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Philip Kiser, Jianye Zhang, Mohsen Badiee, Wuxian Shi, Xuewu Sui, Marcin Golczak, Gregory Tochtrop, Krzysztof Palczewski; Crystal structure of RPE65 in complex with retinoid-mimetics and palmitate. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1699.
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© ARVO (1962-2015); The Authors (2016-present)
RPE65 is the retinoid isomerase of the RPE-based visual cycle, a pathway that regenerates 11-cis-retinal for rod and cone opsins. This membrane-bound enzyme catalyzes the conversion of all-trans-retinyl esters into 11-cis-retinol in an iron(II)-dependent fashion. Despite years of research its catalytic mechanism remains poorly understood. A major barrier to further advancement has been the lack of experimental structural information on how retinyl ester substrates bind in the active site cavity. In fact, no reliable structure of any RPE65-family enzyme in complex with a retinoid or carotenoid molecule has been determined to date, likely due to the poor aqueous solubility these isoprenoid compounds. Thus, we thus sought alternative retinoid-like molecules for studies designed to address the structural basis of RPE65 isomerase activity.
We identified a compound named emixustat, as a potential retinoid-mimetic with improved aqueous solubility for structural studies. This compound is currently in a phase 2b/3a clinical trial for treatment of dry age-related macular degeneration. A derivative of emixustat called MB-001 in which the cyclohexyl moiety is replaced with a beta-ionone ring to improve retinoid-likeness was also synthesized. The ability of these compounds to suppress RPE65 activity in vitro and in vivo was examined in vitro and in vivo. Co-crystallization trials were performed using previously established methods.
We confirmed that both emixustat and MB-001 strongly suppress RPE65 activity both in vitro and in vivo. RPE65 crystals obtained in the presence of the compounds diffracted X-rays to a maximal resolution of 1.8 angstroms. Electron density maps calculated from these data clearly revealed the bound inhibitors within the active site cavity. Additionally, an active site palmitate molecule in complex with the iron cofactor was clearly resolved in both structures. In silico retinoid docking experiments supported our proposed assignment of the retinoid and palmitate binding sites and thus the orientation of bound retinyl esters in the ES complex.
These results allowed us to propose a detailed mechanism of catalysis by RPE65 that reconciles a large body of data on the retinoid isomerization reaction. The structures also reveal at atomic level detail the mechanism of RPE65 inhibition by small molecules, information that can be used for rational drug design.
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