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Angelica Harper, Tim Mather, Jody A Summers Rada; Inhibition of choroidal retinoic acid synthesis by dichloro-all-trans-retinone (DAR).. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5475.
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Choroidal retinaldehyde dehydrogenase 2 (RALDH2) has been identified as a potential pharmaceutical target for the control of postnatal ocular growth. Our lab has recently developed a novel RALDH2 selective inhibitor, dichloro-all-trans-retinone (DAR) to examine the role of RALDH2 in the control of visually guided eye growth. The objective of the current study was to evaluate the selectivity and potency of DAR, in vitro and ex vivo as a foundation for future in vivo experiments on DAR’s efficacy as an ocular growth modulator.
Enzyme specificity and mechanism of inhibition was determined in vitro using NADH assays with recombinant chick and human RALDH1, RALDH2, RALDH3, and ALDH2. In vitro and ex vivo experiments utilized a cell line with doxycycline-inducible RALDH2 [(Dox)RALDH2-eGFP 293], as well as choroidal lysates isolated from chick eyes following 4 days of recovery from form deprivation myopia. Retinoic acid (RA) synthesis by choroidal lysates with and without DAR (0–10 µM) was quantified by HPLC/MS. DAR toxicity was evaluated on chick sclera utilizing an in vitro proteoglycan synthesis assay.
In vitro assays using recombinant protein indicated that DAR inhibits both chick and human RALDH2 with an IC50=52.2 nM and 173 nM, respectively. DAR also inhibited RALDH1 and RALDH3 at higher concentrations (RALDH1, IC50=670 nM; RALDH3, IC50=340 nM), but had no inhibitory effect on human mitochondrial ALDH2. Inhibition of all RALDH enzymes was concentration and time dependent, suggesting irreversible inhibition. Pretreatment of (Dox)RALDH2-eGFP 293 cell lines with DAR (0–2 µM; in the presence of 10 µM retinaldehyde) resulted in significant inhibition of RALDH activity in cell lysates (↓96%, p<0.001, ANOVA) and significant inhibition of RA synthesis in vitro (↓42%, p<0.001, ANOVA). Similarly, following incubation of choroids isolated from control and recovering eyes with DAR (0–5 µM; 18 hrs at 37°C) significant inhibition of RALDH activity in tissue lysates was also observed (↓38%, p<0.05, ANOVA), with an IC50=53.6 nM. Incubation of sclera with DAR resulted in significant inhibition of scleral proteoglycan synthesis at concentrations >10 µM (p<0.001, ANOVA) (IC50=15.5 μM).
Dichloro-all-trans-retinone effectively inhibits RALDH activity and RA synthesis in cultured cells and in isolated choroids.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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