Therapeutic Potential of Partial Retinoid Agonists against Vertebrate Rhodopsin Misfolding Disorders

Abstract

Mutations in rod opsin are a leading cause of inherited retinal degenerative diseases such as retinitis pigmentosa (RP). Pharmacological compounds that stabilize rhodopsin (Rho) and mitigate cellular stress pathways hold promise for therapeutic intervention. Among these, retinoid analogs have shown efficacy in models of autosomal dominant RP (adRP) and age-related macular degeneration (AMD). In this study, we evaluated the pharmacological potential of two partial retinoid agonists, acyclic-retinal and 9-cis-9-demethyl-retinal, as well as newly synthesized retinol and amine derivatives of 9-cis-9-demethyl-retinal. A photoreceptor-derived 661W cell line stably expressing two RP-linked misfolding rod opsin mutants, P23H and T289P, was used to assess the compound activity. We investigated the effects on opsin folding, glycosylation, membrane localization, and pigment regeneration. Both acyclic-retinal and 9-cis-9-demethyl-retinal promoted mature glycosylation and enhanced cell surface trafficking of P23H and T289P rod opsins. Spectroscopic analysis confirmed that these compounds regenerated functional, photosensitive pigments and stabilized the receptor in the Meta-I conformation upon light exposure. Notably, 9-cis-9-demethyl-retinal exhibited higher binding affinity than 9-cis-retinal, without impairing visual signaling postphotoisomerization. Among the derivatives, the amine form of 9-cis-9-demethyl-retinal was most effective in promoting proper folding and localization of misfolded rod opsin, outperforming the corresponding retinol analog. These findings support the therapeutic potential of acyclic-retinal, 9-cis-9-demethyl-retinal, and its derivatives for rescuing misfolded rod opsin and delaying photoreceptor degeneration in RP.

Introduction

Vertebrate rhodopsin (Rho) is a visual G protein-coupled receptor (GPCR) composed of the seven-transmembrane helical domain apoprotein, opsin, covalently bound to an 11-cis-retinal chromophore via a protonated Schiff base linkage. It is highly expressed in rod photoreceptor cells, where it initiates a visual signaling cascade upon light activation. This activation involves the photoisomerization of 11-cis-retinal to all-trans-retinal, triggering conformational changes in the receptor and establishing an equilibrium between the inactive Meta-I and the active Meta-II states. The binding of the G protein transducin to Rho shifts this equilibrium toward the Meta-II conformation, which is essential for signal transduction. ,

Beyond its role in visual signaling, 11-cis-retinal also acts as a molecular chaperone, promoting proper folding of the nascent opsin protein by stabilizing its native ground-state conformation. Over 150 known mutations in the rhodopsin gene (RHO) can cause conformational defects, including protein misfolding and aggregation within the endoplasmic reticulum (ER). These defects lead to Rho degradation and photoreceptor cell death, resulting in the progressive retinal degenerative disease, namely retinitis pigmentosa (RP). − In vitro studies have shown that the folding and membrane trafficking of some misfolded RHO mutants can be partially restored by the natural chromophore 11-cis-retinal or its synthetic analog 9-cis-retinal (9c-R). , In fact, vitamin A therapy, which increases retinal availability, is currently the only pharmacological therapy recommended for patients with RP. However, its efficacy is limited, in part due to the light sensitivity of the retinal and the potential toxicity associated with its long-term administration in this chronic condition. − Moreover, misfolding mutations often destabilize the internal interactions between retinal and its binding pocket, making the chromophore more prone to light-induced decay. , Accumulation of released all-trans-retinal in excessive amounts can further exacerbate retinal degeneration. ,

Interestingly, some synthetic retinal analogs, such as acyclic-retinal (ac-R) and 9-cis-9-demethyl-retinal (9c-dm-R), favor the stabilization of the inactive Meta-I state over the active Meta-II conformation during light activation. − This shift could reduce retinal toxicity and potentially make these analogs safer and more effective pharmacochaperones than 11-cis- or 9-cis-retinals for treating Rho misfolding disorders.

In this study, we investigated the pharmacochaperone properties of ac-R and 9c-dm-R using in vitro binding and thermal stability assays, as well as in situ cell-based assays. We found that 9c-dm-R, in particular, binds effectively to unliganded rod opsin, delays light-induced retinal release from the binding pocket, and enhances plasma membrane expression of two misfolded rod opsin mutants: the most common P23H and the less frequent T289P. While the P23H mutation is the most studied Rho misfolding mutation, the T289P variant, first identified in a Spanish family in 2000, remains largely uncharacterized. It is located on the cytoplasmic side of transmembrane helix 7 (TM7), adjacent to the retinal binding site, and situated opposite the P23H mutation within the Rho molecule. Notably, unlike most mutations on this side of Rho, T289P responds strongly to the recently discovered by us nonretinoid pharmacochaperones JC3 and JC4, making it an intriguing exception and the focus of this current study. Given the potential toxicity associated with the aldehyde group, we also synthesized and evaluated the retinol and amine derivatives of 9c-dm-R for their pharmacological activity. Our results showed that removal of the aldehyde group diminished the compounds’ beneficial effects. However, despite their more modest activity, the amine derivative, 9-cis-9-demethyl-retinyl methylamine (9c-dm-NHCH₃), was able to increase the ratio of mature to immature rod opsin and enhance the cell surface expression of both RP-linked mutants. These findings suggest that this compound merits further investigation for its potential therapeutic efficacy in vivo.

Results

Binding of Retinal Partial Agonists to Rod Opsin

The retinal binding pocket is large (∼312 ų) and flexible enough to accommodate different retinal and nonretinal analogs. Indeed, two partial agonist retinals investigated in this study, namely acyclic-retinal (ac-R) and 9-cis-9-demethyl-retinal (9c-dm-R), could fit the binding cleft occupied by 9-cis-retinal (9c-R) found in the crystal structure of 9-cis-rhodopsin (isoRho) (PDB ID: 2PED) (Figure A–C). Within the binding pocket, retinal is primarily stabilized through a protonated Schiff base linkage with Lys296 and Glu113 acting as the counterion. Additional stabilization arises from interactions with Ile189 and Tyr191 in extracellular loop 2 (ECL-2); Ala117, Thr118, Gly121, and Glu122 in transmembrane helix 3 (TM3); Met207, His211, and Phe212 in TM5; and Phe261, Trp265, and Tyr268 in TM6. ,, The β-ionone ring of retinal interacts mainly with residues in TM5, as well as Gly121 and Glu122, while the polyene chain, particularly near the C9 position, interacts primarily with residues from TM6 and ECL-2. Structural modifications to the retinal molecule, such as those present in ac-R and 9c-dm-R, are expected to alter these interaction networks. Such changes may influence the compound’s ability to bind specific regions of opsin within the binding pocket, thereby affecting overall binding affinity. To determine the binding affinity (K _d) of ac-R and 9c-dm-R partial agonists, we performed titration experiments using rod opsin within disk membranes and monitored the quenching of the tryptophan fluorescence at 330 nm. The results were compared to those obtained with the full agonist 9c-R. The intrinsic fluorescence of Trp265 is quenched upon ligand binding, as observed with native 11-cis-retinal in Rho. Conversely, photoactivation leads to chromophore release and an increase in fluorescence. Similarly, the addition of a ligand to unliganded opsin causes a measurable quenching of Trp fluorescence, indicating binding. The apparent K _d values obtained were 57.6 nM for 9c-R, 100.0 nM for ac-R, and 9.1 nM for 9c-dm-R, indicating that 9c-dm-R has approximately 6-fold greater binding affinity compared to 9c-R (Figure B). The pigment regeneration kinetics monitored at 4 °C by UV–visible spectroscopy revealed that 9c-dm-Rho was formed rapidly with a half-time (t _1/2) of 0.6 min. In comparison, isoRho required approximately three times longer (t _1/2 of 2 min), while ac-Rho regenerated even more slowly with a t _1/2 of 5.1 min (Figure C). These results align well with the calculated binding affinities of these retinal derivatives. The UV–visible spectra of immunoaffinity-purified pigments resulting from the binding of the retinals revealed peak absorption at 460 nm for ac-Rho (regenerated with ac-R) and 464 nm 9c-dm-Rho (regenerated with 9c-dm-R), while isoRho absorbed at 485 nm (Figure D). Next, using UV–visible absorption spectroscopy, we monitored the kinetics of Meta-II formation after the photobleaching of the purified pigments. The measurement was performed at 4 °C and at pH 8.5 to slow this process, as temperature, acidic pH, and detergent accelerate the light-induced transition from the dark to the Meta-II state. The t _1/2 of isoRho Meta-II formation was 0.6 min under these experimental conditions, while for ac-Rho and 9c-dm-Rho this process was slower with t _1/2 = 1.3 min and t _1/2 = 10.8 min, consistent with a shift in conformational equilibrium from the active Meta-II to inactive Meta-I state (Figure E). However, the kinetics of light-induced chromophore release showed slightly faster release of ac-R (t _1/2 = 23 min) and slower release of 9c-dm-R (t _1/2 = 30.7 min) compared to 9c-R (t _1/2 = 23.8 min) (Figure F) in agreement with previous reports.

