Identification of small molecule furin-like protease inhibitors

小分子弗林蛋白酶抑制剂的鉴定

• 批准号: 7747509
• 负责人: Julia Coppola
• 金额: $ 23.72万
• 依托单位: BOISE TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7747509
• 关键词: Address; Alkaline Phosphatase; Alzheimer' s Disease; Anthrax disease; Antigens; Arterioloscleroses; Arteriosclerosis; Arthritis; Avian Influenza; Bacterial Toxins; Biological Assay; Cell Line; Cell Proliferation; Cells; Cellular Assay; Chemicals; Cleaved cell; Collection; Contracts; Degenerative Disorder; Development; Diphtheria; Disease; Dose; Drug usage; Ensure; Family; Family member; Glycoproteins; Goals; Growth Factor; HIV-1; Hormone Receptor; Human Papillomavirus; In Vitro; Influenza A Virus, H5N1 Subtype; Inhibitory Concentration 50; Lead; Life; MMP14 gene; Malignant - descriptor; Malignant Neoplasms; Matrix Metalloproteinases; Measles; Mediating; Metalloproteases; Monitor; Neoplasm Metastasis; Oranges; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Physiological Processes; Plasma Proteins; Poison; Process; Property; Proprotein Convertases; Protease Inhibitor; Proteins; Proteolytic Processing; Pseudomonas; Reporter; Reporting; SARS coronavirus; Sensitivity and Specificity; Serine Protease; Shigella; Solubility; Specificity; System; Testing; Therapeutic; Time; Toxic effect; Validation; Viral; Virus; Virus Diseases; Western Blotting; Yellow Fever; angiogenesis; anthrax protective factor; base; botulinum; cancer cell; cell motility; cell transformation; cytotoxic; cytotoxicity; drug development; high throughput screening; improved; inhibitor/antagonist; member; miniaturize; pathogen; public health relevance; research study; scaffold; small molecule; small molecule libraries; therapeutic development; tumor progression

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a non-invasive, real-time, quantifiable cell-based assay to detect and report on furin-like protease activity to identify small molecule inhibitors of furin-like proteases by high throughput screening (HTS). Furin-like proteolytic enzymes are members of the Proprotein Convertase (PC) family that serve to process immature latent proteins, including growth factors and hormones, receptors, plasma proteins, and matrix metalloproteases containing a specific recognition cleavage motif (RX(K/R)R?), to their mature or functional forms. Processing by furin-like protease family members, such as furin, PACE4, PC5/6, and PC7/8, contributes to development of several degenerative diseases, such as Alzheimer's disease, arteriolosclerosis, and arthritis. Furin-like protease expression and activity is necessary for processing substrates that enhance the cancer phenotype, contributing to cell transformation, tumor progression, metastasis, and angiogenesis. Further, furin-like proteolytic processing of viral coat glycoproteins is required for propagation of infectious viruses such as H5N1 avian influenza, HIV-1, human papillomavirus, ebola, yellow fever, and SARS-CoV. Furin-like proteases activate bacterial toxins found in anthrax, shigella, botulinum, pseudomonas, and diphtheria. Inhibition of furin-like proteolytic activity has been shown to halt toxicity of bacterial toxins, infectivity of viruses, and motility of cancer cells. We hypothesize that inhibiting furin-like proteolytic activity may lead to development of a therapeutic drug that inhibits a broad-spectrum of furin-like protease mediated disease. To aid in experimentation of this hypothesis, in specific aim 1A, we will develop a furin-like protease reporter, which non-invasively and quantitatively senses furin-like protease activity in real time and characterize its specificity and sensitivity to furin-like protease activity. In specific aim 1B, we will miniaturize this assay to adapt it to HTS. In specific aim 1C, we will perform HTS of several specialized small molecule libraries containing 71K compounds to identify furin-like protease inhibitory molecules. In specific aim 2A, a secondary screen will be employed to eliminate false positives, cytotoxic, and non-specific inhibitory molecules. Potency will be assessed by exposing the furin-reporter cells to various concentrations of the candidate compound to determine pIC50 values. In specific aim 2B, we subject the five most efficacious compounds to further validation by determining inhibition (IC50 value) of furin processing of physiological substrates using western blot analysis. Additionally, cytotoxicity will be gauged using cell proliferation assays. In specific aim 2C, the compound's ability to inhibit furin will be confirmed using purified furin in vitro. We will also investigate the molecule's specificity by performing in vitro inhibition assays with other serine proteases. At the conclusion of phase I, we expect to have identified at least one compound or derivative with IC50 < 1uM that will be the subject of further analysis and targeted for drug development to treat furin-mediated diseases such as anthrax and cancer in subsequent years. PUBLIC HEALTH RELEVANCE: Millions of people worldwide are exposed to and/or contract furin-like protease mediated diseases such as HIV-1, ebola, avian influenza, human papillomavirus, yellow fever, SARS-CoV, anthrax, botulinum, measles, pseudomonas, shigella, diphtheria, arthritis, arteriosclerosis, Alzheimer's disease, and malignant cancer. Instead of searching for a therapeutic to address each pathogen and disease individually, targeting a single cellular protease may allow defeat of a broad spectrum of furin-like protease mediated disease. The studies described here will result in identification of a molecule that inhibits furin-like proteases and thus may be used to treat the diseases listed above.

