It traces back to the discovery of a protease from metamorphosed tadpole about 60 years ago by Jerome Gross, which eventually opened a new field of metalloprotease biochemistry (Gross and Lapiere 1962). The protease discovered then was an interstitial collagenase, also known as matrix metalloproteinase-1 (MMP-1) that involved in extracellular matrix (ECM) degradation. The complete protease repertoire of human accounting ∼2% of whole genome holds a great treasure of information on human health and diseases. All proteases are classified into five classes, namely, serine, threonine, cysteine, aspartic, and metalloproteinase proteases. Metalloprotease encompasses metzincin superfamily, which is further divided into five groups, e.g., astacin, serralysin, adamlysin, pappalysin, and matrixins (Lopez-Otin and Matrisian 2007). MMPs (or matrixins) are calcium-dependent zinc containing endopeptidases and members of metzincin consisting of family of enzymes sharing common domain structures. Now, 24 different MMPs are reported from human, and they perform important roles in multiple physiological processes including embryonic development, mammary morphogenesis, and uterine remodeling and in different disease pathogenesis (Page-McCaw et al. 2007). MMPs readily degrade most of the ECM proteins including collagen, gelatin, laminin, fibronectin, as well as some non-ECM proteins, e.g., ligands and cell surface receptor. Thus, cellular events such as apoptosis, proliferation, and differentiation have been associated with MMP-mediated functions (Egeblad and Werb 2002). Majority of the MMPs are secreted as latent pro-form and activated by removal of the prodomain, while few MMPs including membrane-bound MMPs are activated by intercellular processing by furins. The latency of proMMPs is maintained through interaction of cysteine of the prodomain with zinc ion of the catalytic domain, known as “cysteine switch”; activation occurs by disruption of the cysteine-zinc interaction (Nagase et al. 2006). Catalysis is additionally influenced by transcriptional, posttranscriptional, and epigenetic regulation of MMPs. Active MMPs then modulate proteolytic environment in the particulate niche or globally through ECM degradation. Since MMPs are involved in different disease pathogenesis, attempts were made in last two decades to inhibit MMP expressions at different regulatory points. Herein, we review the evidences of MMP responses in inflammation, chronic wounds, arthritis, cancer, cardiovascular disease, periodontitis, etc. (Swarnakar et al. 2011). We present also the inhibition of particular MMP by small molecules that target specific pathway in disease development.
Structure and Function of Matrix Metalloproteinases
MMPs degrade different ECM substrates including collagen, gelatin, fibronectin, laminin, elastin, fibrin, aggrecan, etc. and are directly involved in regulating cellular migration and invasion (Egeblad and Werb 2002). The degradation of non-ECM proteins is associated with different cellular responses, such as apoptosis, proliferation, angiogenesis, immune responses, etc. (Khokha et al. 2013). MMPs promote proliferation by releasing growth factors from its bound form, for example, release of insulin-like growth factor from insulin-like growth factor-binding protein (IGF-BP) or soluble transforming growth factor (TGF)-α from its bound TGF-α form, respectively. MMP-1 and MMP-3 degrade perlecan to release bound fibroblast growth factor (FGF). MMPs modulate inflammatory responses by cleaving tumor necrosis factor (TNF)-α from its precursor molecule (Page-McCaw et al. 2007). ECM degradation by MMPs generates small peptides that act as anti-angiogenesis molecules. For example, endostatin is generated via cleavage of type XVIII collagen by MMPs. Proteolytic cleavage of collagen IVα3 and plasminogen by MMPs generate tumstatin and angiostatin, respectively, that inhibit angiogenesis (Egeblad and Werb 2002). On the contrary, MMPs also promote angiogenesis by elevating soluble levels of proangiogenic growth factors. For example, MMP-9 triggers angiogenic switch in cancer by releasing vascular endothelial growth factor (VEGF) from angiogenic islets (Bergers et al. 2000). MMP-2 regulates the bioavailability of soluble VEGF-A from its bound form and thus regulates vascular patterning in tumors. MMP-9 proteolytically activates TGF-β and promotes tumor invasion and angiogenesis as well as suppresses T-cell responses against cancer cells (Overall and Lopez-Otin 2002). Cleavage of Fas ligand by MMP-7 alters apoptotic responses. MMP also promotes cellular invasion by cleaving cell-cell adjunct proteins, like E-cadherin or cell matrix junctional proteins (Egeblad and Werb 2002). MMPs are involved in epithelial to mesenchymal transition in cancer cells and promote cancer metastasis. MMPs are also involved in modulating cancer immune responses. For example, MMPs inhibit T lymphocyte proliferation by cleaving interleukin-2 receptor-α on T lymphocytes (Egeblad and Werb 2002; Khokha et al. 2013).