1.

Structural models accommodating the retinal analogs within the orthosteric site of rod opsin. (A) The structural model of isoRho (PDB ID: 2PED) with the binding cavity shown in green-orange-red mesh. (B) Close-up of 9-cis-retinal (9c-R), acyclic-retinal (ac-R), and 9-cis-9-demethyl-retinal (9c-dm-R) within the rod opsin orthosteric site. Ac-R and 9c-dm-R were fitted to the orthosteric cavity shown in (A) upon the removal of 9c-R. (C) Two-dimensional representation of the structures obtained from LigPlot. The interactions between the retinal analogs and the residues in the opsin’s polypeptide chain are shown. The structures were visualized with PyMol software.

2.

Binding of retinal analogs to rod opsin, their effects on pigment regeneration, and stability. (A) Chemical structures of 9-cis-retinal (9c-R), acyclic-retinal (ac-R), and 9-cis-9-demethyl-retinal (9c-dm-R). (B) The binding affinity (K _d) of retinal analogs to rod opsin was determined by quenching the opsin’s intrinsic Trp fluorescence in a concentration-dependent manner. Compounds were added to opsin membranes at different concentrations (0–1 μM), and changes in the fluorescence intensity at 330 nm were recorded and plotted as a function of the compound concentration. The binding curves were fitted by using PRISM GraphPad 10 software. The K _d values of each compound were calculated and averaged from triplicates. (C) The kinetics of Rho pigment regeneration in the presence of 5 μM 9c-R, ac-R, and 9c-dm-R. The normalized values of the absorbance maximum were plotted as a function of time. The half-life (t _1/2) of pigment regeneration was calculated from three independent experiments. (D) The UV–visible spectra of immunoaffinity-purified Rho pigments (isoRho, ac-Rho, and 9c-dm-Rho) regenerated with 5 μM 9c-R, ac-R, and 9c-dm-R, respectively. (E) The kinetics of pigment photobleaching. The normalized values of the absorbance maximum were plotted as a function of time. The half-life (t _1/2) of pigment Meta-II state formation was calculated from three independent experiments. (F) The kinetics of retinal release from isoRho, ac-Rho, and 9c-dm-Rho. The normalized values of fluorescence (RFU) measured at 330 nm were plotted as a function of time. The half-life (t _1/2) of retinal release after illumination was calculated from three independent experiments. (G) The thermal stability of Rho pigments regenerated with 9c-R, ac-R, and 9c-dm-R. The temperature of melting (T _m) was determined by using a fluorescent probe BFC. The samples were incubated in the stepwise temperature gradient up to 99.9 °C. The obtained values of fluorescence were plotted as a function of temperature, and the melting temperature was calculated using GraphPad 10 software. The T _m for each pigment is presented as a function of concentration and was calculated from three independent experiments. (H) The kinetics of isoRho, ac-Rho, and 9c-dm-Rho pigment stability to acidic pH. The normalized values of the absorbance maximum were plotted as a function of time. The half-life (t _1/2) of pigment regeneration was calculated from three independent experiments. (I) The chemical stability of isoRho, ac-Rho, and 9c-dm-Rho pigments. The changes in the absorbance maximum were monitored in the dark in the presence of 40 mM hydroxylamine. The error bars shown in each result represent the standard deviation (S.D.).

The Stability of Rho Pigments Regenerated with Partial Agonists

The binding of the natural ligand 11-cis-retinal and its analog 9c-R enhances the stability of the natively folded rod opsin receptor. , To assess whether partial agonists ac-R and 9c-dm-R differ in their ability to stabilize the receptor compared to that of the full agonist 9c-R, we measured the melting temperatures (T _m) of pigments regenerated within the rod outer segment (ROS) disk membranes using a thermal shift assay. Both ac-Rho (T _m = 67.9 °C) and 9c-dm-Rho (T _m = 70.1 °C) showed significantly increased thermal stability compared to unliganded opsin (T _m = 57.9 °C). Notably, the stability of 9c-dm-Rho was comparable to that of isoRho (T _m = 70.5 °C), while ac-Rho was slightly less stable (Figure G). Furthermore, ac-Rho exhibited lower resistance to acidic pH and hydroxylamine treatment compared to that of isoRho and 9c-dm-Rho. At pH 4.0, the half-life (t _1/2) of 9c-dm-Rho was 3.2 min compared to 1.3 min for isoRho and only 0.2 min for ac-Rho (Figure H). When incubated in the dark with 40 mM hydroxylamine at room temperature for 2 h, isoRho showed no retinal release, likely reflecting that the binding pocket remained tight (Figure I). In contrast, within the same time frame, 9c-dm-Rho and ac-Rho released ∼2 and 5% of retinal, respectively, suggesting a looser fit of these analogs within the binding pocket, which may permit hydroxylamine access to promote the Schiff base hydrolysis.

Signaling Activation by Rho Pigments Regenerated with Partial Agonists

Previous studies have shown that the activation kinetics of transducin (G_t) are affected by pigments regenerated with ac-R and 9c-dm-R, likely due to conformational differences in the active form of the receptor. In this study, we similarly assessed G_t activation by monitoring changes in the intrinsic Trp fluorescence of G_tα upon pigment-catalyzed GTPγS uptake. Both ac-Rho and 9c-dm-Rho exhibited slower activation rates compared to isoRho, as indicated by the longer half-time (t _1/2) of activation (Figure A). Nevertheless, G_t was still effectively activated, albeit at a reduced rate, by these partial agonist-bound pigments. Based on this observation, we next investigated whether rod opsin misfolding mutants could also support signaling activation when bound to ac-R or 9c-dm-R in situ. We used cultured 661W cells stably expressing two rod opsin misfolding mutants, P23H and T289P, alongside WT rod opsin as a control. Although transducin itself is absent in the heterologous system, Rho still couples light signals to the G_i/o pathway (as G_t belongs to the inhibitory G_i/o family of heterotrimeric G proteins), leading to a reduction in cellular cAMP levels. To assess signaling, we measured the cAMP concentration in cells incubated with ac-R, 9c-dm-R, or 9c-R in the dark to permit pigment regeneration, followed by light stimulation (Figure B,C). Control cells were kept in the dark. Upon illumination, cAMP levels decreased by ∼50–60% in both WT and mutant-expressing cells, indicating successful activation of signaling. Interestingly, a similar decrease in cAMP was observed in cells incubated with ac-R or 9c-dm-R, suggesting that neither partial agonist impairs light-induced signaling. As expected, no activation was detected in cells maintained in the dark.

3.

