Interrogating the role of complement MAC in the pathogenesis of age-related macular degeneration: Structure-enhanced discovery of probes and leads for novel therapies

探究补体 MAC 在年龄相关性黄斑变性发病机制中的作用：结构增强的探针和新疗法先导化合物的发现

• 批准号: 9010453
• 负责人: ROBERT Colin LIDDINGTON
• 金额: $ 45.3万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9010453
• 关键词: Ablation; Affect; Age related macular degeneration; Back; Bacteria; Binding; Biological Assay; Blindness; Calorimetry; Cells; Collaborations; Complement; Complement 3a; Complement Activation; Complement Membrane Attack Complex; Crystallization; Cytolysis; Data; Degenerative Disorder; Deposition; Development; Disease; Epitopes; Evolution; Future; Gap Junctions; Genetic Polymorphism; Goals; Hemolysis; Housing; Human; In Vitro; Inflammation; Lasers; Libraries; Link; Lytic; Measures; Mediating; Membrane; Modeling; Molecular Conformation; Molecular Genetics; Natural Immunity; Outcome; Pathogenesis; Pathology; Pathway interactions; Phase; Population; Positioning Attribute; Process; Prodrugs; Property; Proteins; Recruitment Activity; Resistance; Resolution; Role; Serum; Severities; Specificity; Structure; Testing; Therapeutic; Titrations; Variant; Work; assay development; base; cohort; complement system; design; drug quality; feeding; gene therapy; high throughput screening; high throughput technology; immune function; in vivo; inhibitor/antagonist; insight; medical schools; mouse model; mutant; novel; novel therapeutics; public health relevance; scaffold; screening; small molecule; small molecule inhibitor; stoichiometry; structural biology

 DESCRIPTION (provided by applicant): This proposal focuses on the role of the membrane-lytic Membrane Attack Complex (MAC) in the pathology of Age-related Macular Degeneration (AMD), a debilitating disease that is a major cause of blindness. In humans, the MAC has been conserved through evolution, but its role now includes the destruction of sensitive host cells. Our prior and ongoing work on the high-resolution structures of MAC components, and the insights they provide into MAC assembly, place us in a unique position to propose and test mechanism-based approaches to discovering novel probes and inhibitors of MAC assembly. In order to approach these goals, we have formed a collaboration with Dr. Kumar-Singh at Tufts Medical School, who is an expert in murine models of AMD; and we have recruited Dr. Sergienko, an expert in high-throughput screening and assay development, who works in a major center for small-molecule discovery housed within the Sanford-Burnham campus (the "Prebys Center"). We have shown that MAC assembly requires conformational changes within each MAC component (C6-C9), switching from a compact auto-inhibited conformation to a highly extended state as it joins (and augments) the nascent pore. For Aim 1, we hypothesize that small molecules that bind to MAC proteins and stabilize their compact conformation could modulate this process and inhibit or promote pore formation. Using a small commercial screen and a high-throughput "Protein Thermal Shift" approach, we have already demonstrated feasibility. Thus, 12 out of 1280 compounds showed a significant ΔTM (≥1.5°C), and we have tested 6 of these in a functional cell-based assay. Of the 6, we discovered 2 inhibitors and 2 activators of MAC pore formation, providing encouraging support for our hypothesis. We will perform a parallel study on C6, the first recruit to the MAC pore, for which we already have a crystal structure. In Aim 2, we will study binding on the macro-scale, using Isothermal Titration Calorimetry, and co-crystallization of the most promising compounds at 3 Å resolution or better, which will provide atomic models of protein with bound compounds. This will reveal the major determinants of binding and specificity, as well as strong clues into mechanism. Structural insights will feed back to Aim 1 to guide further screening, and forward to Aim 3 for selection of compounds for in vivo studies. In Aim 3, we will define the role of the MAC in a mouse model of AMD, using inhibitors and activators of MAC deposition characterized in Aims 1 and 2. The longer-term focus of this Aim will be on small-molecule inhibitors of MAC, as these could provide leads for novel therapeutics to tear AMD, thereby framing the next phase of our studies. The most efficacious compounds will feed back to Aims 1 and 2 to guide the selection of compounds for subsequent rounds of high-throughput screening, and help guide the selection of large-scale screens (~50,000) taken from our main ~700,000 compound library. We expect to discover small molecules with high potency and selectivity, and predict that the mechanistic insights and novel probes generated here will be relevant to other degenerative diseases in which the MAC is implicated.

 描述（由申请人提供）：该提案重点关注膜溶解膜攻击复合物（MAC）在年龄相关性黄斑变性（AMD）病理学中的作用，AMD是一种使人衰弱的疾病，是失明的主要原因。在人类中，MAC 在进化中得到了保留，但它现在的作用包括破坏敏感的宿主细胞。我们之前和正在进行的关于 MAC 组件高分辨率结构的工作，以及它们为 MAC 组装提供的见解，使我们处于独特的地位，可以提出和测试基于机制的方法，以发现 MAC 组装的新型探针和抑制剂。为了实现这些目标，我们与塔夫茨医学院的 Kumar-Singh 博士建立了合作关系，他是 AMD 小鼠模型方面的专家；我们还聘请了 Sergienko 博士，他是高通量筛选和检测开发方面的专家，在桑福德-伯纳姆校区的一个主要小分子发现中心（“Prebys 中心”）工作。我们已经表明，MAC 组装需要每个 MAC 组件（C6-C9）内的构象变化，当它加入（并增强）新生孔时，从紧凑的自动抑制构象切换到高度延伸的状态。对于目标 1，我们假设与 MAC 蛋白结合并稳定其紧凑构象的小分子可以调节该过程并抑制或促进孔形成。使用小型商业屏幕和高通量“蛋白质热转移”方法，我们已经证明了可行性。因此，1280 种化合物中有 12 种显示出显着的 ΔTM (≥1.5°C)，并且我们已在基于功能细胞的测定中测试了其中的 6 种。在这 6 种物质中，我们发现了 2 种 MAC 孔形成抑制剂和 2 种激活剂，为我们的假设提供了令人鼓舞的支持。我们将对 C6 进行平行研究，C6 是 MAC 孔的第一个新成员，我们已经有了它的晶体结构。在目标 2 中，我们将使用等温滴定量热法研究宏观尺度上的结合，并以 3 Å 或更高的分辨率共结晶最有前途的化合物，这将提供具有结合化合物的蛋白质的原子模型。这将揭示结合和特异性的主要决定因素，以及机制的有力线索。结构见解将反馈至目标 1 以指导进一步筛选，并转发至目标 3 以选择用于体内研究的化合物。在目标 3 中，我们将使用目标 1 和 2 中表征的 MAC 沉积抑制剂和激活剂来定义 MAC 在 AMD 小鼠模型中的作用。该目标的长期重点将是 MAC 小分子抑制剂，因为这些抑制剂可以为撕裂 AMD 的新疗法提供线索，从而构建我们下一阶段的研究。最有效的化合物将反馈到目标 1 和 2，以指导后续几轮高通量筛选的化合物选择，并帮助指导从我们的主要约 700,000 种化合物库中选择大规模筛选（约 50,000 个）。我们期望发现具有高效力和选择性的小分子，并预测这里产生的机制见解和新颖的探针将与 MAC 涉及的其他退行性疾病相关。