Dual inhibition of phosphodiesterase 4 and matrix metalloproteinases by an (arylsulfonyl)hydroxamic acid template.
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3',5'-Cyclic-AMP Phosphodiesterases↗
Biomedical subjects
G M McGeehan
Publications and source records attributed to G M McGeehan.
Method to analyze collagenase and gelatinase activity by fibroblasts in culture.
The net amount of collagen produced and deposited by fibroblasts in cell culture is determined by the rate of collagen synthesis as well as the rate of collagen degradation. Although collagen synthesis can be analyzed by several techniques, it is more difficult to measure collagen degradation. Breakdown of collagen depends upon the activity of a family of structurally and catalytically related mammalian enzymes termed matrix metalloproteinases (MMPs). Interstitial collagenase (MMP1) initiates the cleavage of fibrillar collagen, whereas gelatinases (MMP2 and MMP9) digest the denatured collagen fragments. A method has been developed to quantitate the activity of collagenase (MMP1) and gelatinase (MMP9) in conditioned medium from fibroblast cell cultures. The assay, which uses the fluorogenic substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-AlaLys(Nma)NH2, is technically simple and amenable to high throughput analysis. Addition of specific inhibitors of the metalloproteinases allows for simultaneous measurement of both collagenase and gelatinase activity.
Animals↗
Effect of matrix metalloproteinase inhibitors on tumor growth and spontaneous metastasis.
Four potent, synthetic inhibitors of matrix metalloproteinases (MMPs) were assessed as inhibitors of tumor growth and spontaneous metastasis to the lung. Mat Ly Lu rat prostate tumor, LOX human melanoma and M27 murine Lewis lung tumor were implanted subcutaneously (s.c.) in mice and allowed to grow for 3-12 days. The lungs of the tumor-bearing mice were then removed and implanted s.c. into untreated mice, and the outgrowth of secondary tumors from the implanted lungs measured. The incidence and rate of outgrowth of secondary tumors increased with the length of primary tumor growth, validating these measurements as indices of spontaneous metastasis to the lung. Compounds were tested by s.c. implantation of minipumps which delivered compound throughout the period of primary tumor growth and spontaneous metastasis to the lung at steady-state drug concentrations orders of magnitude greater than the concentrations needed to either inhibit collagenase, gelatinase or stromelysin in vitro. Inhibitor treatment slowed the growth of primary s.c. Mat Ly Lu and LOX tumors by 40-60% but had no significant effect on the growth of primary M27 tumors. Surprisingly, inhibitor treatment had no significant effect on the ability of the lung to generate secondary tumors when reimplanted s.c. in untreated mice. Because of the possible importance of cathepsins B, H and L in tumor growth and metastasis, the irreversible inhibitor E-64 was also infused by s.c. minipump. E-64 had no effect on the growth or spontaneous metastasis of Mat Ly Lu or M27 tumors.
Animals↗
Defined substrate mixtures for mapping of proteinase specificities.
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Amino Acid Sequence↗
Monoclonal antibodies against human collagenase and stromelysin.
Mouse monoclonal antibodies against recombinant human fibroblast procollagenase and prostromelysin have been generated and characterized. The epitope-containing domains for the antibodies have been assigned based on their immunoreactivities against recombinant proenzymes, mature enzymes, truncated collagenases, proteolytic fragments of stromelysin, and chimeric molecules constructed from different domains of the two enzymes. These antibodies can be divided into four groups: (1) antibodies that recognize the truncated 19-kDa NH2-terminal collagenase, (2) antibodies that recognize the C-terminal domain of collagenase and stromelysin, (3) antibodies that recognize a 31-kDa NH2-terminal collagenase fragment, and (4) antibodies that recognize the 19-kDa NH2-fragment of stromelysin. The prostromelysin-specific antibody 11N13 is of particular interest; it neutralizes stromelysin activity in a stromelysin peptide substrate assay, with an IC50 value of 75 nM. MAb 11N13 may be useful for in vivo and in vitro studies to validate the roles of stromelysin in tumor cell invasion, metastasis, and connective tissue disorders.
Amino Acid Sequence↗
Characterization of the peptide substrate specificities of interstitial collagenase and 92-kDa gelatinase. Implications for substrate optimization.
The peptide substrate specificities of two matrix metalloproteinases (MMPs), interstitial collagenase (MMP-1), and 92-kDa gelatinase (MMP-9), have been examined. Starting with the parent substrate, Dnp-Pro-Leu-Gly approximately Leu-Trp-Ala-D-Arg-NH2, four separate substrate mixtures were synthesized at subsites P2(Leu) through P2'(Trp). These mixtures contained either naturally occurring L-amino acids, D-amino acids, or either of two distinct sets of miscellaneous amino acids. Combined, these mixtures gave 88 unique substitutions at each position and, over the four subsites, represented 352 potential substrates. Optimal substrates were identified using a combined high performance liquid chromatography/mass spectrometry analysis as previously reported. The results gave an extended profile of the substrate specificities for both MMP-1 and MMP-9 at subsites P2(Leu) through P2'(Trp). Using the data obtained from the mapping, a new peptide substrate, Dnp-Pro-Cha-Abu approximately Smc-His-Ala-D-Arg-NH2 (where Dnp is 2,4-dinitrophenyl, Cha is cyclohexylalanine, Abu is alpha-aminobutyric acid, and Smc is S-methylcysteine) was designed and characterized. This peptide showed a 36-fold improvement in turnover (kcat/Km) versus the parent substrate by interstitial collagenase. In addition, some collagenase subsite specificities described here were found to be different from those previously reported. Experimental data show that the observed selectivity is dependent on the original peptide template employed, which has broader implications for substrate specificity studies.
