The biosynthesis of elastin and collagen involves post-translational, enzyme-catalyzed processing of each of the newly synthesized proteins. Among these essential processes, the oxidation of peptidyl lysine, catalyzed by lysyl oxidase, initiates the formation of intra- and intermolecular lysine-derived crosslinkages which stabilize and insolubilize extracellular fibers of these connective tissue proteins. Since this appears to be the last step in fiber synthesis, it is potentially a key point of biological control in the development of connective tissues. Moreover, lysyl oxidase is a rational chemotherapeutic target of anti-fibrotic agents designed to prevent of limit the development of fibrotic sequellae as in lung fibrosis and atherosclerosis and to limit scar formation. The present proposal intends to define key aspects of the mechanism of action and substrate specificity of lysyl oxidase and, based on such information, to design, synthesize and test at both test-tube and tissue levels new, mechanism- based, peptidyl and non-peptidyl inhibitors of this crosslinking enzyme. These efforts will involve analyses of the roles of the pyrroloquinoline quinone (PQQ) and copper ion cofactors, employing techniques as electron paramagnetic resonance, absorption and fluorescence spectrophotometry, and stopped flow kinetic analyses of the reductive and oxidative half reactions catalyzed by this enzyme. Specificity of the enzyme will be probed by kinetic analyses of oligopeptide substrates assembled by solid phase peptide synthesis. The peptide environment of the active site will be assessed by amino acid sequence analysis and mass spectroscopy of peptides isolated from the enzyme labelled with covalent, radioactive probes of the active site. Chemical and kinetic mechanisms of inhibition by new inhibitors directed at the cofactor or active site nucleophiles will be analyzed. The effect of such new inhibitors on the crosslinking of elastin in chick embryo aortae will be assessed in organ culture as an index of their potential as anti-fibrotic agents in vivo.
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