Myopia is a significant global public health concern. The incidence of myopia is reaching epidemic proportions, as have the serious ocular complications associated with high myopia. It is well-established that postnatal eye growth is controlled via a cascade of locally generated chemical events that are initiated in the retina and ultimately cause changes in scleral extracellular matrix (ECM) remodeling to effect changes in eye size and refraction. All-trans-retinoic acid (atRA) is an important molecular signal for the control of postnatal ocular growth [6-9]. We have recently demonstrated that in response to visual stimuli, ocular atRA synthesis is regulated exclusively via choroidal expression of retinaldehyde dehydrogenase 2 (RALDH2). RALDH2 is a member of a large family of aldehyde dehydrogenases. Surprisingly, specific pharmacological inhibitors have been developed for only 3 of the 19 ALDH isozymes. Other nonspecific inhibitors have been found to be effective against the RALDH isoenzymes; however, these compounds are non-specific and inhibit a range of ALDH family members, resulting in disruption of many cellular processes. We have recently designed and developed a novel compound, dichloro-all- trans-retinone (DAR), that is a potent, irreversible, RALDH inhibitor. Unlike other compounds (e.g. disulfiram, DEAB, citral, and WIN 18,446), our compound does not inhibit mitochondrial ALDH2, thereby eliminating potential adverse side effects. However, large scale synthesis of DAR, was inefficient thus requiring an alternative approach to generate quantities of inhibitor necessary for in vivo studies. Several new compounds have now been developed (CAF164 and N96) that have the same methyl dichlorocarbonyl reactive endgroups as DAR, and these compounds are amenable to industrial scale synthesis. Therefore, the objective of this proposal is to test these compounds in vitro, ex vivo, and in vivo for their selectivity for RALDH2 inhibition. We hypothesize that methyl dichlorocarbonyl portion of CAF164 and N96 is necessary for the formation of a covalent bond between the inhibitor and the catalytic cysteine (Cys 320) of RALDH2, resulting in irreversible inhibition of RALDH2 similar to other compounds, but the presence of multiple cyclic sidegroups enables RALDH selectivity over other members of the ALDH superfamily. We propose to test our central hypothesis and accomplish the objective of this application by pursuing the following three specific aims:
Aim 1. Elucidate the mechanism of RALDH2 inhibition by two methyl dichlorocarbonyl compounds.
Aim 2. Test RALDH?inhibitors for selectivity for RALDH2.
Aim 3. Assess the effects of pharmacologic inhibition of RALDH activity on eye growth.
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