Amino Acid Sequence↗
A high throughput fluorogenic substrate for stromelysin (MMP-3).
Stromelysin, a member of the matrix metalloproteinase family of enzymes, has been implicated in the pathogenesis of tumor metastasis and inflammatory diseases such as rheumatoid arthritis. To screen prospective inhibitors of this protease, we developed a fluorogenic substrate with excitation and emission spectra compatible with commercially available 96-well plate readers. The substrate is based on the addition of 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] hexanoic acid (NBD) (EX467/EM534) and 7-dimethylaminocoumarin-4-acetate (DMC) (EX368/EM459) to the previously reported peptide substrate for stromelysin, Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-NH2. The new substrate, NBD-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-Lys-(DMC)-NH2 is 95% quenched and the fluorescent product, Nva-Trp-Lys(DMC)-NH2 is easily detected (EX350/EM465). In competition assays the new fluorogenic substrate has a relative kcat/Km that is one half that of the parent peptide. The fluorophores NBD and DMC were chosen based on the high fluorescence yield of DMC and the overlap of the emission spectrum of DMC and excitation spectrum of NBD which results in an efficient energy transfer system in the intact substrate. These characteristics make this an excellent substrate for routine determination of in vitro activities of stromelysin inhibitors.
Amino Acid Sequence↗
Regulation of tumour necrosis factor-alpha processing by a metalloproteinase inhibitor.
Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.
Animals↗
Isotope effects and alternative substrate reactivities for tryptophan 2,3-dioxygenase.
Tryptophan 2,3-dioxygenase (EC 1.13.1.12) is a hemoprotein which catalyzes the first step in the oxidative degradation of tryptophan. The reaction is believed to proceed by addition of O2 across the 2,3-bond of the indole ring, followed by decomposition of the resultant dioxetane to give N-formylkynurenine. A primary D2O isotope effect of 4.4 on Vmax/Km was observed at the pH optimum, pH 7.0. This implies that abstraction of the indole proton is at least partially rate-determining. An inverse secondary isotope effect of 0.96 was observed for L-[2-3H]tryptophan at this pH. The secondary isotope effect signals the formation of the C-O bond at C-2. As the rate of proton abstraction increased with increasing pH, the D2O isotope effect decreased to 1.2 at pH 8.5 and the secondary isotope effect increased to 0.92. The rate-determining steps therefore change with increasing pH, and bond formation at C-2 becomes more rate-limiting. The secondary isotope effect did not change significantly with varying O2 concentration so that substrate binding is primarily ordered with O2 binding first. The specificity of the enzyme towards substituted tryptophans shows that substitution of the phenyl ring of the indole is sterically unfavorable. Steric hindrance is highest at the 4- and 7-positions, while the 5- and 6-positions are less sensitive. 6-Fluoro-L-tryptophan was more reactive than tryptophan, and the increased reactivity can be explained by an electronic effect that enhances of the rate of C-O bond formation at C-2.
Animals↗
A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9).
Two members of the matrix metalloproteinase family of enzymes, interstitial collagenase and 92-kDa gelatinase, have been implicated in the pathogenesis of rheumatoid arthritis and tumor metastasis. In order to characterize the activities of these enzymes, we have developed a fluorogenic peptide substrate which is efficiently hydrolyzed by both enzymes. This substrate was developed based on the addition of the fluorescent tag, N-methyl-anthranilic acid (Nma), to several previously synthesized substrates that had been evaluated with respect to their turnover by interstitial collagenase. One substrate, Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys-(Nma)-NH2, had favorable solubility characteristics, was > 98% quenched, and produced a single cleavage product, Dnp-Pro-Cha-Gly, with a high fluorescence yield with both interstitial collagenase and 92-kDa gelatinase. Since the assay depends on measurement of increases in fluorescence, the position of the Nma group also proved to be important for optimization of the fluorescence signal. The assay is free from interference by organomercurial compounds and the cleavage product has excitation and emission spectra compatible with filters commonly available on commercial plate readers. The assay has been adapted to a 96-well format and provides a rapid screening protocol for the evaluation of inhibitors of these enzymes.
Amino Acid Sequence↗
The ribonuclease from an extinct bovid ruminant.
The sequence of the ribonuclease from the ancestor of swamp buffalo, river buffalo, and ox, corresponding approximately to Pachyportax latidens, an extinct ruminant known from the fossil record, has been reconstructed using the rule of 'maximum parsimony'. This protein and two sequences that may have been intermediates in the evolution of modern ribonuclease have been constructed in the laboratory by site-directed mutagenesis, and their properties examined.
Amino Acid Sequence↗
An improved system for expressing pancreatic ribonuclease in Escherichia coli.
An improved method for expressing and purifying bovine pancreatic ribonuclease from a synthetic gene using the lambda promoter controlled by a temperature-sensitive repressor is described. The procedure involves isolation in the presence of a refolding buffer containing oxidized and reduced glutathione, under conditions where RNase can refold, but where proteases presumably do not. Yields are approx. 2 mg purified protein per 1 ferment.
Bacteriophage lambda↗