Matrix metalloproteinases in cancer: from new functions to improved inhibition strategies

Over the last years, the relevance of the matrix metalloproteinase (MMP) family in cancer research has grown considerably. These enzymes were initially associated with the invasive properties of tumour cells, owing to their ability to degrade all major protein components of the extracellular matrix (ECM) and basement membranes. However, further studies have demonstrated the implication of MMPs in early steps of tumour evolution, including stimulation of cell proliferation and modulation of angiogenesis. The establishment of causal relationships between MMP overproduction in tumour or stromal cells and cancer progression has prompted the development of clinical trials with a series of inhibitors designed to block the proteolytic activity of these enzymes. Unfortunately, the results derived from using broad-spectrum MMP inhibitors (MMPIs) for treating patients with advanced cancer have been disappointing in most cases. There are several putative explanations for the lack of success of these MMPIs including the recent finding that some MMPs may play a paradoxical protective role in tumour progression. These observations together with the identification of novel functions for MMPs in early stages of cancer have made necessary a reformulation of MMP inhibition strategies. A better understanding of the functional complexity of this proteolytic system and global approaches to identify the relevant MMPs which must be targeted in each individual cancer patient, will be necessary to clarify whether MMP inhibition may be part of future therapies against cancer.     

The intermediate S1' pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses, and homology modeling

Human matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP and its structure has not been reported. The enzyme active site S1' pocket in MMPs is a well defined substrate P1' amino acid residue-binding site with variable depth. To explore MMP-26 active site structure-activity, a series of new potent mercaptosulfide MMP inhibitors (MMPIs) with Leu or homophenylalanine (Homophe) side chains at the P1' site were selected. The Homephe side chain is designed to probe deep S1' pocket MMPs. These inhibitors were tested against MMP-26 and several MMPs with known x-ray crystal structures to distinguish shallow, intermediate, and deep S1' pocket characteristics. MMP-26 has an inhibition profile most similar to those of MMPs with intermediate S1' pockets. Investigations with hydroxamate MMPIs, including those designed for deep pocket MMPs, also indicated the presence of an intermediate pocket. Protein sequence analysis and homology modeling further verified that MMP-26 has an intermediate S1' pocket formed by Leu-204, His-208, and Tyr-230. Moreover, residue 233 may influence the depth of an MMP S1' pocket. The residue at the equivalent position of MMP-26 residue 233 is hydrophilic in intermediate-pocket MMPs (e.g. MMP-2, -8, and -9) and hydrophobic in deep-pocket MMPs (e.g. MMP-3, -12, and -14). MMP-26 contains a His-233 that renders the S1' pocket to an intermediate size. This study suggests that MMPIs, protein sequence analyses, and molecular modeling are useful tools to understand structure-activity relationships and provides new insight for rational inhibitor design that may distinguish MMPs with deep versus intermediate S1' pockets.     

Synthesis of bicyclic molecular scaffolds (BTAa): an investigation towards new selective MMP-12 inhibitors

Starting from 3-aza-6,8-dioxa-bicyclo[3.2.1]octane scaffold (BTAa) a virtual library of molecules was generated and screened in silico against the crystal structure of the Human Macrophage Metalloelastase (MMP-12). The molecules obtaining high score were synthesized and the affinity for the catalytic domain of MMP-12 was experimentally proved by NMR experiments. A BTAa scaffold 20 having a N-hydroxyurea group in position 3 and a p-phenylbenzylcarboxy amide in position 7 showed a fair inhibition potency (IC50 = 149 microM) for MMP-12 and some selectivity towards five different MMPs. These results, taken together with the X-ray structure of the adduct between MMP-12, the inhibitor 20 and the acetohydroxamic acid (AHA), suggest that bicyclic scaffold derivatives may be exploited for the design of new selective matrix metalloproteinase inhibitors (MMPIs).     

