PEPTIDE INHIBITORS OF EXTRACELLULAR MATRIX PROTEASES

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The broad, long term objective of this proposal is to elucidate the role of each of three types of human collagenases in experimental rheumatoid arthritis and experimental angiogenesis. The three target enzymes are human skin fibroblast collagenase (most active against type III collagen), human skin fibroblast type IV collagenase, and human neutrophil collagenase (most active against type I collagen). These enzymes will be purified from cultured human skin fibroblasts (type III and type IV enzymes), and from purulent human sputum (type I enzyme). The 75 human skin fibroblast collagenase (type III) inhibitors developed in the last renewal of this proposal are tripeptide analogs the best of which have Ki's of 0.02 uM. These inhibitors will be screened against the other two pure enzymes. Amino acid substitutions will be made in these compounds in order to maximize specificity for each of the three collagenases. Variation of the amino acid side chain at the P2 position in a hexapeptide substrate has been shown to differentiate between fibroblast (type III) and tumor (type IV) collagenase. New specific peptide substrates for continuous spectrophotometric or fluorimetric assay of each collagenase will also be developed based on these same amino acid substitutions. Our best specific inhibitors will be tested as antiangiogenic agents against experimental angiogenesis in the rat cornea. Positive results (inhibition) would prove the role of the respective collagenase in this pathological condition and suggest the use of collagenase inhibitors as anti-angiogenic drugs.