PEPTIDE INHIBITORS OF EXTRACELLULAR MATRIX PROTEASES

这项提议的广泛、长期的目标是阐明 三种人胶原酶在实验性类风湿中的表达 关节炎和实验性血管生成。这三种目标酶是 人皮肤成纤维细胞胶原酶(对III型最有活性 胶原蛋白)、人皮肤成纤维细胞IV型胶原酶和人 中性粒细胞胶原酶(抗I型胶原最活跃)。这些 将从培养的人皮肤成纤维细胞(III型)中提纯酶 和IV型酶)，以及来自人类化脓性痰(I型酶)。 75种人皮肤成纤维细胞胶原酶(III型)抑制剂的研制 在这项建议的最后一次更新中，三肽类似物是最好的 其KI为0.02微米。这些抑制物将被筛选出来 另外两种纯酶。氨基酸的替代将在 这些化合物以最大限度地提高这三种化合物的特异性 胶原酶。P2位氨基酸侧链的变异 在六肽底物中已经显示出区别于 成纤维细胞(III型)和肿瘤(IV型)胶原酶。新的特定 连续分光光度法或荧光法的多肽底物 还将在此基础上开发每种胶原酶的检测方法 氨基酸替换。 我们最好的特异性抑制剂将作为抗血管生成剂进行测试 对抗实验性的大鼠角膜血管生成。积极的结果 (抑制)将证明各自的胶原酶在这方面的作用 病理状态，建议使用胶原酶抑制剂作为 抗血管生成药物。

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.