Type IV collagenases (MMP-2 and MMP-9) and their substrates--intracellular proteins, hormones, cytokines, chemokines and their receptors

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave protein components of extracellular matrix such as collagens, laminin, fibronectin, proteoglycans and contribute to cell migration by eliminating the surrounding extracellular matrix and basement membrane barriers. However, the extracellular matrix is not simply an extracellular scaffold because, for example, it contains sites that can bind growth factors; therefore, degradation of the extracellular matrix components by MMPs can alter cellular behavior. MMPs also cleave a variety of non-ECM proteins, including cytokines, chemokines, and growth factors, activating or inactivating them, or generating other products that have biological consequences. The immune system is also influenced by MMPs. For that reason, the function of MMPs is much more complex and subtle than simple demolition. MMPs are essential for embryonic development and morphogenesis, however, exuberant expression of these enzymes has been associated with a variety of destructive diseases, including tumor progression, cardiovascular diseases and autoimmune diseases.     

Matrix metalloproteinase inhibitors as anticancer agents

The important role of matrix metalloproteinases (MMPs) in the process of carcinogenesis is well established. However, despite very promising activity in a plethora of preclinical models, MMP inhibitors (MMPIs) failed to demonstrate a statistically significant survival advantage in advanced stage clinical trials in most human malignancies. Herein, we review the implication of MMPs in carcinogenesis, outline the pharmacology and current status of various MMPIs as anticancer agents and discuss the etiologies for the discrepancy between their preclinical and clinical evaluation. Finally, strategies for effective incorporation of MMPIs in current anticancer therapies are proposed.     

Natural products as a gold mine for selective matrix metalloproteinases inhibitors

Nineteen natural compounds with diverse structures are identified as potential MMPIs using structure-based virtual screening from 4000 natural products. Hydroxycinnamic acid or analogs of natural products are important for potent inhibitory and selectivity against MMPs, and the solvent effect in the S1' pocket can affect the hydrophobic interactions and hydrogen bonds between MMPIs and MMPS, making MMPIs exhibit certain selectivity for a specific MMP isoenzyme. Furthermore, compound 5 can reduce the expression of both MMP-2 and active-MMP-9, and suppress the migration of MDA-MB-231 tumor cell in a wound healing assay, which may be further developed as an anticancer agent.     

Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting

Matrix metalloproteinases (MMPs) consist of a multigene family of zinc-dependent extracellular matrix (ECM) remodeling endopeptidases implicated in pathological processes, such as carcinogenesis. In this regard, their activity plays a pivotal role in tumor growth and the multistep processes of invasion and metastasis, including proteolytic degradation of ECM, alteration of the cell-cell and cell-ECM interactions, migration and angiogenesis. The underlying premise of the current minireview is that MMPs are able to proteolytically process substrates in the extracellular milieu and, in so doing, promote tumor progression. However, certain members of the MMP family exert contradicting roles at different stages during cancer progression, depending among other factors on the tumor stage, tumor site, enzyme localization and substrate profile. MMPs are therefore amenable to therapeutic intervention by synthetic and natural inhibitors, providing perspectives for future studies. Multiple therapeutic agents, called matrix metalloproteinase inhibitors (MMPIs) have been developed to target MMPs, attempting to control their enzymatic activity. Even though clinical trials with these compounds do not show the expected results in most cases, the field of MMPIs is ongoing. This minireview critically evaluates the role of MMPs in relation to cancer progression, and highlights the challenges, as well as future prospects, for the design, development and efficacy of MMPIs.     

Methotrexate gamma-hydroxamate derivatives as potential dual target antitumor drugs

A series of new aminopteroyl-based hydroxamate derivatives were synthesized and tested in vitro in cell culture models as potential dual target drugs. These compounds were designed to target two families of enzymes, matrix metalloproteinases (MMP) and a folate enzyme, dihydrofolate reductase (DHFR). These enzymes are the components of two unrelated cellular pathways and they are often over-expressed in metastasizing tumors. In addition to the synthesis and full structural characterization of the hybrid molecules, we describe their inhibitory activities against a series of MMPs (MMP-2, MMP-7, MMP-9, MMP-14) and DHFR, as well as their antiproliferative activity in three cancer cell lines. The new hydroxamate derivatives of MTX proved to be effective inhibitors of MMPs and DHFR in the micromolar and nanomolar range, respectively. Furthermore, they showed strong antiproliferative activity against A549 cells (non-small cell lung carcinoma), and PPC-1 and Tsu-Pr1 prostate cancer cell lines. Therefore, based on the present results, these bi-functional drugs may be good candidates to target specific tumors in animal models due to potential combined effects on two pathways crucial for tumor development.     

