Design and synthesis of orally active inhibitors of TNF synthesis as anti-rheumatoid arthritis drugs.

A novel series of TNF inhibitors was identified based on the screening of existing MMP inhibitor libraries. Further SAR optimization led to the discovery of a novel lead compound. Its synthesis, efficacy in experimental animal models, and pharmacokinetic data are discussed.

Novel inhibitors of procollagen C-proteinase. Part 2: glutamic acid hydroxamates.

Glutamic acid derived hydroxamates were identified as potent and selective inhibitors of procollagen C-proteinase, an essential enzyme for the processing of procollagens to fibrillar collagens. Such compounds have potential therapeutic application in the treatment of fibrosis.

Novel inhibitors of procollagen C-terminal proteinase. Part 1: diamino Acid hydroxamates.

The parallel synthesis of novel inhibitors of procollagen C-terminal proteinase is described. The synthetic strategy allowed for the facile synthesis of a large number of side-chain diversified diamino acid hydroxamates, of which the D-diaminopropionic acid derivatives were shown to be single digit nanomolar PCP inhibitors.

Amino acid derived sulfonamide hydroxamates as inhibitors of procollagen C-proteinase. Part 2: Solid-phase optimization of side chains.

Optimization of the amino acid side chain and the N-alkyl group of the sulfonamide of amino acid derived sulfonamide hydroxamates is discussed. The solid-phase synthesis of these potent inhibitors of procollagen C-proteinase (PCP) is presented. In addition, novel carboxylic acid sulfonamides were discovered to be PCP inhibitors.

Proteolysis involving matrix metalloproteinase 13 (collagenase-3) is required for chondrocyte differentiation that is associated with matrix mineralization.

Collagenases are involved in cartilage matrix resorption. Using bovine fetal chondrocytes isolated from physeal cartilages and separated into a distinct prehypertrophic subpopulation, we show that in serum-free culture they elaborate an extracellular matrix and differentiate into hypertrophic chondrocytes. This is characterized by expression of type X collagen and the transcription factor Cbfal and increased incorporation of 45Ca2+ in the extracellular matrix, which is associated with matrix calcification.

Amino acid derived sulfonamide hydroxamates as inhibitors of procollagen C-proteinase: solid-phase synthesis of ornithine analogues.

A discussion of the solid-phase synthesis of ornithine derived sulfonamide hydroxamic acids is illustrated. These analogues are shown to be potent, non-peptide inhibitors of procollagen C-proteinase (PCP).

Solid-phase synthesis of di- and tripeptidic hydroxamic acids as inhibitors of procollagen C-proteinase.

A solid-phase approach to the rapid synthesis of di- and tripeptide-like hydroxamic acids is presented. These compounds are shown to be potent inhibitors of procollagen C-proteinase (PCP).

Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1).

Objective: To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]).

Methods: Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium.

Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase.

Objective: To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and type II collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of collagenase 2 and collagenase 3 on these catabolic processes.

Methods: Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of collagenase 2 and collagenase 3 (matrix metalloproteinase 8 [MMP-8] and MMP-13, respectively) but not of collagenase 1 (MMP-1). Immunoassays were used to measure collagenase-generated type II collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total type II collagen content (antibody COL2-3/4m) in articular cartilage and culture media.

Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency.

Crystal structures of MMP-1 and -13 reveal the structural basis for selectivity of collagenase inhibitors.

The X-ray crystal structures of the catalytic domain of human collagenase-3 (MMP-13) and collagenase-1 (MMP-1) with bound inhibitors provides a basis for understanding the selectivity profile of a novel series of matrix metalloprotease (MMP) inhibitors. Differences in the relative size and shape of the MMP S1' pockets suggest that this pocket is a critical determinant of MMP inhibitor selectivity. The collagenase-3 S1' pocket is long and open, easily accommodating large P1' groups, such as diphenylether.

Role of His-224 in the anomalous pH dependence of human stromelysin-1.

A plot of the pH dependence of kcat/KM for human stromelysin-1 (HS) exhibits a narrow range of maximal activity extending from pH 5.75 to 6.25 and a broad shoulder in the pH range of 7.

Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase.

Macrophage elastase (ME) was originally named when metal-dependent elastolytic activity was detected in conditioned media of murine macrophages. Subsequent cDNA cloning of the mouse and human enzyme demonstrated that ME is a distinct member of the matrix metalloproteinase family. To date, the catalytic parameters that describe the hydrolysis of elastin by ME have not been quantified and its activity against other matrix proteins have not been described.

Comparison of peroxidase reaction mechanisms of prostaglandin H synthase-1 containing heme and mangano protoporphyrin IX.

