Light initiates visual perception, but it also exacerbates a subset of blinding diseases in which visual (retinoid) cycle metabolic intermediates contribute to pathophysiology. Small molecule visual cycle modulators (VCMs) have demonstrated efficacy in preclinical models, but have been limited clinically by chronic, indiscriminate visual cycle suppression leading to side effects including night blindness in human subjects. Here, we demonstrate VCMs that are activated via a Z→E photoisomerization of an azobenzene-containing VCM by visible light within the eye. One such VCM photoswitch, (Z)-9, is a weak inhibitor of the visual cycle isomerohydrolase, RPE65, that affords potent, rapid, and on-demand inhibition when photoisomerized to the E-configuration by visible light. (E)-9 protects the retina from visual cycle toxicity and, after oral administration, shows a shorter pharmacodynamic duration than emixustat as measured by electroretinography. These results establish posterior-segment photopharmacology and outline a blueprint for light-activated therapies that mitigate daytime toxicity while sparing night vision.

Rhodopsin, the most intensively studied G protein-coupled receptor (GPCR), is activated by light-induced isomerization of its chromophore 11-cis-retinal. This study employed cryogenic electron microscopy (cryo-EM) to investigate rhodopsin structure using a megabody (Mb7) as a negative allosteric modulator. Three distinct cryo-EM structures were solved: ground-state rhodopsin, photoactivated rhodopsin, and apo-rhodopsin, all in complex with Mb7. Photoactivated rhodopsin and apo-rhodopsin, both in complex with Mb7, maintain a conformation remarkably similar to ground-state rhodopsin rather than adopting a Meta-II-like conformation. Structural elements, including the conserved residues of the NPxxY motif and the ionic lock, remain in positions corresponding to inactive rhodopsin. The megabody forms extensive interactions with rhodopsin's extracellular loop 2, N terminus, and glycans. The findings demonstrate that Mb7 stabilizes photoactivated rhodopsin in a Meta-I-like conformation, preventing progression to the active Meta-II state through specific immobilization of the extracellular domain. This work establishes a foundation for cryo-EM-guided discovery of ligands modulating rhodopsin.

Visual function depends critically on the supply of visual chromophore, 11-cis-retinal, to the photoreceptor cells in the retina. Chromophore deficiency due to aging or mutations affecting its delivery and recycling in the eye by the retinal pigment epithelium (RPE) and the Müller cells cause a wide range of visual disorders. The cellular retinaldehyde-binding protein (CRALBP), expressed in both compartments, crucially aids in accelerating the recycling of visual chromophore. Here, we explored the potential of chromophore-loaded CRALBP in restoring vision in chromophore-deficient mice. We tested wild-type human CRALBP and its redox-sensitive A212C:T250C mutant, both pre-loaded with 9-cis-retinal, for their efficacy in delivering chromophore to the retina and restoring rod photoreceptor function in RPE65-deficient mice that cannot produce visual chromophore. We observed robust restoration of rod function both in the isolated retina treated with chromophore-loaded CRALBP and in vivo after a single intravitreal injection of wild-type or mutant CRALBP proteins. Notably, the recovery of rod visual function after exposure to bright light that photoactivated most of the visual pigment was greatly accelerated in CRALBP-treated RPE65-knockout mice compared to wild-type control mice. Together, our results highlight the therapeutic potential of CRALBP complexes in efficiently delivering visual chromophore to retinal rod photoreceptors.

Carotenoid cleavage dioxygenases (CCDs) are non-heme FeII enzymes that catalyze the oxidative cleavage of alkene bonds in carotenoids, stilbenoids, and related compounds. How these enzymes control the reaction of dioxygen (O2) with their alkene substrates is unclear. Here, we apply spectroscopy in conjunction with X-ray crystallography to define the iron coordination geometry of a model CCD, CAO1 (Neurospora crassa carotenoid oxygenase 1), in its resting state and following substrate binding and coordination sphere substitutions. Resting CAO1 exhibits a five-coordinate (5C), square pyramidal FeII center that undergoes steric distortion toward a trigonal bipyramidal geometry in the presence of piceatannol. Titrations with the O2-analog, nitric oxide, show a >100-fold increase in iron-nitric oxide affinity upon substrate binding, defining a crucial role for the substrate in activating the FeII site for O2 reactivity. The importance of the 5C FeII structure for reactivity was probed through mutagenesis of the second-sphere Thr151 residue of CAO1, which occludes ligand binding at the sixth coordination position. A T151G substitution resulted in the conversion of the iron center to a six-coordinate state and a 135-fold reduction in apparent catalytic efficiency toward piceatannol compared with the wildtype enzyme. Substrate complexation resulted in partial six-coordinate to 5C conversion, indicating solvent dissociation from the iron center. Additional substitutions at this site demonstrated a general functional importance of the occluding residue within the CCD superfamily. Taken together, these data suggest an ordered mechanism of CCD catalysis occurring via substrate-promoted solvent replacement by O2. CCDs thus represent a new class of mononuclear non-heme FeII enzymes.