A quantitative structure-activity relationship study on matrix metalloproteinase inhibitors: Piperidine sulfonamide aryl hydroxamic acid analogs

A quantitative structure-activity relationship (QSAR) study has been made on a series of piperidine sulfonamide aryl hydroxamic acid analogs acting as matrix metalloproteinase (MMP) inhibitors. The inhibitory potencies of the compounds against two MMPs, MMP-2 and MMP-13, are found to be significantly correlated with the hydrophobic properties of the molecules, suggesting that in both enzymes the hydrophobic interaction is playing a dominant role.     

A quantitative structure-activity relationship study on matrix metalloproteinase inhibitors: piperidine sulfonamide aryl hydroxamic acid analogs

A quantitative structure-activity relationship (QSAR) study has been made on a series of piperidine sulfonamide aryl hydroxamic acid analogs acting as matrix metalloproteinase (MMP) inhibitors. The inhibitory potencies of the compounds against two MMPs, MMP-2 and MMP-13, are found to be significantly correlated with the hydrophobic properties of the molecules, suggesting that in both enzymes the hydrophobic interaction is playing a dominant role.     

Matrix metalloproteinases and their function in myocardium

A significant number of myocardial diseases are accompanied by increased synthesis and degradation of the extracellular matrix (ECM) as well as by changed maturation and incorporation of ECM components. Important groups of enzymes responsible for both normal and pathological processes in ECM remodeling are matrix metaloproteinases (MMPs). These enzymes share a relatively conserved structure with a number of identifiable modules linked to their specific functions. The most important function of MMPs is the ability to cleave various ECM components; including such rigid molecules as fibrillar collagen molecules. The amount and activity of MMPs in cardiac tissue are regulated by a range of activating and inhibiting processes. Although MMPs play multifarious roles in many myocardial diseases, here we have focused on their function in ischemic cardiac tissue, dilated cardiomyopathy and hypertrophied cardiac tissue. The inhibition of MMPs by means of synthetic inhibitors seems to be a promising strategy in cardiac disease treatment. Their effects on diseased cardiac tissue have been successfully tested in several experimental studies.     

基质金属蛋白酶

基质金属蛋白酶是一类分解细胞外基质组分的锌蛋白酶⒚它们在有机体生长发育中的细胞外基质逆转与重塑以及疾病中的病理损害起着极为重要的作用⒚基质金属蛋白酶的表达和活性在不同细胞水平受到严密调控,如细胞因子、生长因子以及激素的调节⒚基质金属蛋白酶以酶原形式分泌,随后被其它蛋白酶如胞浆素或非蛋白酶类化学物质如有机汞所激活⒚所有基质金属蛋白酶都受到天然抑制剂 金属蛋白酶组织抑制剂所抑制⒚两者的不平衡导致许多疾病的发生,如肿瘤侵入及转移⒚合成基质金属蛋白酶组织抑制剂所抑制,如 Ｍ ａｒｉｍ ａｓｔａｔ 能控制肿瘤转移的发生及进一步扩散⒚本文将对基质金属蛋白酶的特征、分子区域结构、底物特性、激活机制、调控方式等方面进行最新概述⒚     

Genetic polymorphisms of matrix metalloproteinases and their inhibitors in potentially malignant and malignant lesions of the head and neck

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that are capable of cleaving all extra cellular matrix (ECM) substrates. Degradation of matrix is a key event in progression, invasion and metastasis of potentially malignant and malignant lesions of the head and neck. It might have an important polymorphic association at the promoter regions of several MMPs such as MMP-1 (-1607 1G/2G), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) and TIMP-2 (-418 G/C or C/C). Tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring inhibitors of MMPs, which inhibit the activity of MMPs and control the breakdown of ECM. Currently, many MMP inhibitors (MMPIs) are under development for treating different malignancies. Useful markers associated with molecular aggressiveness might have a role in prognostication of malignancies and to better recognize patient groups that need more antagonistic treatment options. Furthermore, the introduction of novel prognostic markers may also promote exclusively new treatment possibilities, and there is an obvious need to identify markers that could be used as selection criteria for novel therapies. The objective of this review is to discuss the molecular functions and polymorphic association of MMPs and TIMPs and the possible therapeutic aspects of these proteinases in potentially malignant and malignant head and neck lesions. So far, no promising drug target therapy has been developed for MMPs in the lesions of this region. In conclusion, further research is required for the development of their potential diagnostic and therapeutic possibilities.     

