Novel neuroprotective activities of flavonoids against retinal degenerative diseases

摘要 功能性视紫红质（Rho）由脱辅基蛋白视蛋白和共价结合的11-顺式-视网膜发色团组成， 正常的光传导和视力所需的。遗传突变损害了正确的折叠， 11-顺式-视网膜，和Rho的稳定性，导致视网膜色素变性（RP），一种进行性退行性眼睛 导致失明的疾病。目前，没有治疗方案可用于对抗RP。视网膜- 基于药理学伴侣和非视网膜小分子，包括类黄酮，可以稳定某些 体外Rho突变体。然而，有效和安全的治疗RP尚未建立。膳食黄酮 已经报道在眼损伤中显示有益效果。我们的初步研究表明， 常见的黄酮类槲皮素可以稳定致病性Rho，改善其折叠，膜整合， 视网膜结合我们还发现，槲皮素抑制应激诱导的细胞反应， 突变体触发了对视网膜健康有益的ESTA介导的细胞凋亡。 在这里，我们建议研究槲皮素及其与视网膜类似物的组合的治疗潜力， 新的小分子Bax抑制剂，以挽救RP病理。首先，我们将评估槲皮素的积极影响， RP突变体独立于体外遗传背景。然后，我们将评估他们恢复RP的能力 RP小鼠模型的体内表型。了解Rho的潜在分子机制 突变体的稳定提供了槲皮素，我们将进行结构研究，以描绘架构 槲皮素Rho复合物的晶体学和混合质谱技术。此外，本发明还提供了一种方法， 我们将通过高通量筛选来寻找更有效的稳定Rho突变体的化合物， 类黄酮相关化合物库，然后将用系统的生物化学和生物物理学方法进行验证。 分析（目标1）。其次，我们将研究槲皮素作为特定的视网膜类似物的佐剂的作用 稳定地容纳视网膜结合袋。这些视网膜中的一些改善了P23 H的折叠 致病性Rho突变体。我们将检查新的视网膜和脊柱闭锁的潜在矫正效果- 修饰的P23 H Rho的视网膜类似物，并确定这些视网膜的结合是否可以通过 槲皮素，以获得更大的治疗效果RP（目的2）。第三，我们将研究RP中的细胞过程。 为了更好地了解RP病理，我们将寻找细胞应激激活的分子靶点 信号，并评估槲皮素是否可以调节这些细胞过程，以逆转疾病表型 (Aim 3）。首先，我们将检测槲皮素对β-内酰胺酶介导的细胞凋亡的影响，以了解其作用是否与β-内酰胺酶介导的细胞凋亡有关。 与Bax的直接或间接抑制有关，或两者兼而有之。在这些研究中，我们还将使用一种新的生物可利用的 小分子Bax抑制剂。我们将确定这种新的Bax抑制剂及其组合的有效性 槲皮素对Rho相关的RP。总之，从这项研究中获得的知识将为以下方面铺平道路： 设计一种有效的治疗方法，以防止或减缓RP病理。

ABSTRACT Functional rhodopsin (Rho) composed of apoprotein opsin and covalently bound 11-cis-retinal chromophore is required for normal phototransduction and vision. Inherited mutations compromise proper folding, binding of 11-cis-retinal, and stability of Rho, leading to retinitis pigmentosa (RP), a progressive degenerative eye disease that causes blindness. Currently, there are no treatment options available to combat RP. Retinal- based pharmacological chaperones and non-retinal small molecules, including flavonoids, can stabilize certain Rho mutants in vitro. However, effective and safe therapy for RP has yet to be established. Dietary flavonoids have been reported to show beneficial effects in ocular impairments. Our preliminary studies showed that common flavonoid quercetin could stabilize pathogenic Rho, improve its folding, membrane integration, and retinal binding. We also found that quercetin inhibits stress-induced cellular responses related to pathogenic mutants triggering Bax-mediated apoptosis with beneficial consequences to retinal health. Here we propose to study, the therapeutic potential of quercetin and its combinations with retinal analogs, and novel small molecule Bax inhibitor to rescue RP pathology. First, we will evaluate quercetin positive effects on RP mutants independently of the genetic background in vitro. Then, we will assess their ability to revert the RP phenotype in vivo in mouse models of RP. To understand the underlying molecular mechanism of Rho mutant’s stabilization offered by quercetin we will perform structural studies to delineate the architecture of quercetin-Rho complexes by applying crystallographic and hybrid mass-spectrometry techniques. In addition, we will search for more effective compounds stabilizing Rho mutants by high-throughput screening of flavonoid-related compound libraries, which then will be validated with systematic biochemical and biophysical analyses (Aim 1). Second, we will examine the role of quercetin as an adjuvant for the specific retinal analogs stably accommodating the retinal-binding pocket. Some of these retinals improve the folding of the P23H pathogenic Rho mutant. We will examine the potential corrective effect of new locked retinal and backbone- modified retinal analogs for P23H Rho and determine if the binding of these retinals could be enhanced by quercetin to gain a greater therapeutic effect in RP (Aim 2). Third, we will study the cellular processes in RP. To gain a better understanding of RP pathology we will look for molecular targets activated by cellular stress signals in RP and assess if quercetin can modulate these cellular processes to revert the disease phenotype (Aim 3). At first, we will examine the effect of quercetin on Bax-mediated apoptosis to learn if its effect is related to direct or indirect inhibition of Bax, or both. In these studies, we will also use a novel bioavailable small molecule Bax inhibitor. We will determine the effectiveness of this new Bax inhibitor and its combination with quercetin against Rho-related RP. Altogether, the knowledge gained from this study will pave the way to design an effective therapeutic remedy to prevent or slow down RP pathology.