Molecular biophysics of integrin activation by oxysterols and rational discovery of small-molecule modulators

氧甾醇激活整合素的分子生物物理学和小分子调节剂的合理发现

• 批准号: 10255990
• 负责人: Senthil Kumar Natesan
• 金额: $ 29.07万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-10 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10255990
• 关键词: 25-hydroxycholesterol; Affect; Affinity; Anti-Inflammatory Agents; Area; Autoimmune Diseases; Binding; Binding Sites; Biochemical; Biological; Biological Response Modifiers; Biophysics; Brain; Brain Diseases; Cardiovascular Diseases; Cell model; Cells; Central Nervous System Degenerative Diseases; Cholesterol; Communicable Diseases; Computational Technique; Data; Degenerative Disorder; Development; Disease; Docking; Extracellular Matrix; Extracellular Matrix Proteins; Focal Adhesion Kinase 1; Free Energy; Goals; Homeostasis; Human body; Immune; Immune System Diseases; Immune system; Immunotherapy; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Integrin alpha5beta1; Integrins; Interleukin-6; Knowledge; Ligands; Lipids; Malignant Neoplasms; Mediating; Membrane; Mission; Modeling; Modification; Molecular; Molecular Conformation; Natural Immunity; Oxides; Pathologic; Pathologic Processes; Pathway interactions; Pattern recognition receptor; Pharmaceutical Preparations; Physiological Processes; Play; Production; Property; Public Health; Publications; RGD (sequence); Regulation; Research; Role; Signal Transduction; Site; Solvents; Specificity; Structure; Surface; TNF gene; Therapeutic; Thrombosis; Time; United States National Institutes of Health; Virus Activation; Virus Diseases; adaptive immunity; base; biophysical techniques; combat; cytokine; immune activation; in silico; in vivo; in vivo Model; in vivo evaluation; innovation; insight; interdisciplinary approach; macrophage; molecular dynamics; molecular recognition; mouse model; novel; pharmacophore; prevent; response; screening; simulation; small molecule; small molecule therapeutics; therapeutic target; virtual screening

PROJECT SUMMARY/ABSTRACT Oxysterols are oxygenated metabolites of cholesterol formed in the human body and are involved in a plethora of physiological and pathological processes such as lipid homeostasis, inflammation, innate and adaptive immunity, cancer, and brain degenerative diseases. Specifically, 25-hydroxycholesterol (25HC) is now established as an important regulator of the immune system, and is produced by immune cells in response to viral infection and activation of pattern recognition receptors. Recently, we uncovered a novel cellular mechanism of 25HC-mediated regulation of the proinflammatory response. We showed that 25HC amplifies the activation of immune cells and increases the production of immune mediators such as TNF and IL-6, by directly binding to αvβ3 and α5β1 integrins and activating the integrin-focal adhesion kinase pathway. We also discovered that 25HC binds to integrins at a novel binding site (site 2), distinct from the site where the extracellular matrix (ECM) ligands containing an Arg-Gly-ASP (RGD) motif are known to bind. Binding of 25HC at site 2 produces significant conformational changes in the specificity-determining loop (SDL) of integrins, near the RGD-binding site. The effect of such conformational changes in the SDL on the binding of ECM ligands, as well as the basis of 25HC- mediated allosteric signaling mechanism underlying integrin activation, are not known. Our hypothesis is that binding of 25HC to integrins at site 2 triggers conformational changes in the SDL that result in efficient binding of ECM ligands producing further modification of innate inflammatory response. We also hypothesize that small molecule modulators blocking 25HC-integrin interaction would serve as an efficient anti-inflammatory therapeutic strategy to combat various inflammatory diseases. Accordingly, the central objective of this proposal is to elucidate the molecular mechanisms of integrin activation by oxysterols and to identify selective small molecule modulators targeting site 2 of integrins for potential therapeutic applications. The objective of this project will be accomplished by the following three specific aims: 1) elucidate the molecular basis and conformational dynamics of integrin activation by 25HC; 2) examine the molecular recognition of integrins by non-25HC oxysterols; and 3) identify and evaluate small-molecule modulators targeting the 25HC binding site of integrins. We will utilize state-of-the-art computational techniques such as molecular docking, molecular dynamics simulations, and pharmacophore-based virtual screening to delineate the structural basis and conformational dynamics involved in activation of integrins and to identify high affinity ligands for site 2 of integrins. In addition, our well-established in vitro and in vivo models will be employed to validate our in silico findings and evaluate top ligands. Our multidisciplinary approach is innovative and together, the proposed studies will have a broad impact by offering fundamental insights to the interplay between oxysterols and integrins that culminates in amplification of the inflammatory response. Furthermore, this project will identify one or more modulators of integrin-25HC interactions, thereby advancing potential anti-inflammatory therapies for immunologic and infectious diseases.

项目摘要/摘要 氧化酚是在人体中形成​​的胆固醇的氧化代谢物，并参与了很多 物理和病理过程，例如脂质稳态，炎症，先天和适应性 免疫，癌症和脑退化性疾病。具体而言，现在是25-羟基胆固醇（25HC） 作为免疫系统的重要调节剂，由免疫细胞产生 病毒感染和模式识别受体的激活。最近，我们发现了一种新型的细胞机制 促炎反应的25HC介导的调节。我们证明了25hc放大器的激活 免疫细胞并通过直接结合与 αVβ3和α5β1整联蛋白并激活整联蛋白 - 邻核激酶途径。我们还发现 25HC与新型结合位点（位点2）的整合素结合，与细胞外基质（ECM）的位点不同 已知包含arg-gly-asp（RGD）基序的配体已知结合。在现场2的25HC结合产生明显的 RGD结合位点附近整合素的特异性确定环（SDL）的构象变化。这 SDL中这种构象变化对ECM配体结合的影响以及25HC-的基础 整合素激活的基础介导的变构信号传导机制尚不清楚。我们的假设是 在现场2 HC与整联蛋白的结合2触发SDL的咨询变化，从而导致有效结合 ECM配体产生先天炎症反应的进一步修饰。我们还假设那很小 分子调节剂阻断25HC-整合素相互作用将作为有效的抗炎疗法 打击各种炎症性疾病的策略。彼此之间，该提议的核心目标是 阐明氧甲醇激活整合素激活的分子机制，并鉴定选择性小分子 针对整联蛋白的位点2的调节剂，用于潜在的治疗应用。该项目的目的将是 通过以下三个特定目的完成：1）阐明分子基础和构象动力学 25HC的整联蛋白激活； 2）检查非25HC氧甲醇对整联蛋白的分子识别；和 3）识别和评估靶向整合素25HC结合位点的小分子调节剂。我们将利用 最先进的计算技术，例如分子对接，分子动力学模拟和 基于药效团的虚拟筛选，以描绘涉及的结构基础和构象动态 在激活整联蛋白并鉴定整联蛋白的位点2的高亲和力配体。此外，我们已建立的 体外和体内模型将被雇用以验证我们的硅酸盐发现并评估顶部配体。我们的 多学科的方法是创新的，拟议的研究将通过提供广泛的影响。 对氧化酚和整合素之间相互作用的基本见解，这些结合在扩增中达到高潮 炎症反应。此外，该项目将确定一个或多个整合素25HC的调节剂 相互作用，从而推进免疫和传染病的潜在抗炎疗法。