Mechanism of Action of VP1-001 in cryAB(R120G)-Associated and Age-Related Cataracts

Abstract

Purpose We previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent -VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent -VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity. Methods We compared the binding of VP1-001 and ent -VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy. Results Docking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent -VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent -VP1-001 did not. Accordingly, topical treatment of lenses with ent -VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology. Conclusions The ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts.

25-Hydroxycholesterol amplifies microglial IL-1β production in an apoE isoform-dependent manner

“…The reagents used in microglia treatment were LPS (Sigma, L5293, Escherichia coli, 0111:B4); ATP (sigma A2383); 25-hydroxycholesterol (Avanti#700019 or Sigma H1015); cholesterol (Avanti#700100); 7 αhydroxycholesterol (Avanti#700034); VX-765 (Medchemexpress). Ent-25-hydroxycholesterol was synthesized as described [71].…”

“…MYW, ML, YS, AGC, AA, ST, JD, and WL performed all experiments, with help or guidance from PMS, DFC, DMH, and GAP. MQ synthesized ent-25-HC as previously described [71]. All authors read and commented on the manuscript.…”

Come Together: Protein Assemblies, Aggregates and the Sarcostat at the Heart of Cardiac Myocyte Homeostasis

“…Intriguingly, recent studies point to the exciting prospect of employing oxysterols to alter the aggregation properties of cHsps such as the R120 mutant of αB-crystallin (Makley et al, 2015;Molnar et al, 2019), and cataract-causing Y118D mutant in αA-crystallin (Zhao et al, 2015) which were highly effective in restoring protein solubility in the lens to attenuate established cataracts. Understanding how heat shock proteins are regulated via post-translational mechanisms (Gomez-Pastor et al, 2018) will be essential to develop novel therapeutics (such as oxysterols) to therapeutically target them for prevention and treatment of cardiac pathologies.…”

ROP18-Mediated Transcriptional Reprogramming of HEK293T Cell Reveals New Roles of ROP18 in the Interplay Between Toxoplasma gondii and the Host Cell

“…Additionally, SLC38A8 contributes to congenital nystagmus (Weiner et al, 2020), and RIMS1 and CABP4 are associated with dystrophy (Sisodiya et al, 2007) and synaptic disorder of cone-rod (Littink et al, 2009), respectively. Furthermore, alteration of CRYAB is associated with cataract (Molnar et al, 2019), and VCAN is associated with vitreoretinal degeneration (Tang et al, 2019). Most of these eye disease-related genes were upregulated in HEK293T cells by ROP18 ( Supplementary Table S3).…”