Molecular pharmacodynamics of emixustat in protection against retinal degeneration
Jianye Zhang, … , Gregory P. Tochtrop, Krzysztof Palczewski
Jianye Zhang, … , Gregory P. Tochtrop, Krzysztof Palczewski
Published June 15, 2015
Citation Information: J Clin Invest. 2015;125(7):2781-2794. https://doi.org/10.1172/JCI80950.
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Research Article Ophthalmology

Molecular pharmacodynamics of emixustat in protection against retinal degeneration

Abstract

Emixustat is a visual cycle modulator that has entered clinical trials as a treatment for age-related macular degeneration (AMD). This molecule has been proposed to inhibit the visual cycle isomerase RPE65, thereby slowing regeneration of 11-cis-retinal and reducing production of retinaldehyde condensation byproducts that may be involved in AMD pathology. Previously, we reported that all-trans-retinal (atRAL) is directly cytotoxic and that certain primary amine compounds that transiently sequester atRAL via Schiff base formation ameliorate retinal degeneration. Here, we have shown that emixustat stereoselectively inhibits RPE65 by direct active site binding. However, we detected the presence of emixustat-atRAL Schiff base conjugates, indicating that emixustat also acts as a retinal scavenger, which may contribute to its therapeutic effects. Using agents that lack either RPE65 inhibitory activity or the capacity to sequester atRAL, we assessed the relative importance of these 2 modes of action in protection against retinal phototoxicity in mice. The atRAL sequestrant QEA-B-001-NH2 conferred protection against phototoxicity without inhibiting RPE65, whereas an emixustat derivative incapable of atRAL sequestration was minimally protective, despite direct inhibition of RPE65. These data indicate that atRAL sequestration is an essential mechanism underlying the protective effects of emixustat and related compounds against retinal phototoxicity. Moreover, atRAL sequestration should be considered in the design of next-generation visual cycle modulators.

Authors

Jianye Zhang, Philip D. Kiser, Mohsen Badiee, Grazyna Palczewska, Zhiqian Dong, Marcin Golczak, Gregory P. Tochtrop, Krzysztof Palczewski

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Figure 6

Emixustat-atRAL Schiff base formation in vitro and in vivo.

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Emixustat-atRAL Schiff base formation in vitro and in vivo. (A) Chromato...
(A) Chromatographic separation and detection of primary amines, atRAL, and their Schiff base conjugates. Emixustat or Ret-NH2 was incubated with atRAL for 2 hours, and the mixture was separated on a C-18 column. Representative HPLC chromatograms indicate the formation of an emixustat Schiff base (peak “a”) and Ret-NH2 Schiff base (peak “b”) at an aldehyde/amine ratio of 1:1. Peak “c” corresponds to unreacted atRAL. (B) Quantification of Schiff bases formed upon incubation of increasing concentrations of atRAL with either 0.2 mM emixustat (inverted triangles) or Ret-NH2 (circles). (C) The chromatogram illustrates separation of an emixustat Schiff base standard. Peak 1, between 6 and 7 minutes, corresponds to the emixustat retinylidene Schiff base as indicated by its MS spectrum (D) with a dominant ion of m/z = 530.5 corresponding to [MH]+ of the emixustat-retinylidene Schiff base. Peak 2 corresponds to atRAL. Inset in D shows the MS/MS fragmentation pattern of the m/z = 530.5 parent ion. (E) Detection of emixustat-retinylidene Schiff base in vivo; 8 mg/kg emixustat was administered to Abca4–/– Rdh8–/– mice 2 hours before light bleaching. After bleaching, mice were euthanized, and eye extracts were analyzed by LC-MS. Peak 1′ observed in the chromatogram corresponds to an emixustat Schiff base. Its identity was confirmed by its UV/Vis spectrum with a characteristic absorbance maximum of 462 nm (inset) and by MS (F), in which fragmentation of the m/z = 530.5 ion yielded the product ion at m/z = 438.5, identical to that shown in D. mAU, milliabsorbance units.