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

项目摘要/摘要 氧化甾醇是人体内形成的胆固醇的氧化代谢物，与过量的胆固醇有关。 生理和病理过程，如脂类平衡、炎症、先天和适应性 免疫、癌症和脑退行性疾病。具体来说，25-羟基胆固醇(25HC)现在是 作为免疫系统的重要调节器，由免疫细胞产生 病毒感染和模式识别受体的激活。最近，我们发现了一种新的细胞机制 25HC介导的促炎症反应的调节。我们的研究表明，25HC能增强细胞外信号转导通路的活性 免疫细胞，增加免疫介质的产生，如通过直接结合到 αvβ3和α5β1整合素并激活整合素-粘着斑激酶通路。我们还发现， 25HC在一个新的结合部位(部位2)与整合素结合，不同于细胞外基质(ECM)的部位。 已知含有Arg-Gly-ASP(RGD)基序的配体可以结合。25HC在第2位的结合产生了显著的 RGD结合位点附近整合素的特异性决定环(SDL)的构象变化。这个 这种构象变化对ECM配体结合的影响，以及25HC- 整合素激活背后的介导变构信号机制，目前尚不清楚。我们的假设是 25HC与第2位整合素的结合触发了SDL的构象变化，从而导致有效的结合 细胞外基质配体产生对先天炎症反应的进一步修饰。我们还假设小的 阻断25HC-整合素相互作用的分子调节剂将成为有效的抗炎治疗药物 抗击各种炎症性疾病的战略。因此，这项提案的中心目标是 氧化甾醇激活整合素的分子机制研究及选择性小分子鉴定 以整合素第2位为靶点的调节剂潜在的治疗应用。该项目的目标将是 通过以下三个具体目标完成：1)阐明分子基础和构象动力学 2)检测非25HC氧合甾醇对整合素的分子识别；以及 3)鉴定和评价以整合素25HC结合位点为靶点的小分子调节剂。我们将利用 最先进的计算技术，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述所涉及的结构基础和构象动力学 在整合素的激活中，并鉴定整合素第2位的高亲和力配体。此外，我们历史悠久的 体外和体内模型将被用来验证我们的计算机研究结果和评估顶级配体。我们的 多学科方法是创新的，拟议的研究将产生广泛的影响，通过提供 对氧化甾醇和整合素之间相互作用的基本见解，最终导致 炎症反应。此外，该项目将确定整合素-25HC的一个或多个调节子 相互作用，从而推进免疫和传染病的潜在抗炎疗法。

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.