Targeting Allosteric Studies to Modulate Protein Interactions and Function

描述(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干扰蛋白质相互作用和抑制蛋白质功能的小分子。该方法还将提供有关目标蛋白上分子结合位置的关键信息。作为原则证明，我们在疱疹病毒家族中确定了一种二聚体病毒蛋白水解酶的干扰物，它具有重要的临床意义，但到目前为止被证明是一个难以处理的靶点。许多病毒蛋白水解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。我们先前已经使用化学生物学和核磁共振相结合的方法证明了二聚体界面上诱导的螺旋开关结构调节Kaposi肉瘤疱疹病毒(KSHV)蛋白酶的活性。为了测试我们建议的筛选范式，我们将执行以下具体目标：目标1.调整和验证现有的基于荧光的蛋白酶分析，用于高通量筛选(HTS)格式，以识别活性位点抑制物和二聚体干扰物。目的II.用HTS荧光法从几个高潜力库中筛选20多万个KSHV蛋白水解酶抑制物，并鉴定疱疹病毒蛋白水解酶的变构抑制物。目的III.用13C-Met标记的蛋白酶的2D核磁共振表征AIM II中鉴定的HITS的结合部位，并用15N-特异性标记的蛋白酶的2D核磁共振确定新的抑制剂的结合部位。在完成时，我们将在寡聚酶系统中展示强大的原理证明，并将识别出一小部分抑制分子。其中一些是针对某个家庭成员的，而另一些则是对整个家庭的广谱抑制。所有的都将是有效的(微米)，并将根据它们的结合部位(活性部位、二聚体界面或变构部位)进行表征，以供后续开发。合作已经到位，以提供偏向于成功的库。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类药物小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质药物靶点，这些靶点以前被证明是药物发现努力的棘手问题，并将提供一种新的药物发现方法，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.