G_t activation and signal transduction by isoRho, ac-Rho, and 9c-dm-Rho pigments. (A) G_t activation by the illuminated purified pigments was recorded by monitoring changes in the Trp fluorescence at 345 nm upon the addition of 10 μM GTPγS. These changes related to the dissociation of G_tα were plotted as a function of time. The representative plots are shown. The excitation and emission wavelengths were 295 and 345 nm, respectively. Each measurement was performed three times. (B,C) The effect of 9c-R, ac-R, and 9c-dm-R on signal transduction through G_i/o signaling was monitored in photoreceptor-derived 661W cells stably expressing WT, P23H, or T289P rod opsin. Changes in the levels of cAMP were determined upon light stimulation (B) and in the dark (C). Nontreated (NT) cells and cells treated with 9c-R were used as controls. Each condition was performed in triplicate, and the experiment was repeated. The one-way ANOVA and Tukey post hoc tests were used for the statistical analysis. * p < 0.05, ** p < 0.01 indicate statistically significant changes; ns, not statistically significant.

The Effect of Partial Agonists on Proteostasis of RP-Linked Rod Opsin Mutants

Inherited mutations in rod opsin often disrupt its internal interaction network, leading to protein misfolding and aggregation within the ER. This triggers activation of the unfolded protein response (UPR) and ultimately results in photoreceptor cell death. For some mutations, these folding defects can be corrected by pharmacochaperones that stabilize the protein, enabling proper folding and trafficking to the plasma membrane. The P23H rod opsin mutant, for example, can be rescued by the natural chromophore 11-cis-retinal or its analog 9c-R, both in vitro and in vivo. However, the light sensitivity of these retinoids limits their therapeutic utility, as rescue efficacy depends on protection from light exposure. Moreover, free retinal released after photobleaching can exacerbate retinal degeneration. , To overcome these limitations, we hypothesized that retinal analogs capable of stabilizing Rho in its Meta-I photointermediate state may offer a therapeutic advantage over natural retinoids. To test this, we treated 661W cells stably expressing WT human rod opsin or two RP-linked mutants, P23H and T289P, with either 9c-R, ac-R, or 9c-dm-R. Cell surface expression was assessed by fluorescent immunostaining of nonpermeabilized cells using an antibody targeting the N-terminal epitope of rod opsin. All three retinal treatments increased the plasma membrane expression of both WT and mutant rod opsins (Figure A,B). In WT rod opsin-expressing cells, 9c-R and ac-R caused a modest increase in surface expression, while treatment with 9c-dm-R led to an approximately 2-fold increase. In P23H rod opsin-expressing cells, as expected, 9c-R enhanced expression by ∼3-fold, ac-R was less effective and resulted in a ∼2-fold increase, while 9c-dm-R led to a ∼6-fold increase. For the T289P mutant, all three retinals produced a similar ∼6-fold increase in the level of plasma membrane expression. In addition, total receptor expression of either WT or mutant receptors upon treatment with all the retinal analogs increased by approximately 2-fold (Figure C,D). Together, these results suggest that partial agonist retinal analogs, particularly 9c-dm-R, may offer improved pharmacochaperoning efficacy for certain rod opsin mutants linked to RP.

4.

Effect of 9c-R, ac-R, and 9c-dm-R analogs on membrane trafficking of RP-linked rod opsin mutants. (A) Cell surface expression of WT, P23H, and T289P rod opsin stably expressed in 661W cells treated with 9c-R, ac-R, and 9c-dm-R at 2.5 μM for 16 h. The expression of the regenerated pigment receptors on the cell surface (shown in red) was detected by immunolabeling with an antibody recognizing the N-terminus of rod opsin and the Alexa-Fluor 594-conjugated secondary antibody. The nuclei of the cells were labeled with DAPI (blue). The images were taken with a Leica fluorescent microscope at 63× magnification. Scale bar: 10 μm. (B) Quantification of the cell surface fluorescence intensity using ImageJ software. Statistical analysis was performed with the one-way ANOVA and Tukey post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 indicate statistically significant changes; ns, not statistically significant. (C) Immunoblots showing the effect of 9c-R, ac-R, and 9c-dm-R on the expression levels of WT, P23H, and T289P rod opsin in 661W cells stably expressing these receptors and the mutant receptor glycosylation profile. Total cell extracts (25 μg) were loaded and separated using SDS-PAGE gel, followed by transfer to a poly­(vinyl difluoride) (PVDF) membrane. Rod opsin was detected with the antibody, recognizing the C-terminal epitope (1D4). GAPDH was detected with an anti-GAPDH antibody and used as a loading control. (D) Quantification of changes in the total rod opsin expression upon treatment with 9c-R, ac-R, and 9c-dm-R. Band intensities were determined with ImageJ software. The rod opsin expression was normalized to GAPDH. Representative immunoblots are shown.

The Effect of Partial Agonists on Glycosylation of RP-Linked Rod Opsin Mutants

Compromised transport of misfolded RP-linked rod opsin mutants is associated with impaired Golgi processing, leading to reduced levels of the mature glycosylated form of the receptor. This is reflected in a shift of the receptor’s molecular mass from ∼55 kDa in the fully glycosylated WT form to ∼37 kDa in the mutants. Upon treatment with all investigated retinal analogs, an increase in the mature glycosylated species was observed (Figures C and A). Specifically, the P23H mutant showed an approximately 1.5-fold increase in the mature glycoform following treatment with 9c-R and ac-R, and a more pronounced ∼3-fold increase with 9c-dm-R. Similarly, treatment of the T289P mutant resulted in ∼3-fold and ∼2-fold increases with 9c-R and ac-R, respectively, while 9c-dm-R led to a ∼6-fold increase (Figure B). Deglycosylation assays using PNGase F and Endo H confirmed that the observed molecular mass shifts in P23H and T289P mutants treated with retinal analogs were due to enhanced glycosylation. Collectively, these findings demonstrate that immature glycosylation in misfolding-prone mutants can be further processed upon treatment with retinal analogs acting as partial agonists. Notably, 9c-dm-R was significantly more effective than either ac-R or the control analog 9c-R further suggesting that 9c-dm-R may offer enhanced pharmacochaperone efficacy for RP-linked misfolding rod opsin mutants.

5.

Effect of 9c-R, ac-R, and 9c-dm-R analogs on maturation of RP-linked rod opsin mutants. (A) Immunoblots showing the effect of 9c-R, ac-R, and 9c-dm-R on the expression levels of WT, P23H, and T289P rod opsin in 661W cells stably expressing these receptors and the mutant receptor glycosylation profile. To determine the sensitivity of the pigments to PNGase F and Endo H deglycosylases, samples were deglycosylated for 1 h at room temperature prior to loading onto the gel. Total cell extracts (25 μg) were loaded and separated using SDS-PAGE gel, followed by transfer to a polyvinyl difluoride (PVDF) membrane. Rod opsin was detected with the antibody recognizing the C-terminal epitope (1D4). The representative immunoblots are shown. (B) Quantification of band intensities of mature and immature P23H and T289P rod opsin compared to nontreated and 9c-R, ac-R, and 9c-dm-R-treated cells. The error bars shown in each result represent standard deviation (S.D.).

Binding of Nonaldehyde Derivatives of 9c-dm-R to Rod Opsin

Although 9c-dm-R displayed superior pharmacochaperone activity, its potential therapeutic application is limited by the inherent toxicity of its aldehyde group. To address this concern, we designed and synthesized two nonaldehyde analogs: an amine derivative, 9-cis-9-demethyl-retinyl methylamine (9c-dm-NHCH₃), and an alcohol derivative, 9-cis-9-demethyl-retinol (9c-dm-OH). These analogs were evaluated for their opsin-binding and pharmacochaperone properties. Molecular docking into the retinal binding pocket of isoRho (PDB ID: 2PED) showed that both 9c-dm-NHCH₃ and 9c-dm-OH accommodated well within the chromophore-binding site, suggesting favorable binding compatibility (Figure A–C). Docking energies for the best models were −12.487 kcal/mol and −12.476 kcal/mol for 9c-dm-NHCH₃ and 9c-dm-OH, respectively. Both compounds occupied the binding pocket in a similar direction like 9c-R and could make a hydrogen bond with Lys296. 9c-dm-OH also showed two more possible hydrogen linkages with Ser186 and Cys187 in this model. Due to the lack of the covalent Schiff base bond, their polyene tail performs with more flexibility, while their β-ionone ring represents the highest structural alignment to the 9c-R ring and is stabilized by the same hydrophobic interactions with residues from TM5, along with Gly121 and Glu122.

