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Toral P. Parikh, Eugenia Poliakov, Thomas M. Redmond; Spin-traps Are Inhibitors Of Rpe65 Isomerase Activity. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1641.
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A rationale for the inhibition of RPE65 activity occurs in conditions where potentially toxic A2E/bisretinoid byproducts of the visual cycle accumulate, such as in Stargardt’s macular dystrophy, or in lipofuscin accumulation, associated with age-related macular degeneration. The intrinsic properties of the conjugated polyene chain result in easy formation of radical cations in oxidative conditions and our findings support that RPE65 retinol isomerase could employ a retinoid radical cation intermediate in its catalytic mechanism. We hypothesize that these radical intermediates could be stabilized by spin-trap compounds and we tested a variety of hydrophilic and lipophilic spin traps for their ability to inhibit RPE65 isomerase activity.
Human embryonic kidney freestyle suspension cells were grown and transfected with RPE65 and LRAT DNA. Twenty-four hours later, cells were treated with 2.5 microM all-trans retinol and various concentrations of possible inhibitors (spin traps): 2-methyl, 2-nitroso propane monomer (MNP), nitrosobenzene, α-phenyl-N-tert-butyl nitrone (PBN) and others. Retinoids were extracted and analyzed by normal-phase HPLC on a silica column using UV detection at 318nm.
We tested MNP, PBN and nitrosobenzene, which have been successfully employed to stabilize and detect carotenoid and retinoid radicals, and discovered that of these spin-traps only PBN is a potent inhibitor of RPE65 isomerase activity. PBN is a widely used agent that is well tolerated in animals. We found that PBN behaves as an uncompetitive inhibitor of RPE65 isomerase activity. We also tested several recently developed spin traps including DIPPMPO and DMPO. Our results show that several spin traps are effective inhibitors of RPE65 activity.
Spin trapping involves the addition of radical to a nitrone spin trap resulting in the formation of a spin adduct, a nitroxide-based persistent radical. The formation of such spin adducts of retinoid intermediates would result in the inhibition of RPE65 activity. We demonstrate that spin-traps are non-retinoid RPE65 isomerase inhibitors. This would open up a new class of small molecules for treating Stargardt’s macular dystrophy by way of modulating RPE65 activity.
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