Targeting Allosteric Studies to Modulate Protein Interactions and Function

描述（由申请人提供）：我们的目标是开发一种筛选方法，可用于快速有效地识别破坏蛋白质相互作用并抑制蛋白质功能的小分子。该方法还将提供有关分子在靶蛋白上的结合位点的关键信息。作为原理证明，我们在疱疹病毒家族中发现了一种二聚体病毒蛋白酶的干扰物，它具有重要的临床意义，但迄今为止已被证明是一个棘手的靶标。许多病毒蛋白酶药物靶点是二聚体，包括疱疹病毒家族中的那些，也可以被二聚体破坏所抑制。我们之前已经证明，在二聚体界面的螺旋开关的诱导结构调节卡波西肉瘤疱疹病毒（KSHV）蛋白酶的活性，使用化学生物学和核磁共振的组合。为了测试我们提出的筛选范例，我们将执行以下具体目标：目标1 .适应和验证现有的基于荧光的蛋白酶测定，用于高通量筛选（HTS）格式，以识别活性位点抑制剂和二聚体干扰物。目的二世。使用HTS荧光法从几个高潜力文库中筛选超过20万种KSHV蛋白酶抑制剂，并鉴定疱疹病毒蛋白酶的变构抑制剂。第三目标。利用13C-Met标记的蛋白酶的二维核磁共振表征Aim II中发现的hit的结合位点，并利用15n特异性标记的蛋白酶的二维核磁共振确定新型抑制剂的结合位点。在完成后，我们将在寡聚酶系统中展示一个强大的原理证明，并将确定一小部分抑制分子。其中一些“打击”将特定于一个家庭成员，而其他将是整个家庭的广谱抑制剂。所有这些都是有效的（<微米），并将根据其结合位点（活性位点，二聚体界面或变构位点）进行表征，以供后续开发。协作是为了提供偏向于成功的图书馆。我们的长期目标是开发一种通用策略，通过靶向变构位点来调节蛋白质与小药物样分子的相互作用和功能。这里开发的技术将普遍适用于许多可溶性蛋白药物靶点，这些靶点以前被证明是药物发现工作的难点，并将为药物发现提供新的方法，开辟新的治疗途径。

DESCRIPTION (provided by applicant): Our goal is to develop a screening methodology that can be used to rapidly and efficiently identify small molecules that disrupt protein interactions and inhibit the function of the protein. The methodology will also provide critical information regarding the binding site of the molecule on the target protein. As proof-of- principle, we identified a disrupter of a dimeric viral protease in the herpes virus family that has important clinical relevance yet has thus far proven to be an intractable target. Numerous viral protease drug targets are dimeric, including those in the herpes virus family and could also be inhibited by dimer disruption. We have previously shown that induced structure of a helical switch at the dimer interface regulates activity of the Kaposi's sarcoma herpes virus (KSHV) protease using a combination of chemical biology and NMR. To test our proposed screening paradigm we will carry out the following specific aims: Aim I. Adapt and validate existing fluorescence-based protease assays for high throughput screening (HTS) format to identify active site inhibitors and dimer disrupters. Aim II. Screen more than 200,000 compounds from several high potential libraries for KSHV protease inhibitors using HTS fluorescence assays and identify allosteric inhibitors of herpes virus proteases. Aim III. Characterize binding sites of hits identified in Aim II using 2D NMR of 13C-Met labeled protease and determine the binding sites of novel inhibitors using 2D NMR of 15N-specifically labeled protease. At the completion, we will have demonstrated a robust proof-of-principle in an oligomeric enzyme system and will have identified a small subset of inhibitory molecules. Some of these "hits" will be specific for a family member while others will be broad-spectrum inhibitors of the entire family. All will be potent (<microM), and will be characterized in terms of their binding sites (active site, dimer interface, or allosteric site) for subsequent development. Collaborations are in place to provide libraries that are biased for success. Our long-term goal is to develop a general strategy for modulating protein interactions and function with small drug-like molecules by targeting allosteric sites. The techniques developed here will have general applicability to many soluble protein drug targets, which previously have proven intractable to drug discovery efforts and will offer a new approach to drug discovery, opening up new therapeutic avenues.