-16 h; proteolysis by MMP-2 was considerably slower. Cleavage occurred, as predicted, at the Gly-Leu bond to liberate a leucyl drug, and no other intermediates or cleavage products were observed. Although the MMP-9 proteolysis products were equipotent with the parent leucyl drugs, the prodrugs were not differentially active against MMP-2 or -9-expressing versus nonexpressing cell lines during a 4 h exposure. Our data can be interpreted in light of the current understanding of the structural and mechanistic factors governing MMP-2 and -9 proteolysis.
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PMID:Novel antitumor prodrugs designed for activation by matrix metalloproteinases-2 and -9. 1583 97

The present article refers to the mechanism of angiogenesis, a phenomenon which in certain cases is normal, however it usually accompanies the formation of solid cancerous tumors. During the process of angiogenesis, migration, differentiation and proliferation of endothelial cells occurs. In some pathological cases, the most important of which is cancer, the mechanism of angiogenesis is reinforced. It has been observed that a tumor cannot grow without the formation of new blood vessels in the surrounding tissues, which account for blood supply to the tumor. The process of angiogenesis is regulated by chemical signals of the organism, which function as an "angiogenesis switch" regulating the formation of new vasculature. A variety of anti-angiogenic agents which lead to angiogenesis inhibition are found in the clinical trial phase. Among these agents are: i) molecules which inhibit the action of Vascular Endothelial Growth Factors, VEGF, ii) molecules which obstruct migration, differentiation and proliferation of endothelial cells, via their binding to receptors of the alpha(nu)beta(3) integrins and iii) inhibitors of metalloproteinases (MMP). Certain molecules of the above mentioned categories, labeled with radionuclides which emit gamma- radiation or beta- particles or positrons, have been proposed and are being evaluated as possible radiopharmaceuticals, for the visualization of receptors involved in the mechanism of angiogenesis, aiming at the scintigraphic detection of primary or metastatic cancer at an early stage and possibly, aiming at its inhibition/treatment. Regarding the study of angiogenesis the following have been described: a. antibodies targeting VEGF, labeled with radionuclides emitting beta- and/or gamma-radiation, which can be applied for the diagnosis and possibly, for the treatment of cancer, b. peptide derivatives which contain the amino-acid sequence RGD (Arg-Gly-Asp) and compete for the alpha(nu)beta(3) integrins, with the proteins of the stroma. It has been found that these radiolabeled derivatives localize in tumors and can be used for the visualization and, possibly, for the tumor eradication of primary and metastatic cancer.
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PMID:[Angiogenesis in cancer and its detection with radiolabeled biomolecules]. 1588 45

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp(116) to Lys(214) of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly(171)-Leu(187) in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.
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PMID:A monoclonal antibody inhibits gelatinase B/MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains. 1713 15

The turnover of the collagen triple-helical structure (collagenolysis) is a tightly regulated process in normal physiology and has been ascribed to a small number of proteases. Several members of the matrix metalloproteinase (MMPs) family possess collagenolytic activity, and the mechanisms by which these enzymes process triple helices are beginning to be unraveled. The present study has utilized two triple-helical sequences to compare the cleavage-site specificities of 10 MMPs. One substrate featured a continuous Gly-Xxx-Yyy sequence (Pro-Leu-Gly approximately Met-Arg-Gly), while the other incorporated an interruption in the Gly-Xxx-Yyy repeat (Pro-Val-Asn approximately Phe-Arg-Gly). Both sequences were selectively cleaved by MMP-13 while in linear form, but neither proved to be selective within a triple helix. This suggests that the conformational presentation of substrate sequences to a MMP active site is critical for enzyme specificity, in that activities differ when sequences are presented from an unwound triple helix versus an independent single strand. Differences in specificity between secreted and membrane-type (MT) MMPs were also observed for both sequences, where MMP-2 and MT-MMPs showed an ability to hydrolyze a triple helix at an additional site (Gly-Gln bond). Interruption of the triple helix had different effects on secreted MMPs and MT-MMPs, because MT-MMPs could not hydrolyze the Asn-Phe bond but instead cleaved the triple helix closer to the C terminus at a Gly-Gln bond. It is possible that MT-MMPs have a requirement for Gly in the P1 subsite to be able to efficiently process a triple-helical molecule. Analysis of individual kinetic parameters and activation energies indicated different substrate preferences within secreted MMPs, because MMP-13 preferred the interrupted sequence, while MMP-8 showed little discrimination between non-interrupted and interrupted triple helices. On the basis of the present and prior studies, we can assign unique triple-helical peptidase behaviors to the collagenolytic MMPs. Such differences may be significant for understanding MMP mechanisms of action and aid in the development of selective MMP inhibitors.
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PMID:Differentiation of secreted and membrane-type matrix metalloproteinase activities based on substitutions and interruptions of triple-helical sequences. 1733 50


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