YC-001

Cell lines

Transfected 27 adRP-causing hRHO mutants to NIH3T3 cells

Preparation Method

NIH3T3 cells were seeded at 5000 cells/well and cultured in a blackwall, clear bottom, and poly-L-lysine-treated 384-well plate at 37 °C with 5% CO2 on day 1, and then transfected with the plasmids containing hRHO cDNA mutants on day 2. On day 3, cells were treated with 0.1% DMSO, 5 µM 9-cis-retinal (under dim red light), 40 µM YC-001, or 20 µM F5257-0462 in DMEM with 10% FBS for 24 h. On day 4, treated cells were fixed with 4% paraformaldehyde (PFA) in the dark for 20 min and immunostained with 15µL/well of 20 µg/mL Alexa488-conjugated B6-30 anti-RHO antibody and Hoechst 33342 (1:10,000 dilution).

Reaction Conditions

40 µM for 24 h

Applications

YC-001 rescued multiple of 27 adRP-causing RHO mutants to NIH3T3 cells. 9-cis-retinal, F5257-0462, and YC-001 rescued cell surface transport of 16, 11, and 7 mutants, respectively.

Explant

Retinal explants

Preparation Method

Mice were euthanized at P15, and eyes were enucleated and incubated in Ames’ solution containing L-cysteine (0.22 mM) and papain (20 U/mL) at 37°C for 30 minutes, followed by incubation in DMEM with 10% FBS at 4°C for 5 minutes. Each eye cup was made and flattened by 4 cuts, and sclera was removed leaving only RPE attached to retina. Each retinal explant (RPE layer facing down) was cultured in a transwell in a 6-well plate with neurobasal plus medium containing 2% B27 supplement and 5 μg/mL of plasmocin at 37°C with 5% CO2 for 24 hours. The retinal explants were then treated with the same medium containing 40 μM YC-001 or DMSO. Fresh medium with the compound was changed daily.

Reaction Conditions

40 μM YC-001 for 48 h

Applications

YC-001 increases the percentage of ciliary targeted RHOP23H primarily by decreasing the level of misfolded RhoP23H mutant in the the outer nuclear layer, which is contributed by reduced total RHOP23H protein load.

Animal models

Abca4^−/−Rdh8^−/− mice with a 129 Sv/Ev or C57BL/6 mixed background

Preparation Method

Retinal degeneration was initiated by exposing Abca4−/−Rdh8−/− mice for 30 min to white light with an intensity of 10,000 lux. Pupils of mice were dilated with 1% tropicamide 3 min before bright light exposure. YC-001 or DMSO were administered i.p. 30 min before such exposure. The effects of YC-001 were tested at two dosages: 50 and 200 mg kg−1 bw. The volume of each injection was less than 50 µL.

Dosage form

4 μg/μl, 2 μl, lateral cerebral ventricle injection

Applications

DMSO-treated mouse retinas featured significantly diminished outer nuclear layers (ONLs), indicating the loss of photoreceptor cells, YC-001-treated mice evidenced a dose-dependent protection of the ONL from light-induced damage.

References:

[1]. Vats A, Xi Y, Feng B, et al. Nonretinoid chaperones improve rhodopsin homeostasis in a mouse model of retinitis pigmentosa[J]. JCI insight, 2022, 7(10).

[2]. Chen Y, Chen Y, Jastrzebska B, et al. A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration[J]. Nature communications, 2018, 9(1): 1-18.