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: 9206174
• 负责人: ROBERT Colin LIDDINGTON
• 金额: $ 42.92万
• 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/9206174
• 关键词: Ablation; Affect; Age related macular degeneration; Back; Bacteria; Binding; Biological Assay; Biophysics; Blindness; Calorimetry; Cells; Collaborations; Complement; Complement 3a; Complement Activation; Complement Membrane Attack Complex; Crystallization; Cytolysis; Data; Degenerative Disorder; Deposition; Development; Disease; Disease model; Epitopes; Evolution; Future; Gap Junctions; Genetic; Genetic Polymorphism; Goals; Hemolysis; Human; In Vitro; Inflammation; Lasers; Libraries; Link; Lytic; Measures; Mediating; Membrane; Modeling; Molecular; Molecular Conformation; 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; feeding; gene therapy; high throughput screening; high throughput technology; immune function; in vivo; inhibitor/antagonist; insight; medical schools; membrane activity; mouse model; mutant; novel; novel therapeutics; protein complex; 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)：这项提案集中于膜-裂解膜攻击复合体(MAC)在老年性黄斑变性(AMD)病理中的作用，AMD是一种使人衰弱的疾病，是导致失明的主要原因。在人类中，MAC在进化过程中一直是保守的，但它现在的作用包括破坏敏感的宿主细胞。我们先前和正在进行的关于MAC组件的高分辨率结构的工作，以及它们对MAC组装的洞察，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，他是高通量筛查和分析开发方面的专家，他在桑福德-伯纳姆校区内的一个主要小分子发现中心(“Prebys中心”)工作。我们已经证明，MAC组装需要每个MAC成分(C6-C9)中的构象变化，当它加入(和增加)新生的孔隙时，从紧凑的自动抑制构象转换到高度伸展的状态。对于目标1，我们假设与MAC蛋白结合并稳定其紧密构象的小分子可以调节这一过程，抑制或促进孔的形成。使用一个小型的商业筛选和高通量的“蛋白质热转移”方法，我们已经证明了可行性。因此，在1280个化合物中，有12个显示出显著的ΔTm(≥1.5°C)，我们已经在基于细胞的功能测试中测试了其中6个。在这6个化合物中，我们发现了2个MAC孔形成的抑制剂和2个激活剂，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是第一个被招募到MAC孔中的，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，以及最有希望的化合物在3？分辨率或更高分辨率下的共结晶，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，以及对机制的有力线索。结构洞察力将反馈到目标1以指导进一步筛选，并将反馈到目标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和目标2中描述的MAC沉积的抑制剂和激活剂来定义MAC在AMD小鼠模型中的作用。这一目标的长期重点将放在MAC的小分子抑制剂上，因为这些药物可能为撕裂AMD的新疗法提供线索，从而为我们下一阶段的研究奠定基础。最有效的化合物将反馈到AIMS 1和AIMS 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~700,000化合物库中选择大规模筛选(~50,000个)。我们希望发现具有高效率和选择性的小分子，并预测这里产生的机制洞察力和新颖的探针将与其他与MAC有关的退行性疾病相关。