The regioselective Z-isomerization of thermodynamically stable all-trans retinoids remains challenging, and ultimately limits the availability of much needed therapeutics for the treatment of human diseases. We present here a novel, straightforward approach for the catalytic Z-isomerization of retinoids using conventional heat treatment or microwave irradiation. A screen of 20 transition metal-based catalysts identified an optimal approach for the regioselective production of Z-retinoids. The most effective catalytic system was comprised of a palladium complex with labile ligands. Several mechanistic studies, including isotopic H/D exchange and state-of-the-art quantum chemical calculations using coupled cluster methods indicate that the isomerization is initiated by catalyst dimerization followed by the formation of a cyclic, six-membered chloropalladate catalyst-substrate adduct, which eventually opens to produce the desired Z-isomer. The synthetic development described here, combined with thorough mechanistic analysis of the underlying chemistry, highlights the use of readily available transition metal-based catalysts in straightforward formats for gram-scale drug synthesis.

Modulators of the visual cycle have been developed for treatment of various retinal disorders. These agents were designed to inhibit retinoid isomerase [retinal pigment epithelium-specific 65 kDa protein (RPE65)], the rate-limiting enzyme of the visual cycle, based on the idea that attenuation of visual pigment regeneration could reduce formation of toxic retinal conjugates. Of these agents, certain ones that contain primary amine groups can also reversibly form retinaldehyde Schiff base adducts, which contributes to their retinal protective activity. Direct inhibition of RPE65 as a therapeutic strategy is complicated by adverse effects resulting from slowed chromophore regeneration, whereas effective retinal sequestration can require high drug doses with potential off-target effects. We hypothesized that the RPE65-emixustat crystal structure could help guide the design of retinaldehyde-sequestering agents with varying degrees of RPE65 inhibitory activity. We found that addition of an isopropyl group to the central phenyl ring of emixustat and related compounds resulted in agents effectively lacking in vitro retinoid isomerase inhibitory activity, whereas substitution of the terminal 6-membered ring with branched moieties capable of stronger RPE65 interaction potentiated inhibition. The isopropyl derivative series produced discernible visual cycle suppression in vivo, albeit much less potently than compounds with a high affinity for the RPE65 active site. These agents were distributed into the retina and formed Schiff base adducts with retinaldehyde. Except for one compound [3-amino-1-(3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)phenyl)propan-1-ol (MB-007)], these agents conferred protection against retinal phototoxicity, suggesting that both direct RPE65 inhibition and retinal sequestration are mechanisms of potential therapeutic relevance.

Catalytic Z-isomerization of retinoids to their thermodynamically less stable Z-isomer remains a challenge. In this report, we present a photochemical approach for the catalytic Z-isomerization of retinoids using monochromatic wavelength UV irradiation treatment. We have developed a straightforward approach for the synthesis of Z-retinoids in high yield, overcoming common obstacles normally associated with their synthesis. Calculations based on density functional theory (DFT) have allowed us to correlate the experimentally observed Z-isomer distribution of retinoids with the energies of chemically important intermediates, which include ground- and excited-state potential energy surfaces. We also demonstrate the application of the current method by synthesizing gram-scale quantities of 9-cis-retinyl acetate 9Z-a. Operational simplicity and gram-scale ability make this chemistry a very practical solution to the problem of Z-isomer retinoid synthesis.

In the eye, the isomerization of all-trans-retinal to 11-cis-retinal is accomplished by a metabolic pathway termed the visual cycle that is critical for vision. RPE65 is the essential trans-cis isomerase of this pathway. Emixustat, a retinoid-mimetic RPE65 inhibitor, was developed as a therapeutic visual cycle modulator and used for the treatment of retinopathies. However, pharmacokinetic liabilities limit its further development including: (1) metabolic deamination of the γ-amino-α-aryl alcohol, which mediates targeted RPE65 inhibition, and (2) unwanted long-lasting RPE65 inhibition. We sought to address these issues by more broadly defining the structure-activity relationships of the RPE65 recognition motif via the synthesis of a family of novel derivatives, which were tested in vitro and in vivo for RPE65 inhibition. We identified a potent secondary amine derivative with resistance to deamination and preserved RPE65 inhibitory activity. Our data provide insights into activity-preserving modifications of the emixustat molecule that can be employed to tune its pharmacological properties.

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.

Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

Recycling of all-trans-retinal to 11-cis-retinal through the visual cycle is a fundamental metabolic pathway in the eye. A potent retinoid isomerase (RPE65) inhibitor, (R)-emixustat, has been developed and tested in several clinical trials; however, it has not received regulatory approval for use in any specific retinopathy. Rapid clearance of this drug presents challenges to maintaining concentrations in eyes within a therapeutic window. To address this pharmacokinetic inadequacy, we rationally designed and synthesized a series of emixustat derivatives with strategically placed fluorine and deuterium atoms to slow down the key metabolic transformations known for emixustat. Crystal structures and quantum chemical analysis of RPE65 in complex with the most potent emixustat derivatives revealed the structural and electronic bases for how fluoro substituents can be favorably accommodated within the active site pocket of RPE65. We found a close (∼3.0 Å) F-π interaction that is predicted to contribute ∼2.4 kcal/mol to the overall binding energy.

Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged Gt activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates Gt in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.

Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1-/- mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms.

Carotenoid cleavage oxygenases (CCOs) are non-heme iron enzymes that catalyze scission of alkene groups in carotenoids and stilbenoids to form biologically important products. CCOs possess a rare four-His iron center whose resting-state structure and interaction with substrates are incompletely understood. Here, we address this knowledge gap through a comprehensive structural and spectroscopic study of three phyletically diverse CCOs. The crystal structure of a fungal stilbenoid-cleaving CCO, CAO1, reveals strong similarity between its iron center and those of carotenoid-cleaving CCOs, but with a markedly different substrate-binding cleft. These enzymes all possess a five-coordinate high-spin Fe(II) center with resting-state Fe-His bond lengths of ∼2.15 Å. This ligand set generates an iron environment more electropositive than those of other non-heme iron dioxygenases as observed by Mössbauer isomer shifts. Dioxygen (O2) does not coordinate iron in the absence of substrate. Substrates bind away (∼4.7 Å) from the iron and have little impact on its electronic structure, thus excluding coordination-triggered O2 binding. However, substrate binding does perturb the spectral properties of CCO Fe-NO derivatives, indicating proximate organic substrate and O2-binding sites, which might influence Fe-O2 interactions. Together, these data provide a robust description of the CCO iron center and its interactions with substrates and substrate mimetics that illuminates commonalities as well as subtle and profound structural differences within the CCO family.