Pharmacological chaperone for α-crystallin partially restores transparency in cataract models

Abstract

Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins.

Fig 1. High throughput DSF screen identifies pharmacological chaperones for a small heat shock protein

(A) The R120G mutant of cryAB (blue) forms heat resistant amyloids, as judged by electron microscopy (a), light scattering (b) and DSF (c). Wild type cryAB (black) is more resistant to misfolding and has a lower melting transition by DSF, but will form amyloids under harsher conditions (Fig S1). Results are the average of triplicates and error bars represent SEM. Microscopy results are representative of studies on at least three independent samples. Scale bar is 0.25 μm. (B) Summary of DSF screen against Hsp27. The structures of three active sterols are shown. (C) A collection of 32 sterols, based on compound 1, were screened at 20 μM for the ability to restore the Tm of R120G cryAB. Compounds 28 and 29 were 2- to 3-fold more potent than the initial active molecule. (D) Compounds 28 and 29, but not the control compound 16, partially restored the T_m of R120G cryAB and bound to the protein. Results are the average of at least triplicates and error is SEM.

Fig 2. Compound 29 reverses cataract formation in vitro and in the R120G cryAB knock-in mouse

(A) Purified R120G cryAB (20 μM) was treated with compound 29 (100 μM), 16 (100 μM) or a DMSO control (1%) and then aggregated at ambient temperature with shaking for 30 minutes for the aggregation study. For the disaggregation study, amyloid fibrils were formed using the conditions above (40 μM) and then the samples were treated with 100 μM compound. Aliquots were visualized at 44 hours after treatment. Samples were visualized by electron microscopy and then centrifuged to remove insoluble material. The levels of soluble and insoluble R120G cryAB were assessed by absorbance at 280 nm. Results are the average of independent triplicates and the error bars represent SEM. Electron micrographs are representative of independent triplicates. Scale bar is 1 μm. (B) Docking of compound 29 to the crystallin domain of cryAB, based on NMR titrations and chemical shift perturbations. See the Supplemental Material for details. (C) Summary of the treatment of R120G cryAB knock-in mice with compound 29. Slit lamp images and corresponding densitometry plots are shown for wild type mice aged 60–240 days; cryAB R120G heterozygotes aged 66–366 days, treated with vehicle control or 29 (5 mM) every other day for 2 weeks; cryAB R120G heterozygotes aged 260 days–469 days treated with vehicle or 29 (1 mM) daily for 2 weeks. The transparency of the treated mice was measured using a LOCS III scoring system on masked images. In both dosing schedules, 29 significantly improved transparency. p < 0.001. (D) Treatment with compound 29 improved the solubility of mouse and human lens proteins, as measured by BCA assays.