GLUT1 facilitates a continuous flow of glucose across the inner and outer blood-retinal barriers (BRBs) to support vision. To understand the extent to which photoreceptors rely on glucose transport across the outer BRB, we generated a tamoxifen-inducible conditional knockout of Slc2a1 in the retinal pigment epithelium (RPE) (RPE-iΔGlut1). In the RPE-iΔGlut1 mice, rod photoreceptors exhibited impaired outer segment renewal and decreased the expression of proteins involved in phototransduction and ciliary transport. Proteins regulating the retinal stress response increased. Cone photoreceptors were functional and viable 15 months post-tamoxifen treatment in the RPE-iΔGlut1 mice, while 70% of the rods died. When Slc2a1 was genetically deleted from rods (Rod-iΔGlut1 mice), rod degeneration was faster than in the RPE-iΔGlut1 mice. These findings suggest that rods are more dependent on glucose than cones, and that glucose from the deep vascular plexus may support cone function and viability and slow the rate of rod death.

Aims/Purpose: Excessive levels of the intracellular second messengers Ca2+ and cAMP have been linked with photoreceptor cell death during retinal degeneration (RD). We investigated if a combination (TMB) of common clinical drugs; tamsulosin and metoprolol (alpha‐ and beta‐adrenergic antagonists, Gq‐ and Gs‐coupled, respectively), and bromocriptine (a D2‐like dopamine‐receptor agonist, Gi‐coupled), that inhibit intracellular Ca2+ and cAMP signaling could be repurposed to mitigate RD.Methods: Drug efficacy was tested in four distinct RD models: rd10, P23H, and Rpe65−/− mice; and PDE6A−/− dogs. The duration of the drug trials ranged from 1‐wk to 7‐months. Drug serum levels were measured by liquid chromatography‐mass spectrometry (LC‐MS). We used primarily photopic and scotopic electroretinography (ERG) and optical coherence tomography (OCT) to assess drug efficacy. Molecular biology methods such as immunohistochemistry, immunoblotting, and RNA‐sequencing (bulk and single cell) were used to document therapeutic mechanisms, as well as to confirm therapeutic effects.Results: Dietary TMB improved cone function and slowed cone degeneration in P23H mice. In rd10 mice, both rod and cone function were significantly improved by TMB; and cone degeneration was significantly slowed. In dark‐reared rd10 mice, the drug efficacy was associated with decreased lipid peroxidation preceding the onset of cone degeneration. Dietary TMB improved retinal function and optomotor responses in Rpe65−/− mice but did not halt rod or cone degeneration. Seven‐month‐long subcutaneous sustained infusion of TMB into PDE6A−/− dogs led to higher cone counts at the end of the trial. TMB mitigated forskolin‐induced cAMP activity in an ex vivo retina preparation.Conclusions: Our results suggest that simultaneous inhibition of Gs‐ and Gq‐coupled receptors and activation of Gi‐coupled receptors by a combination of existing drugs is a viable therapeutic strategy for RD.

11-cis-Retinal is essential for light perception in mammalian photoreceptors (PRs), and aberrations in retinoid transformations cause severe retinal diseases. Understanding these processes is crucial for combating blinding diseases. The visual cycle, operating within PRs and the retinal pigment epithelium (RPE), regenerates 11-cis-retinal to sustain light sensitivity. Retinoids are also present in Müller glia (MG), hypothesized to supply 11-cis-retinol to cone PRs and retinal ganglion cells (RGCs). To trace retinoid movement through retinal cell types, we used cell-specific knockin of lecithin:retinol acyltransferase (LRAT), which converts retinols into stable retinyl esters (REs). Ectopic LRAT expression in murine PRs, MG, and RGCs resulted in RE synthesis, with REs differing in abundance and isomeric composition across cell types under genetic and light-based perturbations. PR inner segments showed high 11-cis-RE content, suggesting a constant 11-cis-retinoid supply for pigment regeneration. In MG expressing LRAT, all-trans-REs were detected, contrasting with 11-cis-REs in PRs. The MG-specific LRAT phenotype mirrored the RE-rich human neural retina, suggesting human MG may utilize LRAT to maintain retinoid reservoirs. Our findings reveal tightly controlled retinoid flux throughout the mammalian retina that supports sustained vision, expanding understanding of the visual cycle to combat retinal diseases.

