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 是正常的光传导和视力所必需的。遗传突变会影响正确的折叠、结合 11顺式视网膜和Rho的稳定性，导致进行性退行性眼球的视网膜色素变性(RP) 导致失明的疾病。目前，还没有可用的治疗方案来对抗RP。视网膜- 基于药理的伴侣和非视网膜小分子，包括类黄酮类，可以稳定某些 Rho突变体在体外。然而，目前对RP的有效、安全的治疗方法尚未建立。膳食中的类黄酮 已经被报道对眼睛损伤有有益的影响。我们的初步研究表明 常见的黄酮类成分 视网膜捆绑。我们还发现，栎素抑制应激诱导的与致病相关的细胞反应。 突变体触发Bax介导的细胞凋亡，对视网膜健康有利。 在这里，我们建议研究，槲皮素及其与视网膜类似物的联合治疗潜力，以及 新型小分子Bax抑制剂挽救RP病理。首先，我们将评估槲皮素对 Rp突变体在体外不受遗传背景的影响。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 我们将进行结构研究来描绘突变体的结构 应用结晶学和混合质谱学技术研究了栎素-Rho络合物。此外, 我们将通过高通量筛选寻找更有效的稳定Rho突变体的化合物 与类黄酮相关的化合物文库，然后将进行系统的生化和生物物理验证 分析(目标1)。其次，我们将研究作为特定视网膜类似物佐剂的栎素的作用。 稳定地容纳视网膜捆绑袋。其中一些视网膜改善了P23H的折叠 致病性Rho突变体。我们将检查新的视网膜和脊椎锁定的潜在矫正效果- P23H Rho的修饰视网膜类似物，并确定这些视网膜的结合是否可以通过 在RP中获得更好的疗效(目标2)。第三，我们将研究RP中的细胞过程。 为了更好地理解RP病理，我们将寻找由细胞应激激活的分子靶点。 并评估是否可以调节这些细胞过程以逆转疾病的表型。 (目标3)。首先，我们将研究槲皮素对bax介导的细胞凋亡的影响，以了解其作用是否 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利用度 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 用槲皮素对抗Rho相关的RP。总而言之，从这项研究中获得的知识将为 设计有效的治疗药物来预防或减缓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.