6.

Model of accommodation of methylamine and alcohol derivatives of 9c-dm-R. (A) The close-up view of superimposed 9-cis-9-demethyl-retinyl methylamine (9c-dm-NHCH₃) and 9-cis-9-demethyl-retinol (9c-dm-OH) in comparison to 9c-R within the rod opsin orthosteric site. 9c-dm-NHCH₃ and 9c-dm-OH were docked to the isoRho structure (PDB ID: 2PED) using the Galaxy7TM server for flexible GPCR-ligand docking. (B) Three-dimensional representation of ligand interactions within the binding site obtained from the protein–ligand interaction profiler web server for the best docking models. Hydrogen bonds and hydrophobic interactions with side chains are represented by blue solid lines and gray dashed lines, respectively. (C) The predicted interactions between the ligands and the residues in the opsin’s polypeptide chain were obtained with LigPlot⁺. Hydrophobic interactions and hydrogen bonds are represented with red and green dashed lines, respectively. The structures were visualized with the PyMol software.

Trp fluorescence quenching assays confirmed that both analogs (Figure A,B; see UV–visible absorption spectra) could bind to unliganded rod opsin with comparable apparent affinities, showing K _d values of 30.0 nM for 9c-dm-NHCH₃ and 34.9 nM for 9c-dm-OH (Figure C,D). However, their B _max values were approximately 3-fold lower than that of 9c-dm-R, indicating that they function as partial agonists. In addition, thermal stability assays further supported the pharmacochaperone activity. Both compounds increased the thermal stability of rod opsin relative to the unliganded state (T _m = 57.9 °C). Opsin bound to 9c-dm-NHCH₃ exhibited a T _m of 65.9 °C, while 9c-dm-OH-bound opsin had a T _m of 60.9 °C. Although the stabilizing effects were less pronounced than those of 9c-dm-R, these results suggest that both analogs likely possess the ability to stabilize the misfolded rod opsin protein.

7.

Binding of methylamine and alcohol derivatives of 9c-dm-R to rod opsin, their effects on receptor stability and pigment regeneration. (A) Chemical structures of methylamine (9c-dm-NHCH₃) and alcohol (9c-dm-OH) derivatives of 9c-dm-R. (B) UV–visible absorption spectra of 9c-dm-NHCH₃ and 9c-dm-OH. (C) The binding affinity (K _d) of 9c-dm-NHCH₃ and 9c-dm-NHCH₃ was determined by quenching the rod opsin’s intrinsic Trp fluorescence. Compounds were added to opsin membranes at different concentrations (0–1 μM), and changes in the fluorescence intensity at 330 nm were recorded and plotted as a function of the compound concentration. The binding curves were fitted using PRISM GraphPad 10 software. The K _d values of each compound were calculated and averaged from triplicates. (D) The stability of pigments regenerated with 9c-dm-NHCH₃ and 9c-dm-OH. The temperature of melting (T _m) was determined by using a fluorescent probe BFC. The samples were incubated in a stepwise temperature gradient up to 99.9 °C. The obtained values of fluorescence were plotted as a function of temperature, and the melting temperature was calculated using GraphPad 10 software. The T _m for each pigment is presented as a function of the concentration. The error bars shown in each result represent standard deviation (S.D.). (E,F) The UV–visible spectra of isoRho regenerated with 5 μM 9-cis-retinal (9c-R) after treatment of opsin membranes with the 9c-dm-NHCH₃ and 9c-dm-OH compounds at 1 μM, 10 μM, and 100 μM concentrations.

In addition, we investigated the effect of 9c-dm-NHCH₃ and 9c-dm-OH binding on the ability of rod opsin to bind 9c-R and regenerate isoRho by monitoring the appearance of the absorption peak at 485 nm, a signature for the formation of a Schiff base between 9c-R and Lys296 in the protein backbone. Pretreatment of opsin membranes with 9c-dm-NHCH₃ and 9c-dm-OH did not generate a pigment. However, presaturation of the orthosteric pocket with these compounds partially decreased the binding efficiency of 9c-R (Figure E,F). This inhibition was concentration-dependent and more pronounced at 100 μM relative to 1 or 10 μM. Together, these observations confirm that 9c-dm-NHCH₃ and 9c-dm-OH compete with retinal for binding within the rod opsin orthosteric site but do not inhibit the Schiff base formation between the retinal and the receptor.

The Effect of Nonaldehyde Derivatives of 9c-dm-R on Proteostasis of RP-Linked Rod Opsin Mutants and Their Glycosylation Status

The in vitro analysis strongly suggested that both compounds possess pharmacochaperone activity. To further test this, we assessed the effect of 9c-dm-NHCH₃ and 9c-dm-OH on the plasma membrane levels of P23H and T289P rod opsin mutants stably expressed in 661W cells and compared them to the WT control. Treatment with either compound enhanced the cell surface expression of both WT and mutant rod opsins. Notably, 9c-dm-NHCH₃ was more effective, leading to a ∼3-fold increase in plasma membrane expression of P23H mutant and a ∼4-fold increase for T289P mutant expression. In comparison, 9c-dm-OH treatment resulted in a more modest ∼2-fold increase for both mutants (Figure A,B). In addition to cell surface expressions, treatment with both compounds produced a modest increase in total rod opsin levels (between 1.1- and 1.6-fold) for both the WT and RP mutants. However, analysis of receptor glycosylation revealed a limited improvement in maturation. The ratio of mature to immature receptor forms upon 9c-dm-NHCH₃ treatment was ∼1.6 for the P23H mutant and ∼2.0 for T289P, substantially lower than the ratios observed with 9c-dm-R treatment (∼3 and 6, respectively). For 9c-dm-OH, this ratio increased only marginally, indicating a minimal impact on receptor maturation (Figure A–C). Together, these results suggest that while both compounds can enhance surface expression, their ability to promote full maturation of the misfolded receptors is limited when compared to 9c-dm-R.

8.

Effect of amine and alcohol derivatives of 9c-dm-R on membrane trafficking of RP-linked rod opsin mutants. (A) Cell surface of WT, P23H, and T289P rod opsin stably expressed in 661W cells treated with either 9c-dm-NHCH₃ or 9c-dm-OH at 2.5 μM for 16 h. The expression of the regenerated pigment receptors on the cell surface (shown in red) was detected by immunolabeling with an antibody recognizing the N-terminus of rod opsin and the Alexa-Fluor 594-conjugated secondary antibody. The nuclei of the cells were labeled with DAPI (blue). The images were taken with a Leica fluorescent microscope at 63× magnification. Scale bar: 10 μm. (B) Quantification of the cell surface fluorescence intensity was performed using ImageJ software. Statistical analysis was performed with the one-way ANOVA and Tukey post hoc tests. ** p < 0.01, **** p < 0.0001 indicate statistically significant changes.

9.

