Cheminformatic Discovery of Alternative Pathway C3 Pro-Convertase Inhibitors

说明(申请人提供)：虽然补体是先天免疫系统的支柱，但不适当或不受控制的补体激活水平是导致人类迅速增长的炎症性疾病的主要因素。然而，尽管补体反应的治疗操作在临床上非常重要，但只有有限的FDA批准的药物和适应症可以用于特定的靶向 补体系统中的主动成分。补体靶向治疗药物缺乏的一个主要因素是蛋白质的大小和补体活性基础上的分子识别事件的复杂性。这使得科学家很难确定哪些特定的点最容易受到治疗干预的影响。相比之下，细菌病原体金黄色葡萄球菌部署了一系列在宿主/病原体共同进化过程中自然选择和精心优化的蛋白质，以有效地阻止补体级联中的基本事件--特别是驱动补体反应放大的多亚基AP C3转换酶的形成。因此，模拟葡萄球菌毒力蛋白活性的分子有望用于补体导向的抗炎治疗。我们最近研究了使用新的化学信息学工具来识别与金黄色葡萄球菌补体抑制剂SCIN家族相同的C3b位点的小分子的可能性。尽管这项工作是在中试规模上完成的，但这项工作强烈表明，在电子计算机中，方法是筛选新的化学类型的类药物化合物的可行手段，这些化合物专门针对C3b上的重要功能位点。在这项研究中，我们的目标是应用相同的化学信息学方法来筛选高度复杂的(约2000万)化合物文库，并鉴定与C3b位点结合的小分子，这些小分子在AP C3转换酶形成的初始步骤中具有已知的作用。这一步骤产生一种被称为AP C3前转换酶的结构，是被多个分泌型金黄色葡萄球菌补体抑制剂家族(包括SCIN)破坏的途径中的同一点。因此，我们认为AP C3前转换酶的形成是补体介导的炎症反应中药物干预的生物学验证的靶点。本申请中描述的研究计划由三个不同的具体目标组成。每个目标都有一个明确的结果，这些结果要么基于与该项目直接相关的初步研究，要么基于我们以前使用类似方法的经验。在第一个目标中，我们将使用化学信息学来鉴定~100个命中化合物，这些化合物可能是C3b上四个靶点之一的结合子，已知在AP C3前转换酶的形成中发挥作用。然后，在第二个目标中，我们将使用现有的生化和功能工具来验证大约20个既结合C3b又抑制AP活性的候选者。在FINA的目标中，我们将使用结构比较来确定这些经过验证的候选化合物中的药效团，并对我们最有希望的~5化合物系列进行有限的合成扩展。在这个目标的结束时，我们将确定一个单一的，无毒的化合物，将作为我们的AP C3前转换酶组装的主要抑制物。因此，这项工作将为未来开发新的补体靶向抗炎疗法奠定基础。

DESCRIPTION (provided by applicant): Although complement serves as a pillar of the innate immune system, inappropriate or uncontrolled levels of complement activation are a major contributor to a rapidly growing list of human inflammatory diseases. Yet even though therapeutic manipulation of the complement response would be tremendously important in the clinic, only a limited panel of FDA approved drugs and indications exist for specifically targeting an active component within the complement system. A major factor underlying this dearth of complement-targeted therapeutics is the large size of the proteins and complex nature of the molecular recognition events which underlie complement activity. This has made it very challenging for scientists to identify which specific points are most susceptible for therapeutic intervention. By contrast, the bacterial pathogen Staphylococcus aureus deploys an array of proteins that have been naturally selected and exquisitely optimized during the course of host/pathogen co-evolution to efficiently block fundamental events within the complement cascade - in particular formation of the multi-subunit AP C3 convertase that drives amplification of the complement response. As a consequence, molecules which mimic the activities of staphylococcal virulence proteins hold promise for complement-directed, anti-inflammatory therapeutics. We recently investigated the potential of using novel cheminformatic tools for identifying small molecules that bind the same C3b site as the SCIN family of S. aureus complement inhibitors. Although done on a pilot scale, this work strongly suggested that in silico methods are a viable means of screening for new chemotypes of drug-like compounds that specifically target functionally significant sites on C3b. In this investigation, our goal is to aply the same cheminformatic methods to screen a highly elaborated (~20 million) compound library and to identify small molecules that bind C3b sites with known roles in the initial step of AP C3 convertase formation. This step, which generates a structure known as the AP C3 pro-convertase, is the same point in the pathway that is disrupted by multiple families of secreted S. aureus complement inhibitors (including SCINs). Thus, we believe that formation of the AP C3 pro-convertase is a biologically validated target for pharmacological intervention in the complement-mediated inflammatory response. The research plan described in this application consists of three distinct specific aims. Each aim has a defined outcome that is based upon either preliminary studies directly relevant to this project or our previous experience in using similar methodologies. In the first aim, we will use cheminformatics to identify ~100 hit compounds that are likely binders at one of four target sites on C3b with known roles in AP C3 pro-convertase formation. Then, in the second aim, we will use established biochemical and functional tools to validate ~20 candidates that both bind C3b and inhibit AP activity. In the fina aim, we will use structural comparisons to identify the pharmacophores in these validated candidates and conduct a limited synthetic expansion of our most promising ~ 5 compound series. At the conclusion of this aim, we will have identified a single, non-toxic compound that will serve as our lead inhibitor of AP C3 pro-convertase assembly. As a consequence, this work will lay the foundation for future development of new classes of complement-targeted anti-inflammatory therapeutics.