Prostaglandin H synthase (PGHS) is a heme protein that catalyzes both the cyclooxygenase and peroxidase reactions needed to produce prostaglandins G2 and H2 from arachidonic acid. Replacement of the heme group by mangano protoporphyrin IX largely preserves the cyclooxygenase activity, but lowers the steady-state peroxidase activity by 25-fold. Thus, mangano protoporphyrin IX serves as a useful tool to evaluate the function of the heme in PGHS.

Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage.

We demonstrate the direct involvement of increased collagenase activity in the cleavage of type II collagen in osteoarthritic human femoral condylar cartilage by developing and using antibodies reactive to carboxy-terminal (COL2-3/4C(short)) and amino-terminal (COL2-1/4N1) neoepitopes generated by cleavage of native human type II collagen by collagenase matrix metalloproteinase (MMP)-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3). A secondary cleavage followed the initial cleavage produced by these recombinant collagenases. This generated neoepitope COL2-1/4N2.

Understanding the P1' specificity of the matrix metalloproteinases: effect of S1' pocket mutations in matrilysin and stromelysin-1.

Matrilysin (MAT) prefers leucine over residues that have aromatic side chains at the P1' position of peptide and protein substrates, while stromelysin (HFS) has a broader specificity. The X-ray structures of these enzymes show that their respective S1' subsites differ primarily due to the amino acids present at positions 214 and 215. To examine the role that these residues play in determining P1' specificity, the amino acids at these positions in matrilysin have been replaced by those found in stromelysin (MAT: Y214L, MAT:A215V, and MAT:Y214L/A215V).

Zinc content and function in human fibroblast collagenase.

The zinc contents of samples of human fibroblast collagenase (HFC) purified by different procedures and of samples purified by the same procedure but prepared for analysis by different dialysis protocols have been determined by atomic absorption spectroscopy. Both the purification method and dialysis conditions affect the zinc stoichiometry. Samples purified with and without the use of a zinc-chelate chromatography step and prepared by dialysis against 1 mM CaCl2 had zinc to enzyme ratios of 1.

Purification of human matrilysin produced in Escherichia coli and characterization using a new optimized fluorogenic peptide substrate.

Human promatrilysin (matrix metalloproteinase-7) has been produced in Escherichia coli as an N-terminal fusion protein with ubiquitin. The insoluble product was solubilized, refolded, and activated with amino-phenylmercuric acetate. Activation of the fusion protein demonstrated kinetics and intermediates that were very similar to those observed during activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells.

Proteolytic and non-proteolytic activation of human neutrophil progelatinase B.

The activation of human neutrophil progelatinase B (pro-HNG) by a variety of proteolytic and non-proteolytic activators has been investigated. A quantitative comparison of the activation efficiencies of treatments previously reported to activate pro-HNG or the related gelatinase B species produced by other cells demonstrates that stromelysin and trypsin are good activators. HgCl2 is a moderately effective activator, while p-chloromercuribenzoate and NaOCl are poor activators.

Zinc content of promatrilysin, matrilysin and the stromelysin catalytic domain.

Promatrilysin expressed in Escherichia coli and Chinese hamster ovary cells contains 2.36 +/- 0.19 and 2.

Differential effect of halide anions on the hydrolysis of different dansyl substrates by thermolysin.

The effect of sodium halide salts on the hydrolysis of three of the dansyl (Dns) peptide substrates described in the previous paper (Yang & Van Wart, 1994) by thermolysin have been studied. Increasing concentrations of sodium chloride decrease the KM value for the hydrolysis of the tripeptides Dns-Gly-Phe-Ala and Dns-Ala-Phe-Ala but leave kcat unaltered. This kinetic behavior is described by a nonessential activation mechanism in which chloride binds preferentially to the enzyme-substrate complex.

Kinetics of hydrolysis of dansyl peptide substrates by thermolysin: analysis of fluorescence changes and determination of steady-state kinetic parameters.

The stopped-flow fluorescence technique has been used to study the hydrolysis of 10 dansyl peptides by thermolysin. The origin of the fluorescence changes observed during the reactions has been investigated in detail. Depending on the substrate and the excitation wavelength, the dansyl fluorescence changes observed arise either from energy transfer (maximal at lambda ex = 230 and 280 nm) between Trp residues of thermolysin and the dansyl group of the substrate in enzyme-substrate (ES) complexes or from both sources.

Comparative sequence specificities of human 72- and 92-kDa gelatinases (type IV collagenases) and PUMP (matrilysin).

The sequence specificities of human 72-kDa fibroblast gelatinase (type IV collagenase), human 92-kDa neutrophil gelatinase (type IV collagenase), and putative metalloproteinase (PUMP or matrilysin) have been examined by measuring the rate of hydrolysis of over 50 synthetic oligopeptides covering the P4 through P4' subsites of the substrate. The peptides investigated in this paper were those employed in our previous study which systematically examined the sequence specificity of human fibroblast and neutrophil collagenases [Netzel-Arnett et al. (1991) J.