Effect of amine and alcohol derivatives of 9c-dm-R on the maturation of RP-linked rod opsin mutants. (A) Immunoblots showing the effect of 9c-dm-NHCH₃ and 9c-dm-OH on the expression levels of WT, P23H, and T289P rod opsin in 661W cells stably expressing these receptors and the mutant receptor glycosylation profile. To determine the sensitivity of the pigments to PNGase F and Endo H deglycosylases, samples were deglycosylated for 1 h at room temperature prior to loading onto the gel. Total cell extracts (25 μg) were loaded and separated using an SDS-PAGE gel, followed by transfer to a polyvinyl difluoride (PVDF) membrane. Rod opsin was detected with an antibody recognizing the C-terminal epitope (1D4). GAPDH was detected with an anti-GAPDH antibody and used as a loading control. The experiment was repeated three times. The representative immunoblots are shown. (B) Quantification of changes in the total rod opsin expression upon treatment with 9c-dm-NHCH₃ and 9c-dm-OH. Band intensities were determined with ImageJ software. The rod opsin expression was normalized to GAPDH. (C) Quantification of band intensities of mature and immature P23H and T289P rod opsin compared in nontreated and 9c-dm-NHCH₃- and 9c-dm-OH-treated cells. The error bars shown in each result represent standard deviation (S.D.).

Discussion

Rho, the holo-form of opsin, is the most abundant protein in rod photoreceptor cells, which constitutes ∼85% of the protein mass, and is densely packed in the ROS membrane disks, where it plays an irreplaceable role in the process of vision. , Any mutation in the RHO gene that introduces misfolding and mislocalization of Rho or malfunctioning of this receptor after light absorption may lead to photoreceptor death and vision demise. , The mutations in Rho are the most common causes of blinding eye diseases, including autosomal dominant (ad)­RP, congenital stationary night blindness, , and Leber congenital amaurosis (LCA). , The only FDA-approved therapy for this group of retina degeneration is voretigene neparvovec (Luxturna), a gene therapy targeting autosomal recessive (ar)­RP caused by biallelic mutations in retinal pigment epithelium protein 65 (RPE65), a key enzyme for converting all-trans-retinal released upon exposure to light back to the 11-cis form. , In the absence of a standard and approved treatment for Rho mutations, the use of pharmacological chaperones offers a noninvasive approach with potential for early-stage disease intervention and has shown promising therapeutic efficacy across a broad range of rod opsin mutants for both retinoid and nonretinoid compounds. ,− Retinoid compounds significantly aid in stabilizing and correcting misfolded opsin variants through the formation of a Schiff base with opsin’s Lys296, which introduces a dramatic structural rearrangement. However, their therapeutic application is limited due to their toxicity and the risk of accumulation of toxic photoproducts. ,

Therefore, to overcome these concerns, we explored the pharmacochaperone potential of partial retinoid agonists, which, to our knowledge, have not been previously investigated for this purpose. It is reported that both molecules, 9c-dm-R and ac-R, can shift the equilibrium between Meta-I⇋Meta-II toward Meta-I; thus, they can stabilize the Meta-I state after photoexcitation, , prolonging the retention time of all-trans-retinal as bound to the receptor. Biophysically, the methyl group bound to C9 (C19) of retinal, which is surrounded by Ile189 and Tyr191 residues from ECL-2 (Figure ), plays a critical structural and functional role in the photoisomerization process and subsequent Meta-II formation in Rho. , This methyl group on the polyene chain adds crucial steric bulk, ensuring tight contact with specific protein residues during isomerization. Without this methyl, retinal lacks the necessary constraints to induce the correct conformational strain on adjacent bonds (especially near C11C12), which is critical for coupling the photoisomerization event to downstream structural changes in the Rho protein. The C19 methyl does not change photoisomerization chemistry itself, but its presence is vital for maintaining the integrity of the Schiff base linkage and supporting the large-scale conformational transitions necessary for receptor activation, as evidenced by the reduced ability of 9c-dm-Rho to displace TM6, reshape the ECL-2, and efficiently transduce activation despite successful isomerization. ,, Indeed, this wedge-like methyl acts as a hinge, transmitting the strain of the isomerization to downstream structural shifts and mediating the coupling of protonation events. Furthermore, in cone photoreceptors, this modification leads to slower Meta-II decay rates, prolonged receptor activation, and delayed phototransduction quenching, demonstrating the critical role of the C19 methyl group in rapid signal termination and recovery. Complementing the role of the C19 methyl group, the β-ionone ring of retinal is indispensable for the allosteric coupling of protonation switches during Rho activation and Meta-II formation. This ring is surrounded by Gly121 and Glu122 in TM3; Met207, His211, and Phe212 in TM5; and Phe261, Trp265, and Ala269 in TM6 (Figure ). Removal or modification of the β-ionone ring disrupts the tight coupling between the protonated Schiff base and cytoplasmic proton uptake events, which are fundamental for the Meta-I to Meta-II transition. This disruption leads to stabilization of inactive or partially active states, preventing the full conformational rearrangement of transmembrane helices, especially TM6 displacement, crucial for G protein engagement. Structurally, the β-ionone ring anchors retinal within the opsin pocket, influencing the polyene chain’s flexibility and the overall chromophore conformation, as supported by computational and spectroscopic analyses. , These effects extend to spectral tuning and pigment stability, where alterations in the β-ionone ring or its substituents modulate the absorption maxima and photochemical efficiency. Thus, the β-ionone ring functions as a molecular fulcrum that orchestrates the intramolecular signaling events initiating Rho activation, ensuring the precise control of photoisomerization-triggered conformational changes essential for visual signal transduction.

In this study, as shown in Figure F, the half-life of Meta-II formation increased from 0.6 min for 9c-R to 1.3 min for ac-R and 10.8 min for 9c-dm-R. Chromophore release data also provided further proof for prolonged retention time for the attachment of the 9c-dm-R photoproduct to the Meta-II state compared to 9c-R, while this is less significant in the case of the ac-R photoproduct (Figure E). These observations are compatible with previous reports, as discussed above. Furthermore, the 9c-dm-R compound represents the most favored binding properties to opsin, even surpassing those of 9c-R (Figure B,C). This shows that while the β-ionone ring plays a critical role in chromophore binding affinity, the steric hindrance caused by the C19 methyl group may not be favorable for rapid ligand channeling. This property makes 9c-dm-R the most promising candidate for improving the folding of misfolded rod opsin mutants, as it can be administered at lower concentrations compared to 9c-R, an advantage that may help reduce toxicity, especially considering that high doses of 9c-R are known to be harmful to retinal cells. In addition, both compounds exhibit great global stability against temperature or acid denaturation and local stability in the presence of hydroxylamine after incubation with rod opsin (Figure G–I). The effect of 9c-dm-R was comparable to that of 9c-R highlighting the importance of the β-ionone ring in retinal ligands for effective binding to rod opsin.

Here, for the first time, we investigated the effects of these two partial retinoid agonists on the maturation and membrane localization of misfolded Rho. We used two opsin mutants, P23H and T289P, stably expressed in the 661W cell line. Both mutations are located in the intradiscal region of opsin, near the orthosteric binding pocket, and are known to induce ER stress and UPR signaling due to misfolding and aggregation of opsin. − Interestingly, both 9c-dm-R and ac-R, similar to 9c-R, improved misfolded opsin maturation as well as membrane localization of both mutants (Figures A–D and A,B). This observation implies that both compounds are effective in improving the folding of misfolded opsin mutants and stabilizing them. Although stabilization of Meta-I by these compounds may slightly alter the kinetics of G-protein activation by delaying Meta-II formation, potentially affecting the onset or amplitude of signaling (Figures C and A,B), the strong catalytic amplification downstream of Meta-II, together with compensatory factors such as retinal regeneration and pigment stability, likely buffers these modest changes and preserves overall signaling capacity in vivo. Importantly, the primary role of these compounds is to rescue rod opsin folding during translation within the ER to support proper trafficking to the ROS disk membranes. Once opsin is localized there, these ligands are expected to be replaced by the endogenous 11-cis-retinal to form functional Rho and restore the full phototransduction capability. Given that properly folded Rho is stable, turning over only at a rate of ∼10% per day through RPE-mediated shedding, Meta-I accumulation in the densely packed ROS disks is expected to be negligible. Together, these data suggest that both partial retinoid agonists used in this study, most notably 9c-dm-R, are promising therapeutic candidates for diseases associated with rod opsin misfolding. In contrast, the earlier reported 11-cis-7-membered-ring-retinal, despite the ability to improve the maturation and membrane expression of P23H rod opsin, does not activate G-protein in vitro and in vivo. On the other hand, another locked retinal, 11-cis-6-membered-ring-retinal can partially activate G-protein; however, its effect on rod opsin mutant maturation and membrane localization has not been studied yet.

