Allosteric Inhibition of a Family of Proteolytic Enzymes

蛋白水解酶家族的变构抑制

基本信息

批准号：
8698774
负责人：
Charles Scott Craik
金额：
$ 30.52万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-10 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8698774
关键词：
Active Sites Allosteric Site Animals Antiviral Agents Binding Binding Sites Biochemistry Biological Assay Biology Cell Culture Techniques Cell Membrane Permeability Cells Chemicals Chemistry Chickenpox Color Communicable Diseases Complex Computer Simulation Crystallization Crystallography Cytomegalovirus Detection Dimerization Disease Disease Resistance Dissociation Disulfides Enzymes Equilibrium Family Family member Fluorescence Genetic Goals Herpes zoster disease Herpesviridae Human Human Herpesvirus 2 Human Herpesvirus 8 Infection Infectious Mononucleosis Kaposi Sarcoma Knock-out Lead Libraries Life Cycle Stages Maps Measures Modeling Molecular Conformation Monitor NMR Spectroscopy Peptide Hydrolases Permeability Pharmaceutical Chemistry Pharmaceutical Preparations Protease Inhibitor Protein Region Recombinants Resolution Retinitis Serine Protease Specificity Structure Testing Validation Viral Virus Western Blotting X-Ray Crystallography abstracting analog base combinatorial chemistry computational chemistry design dimer effective therapy genital herpes high throughput screening improved inhibitor/antagonist interest interfacial member mimetics monomer novel novel strategies prevent protein protein interaction public health relevance scaffold screening small molecule success therapeutic target therapy resistant

项目摘要

DESCRIPTION (provided by applicant): The goal of this R01 project is to identify allosteric inhibitors that trap inactive conformations of a family of proteolytic enzymes and have sufficient pharmacologic viability to serve as the starting point for lead optimization and animal studies. These studies seek to exploit dynamics associated with monomer-dimer equilibrium to offer a new approach for developing specificity against this class of protease targets that has so far been recalcitrant to selective inhibitors. Protein-protein interactions are ubiquitous in biology ad represent potential therapeutic targets for numerous diseases. The dimeric human herpes virus (HHV) proteases are one such example. HHVs make up one of the most prevalent viral families and are the etiological agents to a variety of devastating human illnesses for which there is lack of specific and effective treatments. As with all infectious diseases, resistance to therapy is constantly evolving and new therapies are needed for this virus family. There is significant interest in new viral protease inhibitors based on the recent success of antiproteolytic therapies. All HHVs express a dimeric serine protease that is essential to the viral life cycle. Genetic knockout of this protease in cell culture prevents viral replication, providing genetic validation f the target. We have identified a small molecule inhibitor of the protease of one member of this family, Kaposi's sarcoma-associated herpes virus (KSHV), by screening a biased helical mimetic library. By integrating multiple chemical-biology approaches we have determined a "dimer disruption via monomer trap" mode of inhibition and mapped the binding site to a previously unreported allosteric pocket at the protease dimer interface. Recent chemistry efforts have improved potency and permeability while further informing mode of binding. Considering the structural and functional homology among HHV proteases, we propose to use diverse screening approaches and structure-based inhibitor design to develop allosteric inhibitor scaffolds that target protease dimerization or other allosteric sites in herpes virus proteases. These assays, including cell culture assays for viral infectivity, are currently in place for KSHV protease and CMV protease and will be established for other HHV proteases. We hypothesize that allosteric inhibitors of HHV proteases that trap inactive protease conformations can be identified and used to develop pharmacologically-viable compounds that prevent viral replication in cell-based assays. Aim 1. Develop inhibitory scaffolds for HHV proteases using screening and structure-guided chemistry to achieve nanomolar inhibition and improved cell membrane permeability. Aim 2. Characterize the specificity and binding mode of screening hits using NMR spectroscopy and crystallography and select allosteric inhibitors with a broad spectrum of activity towards KSHV, CMV, and HSV-2 proteases. Aim 3. Determine the mechanism of action of selected inhibitors in herpes viral cell culture models.

描述（由申请人提供）：该 R01 项目的目标是确定变构抑制剂，这些抑制剂可捕获蛋白水解酶家族的非活性构象，并具有足够的药理学活性，可作为先导化合物优化和动物研究的起点。这些研究试图利用与单体-二聚体平衡相关的动力学，提供一种新方法来开发针对此类蛋白酶靶标的特异性，迄今为止，这些蛋白酶靶标对选择性抑制剂来说是难以抵抗的。蛋白质-蛋白质相互作用在生物学中无处不在，代表着许多疾病的潜在治疗靶点。二聚体人类疱疹病毒 (HHV) 蛋白酶就是这样的例子之一。 HHV 是最流行的病毒家族之一，是多种毁灭性人类疾病的病原体，而这些疾病缺乏特异性和有效的治疗方法。与所有传染病一样，对治疗的耐药性在不断演变，该病毒家族需要新的疗法。基于最近抗蛋白水解疗法的成功，人们对新型病毒蛋白酶抑制剂产生了浓厚的兴趣。所有 HHV 都表达对病毒生命周期至关重要的二聚丝氨酸蛋白酶。在细胞培养物中对这种蛋白酶进行基因敲除可防止病毒复制，从而为目标提供基因验证。我们通过筛选有偏差的螺旋模拟文库，鉴定出了卡波西肉瘤相关疱疹病毒（KSHV）这一家族成员之一的蛋白酶的小分子抑制剂。通过整合多种化学生物学方法，我们确定了“通过单体陷阱破坏二聚体”的抑制模式，并将结合位点映射到蛋白酶二聚体界面处先前未报告的变构口袋。最近的化学努力提高了效力和渗透性，同时进一步了解了结合模式。考虑到HHV蛋白酶之间的结构和功能同源性，我们建议使用不同的筛选方法和基于结构的抑制剂设计来开发针对疱疹病毒蛋白酶中的蛋白酶二聚化或其他变构位点的变构抑制剂支架。这些检测，包括病毒感染性的细胞培养检测，目前已针对 KSHV 蛋白酶和 CMV 蛋白酶进行了检测，并将针对其他 HHV 蛋白酶进行检测。我们假设可以识别捕获非活性蛋白酶构象的 HHV 蛋白酶变构抑制剂，并将其用于开发药理学上可行的化合物，以防止细胞检测中的病毒复制。目标 1. 使用筛选和结构引导化学方法开发 HHV 蛋白酶的抑制支架，以实现纳摩尔级抑制并改善细胞膜通透性。目标 2. 使用 NMR 波谱和晶体学表征筛选命中的特异性和结合模式，并选择对 KSHV、CMV 和 HSV-2 蛋白酶具有广谱活性的变构抑制剂。目标 3. 确定所选抑制剂在疱疹病毒细胞培养模型中的作用机制。