AREA: Synthesis and Evaluation of New Cathepsin B, D, and K Inhibitors

描述（由申请人提供）：组织蛋白酶B、D和K已被认为在几种癌症的转移潜能中发挥重要作用，并与多种疾病的病理有关。大多数人认为组织蛋白酶B、D和K仅限于参与蛋白质降解的溶酶体。然而，许多研究人员发现，在许多疾病条件下，组织蛋白酶被释放到细胞质中并出现在血清中。一些化疗药物的使用已被发现导致组织蛋白酶释放到细胞质中，从而导致半胱天冬酶的激活和细胞凋亡。在骨质疏松症、类风湿关节炎、肺癌、前列腺癌和乳腺癌患者中，组织蛋白酶K与加速骨降解有关。乳腺肿瘤组织中组织蛋白酶D的高活化水平与复发和转移的发生率增加有关。事实上，组织蛋白酶B和D水平已被用作预测乳腺癌和子宫癌患者预后的指标。迫切需要新的、更有效的蛋白酶抑制剂作为治疗剂。在SAR数据的基础上，继续设计和合成（羟乙基）胺异构体作为组织蛋白酶D的抑制剂。在这个项目的初始阶段，这些（羟乙基）胺同分酯中的96种已被证明是非常有效的组织蛋白酶D活性抑制剂。此外，还建议继续设计和合成组织蛋白酶B的醛类和硫代氨基脲类抑制剂。在该项目的初始阶段，已经开发了16种新的组织蛋白酶B抑制剂。作为项目这一阶段的补充，新的组织蛋白酶K抑制剂将被合成和检测。合成抑制剂将在全细胞和全细胞裂解物上进行测试。详细的动力学研究和结构活性相关性将绘制合成抑制剂。这一项目是作为一项研究工作而设计的，它将使本科生在生物医学研究的许多方面得到培训，而这些方面在农村本科院校通常是得不到的。公共卫生相关性：通过分子模型设计和制备组织蛋白酶B、D和K抑制剂。组织蛋白酶D是一种与多种癌症转移相关的天冬氨酸蛋白酶。组织蛋白酶B和K是与肿瘤进展、阿尔茨海默病、骨质疏松症和类风湿关节炎以及与乳腺癌、前列腺癌和肺癌相关的骨降解相关的半胱氨酸蛋白酶。合成的化合物将通过荧光测定技术测试其作为组织蛋白酶D、组织蛋白酶B或组织蛋白酶K抑制剂的效力。在这一阶段，我们建议开发组织蛋白酶K抑制剂，除了改进我们之前在开发有效的组织蛋白酶B和D抑制剂方面的工作。我们拟克隆组织蛋白酶原基因，表达组织蛋白酶原，激活组织蛋白酶原，并在全细胞和细胞裂解物中测定合成化合物对组织蛋白酶B、D和K的抑制作用。速率常数，抑制常数，和其他动力学参数，以及构效关系将被记录。考虑到这种方法背后的原理，一些化合物很可能是这些生物学上重要的蛋白酶的非常有效的抑制剂。这项工作可能导致有用的生化工具，以确定特定的酶在疾病条件下的作用，并可能导致开发新的治疗方法来治疗这些毁灭性的疾病。在本科院校进行这种类型的调查，可以让学生从一开始就参与研究训练。计算机建模组件将允许学生学习当前的技术在合理设计的化合物。然后，学生将学习开发和执行一项研究方案，以合成通过计算机建模设计的化合物。他们将通过当前的化学和仪器技术（即高效液相色谱，核磁共振波谱等）合成，分离，纯化和表征化合物。学生将学习基因克隆技术，蛋白质表达和分离，以及酶激活技术。在测试潜在抑制剂的生物活性时，本科生将学习组织培养技术和酶分析。在这个部分，学生将学习优化条件和修改现有的分析，以适应实验条件。动力学参数的确定要求学生收集、分析并对数据进行统计分析。因此，该项目将为学生提供生物医学研究的许多方面的实践经验。学生在这类研究项目中的参与应该证明化合物的化学结构与其生物活性之间的重要联系。因此，该项目将极大地改善这所农村本科院校的研究氛围和可获得性。

DESCRIPTION (provided by applicant): Cathepsins B, D, and K have been suggested to play important roles in the metastatic potential of several types of cancer, and have been implicated in the pathology of a wide variety of diseases. Most assume cathepsins B, D, and K to be restricted to the lysosomes where they are involved in protein degradation. However, many researchers have found that under many diseased conditions cathepsins are released into the cytosol and appear in the serum. The use of several chemotherapeutic drugs have been found to cause a release of cathepsins into the cytosol, which leads to activation of caspases and apoptosis. Cathepsin K has been associated with accelerated bone degradation in patients with osteoporosis, rheumatoid arthritis, lung cancer, prostate cancer, and breast cancer. A high activated cathepsin D level in breast tumor tissue has been associated with an increased incidence of relapse and metastasis. In fact cathepsin B and D levels have been used as markers to predict the prognosis of breast cancer and uterine cancer patients. There is an urgent need for new, more effective protease inhibitors as therapeutic agents. A continuation of the design and synthesis (hydroxyethyl)amine isosteres as inhibitors as cathepsin D based on SAR data is proposed. In the initial phase of this project 96 of these (hydroxyethyl)amine isosteres have proven to be very potent inhibitors of cathepsin D activity. Also, the continuation of the design and synthesis of aldehyde and thio-semicarbazone inhibitors of cathepsin B is proposed. In the Initial phase of this project 16 new cathepsin B inhibitors have been developed. As an addition in this phase of the project, new inhibitors of cathepsin K will be synthesized and assayed. The synthetic inhibitors will be tested on whole cells and lysates of whole