Cellular retinaldehyde-binding protein (CRALBP) is an 11-cis-retinoid binding protein operating within the visual cycle. CRALBP serves as the terminal acceptor of 11-cis-retinaldehyde (11cRAL) produced within the retinal pigment epithelium (RPE) and mediates 11cRAL transport to the RPE apical microvilli. Crystallographic structures of CRALBP revealed that the 11cRAL-binding pocket is sealed off from bulk solvent, indicating a necessity for conformational changes to allow ligand egress. Here, we performed long timescale all-atom molecular dynamics simulations of CRALBP to elucidate the mechanisms of ligand release. CRALBP exhibits slower diffusive behavior in the presence of membranes containing negatively charged phospholipids, which bind to an exposed cationic pocket in CRALBP. Umbrella sampling calculations revealed thermodynamically likely pathways for 11cRAL egress. Our data suggest that the CRALBP-acidic phospholipid interaction facilitates 11cRAL release through allosteric, conformational changes that perturb the binding site, lowering ligand affinity. These findings offer insights into the molecular pathology of CRALBP-associated retinopathy.

The visual cycle is a metabolic pathway essential for visual function. The bisretinoid byproducts of this pathway can induce retinal toxicity, as occurs in Stargardt disease type 1 (STGD1). Emixustat, which inhibits bisretinoid production, is a visual cycle modulator (VCM) that targets RPE65. However, it causes visual impairment due to its unfavorable duration of action. Here, we report ester-containing analogs of emixustat that are susceptible to hydrolytic clearance and function as short-acting VCMs. We show that the esterase-mediated metabolism of these compounds can be tuned while maintaining high-affinity RPE65 targeting. Compounds 6 (EYE-002) and 7 (EYE-003) containing diethyl acetate and valproate esters, respectively, allowed faster recovery of visual cycle function compared to emixustat. These molecules protected against retinal degeneration in mouse models of photic retinopathy and STGD1. These data demonstrate that shorter attenuation of the visual cycle can therapeutically intervene in retinal diseases with fewer visual side effects compared to emixustat.

The visual cycle is a metabolic pathway that enables continuous vision by regenerating the 11-cis-retinal chromophore for photoreceptors opsins. Although integral to normal visual function, the flux of retinoids through this cycle can contribute to a range of retinal pathologies, including Stargardt disease, age-related macular degeneration, and diabetic retinopathy. In such conditions, intermediates and byproducts of the visual cycle, such as bisretinoid components of lipofuscin, can accumulate, concomitant with cellular damage and eventual photoreceptor loss. This has inspired efforts to modulate the visual cycle, aiming to slow or prevent the formation of these toxic intermediates and thus preserve retinal structure and function. Over the past two decades, multiple strategies to modulate the visual cycle have emerged. These include both intrinsic approaches, targeting key enzymes, retinoid-binding proteins, or receptors within the pigment epithelium or photoreceptors (e.g., RPE65, CRBP1, and rhodopsin inhibitors/antagonists) and extrinsic strategies that indirectly alter retinoid availability within the retina (e.g., RBP4 antagonists). Many of these agents have shown promise in animal models of visual cycle-associated retinal diseases, reducing pathological changes, and improving retinal survival. Several have advanced into clinical studies, although none are currently FDA-approved. Challenges remain in optimizing drug specificity and duration of action while minimizing side effects such as nyctalopia. In this review, we comprehensively examine current and emerging visual cycle modulators, discuss their medicinal chemistry, mechanisms of action, efficacy in preclinical and clinical studies, and highlight future opportunities for drug discovery aimed at safely and effectively preserving vision through modulation of this biochemical pathway.

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

Visual arrestin 1 (Arr1) is an essential protein for termination of the light response in photoreceptors. While mammalian Arr1s form dimers and tetramers at physiological concentrations in vitro, oligomerization in other vertebrates has not been studied. Here we examine self-association of Arr1 from two amphibian species, Xenopus laevis (xArr1) and Ambystoma tigrinum (salArr1). Sedimentation velocity analytical ultracentrifugation showed that xArr1 and salArr1 oligomerization is limited to dimers. The KD for dimer formation was 53 μM for xArr1 and 44 μM for salArr1, similar to the 69 μM KD for bovine Arr1 (bArr1) dimers. Mutations of orthologous amino acids important for mammalian Arr1 oligomerization had no impact on xArr1 dimerization. Crystallography showed that the fold of xArr1 closely resembles that of bArr1 and crystal structures in different space groups revealed two potential xArr1 dimer forms: a symmetric dimer with a C-domain interface (CC dimer), resembling the bArr1 solution dimer, and an asymmetric dimer with an N-domain/C-domain interface. Mutagenesis of residues predicted to interact in either of these two dimer forms yielded modest reduction in dimer affinity, suggesting that the dimer interfaces compete or are not unique. Indeed, small-angle X-ray scattering and protein painting data were consistent with a symmetric anti-parallel solution dimer (AP dimer) distinct from the assemblies observed by crystallography. Finally, a computational model evaluating xArr1 binding to compartment-specific partners and partitioning based on heterogeneity of available cytoplasmic spaces shows that Arr1 distribution in dark-adapted photoreceptors is largely explained by the excluded volume effect together with tuning by oligomerization.

AbstractInherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6βrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.

Enzymes can usually be unambiguously assigned to one of seven classes specifying the basic chemistry of their catalyzed reactions. Less frequently, two or more reaction classes are catalyzed by a single enzyme within one active site. Two examples are an isomerohydrolase and an isomero-oxygenase that catalyze isomerization-coupled reactions crucial for production of vision-supporting 11-cis-retinoids. In these enzymes, isomerization is obligately paired and mechanistically intertwined with a second reaction class. A handful of other enzymes carrying out similarly coupled isomerization reactions have been described, some of which have been subjected to detailed structure-function analyses. Herein we review these rarefied enzymes, focusing on the mechanistic and structural basis of their reaction coupling with the goal of revealing catalytic commonalities.

Arrestins halt signal transduction by binding to the phosphorylated C-termini of activated G protein-coupled receptors. Arrestin-1, the first subtype discovered, binds to rhodopsin in rod cells. Mutations in SAG, the gene encoding Arrestin-1, are linked to Oguchi disease, characterized by delayed dark adaptation. Since the discovery of Arrestin-1, substantial progress has been made in understanding the role of these regulatory proteins in phototransduction, including the characterization of visual phenotypes of animals and humans lacking this protein, discovery of splice variants, and documentation of its binding to inositol-polyphosphates. Arrestin-1 was one of the first structurally characterized proteins in the phototransduction cascade. However, there are knowledge gaps regarding the conformational intermediates leading to its binding to phosphorylated rhodopsin. Among various mammalian Arrestin-1 conformations captured via crystallography, the preactivated state is represented by the mutant R175E-Arrestin-1 and by a C-terminally truncated splice variant (p44). This report describes a novel purification method of Arrestin-1 from bovine retinas followed by limited proteolysis to obtain a protein resembling p44. We solved the crystal structure of this preactivated, shortened 3-367Arrestin-1 at a resolution of 1.40 Å. The structure reveals a more complete picture of the finger loop structure and of the role of the polar core in the activation of Arrestin-1. The structure of 3-367Arrestin-1 captures an intermediate form halfway between the inactive and fully activated conformations of Arrestin-1. Finally, we addressed the question of Arrestin-1 oligomerization by comparing the packing interfaces in different Arrestin-1 crystals and dimer models predicted by AlphaFold 3.

Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding for Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal editing outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused to the protein or as excipients) and that lipid nanoparticles (LNPs) encapsulating RNPs can be optimized for enhanced RNP stability, delivery efficiency and editing potency. Specifically, after screening for suitable ionizable cationic lipids and by optimizing the concentration of the synthetic lipid DMG-PEG 2000, we show that the encapsulation, via microfluidic mixing, of adenine base editor and prime editor RNPs within LNPs using the ionizable lipid SM102 can result in in vivo editing-efficiency enhancements larger than 300-fold (with respect to the delivery of the naked RNP) without detectable off-target edits. We believe that chemically defined LNP formulations optimized for RNP-encapsulation stability and delivery efficiency will lead to safer genome editing. The safety and efficacy of ribonucleoproteins for genome editing can be enhanced by formulations of lipid nanoparticles optimized for enhanced stability, delivery efficiency and editing potency of the protein complexes.

The retinal light response in animals originates from the photoisomerization of an opsin-coupled 11- cis -retinaldehyde chromophore. This visual chromophore is enzymatically produced through the action of carotenoid cleavage dioxygenases. Vertebrates require two carotenoid cleavage dioxygenases, β-carotene oxygenase 1 and retinal pigment epithelium 65 (RPE65), to form 11- cis -retinaldehyde from carotenoid substrates, whereas invertebrates such as insects use a single enzyme known as Neither Inactivation Nor Afterpotential B (NinaB). RPE65 and NinaB couple trans–cis isomerization with hydrolysis and oxygenation, respectively, but the mechanistic relationship of their isomerase activities remains unknown. Here we report the structure of NinaB, revealing details of its active site architecture and mode of membrane binding. Structure-guided mutagenesis studies identify a residue cluster deep within the NinaB substrate-binding cleft that controls its isomerization activity. Our data demonstrate that isomerization activity is mediated by distinct active site regions in NinaB and RPE65—an evolutionary convergence that deepens our understanding of visual system diversity.