Matrix Metalloproteinase and Diseases
Physiological abnormalities associated with specific MMP knockout mice
Null mutant phenotype in mice
Defects mammary gland morphogenesis
Structural changes in neuromuscular junctions
Altered secondary mammary branching morphogenesis
Innate immunity defects
Reduced re-epithelialization in trachea
Increased skin tumors
Defects in bone development
Impaired vascular remodeling and angiogenesis
Altered inflammatory responses
Delayed myelination in corpus callosum
Bone remodeling defects
Defects in skeletal remodeling
Retarded lung alveolar development
Angiogenesis defects and die after few days of birth
Defects in amelogenesis
Decreased nerve fiber sprouting and neural invasion
Altered inflammatory response
Ulcer and Wound
MMPs play key role in ECM remodeling during wound development and resolution. Moreover, MMPs are associated with extravasation of leukocytes out of the blood into the injured tissue. Epithelial and stromal cells of the wounded tissues also express multiple MMPs including MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and MMP-28 (Swarnakar et al. 2011). Increased levels of MMP-9 and MMP-3 activities are reported to be elevated with gastric ulcer, whereas MMP-2 level decreases during pathogenesis (Swarnakar et al. 2005). MMP-3-deficient mice show reduced contraction and delayed wound healing. During wound healing, increased MMP-2 plays the key role for promoting angiogenesis and cellular remodeling (Sharma et al. 2012).
Rheumatoid arthritis is an inflammatory condition of joints where articular destruction leads to joint function disability. This inflammatory condition is associated with elevated MMP-1, MMP-3, MMP-13, and MMP-14 responses (Burrage et al. 2006). MMP-1 and MMP-3 exert strong collagenolytic activities and are destructively involved in degradation of cartilage collagens such as types II, IX, X, etc. (Page-McCaw et al. 2007). Loss of MMP-13 function results in bone formation and remodeling, whereas loss of MMP-14 is reported to develop arthritis. Mutation of MMP-2 gene is recently found to be associated with human inherited osteolysis disease or “vanishing bone” syndrome (Page-McCaw et al. 2007). Furthermore, MMP-19 is expressed on the surface of activated peripheral blood mononuclear cells and is detected as an autoantigen in rheumatoid arthritis (Burrage et al. 2006).
Endometriosis is an invasive gynecological disorder with development of uterine-like structure outside uterus. The endometriotic tissues undergo hormone-dependent periodic remodeling and thus are associated with elevated MMP responses. MMP-2, MMP-3, MMP-9, and MMP-14 are found elevated in endometriosis (Swarnakar et al. 2011). MMP-9 is associated with inflammatory responses, whereas MMP-3 is reported to modulate apoptotic responses (Jana et al. 2012). MMP-2 acts as proangiogenic responses in endometriosis and is regulated through prostaglandin-dependent manner in endothelial cells. Inhibition of total or specific MMP responses was found to regress endometriosis in animal models (Jana et al. 2016).
MMPs are associated with different cancers as well as tumor invasion and metastasis (Egeblad and Werb 2002). There are almost no reports for genetic alterations like mutations in MMPs for cancer development; the only genetic alteration is reported in the gene of MMP-23 (translocation) in neuroblastoma (Gururajan et al. 1998). Thus, the elevated MMP responses in cancer result from transcriptional and proteolytic activations. In cancer progression MMPs are involved in increased cancer cell migration, invasion, and metastasis through proteolytic cleavage of different ECM and basement molecules (Egeblad and Werb 2002). MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13 are well reported in different cancers. Cancer cells as well as inflammatory cells, including macrophages and neutrophils, are well-known resources for MMPs. Genetically modified mice with overexpressing MMP-3 and MMP-14 in mammary glands spontaneously develop breast cancer. Overexpression of MMP-1 and MMP-7 results in increased cancer proliferation and susceptibility in mice. On the other hand, MMP-2, MMP-7, MMP-9, or MMP-11 null mice develop fewer tumors than wild-type mice (Page-McCaw et al. 2007). Apart from the ECM degradation, MMP affects cancer progression by means of modulating angiogenesis, proliferation, apoptosis, immune responses, etc. by degrading non-ECM molecules (Kessenbrock et al. 2010) (described in MMP function).
