Potent mechanism-based inhibitors for matrix metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (k(on) of 10(2) to 10(4) M(-1) s(-1) and the reversal of the process is slow (k(off) of 10(-3) to 10(-4) s(-1)). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.     

Normal mode analysis as a prerequisite for drug design: application to matrix metalloproteinases inhibitors

We demonstrate the utility of normal mode analysis in correctly predicting the binding modes of inhibitors in the active sites of matrix metalloproteinases (MMPs). We show the accuracy in predicting the positions of MMP-3 inhibitors is strongly dependent on which structure is used as the target, especially when it has been energy minimized. This dependency can be overcome by using intermediate structures generated along one of the normal modes previously calculated for a given target. These results may be of prime importance for further in silico drug discovery.     

Structural basis of matrix metalloproteinases and tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of secreted/cell-surface-anchored multidomain zinc endopeptidases, all of which exhibit a catalytic domain of a common metzincin-like topology, and which are involved in degradation of the extracellular matrix but also in a number of other biologic processes. Normally, the proteolytic activity of the MMPs is precisely regulated by their main endogenous protein inhibitors, in particular the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumor growth, and tumor metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and their associations with dysfunctions. Since the reports of the first atomic structures of MMPs and TIMPs in 1994, considerable structural information has become available about both of these families of substances. Many of the MMP structures have been determined as complexes with synthetic inhibitors, facilitating knowledge-based drug design. This review focuses on the currently available 3D structural information about MMPs and TIMPs.     

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

Characterization of plasma gelsolin as a substrate for matrix metalloproteinases

We previously showed that plasma gelsolin, a major component of the extracellular actin scavenging system, is an matrix metalloproteinase (MMP)-14 substrate. Here we confirmed that plasma gelsolin is cleaved by MMP-14 at the plasma level, and found that it was most efficiently digested by MMP-3 followed by MMP-2, MMP-1, MMP-14, and MMP-9, in that order. Plasma gelsolin (90 kDa) was cut into several fragments of 43-48 kDa by MMP-3. The MMP-3 cleavage sites in plasma gelsolin were determined by labeling the C termini generated by in-gel digestion with 50% H2 18O combined with peptide mass mapping, and sequencing of the N-terminal amino acids. Plasma gelsolin was cleaved at Asn416-Val417, Ser51-Met52, and Ala435-Gln436. Proteolytic cleavage by MMP-3 resulted in considerable loss of its actin filament-depolymerizing activity. This suggests that MMPs weaken the extracellular actin-scavenging system by cleaving plasma gelsolin and may, therefore, be involved in pathological conditions induced by extracellular actin, such as endothelial injury, respiratory distress syndrome, hepatic necrosis, and septic shock.     

Structural basis of the matrix metalloproteinases and their physiological inhibitors,the tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of multidomain zinc endopeptidases with a metzincin-like catalytic domain, which are involved in extracellular matrix degradation but also in a number of other important biological processes. Under healthy conditions, their proteolytic activity is precisely regulated by their main endogenous protein inhibitors, the tissue inhibitors of metalloproteinases. Disruption of this balance results in pathophysiological processes such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and for rational drug design. Since the first appearance of atomic MMP structures in 1994, a large amount of structural information has become available on the catalytic domains of MMPs and their substrate specificity, interaction with synthetic inhibitors and the TIMPs, the domain organization, and on complex formation with other proteins. This review will outline our current structural knowledge of the MMPs and the TIMPs.     

Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors

The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is largely responsible for the remodeling of tissues. Deregulation of this balance is a characteristic of extensive tissue degradation in certain degenerative diseases. To analyze the role of MMPs and TIMPs in tissue remodeling under normal and pathological conditions, it is important to have reliable detection methods. This review will focus on zymographical techniques for the analysis of MMPs and TIMPs. MMPs can be analyzed with several zymographical techniques, but substrate zymography is the most commonly used. This technique identifies MMPs by the degradation of their preferential substrate and by their molecular weight. Several substrates that can be used for zymography are described. Reverse zymography, which detects TIMPs by their ability to inhibit MMPs, is also discussed. Finally, in situ zymography is described, which is used to localize MMPs in tissue sections. Common problems encountered during sample preparation, zymography itself and the data analysis are discussed. Hints are given to improve the sensitivity and accuracy of zymographical methods. In conclusion, zymography is a valuable tool for research purposes and for the development of new diagnostic techniques and therapies for pathological conditions such as rheumatoid and osteoarthritis, and tumor progression.     

Identifying simultaneous matrix metalloproteinases/soluble epoxide hydrolase inhibitors

Molecular and Cellular Biochemistry - Matrix metalloproteinase (MMP) and soluble epoxide hydrolase (sEH) have completely unrelated biological functions; however, their dysregulation produce similar...     

Natural products as a screening resource

Natural products have been the most productive source of leads for new drugs, but they are currently out of fashion with the pharmaceutical industry. New approaches to sourcing novel compounds from untapped areas of biodiversity coupled with the technical advances in analytical techniques (such as microcoil NMR and linked LC-MS-NMR) have removed many of the difficulties in using natural products in screening campaigns. As the 'chemical space' occupied by natural products is both more varied and more drug-like than that of combinatorial chemical collections, synthetic and biosynthetic methods are being developed to produce screening libraries of natural product-like compounds. A renaissance of drug discovery inspired by natural products can be predicted.     

Heteroaryl and cycloalkyl sulfonamide hydroxamic acid inhibitors of matrix metalloproteinases

Heteroaryl and cycloalkyl sulfonamide-hydroxamic acid MMP inhibitors were investigated. Of these, the pyridyl analogue 2 is the most potent and selective inhibitor of MMP-9 and MMP-13 in vitro.     

