ABSTRACT
A novel series of imidazolidinone-based matrix metalloproteinase (MMP) inhibitors was discovered by structural modification of pyrrolidinone la. Potent inhibition of MMP-13 was exhibited by the analogues having 4-(4-fluorophenoxy)phenyl (4a, IC50 = 3 nM) and 4-(naphth-2-yloxy)phenyl (4h, IC50 = 4 nM) as P1' groups.
Subject(s)
Amides/chemical synthesis , Amides/pharmacology , Carboxylic Acids/chemical synthesis , Carboxylic Acids/pharmacology , Enzyme Inhibitors/chemical synthesis , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Matrix Metalloproteinase Inhibitors , Drug Design , Enzyme Inhibitors/pharmacology , Matrix Metalloproteinase 13 , StereoisomerismSubject(s)
Benzeneacetamides , Hydroxamic Acids/chemical synthesis , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/chemical synthesis , Pyrrolidinones/chemical synthesis , Collagenases/chemistry , Drug Design , Hydroxamic Acids/chemistry , Matrix Metalloproteinase 13 , Models, Molecular , Protease Inhibitors/chemistry , Pyrrolidinones/chemistry , Structure-Activity RelationshipSubject(s)
Cartilage, Articular/enzymology , Collagenases/genetics , Gene Expression Regulation , Animals , Cartilage, Articular/cytology , Gene Expression Regulation/drug effects , Humans , Interleukin-1/pharmacology , Joints , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 13 , RNA, Messenger/genetics , Rabbits , Recombinant Proteins/pharmacology , Transcription, Genetic/drug effectsABSTRACT
Through the use of empirical and computational methods, phosphinate-based inhibitors of MMP-1 and MMP-13 that bind into the S2 pocket of these enzymes were designed. The synthesis and testing of 2 suggested that binding was occurring as hypothesized. Structure determination of a co-crystal of 2 bound to the catalytic domain of MMP-1 confirmed the binding mode. Substituents binding into S2, S1', S2' and S3', were optimized yielding compounds with low double-digit nM IC50's against these enzymes.
Subject(s)
Matrix Metalloproteinase Inhibitors , Phosphinic Acids/pharmacology , Binding Sites , Collagenases/pharmacokinetics , Computer Simulation , Crystallography, X-Ray , Drug Design , Inhibitory Concentration 50 , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 13 , Models, MolecularABSTRACT
Proteolysis of triple-helical collagen is an important step in the progression toward irreversible tissue damage in osteoarthritis. Earlier work on the expression of enzymes in cartilage suggested that collagenase-1 (MMP-1) contributes to the process. Degenerate reverse transcription polymerase chain reaction experiments, Northern blot analysis, and direct immunodetection have now provided evidence that collagenase-3 (MMP-13), an enzyme recently cloned from human breast carcinoma, is expressed by chondrocytes in human osteoarthritic cartilage. Variable levels of MMP-13 and MMP-1 in cartilage was significantly induced at both the message and protein levels by interleukin-1 alpha. Recombinant MMP-13 cleaved type II collagen to give characteristic 3/4 and 1/4 fragments; however, MMP-13 turned over type II collagen at least 10 times faster than MMP-1. Experiments with intact type II collagen as well as a synthetic peptide suggested that MMP-13 cleaved type II collagen at the same bond as MMP-1, but this was then followed by a secondary cleavage that removed three amino acids from the 1/4 fragment amino terminus. The expression of MMP-13 in osteoarthritic cartilage and its activity against type II collagen suggest that the enzyme plays a significant role in cartilage collagen degradation, and must consequently form part of a complex target for proposed therapeutic interventions based on collagenase inhibition.
Subject(s)
Cartilage/enzymology , Collagen/metabolism , Collagenases/metabolism , Osteoarthritis/enzymology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Collagenases/genetics , Humans , Kinetics , Matrix Metalloproteinase 13 , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Analysis , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate SpecificityABSTRACT
Interleukin-1 alpha (IL-1) stimulated the release of degraded proteoglycan from primary cultures of chondrocyte monolayers in a time- and dose-dependent fashion. Bafilomycin A1, a specific inhibitor of the vacuolar H(+)-ATPase, efficiently blocked acidification of the chondrocyte vacuolar system. Under these conditions IL-1-stimulated proteoglycan degradation was inhibited by bafilomycin A1 with an IC50 of < 10 nM in both chondrocyte monolayers and articular cartilage explants. This concentration was at least 100-fold less than that required to partially inhibit total protein synthesis. In chondrocyte monolayers, bafilomycin A1 could be added several hours after IL-1 and complete inhibition was still observed. Tumor necrosis factor-alpha and retinoic acid also stimulated proteoglycan degradation in chondrocyte monolayers, and in both cases the response was inhibited by bafilomycin A1. These results suggest that maintenance of vacuolar acidity is required for cytokine stimulated proteoglycan degradation and that this requirement is at a point distal to receptor binding and early signal transduction events. IL-1 also stimulated the synthesis and secretion of prostromelysin by chondrocyte monolayers, however, under conditions in which IL-1 stimulated proteoglycan release was totally blocked by bafilomycin A1, there was no effect on IL-1-stimulated stromelysin secretion or stromelysin enzyme activity. These results, in which stromelysin synthesis and proteoglycan degradation were dissociated, suggest that an additional enzyme is responsible for proteoglycan degradation in this chondrocyte monolayer system.
