PROTEASES IN CANCER--BIOLOGY AND DRUG DEVELOPMENT

这项提议有三个主要目标：1)鉴定丝氨酸， 金属、半胱氨酸蛋白酶在上皮性癌中的表达 前列腺、结肠和皮肤，2)描述这些组织的功能 蛋白酶在转化、肿瘤生长和转移中的作用，以及3)到 基于Key抑制作用开发新的化疗先导化合物 1)和2)中鉴定的蛋白酶。我们希望利用差异化 蛋白水解酶在癌组织中的定位与正常组织及活动期的比较 抑制物设计的部位特异性。主要蛋白水解酶和 将确定靶向人类肿瘤中存在的起源细胞 通过免疫组织化学、生物素标记的活性部位探针和酶 组织化学。蛋白水解酶的RT-PCR扩增将用于 确认序列同一性，并亚克隆到表达载体中 试剂产量(核心B/项目1)。假定的小说 小分子蛋白酶抑制剂将通过数据库进行鉴定 使用分子建模和UCSF软件的筛查程序。 此外，还将从以下来源获得小分子蛋白酶抑制剂 公司和/或学术来源，包括我们自己的合成 抑制剂库，用于测试(项目1)。第二种方法将是 利用蛋白质工程技术调控大分子 抑制物的活性和增加特异性。将使用噬菌体展示 采用随机和定向的工程方法。特定的 将在一开始就研究抑制作用的大分子 对蛋白水解酶的活性包括Ecotin。组织抑制因子 金属蛋白酶和半胱氨酸蛋白酶(项目1)。中确定的抑制剂 项目1和我们的合作者，有效地对抗 靶向肿瘤中确定的酶将在体内进行测试 细胞、转基因和免疫缺陷小鼠模型(项目2)。 核心C将开发转基因小鼠，这些小鼠要么过度表达1) 以确定这些蛋白在靶肿瘤组织中的作用 蛋白水解酶或2)这些蛋白酶的抑制剂。通过抑制蛋白酶 通过注射外源蛋白质或小分子，或者 抑制物在肿瘤细胞中的内源性转基因表达， 我们将能够确定这些蛋白酶在肿瘤中的作用 进步。在核心D中，抑制物的代谢和消除 来自项目1和我们的合作者将被研究，以便 改善它们对作用部位的传递，以及它们在体内的作用 药物动力学。在项目3中，时间和空间的表达 皮肤鳞状上皮转化中的蛋白水解酶 下定决心。人乳头瘤病毒K14-HPV16转基因小鼠模型的建立 癌变经历多阶段的肿瘤进展到 侵袭性恶性肿瘤。该模型将用于识别和 确定哪些蛋白水解酶在不同阶段表达 肿瘤的发生以及细胞外基质改变的生物学意义 降解性蛋白水解酶和/或抑制物在肿瘤组织中的表达 通过基因互补进行的进步。通过将这一多方面的， 多学科协调的方法，我们乐观地认为，我们将 1)获得对蛋白酶在转化中的作用的重要见解 以及肿瘤的生长和转移，以及2)在 获得治疗癌症的新疗法。

There are three major goals of this proposal: 1) to identify the serine, metallo-, and cysteine proteases expresses by epithelial cancers of the prostate, colon, and skin, 2) to characterize the function of these proteases in transformation, tumor growth, and metastasis, and 3) to develop new chemotherapeutic leads based on inhibitions of key proteases identified in 1) and 2). We hope to exploit differential location of proteases in cancer versus normal issue as well as active site specificity for inhibitor design. Predominant proteases and the cell of origin present in the targeted human tumors will be identified by immunohistochemistry, biotinylated active site probes, and enzyme histochemistry. RT-PCR amplification of the proteases will be used to confirm sequence identity, and to subclone into expression vectors for production of reagent quantities (Core B/Project 1). Putative novel small molecule protease inhibitors will be identified by database screening procedures using molecular modeling and UCSF software. In addition, small molecule protease inhibitors will be obtained from corporate and/or academic sources including our own synthetic inhibitor, library, for testing (Project 1). A second approach will be to use protein engineering techniques to modulate macromolecular inhibitor activity and increase specificity. Phage display will be used incorporating random and directed engineering approaches. Specific macromolecules which will be investigated at the outset for inhibitory activity against proteases include Ecotin. Tissue Inhibitor of Metalloprotease, and Cystatin (Project 1). Inhibitors identified in Project 1 and by our collaborators which are potent against the enzymes identified in the targeted tumors will be tested in in vivo cellular and transgenic and immunodeficient mouse models (Project 2). Core C will develop transgenic mice which either overexpress 1) proteases in targeted tumor tissue to determine the role of these proteases or 2) inhibitors of these proteases. By inhibiting proteases either by administration of exogenous proteins or small molecules, or endogenous transgenic expression of the inhibitors in the tumor cells, we will be able to determine the role of these proteases in tumor progression. In Core D, the metabolism and elimination of inhibitors from Project 1 and our collaborators will be studied in order to improve their delivery to sites of action, and their in vivo pharmacokinetics. In Project 3, the temporal and spatial expression of proteases in transformation of squamous epithelium of the skin will be determined. In the K14-HPV16 transgenic mouse model of epithelial carcinogenesis that undergoes multistage neoplastic progression to invasive malignancy. This model will be used to identify and characterize which proteases are expressed at various stages of neoplasia as well as the biological significance of altered ECM- degrading proteases and/or inhibitor expression on neoplastic progression by genetic complementation. By taking this multifaceted, multidisciplinary coordinated approach, we are optimistic that we will 1) obtain important insights into the role of proteases in transformation as well as tumor growth and metastasis, and 2) be successful in obtaining novel therapies for treating cancer.