Prevention of retinal diseases: Screening of molecules to stabilise protein folding

Abstract

Bestrophin 1 (Best1) is a Ca2+-dependent Cl- channel that localises to the basolateral plasma membrane of retinal pigment epithelium (RPE) cells. Mutations in the BEST1 gene are associated with a group of inherited retinal dystrophies (IRDs) called bestrophinopathies, caused by protein instability and loss-of-function of the Best1 protein. Bestrophinopathies, including Autosomal Dominant Best Vitelliform Macular Dystrophy (BVMD) and Autosomal Recessive Bestrophinopathy (ARB), cause blindness and are currently incurable. Two small molecules, sodium phenylbutyrate (4PBA) and 2-naphthoxyacetic acid (2-NOAA), can increase the function and expression of Best1 mutants, but at a concentration (2.5 mM) that is too high for therapeutic use. The aim of this study was to find more potent analogues that could potentially be of therapeutic use against bestrophinopathies. The calcium binding regions of Best1 and Best2 were firstly evaluated, as they are important in distinguishing mutation types. Due to a significant difference between the location of ARB and BMVD mutants, and as similar calcium sites mediate pH-dependence in other proteins, an investigation into the pH independence of hBest1 and other systems was undertaken. A hypothesis was derived based on whether the calcium binding loop of the protein tested can structurally relax (pH-independent), or not (pH-dependent), in the absence of calcium. Calcium binding proteins calbindin-d9k, calmodulin and human Cl- channels Best1, Best2, TMEM16a, ClC-2, GABAA and GlyR were studied using the pKa Protein-Sol tool. The mechanism of 4PBA action is unknown; it may promote the packaging of mutant proteins into COPII vesicles, reducing the stringency of their retention in the endoplasmic reticulum (ER). A virtual docking model of the COPII Sec24a B site, where 4PBA has been shown to bind, was generated and a library of FDA approved compounds was screened at the site. The top binding compounds were selected and tested in vitro by whole-cell patch-clamp of HEK293T cells expressing mutant Best1. Addition of 25 uM tadalafil resulted in full rescue of Cl- conductance, comparable to wild type Best1 levels, for the ARB mutant p.M325T but not for p.R141H (ARB) or p.L234V (BVMD) mutants. A yellow fluorescence protein (YFP)-based assay for cells expressing the YFP -H148Q/I152L sensor and mutant Best1 was also developed to allow the efficient screening of large libraries of compounds. Attempts to generate a CHO-YFP Best1 stable cell line with either IRES or T2A vectors were unsuccessful. Consequently, the assay was performed by transiently transfecting CHO-YFP stable cells with Best1. A selection of FDA approved compounds were tested in the assay at a concentration of 25 uM in a preliminary study of their effect on mutant Best1 function.

Date of Award	21 Apr 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	James Warwicker (Co Supervisor), Sally Freeman (Co Supervisor) & Forbes Manson (Main Supervisor)