Non-retinoid pharmacochaperones and uses thereof

Abstract

A non-retinoid pharmacochaperone includes a compound of formula (I), (II), (III) or a pharmaceutically acceptable salt thereof.

Description

PATENT NON-RETINOID PHARMACOCHAPERONES AND USES THEREOF RELATED APPLICATION

[0001] This application claims priority from U. S. Provisional ApplicationNo. 63 / 719,963, filed November 13, 2024, the subject matter of which is incorporated herein by reference in its entirety.GOVERNMENT FUNDING

[0002] This invention was made with government support under EY025214, EY032874, EY011373 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Inherited mutations that disrupt protein folding are responsible for a variety of protein conformational diseases (PCDs). Membrane proteins, including G protein-coupled receptors (GPCRs), are especially prone to misfolding. Misfolded membrane proteins that are detected by cellular quality control machinery are retained and degraded in the endoplasmic reticulum (ER), which typically results in a loss of function. One of the most important known PCDs is retinitis pigmentosa (RP), which is a blinding disease associated with mutations in the rod opsin (RHO) gene among other retina-specific genes. Numerous mutations in RHO are known to enhance the misfolding of the nascent rhodopsin (Rho) protein in the ER, which compromises its trafficking, stability, and / or binding of its native 11 -cA-retinal chromophore.

[0004] Pharmacological chaperones that target misfolded proteins and decrease folding energy barriers may correct protein misfolding and promote the proper routing, ameliorating the underlying mechanism of the disease. The expression and trafficking of various misfolded Rho variants can be corrected by both retinoid and non-retinoid pharmacochaperones that bind and stabilize the unliganded opsin protein. 1 1 - -relinal and its analog 9- -relinal are particularly effective at improving the folding and membrane targeting of pathogenic Rho mutants in vitro. However, their therapeutic utility is limited due to their sensitivity to light and chemical reactivity of retinal photo-metabolites. For these reasons, the development of pharmacological chaperones may require the discovery of non-retinoid compounds that bind to ligand-free opsin. Such chaperones may help suppress thedegradation of destabilized rod opsin variants within the secretory pathway of rod cells where the supply of the endogenous stabilizing retinoids is limited.SUMMARY

[0005] Embodiments described herein relate to non-retinoid compounds that act as pharmacochaperones to modulate opsin protein properties, and particularly to the use of the non-retinoid compounds in methods of treating retinal degeneration, such as retinal degeneration associated with inherited rhodopsin mutations in ocular tissue of a subject. The non-retinoid compounds described herein are insensitive to light and reversibly bind to ligand-free opsin. Once bound, the non-retinoid compounds increased the stability of opsin and substantially improved the maturation and expression of heterologously expressed P23H rod opsin, resulting in protection of retinas against light-induced degeneration and prolonged survival of photoreceptors in a retinitis pigmentosa model for rod opsin misfolding.

[0006] In some embodiments, a non-retinoid pharmacochaperone described herein can include a compound of formula (I):R1a(I) or a pharmaceutically acceptable salt thereof; whereinthe dashed line is an optional bond;X is -N-, -C-, or -C(H)-;X1is -C(H)-, -C(H2)-, or -O-;X2is -C(R4)-, -C(R4)2-. or -S-;X3is absent, -N-, -N(H)-, -C(H)-, or -C(H2)-;R1is aryl, heterocyclyl, or heteroaryl, each of which is optionally substituted with one or more R5;Rlais phenyl or benzyl, each of which is optionally substituted with one or more R2;R4is H, halogen, alkyl, or haloalkyl;each R5is halogen, -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m( alkyl), -C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, alkoxy, haloalkyl, cycloalkyl, or heterocyclyl; andm is 0, 1, or 2.

[0007] In other embodiments, a non-retinoid pharmacochaperone described herein can include a compound of formula (11):XCX20^=^ 'A^R1IR1a(II) or a pharmaceutically acceptable salt thereof; whereinthe dashed line is an optional bond;X is -N-, -C-, or -C(H)-;X1is -C(H)-, -C(H2)-, or -O-;X2is -C(R4)-, -C(R4)2-, or -S-;R1is aryl, heterocyclyl, or heteroaryl, each of which is optionally substituted with one or more R5;Rlais a phenyl or a benzyl, each of which is optionally substituted with one or more RR4is H, halogen, alkyl, or haloalkyl;each R5is halogen, -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m( alkyl), -C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, alkoxy, haloalkyl, cycloalkyl, or heterocyclyl; and m is 0, 1, or 2.