cells. Detailed kinetic studies and structure activity correlations will then be drawn for the synthetic inhibitors. This project is designed as a research effort which will allow undergraduate students training in many aspects of biomedical research not normally available at a rural undergraduate institution. PUBLIC HEALTH RELEVANCE: The design, via molecular modeling, and preparation of cathepsin B, D, and K inhibitors is proposed. Cathepsin D is an aspartyl protease associated with metastasis of several types of cancer. Cathepsin B and K are cysteinyl proteases associated with tumor progression, Alzheimer's disease, osteoporosis, and rheumatoid arthritis, as well as bone degradation associated with breast cancer, prostate cancer, and lung cancer. The synthetic compounds will be tested for their potency as protease inhibitors of cathepsin D, cathepsin B, or cathepsin K by fluorometric assay techniques. In this phase of the grant we propose to develop cathepsin K inhibitors, in addition to improving upon our previous work in the development of potent cathepsin B and D inhibitors. We propose to clone the genes for the proenzymes, express the procathepsins, activate the enzymes and assay the synthetic compounds for their inhibition of cathepsins B, D, and K in whole cells and cell lysates. Rate constants, inhibition constants, and other kinetic parameters, as well as structure-activity correlations will be documented. Considering the rationale behind this approach, some of the compounds may well be very potent inhibitors of these biologically important proteases. This work could lead to useful biochemical tools for determining the role of specific enzymes in diseased conditions, and may result in the development of new therapeutics for the treatment of these devastating diseases. Investigations of this type in undergraduate institutions will allow students to participate in research training from the inception of the idea. The computer modeling component will allow students to learn the current techniques in the rational design of chemical compounds. The students will then learn to develop and perform a research protocol in synthesizing the compounds designed by computer modeling. They will synthesize, isolate, purify, and characterize the compounds by current chemical and instrumental techniques (i.e. HPLC chromatography, NMR spectroscopy, etc.). The students will learn gene cloning techniques, protein expression and isolation, and enzyme activation techniques. In testing the biological activities of the potential inhibitors, the undergraduate students will learn tissue culture techniques and enzyme analysis. In this component the students will learn to optimize conditions and modify existing assays to fit experimental conditions. Determination of kinetic parameters will require the students to collect, analyze, and conduct statistical analysis of the data. Therefore, the project will provide students with practical experience with many aspects of biomedical research. Student participation in this type of research project should demonstrate the vital link between the chemical structures of compounds and their biological activities. Therefore, this project would greatly improve the atmosphere for and the availability of research in this rural undergraduate institution.