Abstract
Purpose :
In the pursuit of developing efficient methods for treating inherited retinal degenerative disorders, our study focuses on identifying compounds (both from natural and synthetic sources) capable of stabilizing rhodopsin mutants associated with retinitis pigmentosa (RP). To this aim, we have investigated the effect of geraniol and retigabine on rhodopsin conformational stability.
Methods :
We purified native rhodopsin from bovine retinal rod outer segments, and recombinant rhodopsin (wild type and mutant) expressed in COS-1 cells, by immunoaffinity chromatography. The purified rhodopsin was used for thermal stability experiments, in the dark state, by means of UV-visible spectrophotometry. The experiment was divided into an untreated control group and samples treated with geraniol, or retigabine, at a final concentration of 50 μM and 100 μM, respectively. The decay of the visible absorbance band was monitored for both groups and the results analyzed for statistical significance (N=3). Fluorescence spectroscopy was used to detect binding of the compounds to rhodopsin after the active Meta II conformation was formed upon illuminating with light of λ>495nm.
Results :
Native rhodopsin isolated from bovine rod outer segments, and recombinant wild-type rhodopsin, thermal stabilities were improved in the geraniol-treated samples. Geraniol effective binding to rhodopsin was confirmed by means of fluorescence spectroscopy. In addition, the RP rhodopsin mutant M39R showed increased stability in the geraniol-treated sample. In the case of retigabine, a three-fold increase was observed in the thermal stability of purified native rhodopsin, upon treatment of the protein sample with this compound. The difference observed in the thermal stability between the retigabine-treated rhodopsin and the control sample, with no retigabine, was determined to be statistically significant (p=0.004, N=3).
Conclusions :
Our findings suggest that geraniol and retigabine have the potential to stabilize native rhodopsin and may be useful in stabilizing RP-associated mutant variants. Moreover, the observed stabilizing effect could serve for repurposing these compounds to address retinal degeneration linked to RP caused by rhodopsin mutations.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.