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：
9039629
负责人：
Charles Scott Craik
金额：
$ 30.67万
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 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9039629
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 Health 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 Virus Replication 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 nanomolar novel novel strategies novel therapeutics prevent protein protein interaction scaffold screening small molecule inhibitor 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项目的目的是确定蛋白水解酶家族的无效构象的变构抑制剂，并具有足够的药理生存能力，可以作为铅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，以提供针对此类蛋白酶靶标的新方法，这些蛋白酶靶标一直是选择性抑制剂的顽固性。蛋白质 - 蛋白质相互作用在生物学广告中无处不在，代表了许多疾病的潜在治疗靶标。二聚体人疱疹病毒（HHV）蛋白酶就是这样的例子。 HHV构成了最普遍的病毒家族之一，是各种毁灭性人类疾病的病因学药，缺乏特定有效的治疗方法。与所有感染性疾病一样，对治疗的抵抗力不断发展，该病毒家族需要新的疗法。基于最近的抗蛋白水解疗法的成功，人们对新的病毒蛋白酶抑制剂引起了浓厚的兴趣。所有HHV都表示对病毒生命周期至关重要的二聚体丝氨酸蛋白酶。该蛋白酶在细胞培养中的遗传敲除可以防止病毒复制，从而提供了遗传验证的靶标。我们通过筛选一个有偏见的螺旋模拟物库，确定了该家族一个家族成员蛋白酶（KAPOSI）相关的疱疹病毒（KSHV）的蛋白酶的小分子抑制剂。通过整合多种化学生物学方法，我们确定了抑制作用的“通过单体陷阱的二聚体破坏”模式，并将结合位点映射到蛋白酶二聚体界面的先前未报告的变构袋中。最近的化学工作提高了效力和渗透性，同时进一步告知了结合方式。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靶向蛋白酶二聚化或疱疹病毒蛋白酶中其他变构位点的变构抑制剂支架。这些测定法，包括病毒感染性的细胞培养试验，目前已针对KSHV蛋白酶和CMV蛋白酶进行，并将用于其他HHV蛋白酶。我们假设可以鉴定出诱捕非活性蛋白酶构型的HHV蛋白酶的变构抑制剂，并用于开发可预防基于细胞的测定中病毒复制的药理学可行化合物。 AIM 1。使用筛选和结构引导的化学为HHV蛋白酶开发抑制性支架，以实现纳摩尔抑制和改善的细胞膜渗透性。 AIM 2。使用NMR光谱和晶体学表征筛选的特异性和结合模式，并选择具有针对KSHV，CMV和HSV-2蛋白酶活性广泛的变构抑制剂。目标3。确定疱疹病毒细胞培养模型中选定抑制剂的作用机理。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

DOI：
10.1371/journal.pntd.0006446
发表时间：
2018-04
期刊：
PLoS neglected tropical diseases
影响因子：
3.8
作者：
Leontovyč A;Ulrychová L;O'Donoghue AJ;Vondrášek J;Marešová L;Hubálek M;Fajtová P;Chanová M;Jiang Z;Craik CS;Caffrey CR;Mareš M;Dvořák J;Horn M
通讯作者：
Horn M