Purchase this article with an account.
Yevgeniy Izrayelit, Brittney McGlasson, Paul David Hamilton, Mingxing Qian, Kathleen Molnar, Douglas Covey, Jason Gestwicki, Leah Makley, Usha P Andley; Towards a Mechanism of Action of VP1-001 in R120G cryAB-Associated Cataracts. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5302.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We previously reported the identification of an oxysterol, VP1-001, that partially restores transparency to lenses of R120G cryAB knock-in mice. We proposed that VP1-001 acts via direct and stereospecific binding to a defined cleft in the core domain of cryAB. An alternative, non-stereospecific mechanism of action of VP1-001 might involve its partitioning into lipid membranes. We set out to test the hypothesis that VP1-001’s anti-cataract activity is via cryAB binding by comparing the activity of VP1-001 with its enantiomer (ent-VP1-001) comprising a non-superimposable mirror-image structure.
We utilized differential scanning fluorimetry and protein-observed nuclear magnetic resonance studies to compare VP1-001 and ent-VP1-001’s ability to bind R120G and wild type (WT) cryAB in vitro. Each compound’s functional activity was characterized in aggregation assays using R120G cryAB. Finally, VP1-001 and ent-VP1-001 were administered via an ophthalmic eyedrop preparation to R120G cryAB knock-in mice for a period of two weeks. Slit lamp biomicroscopy was used to assess lens transparency, and gel permeation chromatography and protein solubility assays were used to quantify the observed effect. Transmission electron microscopy was used to visualize the effect of the drug on the ultrastructure of treated lenses.
In heterozygous R120G cryAB mice, 28 of 33 mice (85%) exhibited improved lens transparency. Conversely, ent-VP1-001 had no effect on lens transparency in heterozygous R120G cryAB animals (n=9). The solubility of α-crystallins increased by 63% in the VP1-001-treated relative to the vehicle-only control eyes, and total lens protein solubility increased by 16 ± 5% (n=7). In contrast, ent-VP-001-treated mice did not acquire any improvement in lens transparency relative to the vehicle-treated contralateral eye. As well, ent-VP-001-treated lenses showed no improvement in protein water-solubility (n=5) or molecular weight of α-crystallin in the soluble fraction of the treated lenses (n=5) relative to vehicle-only mice. Finally, there was a lack of observed binding of ent-VP1-001 to the core domain of cryAB.
Topical administration of ent-VP1-001 was not effective in correcting lens transparency in the R120G cryAB model of cataracts. These observations support the proposed mechanism of action of VP1-001 via cryAB protein binding and stabilization.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only