PROTEASES IN CANCER--BIOLOGY AND DRUG DEVELOPMENT

本提案有三个主要目标：1）鉴定丝氨酸， 金属和半胱氨酸蛋白酶表达的上皮癌， 前列腺，结肠和皮肤，2）表征这些功能 蛋白酶在转化、肿瘤生长和转移中的作用，和3） 开发基于抑制关键的化学治疗先导物， 在1）和2）中鉴定的蛋白酶。 我们希望利用微分 蛋白酶在癌症与正常组织中的位置以及活性 抑制剂设计的位点特异性。 优势蛋白酶和 将鉴定靶向人肿瘤中存在的起源细胞 通过免疫组织化学、生物素化活性位点探针和酶 组织化学 蛋白酶的RT-PCR扩增将用于 确认序列同一性，并亚克隆到表达载体中， 试剂量的生产（核心B/项目1）。 假定小说 将通过数据库鉴定小分子蛋白酶抑制剂 使用分子建模和UCSF软件进行筛选程序。 此外，小分子蛋白酶抑制剂将从以下获得： 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）。 第二种方法是 利用蛋白质工程技术调节大分子 抑制剂活性和增加特异性。 将使用噬菌体展示 结合随机和定向工程方法。 具体 大分子，这将是研究在一开始的抑制 针对蛋白酶的活性包括Ecotin。 组织抑制 金属蛋白酶和胱抑素（项目1）。 抑制剂鉴定 项目1和我们的合作者，这是有力的反对 将在体内测试在靶向肿瘤中鉴定的酶 细胞和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，其过表达1） 蛋白酶在靶向肿瘤组织中的作用，以确定这些 蛋白酶或2）这些蛋白酶的抑制剂。 通过抑制蛋白酶 通过施用外源蛋白或小分子，或 抑制剂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， 进展 在核心D中，抑制剂的代谢和消除 项目1和我们的合作者将被研究， 改善它们向作用部位的递送，以及它们在体内 药代动力学 在项目3中， 蛋白酶在皮肤鳞状上皮转化中的作用将被 测定 在K14-HPV 16转基因小鼠上皮细胞模型中， 经历多阶段肿瘤进展的癌发生， 侵袭性恶性肿瘤 该模型将用于识别和 表征哪些蛋白酶在不同阶段表达， 肿瘤以及改变ECM的生物学意义- 降解蛋白酶和/或抑制剂在肿瘤细胞上的表达 通过遗传互补的进展。 通过采取这种多方面的， 多学科协调方法，我们乐观地认为，我们将 1)对蛋白酶在转化中的作用有重要的认识 以及肿瘤生长和转移，以及2）成功地 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.