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、可量化的基于细胞的检测和报告furin样蛋白酶活性的方法，通过高通量筛选（HTS）鉴定furin样蛋白酶的小分子抑制剂。furin样蛋白水解酶是Proprotein Convertase （PC）家族的成员，用于将未成熟的潜在蛋白（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元（RX(K/R)R?）的基质金属蛋白酶）加工成成熟或功能形式。furin样蛋白酶家族成员（如furin、PACE4、PC5/6和PC7/8）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。furin样蛋白酶的表达和活性是处理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，H5N1禽流感、HIV-1、人乳头状瘤病毒、埃博拉病毒、黄热病和sars冠状病毒等传染性病毒的繁殖需要对病毒外壳糖蛋白进行类似糠蛋白的蛋白水解处理。类似呋喃的蛋白酶可以激活炭疽、志贺氏菌、肉毒杆菌、假单胞菌和白喉中的细菌毒素。抑制呋喃样蛋白水解活性已被证明可以抑制细菌毒素的毒性、病毒的传染性和癌细胞的运动。我们假设，抑制糠蛋白样蛋白水解活性可能会导致一种治疗药物的开发，这种药物可以抑制广谱的糠蛋白样蛋白酶介导的疾病。为了帮助这一假设的实验，在特定的目标1A中，我们将开发一种类糠蛋白蛋白酶报告器，它可以无创地、定量地实时感知类糠蛋白蛋白酶的活性，并表征其对类糠蛋白蛋白酶活性的特异性和敏感性。在特定目标1B中，我们将使该试验小型化以适应HTS。在具体目标1C中，我们将对含有71K化合物的几个专门的小分子文库进行HTS，以鉴定furin样蛋白酶抑制分子。在特异性靶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。效价将通过将furin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。在特定的目标2B中，我们使用western blot分析，通过测定furin处理生理底物的抑制作用（IC50值），对五种最有效的化合物进行进一步验证。此外，细胞毒性将使用细胞增殖试验进行测量。在特定目标2C中，该化合物抑制呋喃的能力将在体外使用纯化的呋喃来证实。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I期结束时，我们预计将确定至少一种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呋喃介导的疾病（如炭疽和癌症）的药物开发的目标。

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.