Fig 1.
Scheme of visual cycle and structure of inhibitor (±)-RPE65-61.
(A) Schematic representation of the visual cycle and the target of (±)-RPE65-61. The visual cycle includes a series of biochemical reactions, one of the key steps is the conversion of all-trans-retinyl ester to 11-cis-retinol by RPE65 isomerase. All-trans-retinol converted through a cascade of enzymatic reactions in the retinal pigmented epithelium (RPE), producing a light sensitive chromophore 11-cis-retinal, which binds with opsin and allows photon capture in photoreceptor outer segment. All-trans-retinal and 11-cis-retinal, when accumulated in excessive amounts, can lead to production of A2E which causes oxidative stress and ocular diseases. RPE65-61 selectively inhibits isomerase activity of RPE65 and prevents the retinal damage by slowing down the accumulation of all-trans-retinal in light-induced retinal damage animal model. (B) (±)-RPE65-61 chemical structure.
Scheme 1.
Preparation of (±)-(2-aminoethyl)(3-(cyclohexylmethoxy)phenyl)(imino)-ƛ6-sulfanone ((±)-9, (±)-RPE65-61).
Fig 2.
(±)-RPE65-61 inhibits retinol isomerase activity in vitro.
Bovine RPE microsomes (25 μg) were incubated with 0.2 μM of all-trans- [3H]-retinol in the presence or absence of (±)-RPE65-61 for 1h at 37°C. The generated retinoids were analyzed by HPLC coupled with flow scintillation analyzer. (A) HPLC elution profile without inhibitor; (B) with 375 nM of RPE65-61. Peak 1, retinyl esters; 2, 11-cis-retinol; 3 all-trans-retinol. (C) (±)-RPE65-61 concentration-dependent inhibition of 11-cis-[3H]-retinol generation (mean ± SEM, n = 3).
Fig 3.
Uncompetitive inhibition of RPE65 isomerase by (±)-RPE65-61 in the liposome-based assay.
All-trans-retinyl ester incorporated in liposomes was used as a substrate for purified RPE65 in the isomerase assay. (A). HPLC elution profile of the reaction products without inhibitor. (B) with 180 nM of (±)-RPE65-61; Peak 1, retinyl esters; 2, 11-cis-retinol; 3, all-trans-retinol. (C) Lineweaver-Burk plot of 11-cis-retinol generated by RPE65. Liposomes with increasing concentrations (S) of all-trans-retinyl palmitate were incubated with 25 μg of purified chicken RPE65 in the absence (♦) or in the presence of (±)-RPE65-61 (180 nM) (■).
Fig 4.
Systemic treatment by inhibitor (±)-RPE65-61 delays chromophore regeneration in BALB/cJ mice.
(A) Schematic representation of the experimental design for the study of (±)-RPE65-61 retina protection against light damage in vivo. (B-D) Retinoids levels in the inhibitor treated mouse eyes during dark adaptation after the photobleach. Dark-adapted mice were injected with varying amounts of (±)-RPE65-61, and subjected to light-adaptation under 5000 lx of fluorescent light for 30 min. The eyes were harvested for HPLC retinoid profiling after 30 min of dark-adaptation, to measure levels of (B) 11-cis-retinal, (C) retinyl ester, and (D) all-trans-retinal. Student’s t-test. ** P< 0.01, *** < 0.001 and **** < 0.0001. (Mean ± SEM, n = 8).
Fig 5.
Effect of (±)-RPE65-61 on protection against retinal degeneration.
LIRD caused the degeneration in the retina and RPE, which was rescued by inhibitor at the dose of 2 mg/kg in BALB/c mice. (A) Representative histological analysis for the retinas of (±)-RPE65-61 injected mice 5 days post-LIRD. Retinal cross-sections from three groups were stained with H&E for morphological comparison, 40× magnification. Scale bar, 50μm. (B) Representative OCT images of mice treated with RPE65-61. (C-E) Quantification of the protective effects of (±)-RPE65-61 from OCT measurements are shown by measuring the average total thickness of retina (C), outer retina (D) and inner retina (E). RPE: retinal pigment epithelium; IS: outer segment; OS: outer segment; ONL: outer nuclear layer; OPN: outer plexiform layer; INL: inner nuclear layer; IPN: inner plexiform layer; RNFL: The retinal nerve fiber layer. The three groups: No LIRD control, LIRD + Vehicle or LIRD + (±)-RPE65-61. Data expressed as mean ± SEM. n = 8 per group. Student’s t-test. *P < 0.05, ** < 0.01 and *** < 0.001, **** < 0.0001.
Fig 6.
Analysis of rhodopsin level in LIRD mice treated with (±)-RPE65-61.
LIRD caused the degeneration in the retinal photoreceptor cells and decrease of rhodopsin levels, which was preserved by inhibitor at the dose of 2 mg/kg in BALB/c mice. (A) Expression levels of opsin in the retinas of control and LIRD mice were measured using anti-rhodopsin (1D4) antibody. (B) Opsin monomer level was analyzed by densitometry and normalized by β-actin level. Data expressed as mean ± SEM. n = 4 per group. Student’s t-test. *P < 0.05, ** < 0.01.
Fig 7.
ERG responses of (±)-RPE65-61 treated LIRD mice.
Retina photoreceptor function was measured by ERG 5 days after light exposure. (A) Comparison of averaged scotopic a-wave amplitudes from three mouse groups. Seven different stimulus intensities were used, ranging from 0.002 to 400 cd s/m2. (B) Comparison of the averaged scotopic b-wave amplitude from the three groups. (C) Comparison of the averaged photopic b-wave amplitude from the three groups: No LIRD control, LIRD + Vehicle or LIRD + (±)-RPE65-61, Student’s t-test was used (n = 8, mean ± SEM, *P < 0.05, ** < 0.01 and *** < 0.001).
Fig 8.
Analyses of cGAS-STING pathway and retina cells apoptosis in LIRD mice treated with (±)-RPE65-61.
(A-C) cGAS and STING protein levels in retina were measured by Western blot analysis with β-actin as loading control and densitometry quantification. (D, E) Levels of phosphorylated NF-κB (p-p65) were determined by Western blotting. Total NF-κB (p65) was used as an internal control and for densitometry quantification. Each lane represents an individual mouse. (F, G) Photoreceptor cell apoptosis was evaluated by TUNEL staining on the retinal sections at the dose of 2 mg/kg in BALB/c mice and correlating quantitative analysis. 20X magnification, Scale bar, 100μm. Student’s t-test was used (n = 5, mean ± SEM, *P < 0.05, ** < 0.01).