Allosteric Inhibition of a Family of Proteolytic Enzymes

描述（由申请人提供）：该R 01项目的目标是鉴定变构抑制剂，这些抑制剂可以捕获蛋白水解酶家族的失活构象，并具有足够的药理学活性，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，以提供一种新的方法来开发针对这类蛋白酶靶标的特异性，这类蛋白酶靶标迄今为止一直被选择性抑制剂所排斥。蛋白质-蛋白质相互作用在生物学中是普遍存在的，并且是许多疾病的潜在治疗靶点。二聚体人疱疹病毒（HHV）蛋白酶就是一个这样的例子。HHV构成最普遍的病毒家族之一，并且是各种破坏性人类疾病的病原体，对于这些疾病缺乏特异性和有效的治疗。与所有感染性疾病一样，对治疗的耐药性不断演变，需要针对该病毒家族的新疗法。基于最近抗蛋白水解疗法的成功，人们对新的病毒蛋白酶抑制剂产生了极大的兴趣。 所有HHV都表达病毒生命周期所必需的二聚丝氨酸蛋白酶。在细胞培养物中基因敲除这种蛋白酶可以防止病毒复制，为靶标提供基因验证。我们已经确定了一个小分子抑制剂的蛋白酶的一个成员，卡波西肉瘤相关的疱疹病毒（KSHV），通过筛选一个有偏见的螺旋模拟库。通过整合多种化学-生物学方法，我们已经确定了“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道的变构口袋。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开发变构抑制剂支架，靶向蛋白酶二聚化或其他变构位点的疱疹病毒蛋白酶。这些试验，包括用于病毒感染性的细胞培养试验，目前已用于KSHV蛋白酶和CMV蛋白酶，并将用于其他HHV蛋白酶。我们假设，可以鉴定出捕获非活性蛋白酶构象的HHV蛋白酶的变构抑制剂，并用于开发在基于细胞的测定中防止病毒复制的药理学上可行的化合物。目标1。使用筛选和结构指导化学开发HHV蛋白酶的抑制性支架，以实现纳摩尔抑制和改善细胞膜通透性。目标2.使用NMR光谱学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：确定选定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.