Pro-MMP-9 is a specific macrophage product and is activated by osteoarthritic chondrocytes via MMP-3 or a MT1-MMP/MMP-13 cascade

In joint diseases of both the inflammatory (rheumatoid arthritis, or RA) or the degenerative variety (osteoarthritis, or OA), matrix metalloproteinases (MMPs) are essential mediators of irreversible tissue destruction. MMP-9 is secreted as a stable, inactive zymogen and is proteolytically converted to the active enzyme. To understand the activation mechanism of MMP-9 in joint diseases, the process was investigated in serum-free cocultures of human articular chondrocytes and macrophages. Macrophages extensively expressed and secreted pro-MMP-9 whereas chondrocytes failed to produce the enzyme. However, efficient activation of pro-MMP-9 required soluble and membrane-associated chondrocyte proteinases. Two alternative activation pathways mainly involved MMPs and, marginally, serine or cysteine proteinases. MT1-MMP (MMP-14), the only MT-MMP expressed in chondrocytes, converted pro-MMP-13 which, in turn, cleaved pro-MMP-9. Alternatively, pro-MMP-9 was activated less efficiently by MMP-3, which was converted by autocatalysis or by serine or cysteine proteinases. Both pathways were triggered by chondrocytes from OA, but not normal joints. Therefore, articular chondrocytes are not innocent bystanders in joint diseases. They not only produce destructive enzymes guided by environmental cues but also they can instruct inflammatory cells or cells from surrounding tissues to do so by converting in several ways zymogens produced but not activated by these cells themselves.     

Gelatinase-mediated migration and invasion of cancer cells

The matrix metalloproteinases(MMP)-2 and -9, also known as the gelatinases have been long recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. In the recent years, a plethora of non-matrix proteins have also been identified as gelatinase substrates thus significantly broadening our understanding of these enzymes as proteolytic executors and regulators in various physiological and pathological states including embryonic growth and development, angiogenesis and tumor progression, inflammation, infective diseases, degenerative diseases of the brain and vascular diseases. Although the effect of broad-spectrum inhibitors of MMPs in the treatment of cancer has been disappointing in clinical trials, novel mechanisms of gelatinase inhibition have been now identified. Inhibition of the association of the gelatinases with cell-surface integrins appears to offer highly specific means to target these enzymes without inhibiting their catalytic activity in multiple cell types including endothelial cells, tumor cells and leukocytes. Here, we review the multiple functions of the gelatinases in cancer, and especially their role in the tumor cell migration and invasion.     

Co-localization of integrins and matrix metalloproteinases in the extracellular matrix of chondrocyte cultures

Beta1-integrins were found in the cartilage matrix, suggesting their implication in the assembly of its architectural scaffold, but the mechanism for this event is not yet clear. Matrix metalloproteinases (MMPs) may be involved in an integrin-shedding mechanism and matrix beta1-integrins may act to alter MMP activity. To begin to address this question, this study was designed to determine whether beta1-integrins and MMPs are colocalized in the chondrocytes or in the extracellular matrix of cartilage. We investigated high-density cultures of limb buds of 12-day-old mouse embryos by double immunofluorescence, immunoelectron microscopy and by coimmunoprecipitation assays in order to examine the localization of beta1-integrins and matrix metalloproteinases (MMP-1, MMP-3 and MMP-9) in cartilage. It was found, that all investigated MMPs and beta1-integrins were specifically co-localized in high-density cartilage cultures. Immunogold and immunofluorescence labelling of both beta1-integrins and MMPs were observed not only at the surface of chondrocytes but mainly also in the pericellular space and distributed between collagen fibrils in the extracellular matrix (ECM) as well. Results of immunoprecipitation experiments suggest a functional association of MMPs and beta1-integrins in chondrocytes as already described for other cell types. Further investigations are needed to elucidate the functional association between beta1-integrins and MMPs in chondrocytes.     