Several neuronal diseases are associated with elevated MMP responses, although whether MMP is the direct cause for the disease manifestation is still unknown. Neurodegenerative diseases are associated with elevated MMPs responses (Mukherjee and Swarnakar 2015). Alzheimer’s disease develops due to accumulation of β-amyloid plaques in the hippocampus and cerebral cortex and results in neuronal deaths. MMP-1, MMP-3, MMP-9, and MMP-14 are reported to be associated with Alzheimer’s disease. Moreover, MMP-9 is found to cleave β-amyloid plaques, and therefore, inhibition of MMP-9 improves β-amyloid-mediated cognitive impairment and neurotoxicity in mice (Brkic et al. 2015). Another neurodegenerative disease, Parkinson’s disease, is associated with elevated responses for MMP-1, MMP-2, MMP-3, MMP-9, MMP-14, etc. MMPs cleave α-synuclein and promote aggregate formation in dopaminergic neuronal cells. MMP-9 and MMP-3 are involved with inflammatory responses and subsequent apoptotic responses. MMP-3-deficient mice showed reduced MPTP-induced degeneration of nigrostriatal dopaminergic neurons in the brain (Brkic et al. 2015). Huntington’s disease results from production of mutant huntingtin (mHTT) protein which causes damage and death to neuronal cells in the dorsal striatum which results in difficulties motor functions. Huntington’s disease is associated with elevated MMP-9, MMP-10, MMP-14, and MMP-23 responses. MMP-10 is reported to cleave HTT proteins; knocking down of MMP-10 reduces mHTT-mediated toxicity to striatal cells. Among other neuronal diseases, multiple and amyotrophic lateral sclerosis show elevated MMP-2, MMP-3, and MMP-9 levels. Elevated MMP-9 is involved in cerebral ischemia, and MMP-9 knockout mice show protection against blood–brain barrier disruption and ischemic injury (Brkic et al. 2015).
Elevated MMP responses are reported with several cardiovascular diseases, including atherosclerosis, hypertension, myocardial infarction, and heart failure. Inflammation and associated increase in MMP activities are reported in plaque-forming events of atherosclerosis (Hu et al. 2007). Mice having ApoE−/− and MMP-3−/− together showed increased lesions in the thoracic artery than only ApoE−/− mice. On the other hand, MMP-9 knockout mice showed decrease in intimal hyperplasia, lumen loss, and increased collagen deposition. MMP-13 deletion promotes collagen accumulation and organization in mouse atherosclerotic plaques (Hu et al. 2007). In experimental hypertension model, MMP-9 knockout mice showed vessel stiffness and increased pulse pressure. MMP-2-deficient mice exhibit improved survival rate and attenuated left ventricular enlargement and cardiac rupture in myocardial infarction. During heart failure, MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and MMP-14 responses were upregulated and correlated with the intensity of fibrosis and hypertrophy.
Several pulmonary diseases are reported with elevated MMP responses, including asthma, chronic obstructive pulmonary disorder (COPD), pulmonary fibrosis, etc. (Hu et al. 2007). In COPD patients, MMP-1, MMP-2, MMP-8, and MMP-9 are found upregulated in bronchoalveolar lavage (BAL) fluids (Churg et al. 2012). MMP-12 plays a critical role in tissue degradation during emphysema; MMP-12 knockout mice are resistant to smoke-induced emphysema. MMP-12 is secreted by pulmonary epithelial and macrophage cells and found to be elevated in COPD patients. Airway macrophages overexpress MMP-9 upon smoking and inflammatory responses, whereas for bronchial asthma, infiltrating neutrophils and eosinophils are the major source for MMP-9 responses. MMP-8 plays protective role in asthma, as MMP-8 deficiency promotes granulocytic allergen-induced airway inflammation (Churg et al. 2012). Pulmonary fibrosis develops from loss of lung epithelial cells, which are replaced by myofibroblasts and deposit abnormal amount of ECM proteins into lung interstitium. Elevated MMP-9 activity is severely associated with the pathogenesis of pulmonary fibrosis and promotes myofibroblast invasion. Moreover, MMP-7 is found to be overexpressed in microarray gene analysis of patients with idiopathic pulmonary fibrosis, and MMP-7 deficiency protects from pulmonary fibrosis (Swarnakar et al. 2011).
