Allosteric Inhibition of a Family of Proteolytic Enzymes

描述(由申请人提供)：该R01项目的目标是确定能捕获一系列蛋白水解酶的非活性构象并具有足够的药理活性的变构抑制剂，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来提供一种新的方法来开发针对这类到目前为止一直对选择性抑制剂顽固的蛋白酶靶标的特异性。蛋白质-蛋白质相互作用在生物学中普遍存在，是许多疾病的潜在治疗靶点。二聚体人类疱疹病毒(HHV)蛋白水解酶就是这样一个例子。HHV是最流行的病毒家族之一，是各种破坏性人类疾病的病原体，目前缺乏具体和有效的治疗方法。与所有传染病一样，对治疗的抵抗力在不断演变，这一病毒家族需要新的治疗方法。基于最近抗蛋白水解酶治疗的成功，人们对新的病毒蛋白水解酶抑制剂产生了浓厚的兴趣。 所有的人类疱疹病毒都表达一种对病毒生命周期至关重要的二聚体丝氨酸蛋白酶。在细胞培养中，这种蛋白水解酶的基因敲除可以防止病毒复制，从而提供靶标的基因验证。我们通过筛选一个偏向的螺旋模拟文库，鉴定了该家族中的一个成员--卡波西肉瘤相关疱疹病毒(KSHV)的蛋白水解酶的小分子抑制物。通过整合多种化学-生物学方法，我们确定了一种“通过单体陷阱破坏二聚体”的抑制模式，并将结合位置映射到以前未报道的蛋白酶二聚体界面的变构口袋。最近的化学努力提高了效力和渗透性，同时进一步告知结合模式。考虑到HHV蛋白酶在结构和功能上的同源性，我们建议采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，目前已经用于KSHV和CMV蛋白酶，并将建立用于其他HHV蛋白酶的检测方法。我们假设，捕获非活性蛋白酶构象的HHV蛋白酶的变构抑制剂可以被识别出来，并用于开发药理上可行的化合物，在基于细胞的检测中防止病毒复制。目的1.利用筛选和结构导向化学方法开发HHV蛋白水解酶抑制支架，以实现纳米分子抑制和改善细胞膜通透性。目的2.用核磁共振波谱和结晶学表征筛选HITS的特异性和结合模式，筛选对KSHV、CMV和HSV-2酶具有广谱活性的变构抑制剂。目的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。

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.