Novel neuroprotective activities of flavonoids against retinal degenerative diseases

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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.