Several other diseases are associated with MMP responses, including hepatic diseases, diabetes, periodontitis, etc. Hepatic cirrhosis and fibrosis are associated with elevated MMP-2, MMP-9, and MMP-13 responses; MMPs are also involved with activation of hepatic stellate cells. In TNF-mediated hepatitis models, MMP-2-, MMP-3-, MMP-8-, or MMP-9-deficient mice exhibit lower levels of apoptosis and necrosis of hepatocytes and show better survival. In diabetic mice, increased blood glucose levels are found associated with upregulated MMP-2, MMP-9, and MMP-14 levels. MT5MMP is reported to be elevated in kidney of diabetes patients (Hu et al. 2007; Swarnakar et al. 2011).
Matrix Metalloproteinase and Small Molecule Interactions
Structure–activity relationship (SAR) analysis is utilized to design MMP inhibitors for understanding the potency of interaction between MMP structure and small molecule. It also allows to recognize required modification for improving the potency of a drug by altering the chemical structure. Recent studies are utilizing quantitative SAR (QSPR) to predict the biological activity of new and untested compounds against MMP from the knowledge of their molecular structures.
The critical point for cancer progression is the invasion and metastasis of the cancer stem cells, and MMPs play the grieving role of ECM degradation and basement membrane cleavage. Therefore, inhibiting MMPs was considered as a key factor for regulation of cancer progression. In last few decades, several small molecules were targeted against MMPs for its antitumor and antimetastatic effects. MMP responses can be targeted therapeutically at several steps, including transcription and protein synthesis, and at levels of secretion, intracellular trafficking, subcellular or extracellular localization, pro-zymogen activation and activity inhibition, etc. Majority of the small molecules or drugs are designed targeting at the steps of transcription or activation or direct inhibition of activity (Overall and Lopez-Otin 2002).
Since MMP is induced by different growth and inflammatory factors in different diseases, hence, inhibiting the stimulatory signal for MMP induction was studied in preventing different diseases. Treatment with monoclonal antibodies for TNF-α or soluble forms of TNF receptors found to be effective in reducing MMP production in rheumatoid arthritis. Blockage of the TGF-β pathway using TGF receptor antagonists regresses gelatinase activities in mouse model of breast carcinoma. Inhibition of MAP kinase signaling pathways with specific small molecular inhibitors blocks several MMP productions. RAS signaling pathway inhibitors, for example, malolactomycin D, manumycin A etc., are found to reduce MMP-1, MMP-2 and MMP-9 transcriptions along with cellular transformation. Inhibition of nuclear factors that attribute to MMP gene transcription including AP-1 and NF-κB also regresses MMP responses (Overall and Lopez-Otin 2002).
Furin sequence plays an important role in activation of several MMPs; thus small molecular inhibitors were designed targeting furin recognition site. One furin inhibitor, α1 PDX, inhibited activation of MT1MMP and subsequently reduced MMP-2 processing along with preventing the tumor growth and invasiveness. Anti-angiogenesis molecules like thrombospondin-1 and endostatin are also found to inhibit MMP-2 activation. Proteoglycans, such as testican-3 and its splice-variant gene product N-Tes, are also reported to suppress proMMP-2 activation (Overall and Lopez-Otin 2002).