Design and synthesis of thiol containing inhibitors of matrix metalloproteinases

A series of thiol containing derivatives was prepared. Several of these compounds were found to inhibit matrix metalloproteinases 1, 3, and 9 with selectivity towards 3 and 9. Compounds 15, 20, and 22 were administered to rats orally at 75 mumol/kg. Drug levels of compounds 20 and 22 in the plasma were found to exceed the IC50 values for MMP 3 and 9 four hours after administration.     

Macrocyclic hydroxamate inhibitors of matrix metalloproteinases and TNF-alpha production.

Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.     

Natural products as a hunting ground for combinatorial chemistry

Natural products have a long history of success as biologically active leads for therapeutic agents. The ability to prepare analogues and to discover structure-activity relationships is necessary to truly harness the potential of natural products. Recently, combinatorial chemistry has risen to this challenge, and even fairly complex natural products can be targeted for parallel synthesis. Academic and industrial efforts have employed natural products from the peptide, alkaloid, polyketide, and terpenoid and steroid classes in combinatorial chemistry approaches for the production of medicinally important compounds.     

Dynamic interdomain interactions contribute to the inhibition of matrix metalloproteinases by tissue inhibitors of metalloproteinases

Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.     

The molecular biology of matrix metalloproteinases and tissue inhibitors of metalloproteinases in inflammatory bowel diseases

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be predominant proteases and protease inhibitors involved in the pathogenesis of inflammatory bowel diseases (IBD) through their ability to remodel the extracellular matrix (ECM) in response to inflammatory stimuli and by their immunomodulating effects. An imbalance between MMPs and TIMPs has been linked with acute and chronic inflammation and aberrant tissue remodeling, as seen in IBD. Moreover, recurrent phases of tissue destruction and subsequent tissue repair can cause serious complications in IBD patients such as fistulas and fibrosis. The aims of this review are (i) to summarize current literature on genetic association, mRNA, and protein expression studies with regard to MMPs and TIMPs in IBD patients and various animal models, including those with transgenic and knockout mice; (ii) to compare biochemical and molecular biological data in humans with those obtained in animal model studies and (iii) to critically evaluate and translate how this knowledge may be used in practical terms to understand better the pathophysiology and mechanisms operating in IBD and to apply this for improvement of clinical outcomes at diagnostic, prognostic and therapeutic levels.     

Implications for matrix metalloproteinases as modulators of pediatric lung disease

Matrix metalloproteinases (MMPs) are a large family (>20) of cation-dependent proteinases believed to be important modulators of normal human lung development and potentially harmful mediators of lung damage. Little is known about MMP production and secretion by the lung during childhood or how alterations in MMP levels may be involved in lung damage. We examined endotracheal aspirates from children (<19 years) without lung disease for the presence of MMP activity. Only gelatinase activity was detectable, and inhibitor profiles suggest they represented one or more MMPs. Comparison of gelatinase activity, MMP expression, and MMP activity in children without pulmonary disease with children who required mechanical ventilation for respiratory failure show: 1) gelatinase activity was approximately five- to sixfold higher in respiratory failure; 2) MMP-7, MMP-8, and MMP-9 concentrations and MMP-8 and MMP-9 activities were markedly elevated in respiratory failure; and 3) MMP-7, MMP-8, and MMP-9 levels were significantly correlated in children with lung disease. These studies provide compelling evidence that specific MMPs are present in the diseased lung and may participate in the pathogenesis of pediatric respiratory failure.     

Expression of bone morphogenetic proteins, matrix metalloproteinases and inhibitors of matrix metalloproteinases in lung cancers and their prognostic significance

Matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) are one of the molecules that have become a topic of great interest among scientists studying lung cancers. There is a distinct tendency toward higher expression of selected MMP and TIMP in tumor lung tissue. Furthermore, there is a significant correlation between high expression of TIMP-1 or MMP-2 in lung cancer and shortened survival and between high expression of TIMP-1 or MMP-7 in lung cancer and higher stage of disease. There have been only a few articles about the role of bone morphogenetic proteins (BMP) in lung cancer pathogenesis published so far in which BMP-2 or BMP-4 were overexpressed. It was also shown that BMP-2 stimulates tumor growth while BMP-4 inhibits it. This article is mainly concentrated on the expression of MMP, TIMP and BMP in lung cancers, but also it shows the significance of these proteins.     

Highly water-soluble matrix metalloproteinases inhibitors and their effects in a rat adjuvant-induced arthritis model

A new series of succinate-based dual inhibitors against matrix metalloproteinases (MMPs) and tumor necrosis factor alpha converting enzyme (TACE) possessing highly-water solubility was designed, synthesized, and evaluated for enzyme inhibition. Incorporating of acidic or basic functional groups at the P(2)' position afforded sufficient water solubility without significant loss of inhibitory potencies. Compound 18e, which had a guanidino group at the P(2)' position as the basic functional group, exhibited broad inhibition against target enzymes for a relatively long period in rat plasma (beta t(1/2); 2.0h) after sc administration when compared with compounds possessing acidic functional groups (18a and 18b). Consequently, the representative compound 18e together with compound 18b, Marimastat and Trocade were evaluated in the rat adjuvant-induced arthritis model, a model of chronic cartilage destruction. It is concluded that the newly synthesized highly water-soluble compound 18e showed significant activity in suppressing hindpaw swelling and the bone destruction with a minimal administration period (days 3-7).