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

领域：新型组织蛋白酶 B、D 和 K 抑制剂的合成和评价

• 批准号: 7847053
• 负责人: ROSE M MCCONNELL
• 金额: $ 12.28万
• 依托单位: WESTERN ILLINOIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-19 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7847053
• 关键词: Aldehydes; Alzheimer' s Disease; Amines; Apoptosis; Aspartic Endopeptidases; Biochemical; Biological; Biological Assay; Biological Process; Biological Testing; Biology; Biomedical Research; Bone Resorption; C-terminal; Cancer Patient; Carcinoma; Caspase; Cathepsins; Cathepsins B; Cell Death; Cells; Chemical Structure; Chemicals; Chemistry; Chromatography; Computer Simulation; Cysteine; Cysteine Protease; Cytosol; Data; Development; Disease; Environment; Enzyme Activation; Enzyme Precursors; Enzymes; Epithelial Cells; Evaluation; Grant; HIV-1 protease; High Pressure Liquid Chromatography; Human; Incidence; Institution; Investigation; Ketones; Kinetics; Lead; Learning; Link; Lysosomes; Malignant neoplasm of lung; Malignant neoplasm of prostate; Mammary Neoplasms; Molecular Models; NMR Spectroscopy; Neoplasm Metastasis; Osteoporosis; Pathology; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Play; Preparation; Protease Inhibitor; Protocols documentation; Relapse; Research; Research Personnel; Research Project Grants; Research Training; Rheumatoid Arthritis; Role; Rural; Science; Serum; Statistical Data Interpretation; Structure; Students; Substrate Specificity; Techniques; Testing; Therapeutic Agents; Thiones; Tissue Culture Techniques; Training; Tumor Tissue; Uterine Cancer; Work; base; bone; cancer cell; cancer type; cathepsin K; cytochrome c; design; experience; gene cloning; improved; inhibitor/antagonist; malignant breast neoplasm; molecular modeling; novel therapeutics; outcome forecast; overexpression; planetary Atmosphere; protein degradation; protein expression; public health relevance; statistics; tool; tumor progression

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.

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癌症的转移潜力中发挥重要作用，并且与多种疾病的病理学有关。大多数人认为组织蛋白酶 B、D 和 K 仅限于溶酶体，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，组织蛋白酶会释放到细胞质中并出现在血清中。已发现几种化疗药物的使用会导致组织蛋白酶释放到细胞质中，从而导致半胱天冬酶激活和细胞凋亡。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退化加速有关。乳腺肿瘤组织中高活性组织蛋白酶 D 水平与复发和转移发生率增加有关。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标志物。迫切需要新的、更有效的蛋白酶抑制剂作为治疗剂。提出了基于 SAR 数据的组织蛋白酶 D 抑制剂的（羟乙基）胺电子等排体的设计和合成的延续。在该项目的初始阶段，96 个（羟乙基）胺电子等排体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 的醛类和缩氨基硫脲抑制剂。在该项目的初始阶段，已开发出 16 种新的组织蛋白酶 B 抑制剂。作为该项目此阶段的补充，将合成并分析新的组织蛋白酶 K 抑制剂。合成抑制剂将在全细胞和全细胞裂解物上进行测试。然后将对合成抑制剂进行详细的动力学研究和结构活性相关性。该项目旨在作为一项研究工作，让本科生接受生物医学研究许多方面的培训，而农村本科院校通常无法提供这些培训。公共健康相关性：建议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 是一种天冬氨酰蛋白酶，与多种癌症的转移相关。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退化相关的半胱氨酰蛋白酶。将通过荧光测定技术测试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。在这一阶段的资助中，除了改进我们之前开发强效组织蛋白酶 B 和 D 抑制剂的工作外，我们还建议开发组织蛋白酶 K 抑制剂。我们建议克隆酶原基因，表达组织蛋白酶原，激活酶并测定合成化合物对全细胞和细胞裂解物中组织蛋白酶 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结构-活性相关性将被记录。考虑到这种方法背后的基本原理，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。这项工作可能会产生有用的生化工具来确定特定酶在疾病中的作用，并可能导致开发出治疗这些破坏性疾病的新疗法。在本科院校进行此类调查将使学生从想法一开始就参与研究培训。计算机建模部分将使学生能够学习化合物合理设计的最新技术。然后，学生将学习开发和执行合成计算机建模设计的化合物的研究方案。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。在本部分中，学生将学习优化条件并修改现有测定法以适应实验条件。动力学参数的确定需要学生收集、分析数据并进行统计分析。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。学生参与此类研究项目应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用性。

项目成果

New cathepsin D inhibitor library utilizing hydroxyethyl isosteres with cyclic tertiary amines.

利用羟乙基等排物和环状叔胺的新组织蛋白酶 D 抑制剂库。

• DOI: 10.2174/1573406411208061146
• 发表时间: 2012
• 期刊: Medicinal chemistry (Shariqah (United Arab Emirates))
• 作者: McConnell,RoseM;Inapudi,Kalyani;Kadasala,Naveen;Yarlagadda,Karthika;Velusamy,Priya;McConnell,MatthewS;Green,Adam;Trana,Carol;Sayyar,Kelley;McConnell,JamesS
• 通讯作者: McConnell,JamesS