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Bryan Dunyak, Bonnie Su, Kathleen Molnar, Paul Hamilton, Stephanie Bozeman, Stacy Li, Linda Liu, Usha P Andley, Leah Makley; Discovery of non-sterol αB-crystallin ligands as potential cataract therapeutics. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5691.
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Cataracts are caused by damage and subsequent aggregation of the major lens proteins, including αB-crystallin (cryAB), a chaperone protein critical for maintaining lens homeostasis. We have previously reported on VP1-001, an oxysterol that solubilizes misfolded cryAB and can partially restore lens transparency in preclinical models of cataracts. While VP1-001 continues in development as a potential clinical treatment, we sought to identify non-sterol ligands for cryAB with improved properties. We conducted a computational scaffold-hopping screen and a subsequent medicinal chemistry effort guided by biophysical assays. This led to the discovery of novel scaffolds possessing improved drug-like properties, comparable or better in vitro activity as VP1-001, and efficacy in mouse models of cataracts.
A virtual screen of >6 million compounds in the ZINC database was performed (UCSF Dock) on cryAB (PDB: 2WJ7). Purchased hits were confirmed for binding using differential scanning fluorimetry (DSF). Binding sites from multiple scaffolds were verified by X-ray crystallography. Affinities of optimized compounds were measured with microscale thermophoresis (MST) and anti-aggregation activity was characterized in a turbidity assay using cryAB(R120G). Active compounds were tested in mouse models of cataracts (aged WT and hereditary cryAB(R120G)+/-). Further lead optimization is being guided by affinity, functional activity assays, and ocular pharmacokinetics.
Top scoring hits in the virtual screen were triaged and ~100 compounds were purchased to confirm binding by DSF (ΔTm>1°C). A representative compound, VP-A1, stabilized cryAB by DSF (ΔTm=5.7°C) and bound to cryAB(R120G) with comparable affinity (115±10μM) to VP1-001 by MST. In a cryAB aggregation assay, VP-A1 completely suppressed cryAB(R120G) aggregation (EC50=156±5μM), whereas VP1-001 only slowed aggregation rate. VP-A1 reduced cataract severity in aged wild-type (Δ=1.5±0.5, mean age 480 days, N=8) and cryAB(R120G)+/- (Δ=1.3±0.2, mean age 370 days, N=8) mice using a LOCS III grading scale.
Using a virtual screening strategy and biophysical assay workflow we identified non-sterol ligands for cryAB with enhanced drug-like properties. Importantly, the discovery of these non-sterols lends support to the hypothesis that pharmacological chaperones targeting cryAB can prevent or reverse cataracts. Efforts to optimize these compounds are ongoing.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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