Human matrix metalloproteinases: an ubiquitarian class of enzymes involved in several pathological processes

Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes.     

4-Alkyliden-beta-lactams conjugated to polyphenols: synthesis and inhibitory activity

A series of compounds combining the beta-lactam and polyphenol scaffold have been prepared and evaluated for inhibition of human leukocyte elastase and matrix metallo-proteases MMP-2 and MMP-9. The design of these compounds has been based on the 'overlapping-type' strategy where two pharmacophores are linked in a single molecule. The most powerful compound against elastase was an N-galloyl-4-alkyliden beta-lactam, [3-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4-oxo-1-(3,4,5-tris-benzyloxy-benzoyl)-azetidin-2-ylidene]-acetic acid ethylester, with an IC50 of 0.5 microM; while the most powerful against MMP-2 was a 4-alkyliden beta-lactam arylated on the C-3 hydroxy side chain (3,5-bis-benzyloxy-4-hydroxy-benzoic acid 1-(2-benzyloxycarbonylmethylene-4-oxo-azetidin-3-yl)-ethyl ester) with an IC50 of 4 microM. Of the total 35 compounds tested, high levels of inhibition of elastase and of MMPs were separately exerted by distinct molecules.     

Enzymatic detachment of spore germlings in Magnaporthe oryzae

Magnaporthe oryzae germlings tightly attach to the host surface by producing extracellular matrix (ECM) from germ tubes and appressoria, which are important for the early infection process. To understand the adhesion mechanisms of ECM during differentiation of infection structure, we evaluated the effects of various enzymes on M. oryzae germlings and the disease symptoms of the host plant, wheat. Treatment with β-mannosidase, collagenase N-2, collagenase S-1, or gelatinase B at 1-h postinoculation (hpi) resulted in germling detachment, although producing normal appressoria. Treatment with matrix metalloproteinases (MMPs) at 6 hpi also caused germling detachment. Furthermore, we confirmed by the inoculation tests and scanning electron microscopy that the germlings on the wheat plant were removed and did not manifest pathogenicity on treatment with MMPs. The most effective MMPs were crude collagenase, collagenase S-1, and gelatinase B, suggesting that the application of MMPs is promising for crop protection from fungal diseases by its detachment action.     

Matrix metalloproteinases (MMPs): chemical-biological functions and (Q)SARs

Matrix metalloproteinases (MMPs) are a large family of calcium-dependent zinc-containing endopeptidases, which are responsible for the tissue remodeling and degradation of the extracellular matrix (ECM), including collagens, elastins, gelatin, matrix glycoproteins, and proteoglycan. They are regulated by hormones, growth factors, and cytokines, and are involved in ovarian functions. MMPs are excreted by a variety of connective tissue and pro-inflammatory cells including fibroblasts, osteoblasts, endothelial cells, macrophages, neutrophils, and lymphocytes. These enzymes are expressed as zymogens, which are subsequently processed by other proteolytic enzymes (such as serine proteases, furin, plasmin, and others) to generate the active forms. Matrix metalloproteinases are considered as promising targets for the treatment of cancer due to their strong involvement in malignant pathologies. Clinical/preclinical studies on MMP inhibition in tumor models brought positive results raising the idea that the development of strategies to inhibit MMPs may be proved to be a powerful tool to fight against cancer. However, the presence of an inherent flexibility in the MMP active-site limits dramatically the accurate modeling of MMP-inhibitor complexes. The interest in the application of quantitative structure-activity relationships (QSARs) has steadily increased in recent decades and we hope it may be useful in elucidating the mechanisms of chemical-biological interactions for this enzyme. In the present review, an attempt has been made to explore the in-depth knowledge from the classification of this enzyme to the clinical trials of their inhibitors. A total number of 92 QSAR models (44 published and 48 new formulated QSAR models) have also been presented to understand the chemical-biological interactions. QSAR results on the inhibition of various compound series against MMP-1, -2, -3, -7, -8, -9, -12, -13, and -14 reveal a number of interesting points. The most important of these are hydrophobicity and molar refractivity, which are the most important determinants of the activity.