Identification of small molecule furin-like protease inhibitors

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）鉴定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，其用于加工不成熟的潜伏蛋白，包括生长因子和激素、受体、血浆蛋白和含有特异性识别切割基序（RX（K/R）R？）的基质金属蛋白酶，到它们的成熟或功能形式。弗林蛋白酶样蛋白酶家族成员，如弗林蛋白酶、PACE 4、PC 5/6和PC 7/8的加工有助于几种退行性疾病的发展，如阿尔茨海默病、小动脉硬化和关节炎。弗林蛋白酶样蛋白酶的表达和活性是加工增强癌症表型的底物所必需的，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蛋白酶样蛋白水解加工是感染性病毒如H5 N1禽流感、HIV-1、人乳头瘤病毒、埃博拉病毒、黄热病和SARS-CoV繁殖所必需的。弗林蛋白酶样蛋白酶激活炭疽、志贺氏菌、肉毒杆菌、假单胞菌和白喉中发现的细菌毒素。弗林蛋白酶样蛋白水解活性的抑制已经显示出停止细菌毒素的毒性、病毒的感染性和癌细胞的运动性。我们推测，抑制弗林蛋白酶样蛋白水解活性可能会导致开发一种治疗药物，抑制广谱弗林蛋白酶样蛋白酶介导的疾病。为了帮助该假设的实验，在具体的目标1A中，我们将开发弗林蛋白酶样蛋白酶报告基因，其非侵入性地和定量地在真实的时间内感测弗林蛋白酶样蛋白酶活性，并表征其对弗林蛋白酶样蛋白酶活性的特异性和灵敏度。在具体目标1B中，我们将对该测定进行验证以使其适用于HTS。在具体目标1C中，我们将对含有71 K化合物的几个专门的小分子文库进行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pIC 50值来评估效价。在具体目标2B中，我们通过使用蛋白质印迹分析确定弗林蛋白酶处理生理底物的抑制（IC 50值）来进一步验证五种最有效的化合物。此外，将使用细胞增殖试验测定细胞毒性。在具体目标2C中，将使用纯化的弗林蛋白酶在体外证实化合物抑制弗林蛋白酶的能力。我们还将通过与其他丝氨酸蛋白酶进行体外抑制试验来研究该分子的特异性。在第一阶段结束时，我们预计已经确定了至少一种IC 50 <1 μ M的化合物或衍生物，这将是进一步分析的主题，并有针对性地用于药物开发，以治疗随后几年的弗林介导的疾病，如炭疽和癌症。 公共卫生相关性：全世界数百万人暴露于和/或感染弗林蛋白酶样蛋白酶介导的疾病，例如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癌症。靶向单个细胞蛋白酶可以允许击败广谱弗林蛋白酶样蛋白酶介导的疾病，而不是寻找单独解决每种病原体和疾病的治疗剂。本文所述的研究将鉴定出抑制弗林蛋白酶样蛋白酶的分子，因此可用于治疗上述疾病。

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.