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。亲环素和FKBP是一大类肽基脯氨酸异构酶的成员 （PPIases），并在现代医学中发挥核心作用，临床应用于免疫抑制， 传染病和化疗。尽管几十年的努力揭示了 由于亲免素酶的特性，这个家族的许多成员，包括Cyp 40，还没有达到关键的 治疗检查水平。最近，我们的团队描述了一种使用珠子快速增强 发现针对蛋白质内特定结构域的探针。在这个项目中，我们的团队开发了 方法，作为一种技术，发现分子探针，研究神经作用的免疫亲。的 细胞外淀粉样蛋白斑块和细胞内神经纤维缠结（tau），它们定义了 阿尔茨海默病都是由大的免疫亲。亲环素40（Cyp 40），FK 506结合 蛋白FKBP 51和FKBP 52起重要但拮抗的作用。Cyp 40解聚α-突触核蛋白， 过表达挽救了tau诱导的认知缺陷并解开了神经毒性斑块。Cyp 40和 FKBP 51促进微管的稳定，而FKBP 52已被证明会引起记忆 在tau转基因小鼠模型中的损伤和促进tau发病机制。一个可能的机制包括 Cyp 40和FKPB 52在调节微管解聚中所起的相反作用。FKBP 52具有 已显示通过其TPR结构域的直接结合来结合和抑制微管蛋白形成。作为氨基酸 如果这三个大的亲免蛋白TPR结构域之间的同一性小于26%，则将有可能鉴定 区分Cyp 40、FKBP 51和FKBP 52的TPR结构域的小分子结合剂。 调节大的免疫亲素的作用将为治疗神经退行性疾病提供一种新的治疗形式。 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 使用微珠的工作流程，称为共聚焦纳米扫描（CONA），作为识别和验证荧光 以高或中等亲和力结合Cyp 40、FKBP 51或FKBP 52的天然产物。所得材料 然后用于建立细胞成像的方法，并为每种细胞建立高通量筛选。 这些大的免疫亲素。

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