Since 9c-dm-R retains a reactive aldehyde group, which still may raise systemic safety concerns, , we developed and tested two nonaldehyde derivatives of 9c-dm-R. By replacing the aldehyde moiety with either a hydroxyl or a methylamine group, these new derivatives are no longer capable of forming a covalent Schiff base bond. However, as shown in the docking results (Figure A–C), both compounds could accommodate within the orthosteric site, similarly to 9c-R, and form distinct hydrogen bonds with residues Lys296, Ser186, and Cys187 through their polyene tail. These linkages, along with the conserved hydrophobic interaction of their β-ionone ring and Trp265, contribute to their ability to bind opsin with a nanomolar-range affinity. As compared to the original compound, 9c-dm-R, both 9c-dm-OH and 9c-dm-NHCH₃ exhibit a reduction in maximum binding capacity (B _max) and diminished receptor-stabilizing effect (Figure C,D), which may be attributed to their increased hydrophilicity resulting from the hydrogen-donating hydroxyl or methylamine groups, respectively, and their inability to form the covalent linkage. This increased polarity could impair efficient ligand channeling to the hydrophobic binding pocket. Unlike 11-cis-retinal, which efficiently enters the hydrophobic ligand channel and covalently binds opsin, polar nonaldehyde derivatives may diffuse less readily and thus show lower occupancy in vivo. Nonetheless, under conditions of limited chromophore availability or impaired retinal binding, such compounds could still stabilize folding intermediates, underscoring the importance of balancing polarity with channel permeability in future scaffold design. Indeed, both the hydroxyl and methylamine derivatives of 9c-dm-R were effective in promoting the maturation and membrane localization of the misfolded opsins P23H and T289P. This effect was more pronounced with 9c-dm-NHCH₃, highlighting it as a lead candidate for further optimization aiming to correct misfolded rod opsin. Interestingly, previous studies have reported that synthetic retinal derivatives lacking a reactive aldehyde group, such as 8-epoxy-13-cis-retinoic acid (13-cis-5,8-ERA) and SRD005825, can act as pharmacochaperones. These compounds promoted proper folding and membrane trafficking of rod opsin mutants in vitro. , Notably, SRD005825 also improved photoreceptor survival in the T17M Rho mouse model of RP.

In addition, RP-linked misfolding mutations in Rho are typically dominant due to a toxic gain-of-function effect caused by mutant receptors rather than simply reduced overall protein levels. Even though the wild-type allele continues to produce correctly folded Rho, the presence of normal Rho cannot compensate for the chronic ER stress caused by misfolded protein toxicity. Thus, therapeutic approaches must go beyond simply supplementing the functional receptor. Importantly, strategies involving pharmacological chaperones may counteract misfolding and reduce cellular stress, thereby prolonging photoreceptor survival. While these agents cannot cure RP, they represent a disease-modifying therapy capable of delaying vision loss.

In this study, we evaluated the therapeutic potential of partial retinoid agonists in targeting diseases associated with misfolded rod opsin. Our findings identify 9c-dm-R as a promising lead compound, offering effective stabilization of misfolded rod opsin mutants with reduced production of photoisomerization byproduct. Among its derivatives, the methylamine-substituted form, 9c-dm-NHCH₃, demonstrated particularly favorable activity, highlighting its potential for further development. Future studies will focus on assessing its therapeutic efficacy in animal models of RP.

Experimental Procedures

Chemical Reagents

BODIPY FL l-Cystine (BFC) was obtained from Invitrogen (B20340). 4969-Diamidino-2-phenylindole (DAPI) Fluoromount-G was purchased from SouthernBiotech (Miami, FL). n-Dodecyl-β-D-maltoside (DDM) was purchased from Affymetrix Inc. (Maumee, OH). The Direct Cyclic AMP ELISA Kit was obtained from Arbor Assays (Ann Arbor, MI). Dimethyl sulfoxide (DMSO), 9-cis-retinal and phosphodiesterase inhibitor (Ro-20-1724) were obtained from Sigma (St. Louis, MO). EDTA-free protease inhibitor cocktail tablets were purchased from Roche (Basel, Switzerland). Forskolin, purchased from Sigma, was dissolved in DMSO to obtain a 24 mM stock solution stored at −20 °C. MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide was purchased from Sigma. Polyethylenimine, Linear (M.W. 25,000), was purchased from Polysciences, IL. Polyvinylidene difluoride (PVDF) membrane was obtained from Millipore (Burlington, MA). PNGase F and Endo H were purchased from New England Biolabs (Ipswich, MA). Sodium cyanoborohydride (NaBH₃CN) and sodium borohydride (NaBH₄) were purchased from Sigma.

List of Commercial Antibodies

Anti-GAPDH, mouse monoclonal, Abclonal, No: AC002, dilution 1:10,000.

Anti-mouse IgG Alexa Fluor 555-conjugated, goat, Thermo Fisher, No: A30677, dilution 1:400.

HRP-conjugated Anti-Mouse IgG (H+L), Promega, No: W4021, dilution 1:10,000.

Preparation of Retinal Compounds

9-cis-9-demethyl-retinal (9c-dm-R) was synthesized as previously described.

9-cis-9-demethyl-retinyl methylamine (9c-dm-NHCH₃) and 9-cis-9-demethyl-retinol (9c-dm-OH) were prepared from 9c-dm-R.

9c-dm-NHCH₃ was prepared by reacting an aqueous solution of methylamine (MeNH₂) with 9c-dm-R at 25 °C to form the corresponding imine intermediate. The reaction mixture was then gradually acidified with concentrated hydrochloric acid (12 N) to protonate the imine, as confirmed by UV–visible spectroscopy, thereby facilitating the formation of the protonated Schiff base. Sodium cyanoborohydride (NaBH₃CN) was subsequently added to reduce the imine bond length to the corresponding secondary amine. Upon completion of the reaction, the mixture was filtered, and the product was analyzed by UV–visible spectroscopy.

9c-dm-OH was synthesized by reducing 9c-dm-R with sodium borohydride (NaBH₄) in an anhydrous ethanol medium, followed by filtration and spectroscopic characterization.

Molecular Docking

The coordinates of the 9-cis-retinal (9c-R) bound bovine opsin, PDB ID: 2PED, at 2.95 Å resolution were obtained from the Protein Data Bank. One monomeric unit of the protein was selected, and the cocrystallized molecules and all crystallographic water molecules were removed from the coordinate set; hydrogen atoms were added, and partial charges were assigned to all atoms. The 3D structures of the compounds used in docking were generated using Avogadro, and their geometries were optimized. Before docking, the ligand-free structure of the opsin model was subjected to a 10 ns molecular dynamics equilibration with the Memprot.GPCR-ModSim web server (https://memprot.gpcr-modsim.org/). Then, the molecular docking of 9c-dm-NHCH₃ and 9c-dm-OH into the opsin orthosteric binding pocket was performed with the Galaxy7TM web server (https://galaxy.seoklab.org/cgi-bin/submit.cgi?type=7TM) to perform a GPCR side chain flexible docking. For each compound, the best-generated model was used for further structural analysis.

