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已被认为在几种类型的癌症的转移潜力中起重要作用，并且已涉及多种疾病的病理学。大多数假设组织蛋白酶B、D和K局限于溶酶体，在那里它们参与蛋白质降解。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。已经发现几种化疗药物的使用引起组织蛋白酶释放到胞质溶胶中，这导致半胱天冬酶的激活和细胞凋亡。组织蛋白酶K与骨质疏松症、类风湿性关节炎、肺癌、前列腺癌和乳腺癌患者的骨降解加速有关。乳腺肿瘤组织中高活化组织蛋白酶D水平与复发和转移的发生率增加相关。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标志物。迫切需要新的、更有效的蛋白酶抑制剂作为治疗剂。继续的设计和合成（羟乙基）胺电子等排体作为抑制剂的组织蛋白酶D的SAR数据的基础上提出。在该项目的初始阶段，这些（羟乙基）胺电子等排体中的96种已被证明是组织蛋白酶D活性的非常有效的抑制剂。此外，醛和缩氨基硫脲抑制剂的组织蛋白酶B的设计和合成的继续提出。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。作为该项目这一阶段的补充，将合成和测定新的组织蛋白酶K抑制剂。将在全细胞和全细胞裂解物上检测合成抑制剂。详细的动力学研究和结构活性相关性，然后绘制合成抑制剂。该项目的目的是作为一项研究工作，这将使本科生在生物医学研究的许多方面的培训通常不提供在农村本科院校。公共卫生相关性：通过分子建模设计和制备组织蛋白酶B、D和K抑制剂。组织蛋白酶D是与几种类型的癌症的转移相关的乙酰基蛋白酶。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性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酰蛋白酶。将通过荧光测定技术检测合成化合物作为组织蛋白酶D、组织蛋白酶B或组织蛋白酶K的蛋白酶抑制剂的效力。在这一阶段的赠款，我们建议开发组织蛋白酶K抑制剂，除了改善我们以前的工作，在开发有效的组织蛋白酶B和D抑制剂。我们建议克隆酶原的基因，表达的组织蛋白酶原，激活酶和测定合成化合物的抑制组织蛋白酶B，D和K在整个细胞和细胞裂解物。将记录速率常数、抑制常数和其他动力学参数以及结构-活性相关性。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。这项工作可能会导致有用的生化工具，用于确定特定酶在疾病中的作用，并可能导致开发新的治疗方法来治疗这些毁灭性疾病。在本科院校进行这种类型的调查将使学生从一开始就参与研究培训。计算机建模部分将允许学生学习化学化合物的合理设计的当前技术。然后，学生将学习开发和执行一个研究协议，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HPLC色谱法，NMR光谱法等）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。在这个部分中，学生将学习优化条件和修改现有的分析，以适应实验条件。动力学参数的确定将要求学生收集，分析，并进行数据的统计分析。因此，该项目将为学生提供生物医学研究许多方面的实践经验。学生参与这种类型的研究项目应该证明化合物的化学结构和它们的生物活性之间的重要联系。因此，该项目将大大改善这所农村本科院校的研究氛围和可用性。

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.