Low Molecular Weight Inhibitors in Corneal Ulceration<sup>a</sup>

Galardy, Richard E.; Cassabonne, Marie E.; Giese, Carlanne; Gilbert, James H.; Lopez, Henry; Schaefer, Michele L.; Stack, Robert J.; Sullivan, Michael; Summers, Brent; Tressler, Rob; Tyrrell, Dave; Wee, Jennifer; Allen, Scott D.; Castellot, John J.; Barletta, John P.; Schultz, Gregory S.; Fernandez, Leonardo A.; Fisher, Susan M.; Cui, Tian‐Yi; Foellmer, Harald G.; Grobelny, Damian; Holleran, Walter M.

doi:10.1111/j.1749-6632.1994.tb24746.x

Cited by 152 publications

(116 citation statements)

References 5 publications

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“…The recombinant catalytic domain of MMP-9 was purified on gelatin-Sepharose, followed by ion exchange chromatography (29). The protease was activated with 2 mM APMA for 16 h at room temperature, and the active site was titrated with the hydroxamate inhibitor Ilomastat (29,30). Phage selections were performed with 2.5 g/ml (56 nM) active MMP-9.…”

Section: Resultsmentioning

confidence: 99%

“…Much of the effort in this area has focused on the design of small molecule antagonists with two primary features; 1) a hydroxamate moiety that binds to the proteases catalytic zinc, and 2) a rather large hydrophobic moiety that fits into the deep S 1Ј pocket present in all MMPs (30). This synthetic strategy has focused structure-activity studies to essentially two positions within the catalytic pocket.…”

Section: Discussionmentioning

confidence: 99%

“…The active site of MMP-9 was titrated using the hydroxamate inhibitor Ilomastat (30). Briefly, 25 nM MMP-9 was incubated with a range of Ilomastat.…”

Section: Methodsmentioning

confidence: 99%

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Substrate Hydrolysis by Matrix Metalloproteinase-9*

Kridel

Chen

Kotra

et al. 2001

Journal of Biological Chemistry

173

140

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The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P 3 through P 2 . This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P 3 and P 1 . This study shows that MMP-9 has a unique preference for Arg at both P 2 and P 1 , and a preference for Ser/Thr at P 2 . Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.Matrix metalloproteinase-9 (MMP-9) 1 is a member of the matrixin family of metallo-endopeptidases (1-3). MMP-9 is historically referred to as gelatinase B because of its ability to cleave gelatin, a denatured form of collagen, in vitro. Along with MMP-2, MMP-9 differs from other MMPs because it contains three fibronectin type II repeats that have high binding affinity for collagen. These repeats are thought to mediate the binding of MMP-2 and -9 to collagen (1, 2). This binding interaction brings the catalytic pocket of the MMP in proximity to collagen, thereby enhancing its rate of hydrolysis. Despite these well characterized biochemical interactions, it is now clear that MMP-9 is also able to cleave a number of other proteins and may have a rather wide range of physiologic substrates (4 -8).Much of our understanding of the biological function of MMP-9 comes from the study of mice lacking this gene. For example, MMP-9-deficient mice have impaired ossification of the skeletal growth plate, a defect that has been partially attributed to poor vascularization of developing bone (9). Studies on these mice also show that MMP-9 is essential for the recruitment of osteoclasts into developing bones (10). Other work indicates that MMP-9-deficient mice are resistant to dermal blistering in a bullous pemphigoid model, an effect that has been attributed to the inability of these mice to cleave the SERPIN ␣1-proteinase inhibitor (5). Finally, recent studies in the RIP1-Tag2 transgenic mouse model of multistage carcinogenesis indicate that MMP-9 is part of the angiogenic "switch" that is essential for tumor growth (11,12). Other reports suggests that MMP-9 may play a role in inflammation in the nervous system. MMP-9 is elevated in enceph...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Substrate Hydrolysis by Matrix Metalloproteinase-9*

Kridel

Chen

Kotra

et al. 2001

Journal of Biological Chemistry

173

140

View full text Add to dashboard Cite

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“…To ensure proper quantification of active proteases, their active sites were titrated with active site inhibitors. MMP-2 and -9 were titrated with GM6001 (Ilomastat) (65), and the AG3340 (66) was used to titrate the other MMPs.…”