Preparation of Opsin Membranes

The opsin membranes were prepared as described previously. ,, Briefly, rod outer segment (ROS) membranes were isolated from frozen bovine retinas under dim red light as described in ref. . Membranes were washed four times with a hypotonic buffer containing 5 mM HEPES, pH 7.5, 1 mM EDTA, and 1 mM DTT, then pelleted by centrifugation at 25,000 × g for 25 min. The membrane pellet was resuspended in a buffer composed of 10 mM sodium phosphate, pH 7.5, and 20 mM hydroxylamine and exposed to white light with a 150 W bulb for 1 h at 0 °C. These membranes were then pelleted by centrifugation at 16,000 × g for 10 min. The supernatant was discarded, and the membrane pellet was washed four times with 10 mM sodium phosphate, pH 6.5, and 2% BSA, followed by four washes with 10 mM sodium phosphate, pH 6.5, and two washes with 20 mM bis-tris propane (BTP), pH 7.5, and 120 mM NaCl. Finally, the membranes were suspended in 20 mM BTP at pH 7.5 and 120 mM NaCl, aliquoted, and stored at −80 °C for future experiments.

The concentrations of Rho or opsin within the ROS membranes were measured after membrane solubilization with 20 mM dodecyl-β-D-maltoside (DDM) and pelleting insoluble material by centrifugation at 16,000 × g for 15 min at 4 °C. A UV–visible spectrophotometer (Cary 60, Varian, Palo Alto, CA) and the absorption coefficients ε₅₀₀ nm = 40,600 M^–1 cm^–1 and ε₂₈₀ nm = 81,200 M^–1 cm^–1 were used, respectively, for Rho and opsin.

Ligand Binding Affinity

The quenching of intrinsic tryptophan (Trp) fluorescence assay was used to determine the binding affinity of retinal analogs into the rod opsin orthosteric site. Opsin membranes at a final concentration of 4.2 nM in the buffer containing 20 mM BTP (pH 7.5), 100 mM NaCl, and 1 mM DDM were used. Retinals: 9-cis-retinal (9c-R), acyclic-retinal (ac-R), and 9-cis-9-demethyl-retinal (9c-dm-R) were added at increasing concentrations (0–1 μM), and the emission spectra were recorded with an FL 6500 Fluorescence Spectrometer (PerkinElmer) at 20 °C between 300 and 420 nm after excitation at 295 nm. The excitation and emission slit bands were set at 5 and 10 nm, respectively. The changes in the intrinsic Trp fluorescence at 330 nm (ΔF/F ₀, where ΔF is the difference between the initial Trp fluorescence (F ₀) and fluorescence recorded upon addition of the compound) were plotted as a function of the ligand concentration. GraphPad Prism 10 software was used to fit the ligand-binding curves to calculate the binding affinities (K _d). All measurements were performed in triplicate. The experimental data were corrected for the samples’ background and self-absorption at excitation and emission wavelengths (inner filter effect correction).

Pigment Regeneration Assay

Opsin membranes at a 2.5 μM concentration in 20 mM BTP at pH 7.5 and 120 mM NaCl were incubated with 10 mM DDM for 10 min at room temperature. Solubilized opsin was cleared by centrifugation at 16,000 × g for 5 min at 4 °C. Then, each retinal compound at a 5 μM final concentration was added, and the UV–visible spectrum was recorded every 1 min for 30 min at 20 °C. Alternatively, for pigment regeneration in the presence of 9c-dm-NHCH₃ and 9c-dm-OH, initially the solubilized opsin membrane at a 2.5 μM concentration was incubated with 1 μM, 10 μM, and 100 μM of each 9c-dm-R derivative for 1 h at 4 °C. Then, 9c-R was added to a final concentration of 5 μM, and the UV–visible spectrum was recorded after 30 min. Each condition was repeated three times. The absorbance at 485 nm for 9c-R, 460 nm for ac-R, and 464 nm for 9c-dm-R was plotted as a function of time, and a time course of pigment regeneration was fitted to an exponential rise-to-maximum equation using SigmaPlot 11 to calculate the apparent half-lives (t _1/2) of pigment regeneration.

Thermal Stability, Chemical Stability, and Acid Denaturation Assay

The pigments were regenerated according to the method described above.

Thermal stability was assessed using a thermal shift assay. The BFC probe was dissolved at a stock concentration of 10 mM in DMSO. A final working concentration of 2 μM BFC in 20 mM BTP at pH 7.5 and 120 mM NaCl was used in all experiments. The opsin membranes (40 μL) at a concentration of 100 nM were loaded into a 96-well plate (Applied Biosystems). Then, a specific retinal compound was added at final 0.78, 1.56, 3.12, 6.25, 12.50, 25, and 50 μM concentrations, and the plate was incubated for 1 h at 4 °C. Next, 1 μL of the BFC probe was added to each well. The plate was sealed with a ClearSeal film (HR4-521) from Hampton Research and incubated for 10 min on ice prior to the measurement of their fluorescence. All measurements were performed with a StepOnePlus Real-Time PCR System (Applied Biosystems), and melting curve experiments were recorded using StepOne software, version 2.3. The fluorescence in the SYBR, FAM, and ROX channels was recorded for each sample. The run was set to cool the plate to 4 °C within 10 s, kept at 4 °C for 3 min, and then increased by 3 °C per min in a step-and-hold manner up to 99.9 °C. The multicomponent data were exported to a Microsoft Excel sheet and analyzed with GraphPad Prism 10 software.

The stability against reactive chemical compounds such as hydroxylamine and acid was monitored following the addition of hydroxylamine at 40 mM or HCl at 20 mM final concentration at room temperature. Each condition was repeated three times. Then, the maximum absorbance intensity at 485 nm for 9c-R, 460 nm for ac-R, and 464 nm for 9c-dm-R, respectively, was measured at 2-min intervals for 120 and 28 min, respectively. Absorbance at the maximum wavelength at the initial time point was assumed to be 100%. The percentages of remaining pigments were normalized to their initial concentrations and then plotted as a function of time using the SigmaPlot 11 software. These plots were fitted to an exponential decay function to calculate the decay rates and the apparent half-lives (t _1/2) of chromophore release.

Pigments Purification

Bovine opsin membranes were resuspended in 20 mM BTP (pH 7.5) containing 120 mM NaCl and incubated with a retinal compound (9c-R, ac-R, or 9c-dm-R) at a 10 μM concentration for 1 h at 4 °C on a nutator. Then, DDM was added to 20 mM final concentration to solubilize the membranes and incubated for 1 h at 4 °C on a nutator, followed by lysate centrifugation at 16,000 × g for 1 h at 4 °C. The soluble fraction was combined with 250 μL of 2 mg 1D4/mL agarose beads (anti-Rho C-terminal 1D4 antibody immobilized on cyanogen bromide (CNBr)-activated agarose) and incubated for 1 h at 4 °C on a nutator. Then, the resin was transferred to a column and washed with 10 mL of 20 mM BTP, 120 mM NaCl, containing 2 mM DDM, pH 7.5. Pigments were eluted with the same buffer, supplemented with 0.6 mg/mL of the 1D4 peptide (TETSQVAPA). All of the above steps were carried out in the dark conditions. The protein concentrations were measured with the UV–visible spectrophotometer (Cary 60, Varian, Palo Alto, CA).

Photobleaching

The immuno-affinity-purified pigments were illuminated through a 420–520 nm band-pass filter (Chroma Technology Corporation, Bellows Falls, VT) for 5, 10, 20, 30, 60, and 120 s for isoRho (regenerated with 9c-R) from a distance of 15 cm. The illumination was carried out for up to 7 min for ac-Rho (regenerated with ac-R) and for up to 15 min for 9c-dm-Rho (regenerated with 9c-dm-R). The absorbance at the maximum wavelength (485 nm for 9c-R, 460 nm for ac-R, and 464 nm for 9c-dm-R) at the initial time point was assumed to be 100%. The values of maximum absorbance intensity were plotted as a function of time using SigmaPlot 11 software. These plots were fitted to the exponential decay function to calculate the decay rates and the apparent half-lives (t _1/2) of chromophore release.