Initially, MMP inhibitors were designed as small peptides based on the mimics of collagen amino acid sequences. Later, with the discovery of new MMPs, the focus shifted toward small synthetic molecules targeting specific MMPs in different diseases, including cancer. The first-generation MMP inhibitors were designed based on the hydroxamate-based inhibitors to chelate the enzyme. The substrate-binding groove for MMP opens at S3-S1 and S3′ pocket and narrows at the position of S1′, S2′ developing a well-defined S1′ pocket with the presence of structural zinc and two calcium ions. Interactions between MMP and small inhibitors take place in S1′ subunit and P1′ residue. Hydroxamate acts as a ligand to MMPs and binds to the catalytic Zn2+ ion of the enzyme developing a distorted trigonal–bipyramidal geometry around the Zn2+ ion. The –NH group of the hydroxamate forms a hydrogen bond with the adjacent carbonyl oxygen, and hydrophobic contacts support to stabilize the inhibitor–enzyme complex (Hu et al. 2007). Such inhibitors are marimastat, ilomastat (also known as GM6001), and batimastat. Marimastat and batimastat were the first among few MMP inhibitors that went through clinical trials. Although these inhibitors improved the overall survival of cancer patients, the affectivity was associated with side effects, such as musculoskeletal toxicity (Overall and Lopez-Otin 2002). New-generation hydroxamate-based MMP inhibitors are equipped with aryl, sulfonamide groups along with hydroxamate zinc-binding group. MMI-270 is water soluble and orally available and acts as a broad-spectrum MMP inhibitor. Since the S1′ pocket in part formed the selectivity loop which is of various lengths for different MMPs, the differences in S1′ pocket were utilized to develop specific or narrow-range MMP inhibitors. Selective MMP inhibitory molecules include MMI-166 that inhibits MMP-2, MMP-9, and MMP-14. Prinomastat and ABT-770 inhibitors were designed in such a way that it can avoid “shallow pocket” binding for MMP-1, which will reduce the musculoskeletal toxicity. Cipemastat is an inhibitor for MMP-1, MMP-3, and MMP-9 and clinically trialed against rheumatoid and osteoarthritis (Hu et al. 2007).
Several other non-hydroxamate MMP inhibitors are present which includes carboxylates, sulfhydryls, phosphoric acid derivatives, hydantoins, etc. Rebimastat and tanimastat are zinc-binding group and are broad-spectrum MMP inhibitors. SB-3CT is a thiol-based specific inhibitor for MMP-2 and MMP-9. Ro 28–2653 is a pyrimidine-based inhibitor specific to MMP-2, MMP-9, and MT1MMP. Hydantoin is an alternative zinc-binding drug specific to MMP-13. Phosphorous-based MMP inhibitors mimic the tetrahedral transition state of amide hydrolysis, where the phosphinic group is the zinc-binding group. Phosphorous-based MMP inhibitors are 582311-81-7 for MMP13 and RXP-03 for MMP-11. Tetracycline antibiotics (minocycline and doxycycline) also exhibit innate MMP inhibitory activity and approved by FDA for clinical practice (Hu et al. 2007; Mishra et al. 2011).
The spectrum of diverse functional roles of MMPs in physiological and pathological responses that resulted in disease outcome is revealed from various reports (Page-McCaw et al. 2007). However, understanding the underlying mechanism of discordant action of MMPs to specific tissue site during disease development remains the major challenging task. In addition, MMPs is a growing family of proteases, and structure–activity relation is a dynamic field of research. By the advent of newer modalities of assaying, particular MMP could be exploited as independent prognostic marker and as drug target for particular disease. Moreover, MMPs play key role in controlling several signaling pathways through proteolytic cleavage of ECM and non-ECM proteins. Previously, the major function of MMPs was believed to be restricted to metastasis owing to their degrading action on basement membrane and ECM. However, recent advances on MMPs showed multitude of activities that are far more vast and complex. MMPs are now implicated from bone formation and angiogenesis to inducer of neurodegenerative diseases. Broad-spectrum MMP inhibitors as well as selective MMP inhibitor having different modes of actions are coming up to mitigate harms caused by dysregulation of MMP activity. In addition, the new concept in stability of functional conformation of MMPs encompasses the cellular events affecting disease evolution. However, the major drawback for MMP inhibitors in clinical trial comes from the lack of specificity for particular MMP and insufficiency in knowledge about complexity of MMP function. Thus, the challenge of scientific community is to find new approaches which will not only inhibit targeted MMPs but also the compensating ability of other MMPs. Stabilizing the MMPs in inactive pro-form through small molecule for particular time span might be an alternative approach for disease management. It is possible that prognostication of a patient with the help of specific MMP level allows clinician to evaluate high- and low-risk groups and more likely to succeed overall survival of patients.