Section: Methodsmentioning

confidence: 99%

Basis for substrate recognition and distinction by matrix metalloproteinases

Ratnikov¹,

Cieplak²,

Gramatikoff³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Genomic sequencing and structural genomics produced a vast amount of sequence and structural data, creating an opportunity for structure-function analysis in silico [Radivojac P, et al. (2013) Nat Methods 10(3):221-227]. Unfortunately, only a few large experimental datasets exist to serve as benchmarks for functionrelated predictions. Furthermore, currently there are no reliable means to predict the extent of functional similarity among proteins. Here, we quantify structure-function relationships among three phylogenetic branches of the matrix metalloproteinase (MMP) family by comparing their cleavage efficiencies toward an extended set of phage peptide substrates that were selected from ∼64 million peptide sequences (i.e., a large unbiased representation of substrate space). The observed second-order rate constants [k (obs) ] across the substrate space provide a distance measure of functional similarity among the MMPs. These functional distances directly correlate with MMP phylogenetic distance. There is also a remarkable and near-perfect correlation between the MMP substrate preference and sequence identity of 50-57 discontinuous residues surrounding the catalytic groove. We conclude that these residues represent the specificity-determining positions (SDPs) that allowed for the expansion of MMP proteolytic function during evolution. A transmutation of only a few selected SDPs proximal to the bound substrate peptide, and contributing the most to selectivity among the MMPs, is sufficient to enact a global change in the substrate preference of one MMP to that of another, indicating the potential for the rational and focused redesign of cleavage specificity in MMPs.protease | specificity-determining positions | MMPs A paramount objective of biological research is to understand how sequence encodes function. Previously, functional regions in proteins were identified using large-scale mutagenesis (e.g., alanine scanning) (1). More recently, our insights are largely gained by computational approaches aimed at comparing sequences and structures of large protein sets across multiple genomes. The vast increase in the number of available sequences makes it possible to compare homology between sequences from genome projects to proteins of known structure and function and, as a result, identify functional similarities in silico (2-4). Because the global fold of most ordered proteins can be reliably predicted (5, 6) and because the catalytic residues of most classes of enzymes are either known or can be inferred (7,8), protein sequences can now be directly used to elucidate and classify major protein functions (9-12), and are even being extended to predict enzyme substrates (13).However, such classifications fail to explain the specialization and expansion of function that is required for organismal plasticity, complexity, and adaptability, all of which are normally driven by gene duplications and subsequent divergence. Computational approaches aimed at identifying functional distinctions across protein families have pri...

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“…These MMPIs are not orally bioavailable but can be applied topically. In fact, one of the first MMPIs to go into clinical trials, GM6001, was used in topical treatment of corneal ulcers resulting from bacterial keratitis [61]. The second generation of MMPIs can be administered orally and show some increased specificity.…”

Section: Mmp Inhibitors As Therapeutics For Managing Asc and Pcomentioning

confidence: 99%

Matrix metalloproteinases as mediators of primary and secondary cataracts

West‐Mays

Pino

2007

Expert Review of Ophthalmology

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The matrix metalloproteinases (MMPs) are a family of endopeptidases involved in numerous remodeling and fibrotic disorders. Although MMPs have been shown to play important roles in regenerative and disease processes in many parts of the eye, including the cornea, retina and trabecular meshwork, the role of MMPs in the normal and cataractous lens has only recently been studied. These investigations have shown that multiple MMPs are expressed in the lens and their expression is altered in a number of cataract phenotypes. However, anterior subcapsular cataract and posterior capsular opacification, cataracts of a fibrotic nature, show a particular involvement of MMPs. This review will highlight recent findings that suggest a causative role for MMPs in these fibrotic cataract phenotypes. Keywords cataract; EMT; fibrosis; lens; MMPIs; matrix metalloproteinase Cataract is the leading cause of blindness worldwide despite the availability of effective surgery in the developed countries [1,2]. The etiology of cataract is diverse (see Allen [3] for review) with the majority of cataracts related to aging. Extracapsular cataract extraction provides quick restoration of vision and is the most frequently performed surgical procedure in the developed world, costing over US$3.5 billion each year in the USA alone [4]. However, it is not without its problems and can lead to complications such as the development of secondary cataract (up to 40-50% incidence of patients), also known as posterior capsular opacification (PCO) [5,6]. PCO is a major medical problem with profound consequences to the patient's well-being and is a significant financial burden. PCO is considered a proliferative, fibrotic disorder that involves the aberrant deposition of matrix and wrinkling of the lens capsule [7]. Another cataract type, also of a fibrotic nature, is anterior subcapsular cataract (ASC) [6,8,9]. ASC, unlike PCO, is a primary cataract that occurs when lens epithelial cells (LECs), in situ, are stimulated to transition into myofibroblasts. These fibroblasts form subcapsular plaques directly beneath the lens capsule. Despite these differences, recent evidence has shown that in both cataract phenotypes the matrix metalloproteinases (MMPs), matrix-degrading enzymes, †Author

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Low Molecular Weight Inhibitors in Corneal Ulceration^a

Cited by 152 publications

References 5 publications

Substrate Hydrolysis by Matrix Metalloproteinase-9*

Substrate Hydrolysis by Matrix Metalloproteinase-9*

Basis for substrate recognition and distinction by matrix metalloproteinases

Matrix metalloproteinases as mediators of primary and secondary cataracts

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Low Molecular Weight Inhibitors in Corneal Ulcerationa

Cited by 152 publications

References 5 publications

Substrate Hydrolysis by Matrix Metalloproteinase-9*

Substrate Hydrolysis by Matrix Metalloproteinase-9*

Basis for substrate recognition and distinction by matrix metalloproteinases

Matrix metalloproteinases as mediators of primary and secondary cataracts

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Product

Resources

About

Low Molecular Weight Inhibitors in Corneal Ulceration^a