Abstract
Purpose :
Risuteganib (RSG) is a synthetic peptide that has shown promising efficacy in an intermediate dry age-related macular degeneration (AMD) phase 2 clinical study. We previously reported that RSG protected against retinal pigment epithelial (RPE) cell injury induced by hydroquinone (HQ), a major oxidant in cigarette smoke and atmospheric pollutants agents implicated in AMD pathogenesis. Herein, we investigate whether RPE cells take up RSG.
Methods :
Cultured human RPE cells were transduced overnight with late endosome-GFP, mitochondria-GFP, or endoplasmic reticulum-GFP using CellLight Reagents (BacMam 2.0) at 40 particles per cell. Cells were washed, incubated with Alexa Fluor™ 555 alkyne, triethylammonium salt (AF555, 50uM, control) or RSG-Alexa Fluor™ 555 (RSG-AF555, 50uM, synthesized using Fmoc [the fluorenylmethoxycarbonyl protecting group] solid-phase peptide synthesis and N-Hydroxysuccinimidyl ester-amine reaction) for various times at 37°C or for 2.5 hours at 4°C. RSG co-localization with cell organelles were examined by confocal microscopy.
Results :
A large amount of cytoplasmic dot-like staining was detected in RSG-AF555-treated cells while little staining was seen in AF555-treated cells 2.5 hours post-treatment. The cytoplasmic dot-like staining pattern in RSG-AF555-treated cells was dramatically reduced at 4°C when compared to 37°C at 2.5 hours post-treatment. RSG-AF555 accumulated in the cells as early as 30 minutes and remained for at least 21 hours. In RSG-AF555-treated cells, some RSG-AF555 dots were co-stained with late endosome and additional RSG-AF555 dots were co-stained with mitochondrial and endoplasmic reticulum 2.5 hours post-treatment.
Conclusions :
Experiments at temperatures that permit and inhibit endocytosis suggest that cultured RPE cells take up RSG via endocytosis. Co-localization of RSG with cell organelles may be involved in RSG’s protection against HQ-induced cell damage.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.