描述（由申请人提供）：本提案的总体目标是开发一种非侵入性、实时、可定量的基于细胞的测定法，以检测和报告弗林蛋白酶样蛋白酶活性，从而通过高通量筛选（HTS）识别弗林蛋白酶样蛋白酶的小分子抑制剂。弗林蛋白酶样蛋白水解酶是前蛋白转化酶 (PC) 家族的成员，用于将不成熟的潜在蛋白（包括生长因子和激素、受体、血浆蛋白和含有特定识别切割基序 (RX(K/R)R?) 的基质金属蛋白酶）加工成成熟或功能形式。弗林蛋白酶样蛋白酶家族成员（例如弗林蛋白酶、PACE4、PC5/6 和 PC7/8）的加工有助于多种退行性疾病的发生，例如阿尔茨海默病、动脉硬化和关节炎。弗林蛋白酶样蛋白酶的表达和活性对于处理增强癌症表型的底物是必需的，有助于细胞转化、肿瘤进展、转移和血管生成。此外，病毒外壳糖蛋白的弗林样蛋白水解加工是传染性病毒（例如 H5N1 禽流感、HIV-1、人乳头瘤病毒、埃博拉病毒、黄热病和 SARS-CoV）的繁殖所必需的。弗林蛋白酶样蛋白酶可激活炭疽、志贺氏菌、肉毒杆菌、假单胞菌和白喉中发现的细菌毒素。抑制弗林蛋白酶样蛋白水解活性已被证明可以阻止细菌毒素的毒性、病毒的感染性和癌细胞的运动性。我们假设抑制弗林蛋白酶样蛋白水解活性可能会导致开发出一种治疗药物，该药物可抑制广谱弗林蛋白酶样蛋白酶介导的疾病。为了帮助实验这一假设，在具体目标 1A 中，我们将开发一种弗林蛋白酶样报告基因，它可以非侵入性地实时定量检测弗林蛋白酶样活性，并表征其对弗林蛋白酶样蛋白酶活性的特异性和敏感性。在具体目标 1B 中，我们将小型化该测定以适应 HTS。在具体目标 1C 中，我们将对几个包含 71K 化合物的专门小分子库进行 HTS，以鉴定弗林蛋白酶样蛋白酶抑制分子。在具体目标 2A 中，将采用二次筛选来消除假阳性、细胞毒性和非特异性抑制分子。通过将弗林蛋白酶报告细胞暴露于不同浓度的候选化合物以确定 pIC50 值来评估效力。在具体目标 2B 中，我们通过使用蛋白质印迹分析确定生理底物弗林蛋白酶加工的抑制（IC50 值），对五种最有效的化合物进行进一步验证。此外，将使用细胞增殖测定来测量细胞毒性。在具体目标2C中，将使用体外纯化的弗林蛋白酶来证实该化合物抑制弗林蛋白酶的能力。我们还将通过使用其他丝氨酸蛋白酶进行体外抑制测定来研究该分子的特异性。在第一阶段结束时，我们预计已经鉴定出至少一种 IC50 < 1uM 的化合物或衍生物，这些化合物或衍生物将成为进一步分析的主题，并在随后的几年中用于治疗弗林蛋白酶介导的疾病（如炭疽和癌症）的药物开发。 公共卫生相关性：全世界有数百万人暴露于和/或感染弗林蛋白酶样蛋白酶介导的疾病，如 HIV-1、埃博拉、禽流感、人乳头瘤病毒、黄热病、SARS-CoV、炭疽、肉毒杆菌、麻疹、假单胞菌、志贺氏菌、白喉、关节炎、动脉硬化、阿尔茨海默病和恶性肿瘤 癌症。无需寻找单独治疗每种病原体和疾病的治疗方法，而是针对单一细胞蛋白酶可以战胜广泛的弗林蛋白酶样蛋白酶介导的疾病。这里描述的研究将鉴定出抑制弗林样蛋白酶的分子，因此可用于治疗上面列出的疾病。