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

 描述（由申请人提供）：该提案重点关注膜溶解性膜攻击复合物（MAC）在视网膜相关黄斑变性（AMD）病理学中的作用，AMD是一种使人衰弱的疾病，是致盲的主要原因。在人类中，MAC在进化过程中得到了保护，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工作的高分辨率结构的MAC组件，以及他们提供的见解到MAC组装，使我们在一个独特的位置，提出和测试机制为基础的方法，发现新的探针和抑制剂的MAC组装。为了实现这些目标，我们与塔夫茨医学院的Kumar-Singh博士合作，他是AMD小鼠模型的专家;我们还招募了Sergienko博士，他是高通量筛选和检测开发方面的专家，他在Sanford-Burnham校园内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，从一个紧凑的自抑制构象切换到一个高度扩展的状态，因为它加入（和扩增）新生孔。对于目标1，我们假设结合MAC蛋白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。使用小型商业筛选和高通量“蛋白质热位移”方法，我们已经证明了可行性。因此，1280种化合物中有12种显示出显著的ΔTM（≥1.5°C），我们在基于功能细胞的测定中测试了其中6种。在这6种中，我们发现了2种MAC孔形成的抑制剂和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孔的第一个补充物，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，以及最有前途的化合物在3 μ m分辨率或更好的条件下的共结晶，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，以及机制的有力线索。结构见解将反馈到目标1以指导进一步筛选，并转发到目标3以选择用于体内研究的化合物。在目的3中，我们将使用目的1和2中表征的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。这个目标的长期重点将是MAC的小分子抑制剂，因为这些可以为新的治疗方法提供线索，从而为我们的下一阶段研究奠定基础。最有效的化合物将反馈到目标1和2，以指导后续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化合物库中选择大规模筛选（约50，000）。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型探针将与MAC所涉及的其他退行性疾病相关。

 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.