Targeting the large cyclophilins through an autonomous CONA assay

通过自主 CONA 测定靶向大亲环蛋白

• 批准号: 10509443
• 负责人: James J La Clair
• 金额: $ 27.5万
• 依托单位: XENOBE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-02 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10509443
• 关键词: Affinity; Alzheimer' s Disease; Amino Acids; Binding; Biological; Biological Assay; Cells; Clinical; Cognitive deficits; Communicable Diseases; Cyclophilins; Development; Enzymes; Equipment; FK506 binding protein 5; Family; Family member; Fluorescence; Fluorescent Dyes; Fluorescent Probes; Goals; Human; Imaging Device; Immunophilins; Immunosuppression; Isomerase; Ligands; Measurement; Memory impairment; Methods; Microtubule Stabilization; Microtubules; Modern Medicine; Modernization; Molecular; Molecular Probes; Natural Products; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurologic; Neurons; Pathogenesis; Pharmaceutical Preparations; Play; Proteins; Reagent; Reporting; Resources; Role; Senile Plaques; Series; System; Tacrolimus Binding Proteins; Techniques; Testing; Therapeutic; Transgenic Mice; Translating; Tubulin; alpha synuclein; base; brain tissue; cellular imaging; chemotherapy; clinical application; cyclophilin D; extracellular; high reward; high risk; high throughput screening; imaging agent; inhibitor; member; mouse model; nervous system disorder; neuropathology; neurotoxic; overexpression; programs; screening; small molecule; survivin; tacrolimus binding protein 4; targeted agent; tau Proteins; therapeutic target; tool

Project Summary. Cyclophilins and FKBPs are members of a large class of Peptidyl-Proline Isomerases (PPIases) and play a central role in modern medicine with clinical applications in immunosuppression, infectious disease, and chemotherapy. Although decades of effort have revealed the remarkable structural features of immunophilin enzymes, many members of this family, including Cyp40, have yet to meet the critical level of therapeutic examination. Recently our team described a system that uses beads to rapidly enhance the discovery of probes that target specific domains within a protein. In this program our team develops this method, as a technique to discover molecular probes to study the neurological roles of the immunophilins. The extracellular amyloid plaques and the intracellular neurofibrillary tangles (tau) that define the neuropathology of Alzheimer's disease are both regulated by the large immunophilins. Cyclophilin 40 (Cyp40), FK506-binding proteins FKBP51 and FKBP52 play important, but antagonistic roles. Cyp40 disaggregates α-synuclein and overexpression rescues tau-induced cognitive deficits and unravels neurotoxic plaques. Both Cyp40 and FKBP51 promote the stabilisation of microtubules while FKBP52 has been shown to cause memory impairments in a tau transgenic mouse model and to promote tau pathogenesis. A likely mechanism involves the opposing roles played by Cyp40 and FKPB52 in regulating microtubule depolymerisation. FKBP52 has been shown to bind and inhibit tubulin formation via direct binding by its TPR domain. As the amino acid identity between these three large immunophilin TPR domains is less than 26%, it will be possible to identify small molecule binders that distinguish between the TPR domains of Cyp40, FKBP51 and FKBP52. Modulating the effects of the large immunophilins will provide a new form of therapy to treat neurodegenerative diseases caused by plaque formation including Alzheimer's disease. Here, our team adapts an optimized workflow that uses beads, called Confocal Nanoscanning (CONA) as a tool to identify and validate fluorescent natural products that bind with high or medium affinity to Cyp40, FKBP51 or FKBP52. The resulting materials are then used to establish methods for cellular imaging as well as establish a high-throughput screen for each of these large immunophilins.

项目摘要。亲环蛋白和 FKBP 是一大类肽基脯氨酸异构酶的成员 （PPIs）并在现代医学中发挥着核心作用，在免疫抑制方面具有临床应用， 传染病、化疗。尽管几十年的努力已经揭示了非凡的结构 尽管亲免素酶的特性，但该家族的许多成员，包括 Cyp40，尚未满足关键要求 治疗检查水平。最近，我们的团队描述了一种使用珠子来快速增强 发现针对蛋白质内特定结构域的探针。在这个程序中，我们的团队开发了这个 方法，作为一种发现分子探针以研究亲免素的神经学作用的技术。这 细胞外淀粉样斑块和细胞内神经原纤维缠结 (tau) 定义了神经病理学 阿尔茨海默氏病均受大亲免素调节。亲环蛋白 40 (Cyp40)，FK506 结合 蛋白质 FKBP51 和 FKBP52 发挥着重要但相互拮抗的作用。 Cyp40 分解 α-突触核蛋白并 过度表达可以挽救 tau 诱导的认知缺陷并消除神经毒性斑块。 Cyp40 和 FKBP51 促进微管的稳定，而 FKBP52 已被证明可以引起记忆 tau 转基因小鼠模型中的损伤并促进 tau 发病机制。一个可能的机制涉及 Cyp40 和 FKPB52 在调节微管解聚中发挥相反的作用。 FKBP52有 已被证明可通过其 TPR 结构域直接结合来结合并抑制微管蛋白形成。作为氨基酸 这三个大的亲免素TPR结构域之间的同一性小于26%，将有可能识别 区分 Cyp40、FKBP51 和 FKBP52 的 TPR 结构域的小分子结合物。 调节大亲免素的作用将为治疗神经退行性疾病提供一种新的疗法 由斑块形成引起的疾病，包括阿尔茨海默病。在这里，我们的团队采用了优化的 使用称为共焦纳米扫描 (CONA) 的珠子作为识别和验证荧光的工具的工作流程 以高或中等亲和力与 Cyp40、FKBP51 或 FKBP52 结合的天然产物。所得材料 然后用于建立细胞成像方法以及为每个细胞建立高通量筛选 这些大的亲免蛋白。

项目成果

Nanoscaled Discovery of a Shunt Rifamycin from Salinispora arenicola Using a Three-Color GFP-Tagged Staphylococcus aureus Macrophage Infection Assay.

• DOI: 10.1021/acsinfecdis.3c00049
• 发表时间: 2023-08-11
• 期刊: ACS INFECTIOUS DISEASES
• 影响因子: 5.3
• 作者: Pham, Nhan T.;Alves, Joana;Sargison, Fiona A.;Cullum, Reiko;Wildenhain, Jan;Fenical, William;Butler, Mark S.;Mead, David A.;Duggan, Brendan M.;Fitzgerald, J. Ross;La Clair, James J.;Auer, Manfred
• 通讯作者: Auer, Manfred