Light-Induced Chromophore Release

To determine the light-induced chromophore release rates and half-lives (t _1/2) in pigments regenerated with 9c-R, ac-R, and 9c-dm-R changes in the intrinsic Trp fluorescence were measured. Purified pigments at a 50 nM concentration in a buffer composed of 10 mM BTP (pH 6.0), 100 mM NaCl, and 1 mM DDM were exposed to light with a Fiber-Light illuminator (Dolan Jenner Industries Inc., Boxborough, MA) through a 420–520 nm band-pass filter (Chroma Technology Corporation, Bellows Falls, VT) for 15 s at a distance of 15 cm. Changes in Trp fluorescence, which correlate with the decrease in the protonated Schiff base concentration, were recorded for 60 min at 20 °C with an FL 6500 Fluorescence Spectrometer (PerkinElmer). The slits were set to 8 at 295 nm for excitation and 10 at 330 nm for emission. The recorded fluorescence data was normalized (to 1 at the final time point) and plotted as a function of time using the SigmaPlot 11 software. These plots were fitted to the exponential decay function to calculate the decay rates and the apparent half-lives (t _1/2) of chromophore release.

G_t Activation

One hundred dark-adapted bovine retinas were used to isolate ROS membranes, followed by the extraction of G_t and its purification as described in refs. , . Activation properties of the purified pigments regenerated with 9c-R, ac-R, and 9c-dm-R were tested in a Trp fluorescence G_t activation assay. Briefly, G_t (500 nM) and pigment (50 nM) were mixed in a buffer composed of 10 mM BTP at pH 7.0, 120 mM NaCl, and 1 mM DDM, and illuminated with a Fiber-Light illuminator through a band-pass wavelength filter (480–520 nm) for 30 s. Next, 10 μM GTPγS was added, and the measurement was performed for 1200 s with an FL 6500 Fluorescence Spectrometer. Excitation and emission wavelengths were set at 300 and 345 nm, respectively. − The recorded fluorescence data was normalized (to 1 at the final time point) and plotted as a function of time using the SigmaPlot 11 software. These plots were fitted to the exponential rise-to-maximum function to calculate the rates and apparent half-lives (t _1/2) of G_t activation.

Cell Culture and Stable Cell Line Generation

The 661W cells, murine photoreceptor-derived cells, were obtained by Dr. Muayyad Al-Ubaidi, University of Houston, and they were cultured according to the provided instruction in DMEM with 10% FBS (Hyclone, Logan, UT), 0.04 mg/mL progesterone, 0.04 mg/mL hydrocortisone, 0.032 mg/mL putrescine, 0.04% β-mercaptoethanol, and 1 unit/mL penicillin with 1 μg/mL streptomycin (Life Technologies) at 37 °C under 5% CO₂.

The 661W cells stably expressing WT, P23H, and T289P human rod opsin were generated by transduction of 661W cells with the appropriate virus construct obtained from the Phoenix-Ampho packaging cell line (CRL-3213, ATCC, VA, USA), followed by selection with puromycin. Phoenix-Ampho cells were transfected with 10 μg of pMXs-hOpsin-IRES, pMXs-hOpsinP23H-IRES, and pMXs-hOpsinT289P-IRES using polyethylenimine M.W. 25,000 (Polysciences, IL). The conditional medium was collected 48 h later and either used immediately to infect the 661W cells or flash-frozen and kept at −80 °C.

Immunoblotting

The 661W cells stably expressing WT, P23H, and T289P human opsin were plated in a six-well plate at a density of 500 × 10³ cells/well in the morning. In the afternoon, these cells were treated with retinal compounds at a final concentration of 2.5 μM and cultured overnight in the dark. The next day, the cells were washed with PBS, collected, and proteins were extracted with the buffer containing 20 mM BTP (pH 7.5), 120 mM NaCl, 20 mM DDM, and protease inhibitors. After 1 h of incubation, the protein extract was centrifuged at 16,000 × g for 30 min using a top bench centrifuge. The soluble fractions were collected, and the total protein concentration was measured at 280 nm using a Nanodrop spectrophotometer. The proteins were separated by using a 10% polyacrylamide SDS-PAGE gel and then transferred to a PVDF membrane. After blocking the membrane with 5% milk, the expression of rod opsin was probed with the 1D4 antibody. GAPDH was used as a loading control.

cAMP Detection

The 661W cells stably expressing WT, P23H, and T289P human opsin were plated in two 96-well plates at a density of 100 × 10³ cells per well in 85 μL of media in the morning. In the afternoon, these cells were treated with 5 μL of each compound at a final concentration of 2.5 μM and cultured overnight in the dark. The next day, while keeping in the dark, 5 μL of phosphodiesterase inhibitor (1.95 mM) and 5 μL of forskolin (100 μM) were added to maximize the concentration of total cAMP within the cells. Two minutes after forskolin addition, one plate was exposed to white light with a 150-W bulb for 15 min at RT and assigned as “Light Condition”, while the other plate was assigned as “Dark Condition”. The level of cAMP was measured using DetectX (Direct Cyclic AMP ELISA Kit, Arbor Assays) following the manufacturer’s protocol.

Membrane Localization of Rod Opsins in 661W Cells

The 661W cells stably expressing WT, P23H, and T289P mutant opsin were cultured on a collagen-coated coverslip in a 24-well plate at a density of 60 × 10³ cells/well, and then the cells were treated with retinal compounds at a final concentration of 2.5 μM and cultured overnight in the dark. The next day, the medium was removed, and the cells were washed with PBS three times. Then, the cells were fixed with 3% paraformaldehyde freshly prepared in PBS for 20 min at room temperature, followed by two washes with PBS. Next, the cells were incubated in 10% normal goat serum diluted in PBS for 1 h at room temperature. To detect opsin, the cells were incubated with the B6-30 antibody that recognizes the N-terminal epitope of rod opsin for 3 h at room temperature. Then, cells were washed three times with PBS for 10 min. Opsin immunostaining was visualized by incubating the cells with an anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo Fisher) at a 1:400 dilution for 1 h at room temperature. After that, the cells were washed three times with PBS for 10 min. The cell nuclei were stained with DAPI (1 mg/mL) for 5 min at room temperature, followed by mounting with Fluoromount-G. The cells were imaged with a Leica fluorescent microscope at 63× magnification.

Statistical Analysis

Thermal shift assay, cAMP quantification, and compound cytotoxicity experiments were performed in triplicate and repeated. Each assay included positive and negative controls. The following assays, ligand binding, pigment regeneration, photobleaching, chromophore release, G_t activation, immunostaining, and immunoblotting, were performed in triplicate. The parameters derived from these measurements are shown as an average and standard deviation (S.D.). The Student’s t-test or one-way ANOVA with Tukey post hoc test was used for hypothesis testing. All statistical calculations were performed using Prism GraphPad 10 software. Type 1 error tolerance for the experiments was established at 5%. Differences were considered statistically significant for a p-value of <0.05 (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). A different person from the experimenter performed the analysis.

The data that support the findings of this study are included in the manuscript.

Participated in research design: B.J. and Z.P. Conducted experiments: B.J., Z.P., M.L., and M.A. Chemical synthesis: S.D. and M.S. Performed data analysis: B.J., Z.P., M.L., and M.A. Wrote or contributed to the writing of the manuscript: B.J., Z.P., M.L., M.A., and M.S.

This research was supported by the National Institutes of Health (NIH) (R01EY032874 to B.J.) and the Visual Sciences Research Center Core Facilities at Case, supported by NIH grant P30 core grant (P30EY011373). In addition, this research was in part supported by the Cleveland Clinic Foundation’s Cole Eye Institute T32 (5T32EY024236-09) and the Molecular Pharmacology Training Program T32 (1T32GM158814-01), granted to the graduate student M.L.

The authors declare no competing financial interest.