[0008] In some embodiments, Rlais a phenyl, which is optionally substituted with one or more R5.R6

[0009] In other embodiments, R1is ' —> J R, wherein R6and R7are each independently absent, halogen, -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m( alkyl), -C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, haloalkyl, cycloalkyl, or heterocyclyl; or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N, and wherein the heterocycle is optionally substituted with R8; and R8is halogen, alkyl, or alkoxy.

[0010] wherein R9is a halogen, C^C6alkyl, or C^C6alkoxy.

[0011] In other embodiments, a non-retinoid phamiacochaperone described herein can include a compound of formula (III):-X2R®OR- (III) or a pharmaceutically acceptable salt thereof; whereinthe dashed line is an optional bond;X2is -C(R4)-, -C(R4)2-, or -S-;R2and R3are each independently absent or halogen, alkyl, haloalkyl, or alkoxy;R4is H, halogen, alkyl, or haloalkyl;R6and R7are each independently absent, halogen, -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m( alkyl), -C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, haloalkyl, cycloalkyl, or heterocyclyl;or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N, and wherein the heterocycle is optionally substituted with R8;R8is halogen, alkyl, or alkoxy; andm is 0, 1, or 2.

[0012] In some embodiments, X2is S; R2and R3are each independently absent, a halogen, Ci-Ce alkyl, or Ci-Ce alkoxy; and R6and R7are each independently absent, halogen, Ci-Ce alkyl, or C'l-C'e alkoxy; or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N.

[0013] For example, the compound or the pharmaceutically acceptable salt thereof can include a compound selected from:acceptable salt thereof.

[0014] In other embodiments, X2is -C(R4)-; R2and R3are each independently absent, a halogen, Ci-Ce alkyl, or Ci-Ce alkoxy; and R6and R7arc each independently absent, halogen, Ci-C6alkyl, or Ci-Ce alkoxy.

[0015] For example, the compound or the pharmaceutically acceptable salt thereof can include a compound selected from:Cl

[0016] In other embodiments, the compound or the pharmaceutically acceptable salt thereof can include a compound selected from:or a pharmaceutically acceptable salt thereof.

[0017] In other embodiments, the compound or the pharmaceutically acceptable salt thereof can be selected from:an (R) enantiomerofan (S) enantiomer ofan (R) enantiomer ofan (S) enantiomer of salt thereof.

[0018] Other embodiments relate to a method of suppressing the pathogenic effects of misfolded rhodopsin variants in a subject in need thereof. The method can includeadministering to the subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof as described herein.

[0019] Still other embodiments relate to a method of treating retinal degeneration in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof as described herein.

[0020] In some embodiments, the retinal degeneration is selected from the group consisting of Leber congenital amaurosis, Stargardt disease, and retinitis pigmentosa.

[0021] In some embodiments, the retinitis pigmentosa can include autosomal dominant retinitis pigmentosa associated with a P23II RIIO mutation.

[0022] In some embodiments, the therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof is an amount effective to inhibit photoreceptor cell death in the subject.

[0023] In other embodiments, the therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof is an amount effective to inhibit bright light-induced retinal degeneration in a Abca4- / -Rdh8- / -mouse.

[0024] In some embodiments, administration of the compound or the pharmaceutically acceptable salt thereof to the subject stabilizes P23H rod opsin mutant proteins.

[0025] In some embodiments, administration of the compound or the pharmaceutically acceptable salt thereof to the subject promotes rod photoreceptor cell homeostasis in the subject.

[0026] In some embodiments, administration of the compound or the pharmaceutically acceptable salt thereof to the subject mobilizes the P23H opsin from the endoplasmic reticulum to the plasma membrane of photoreceptor cells

[0027] In some embodiments, the compound or the pharmaceutically acceptable salt thereof can be used in a method of stabilizing unliganded or ligand-free opsin.

[0028] In some embodiments, the compound or the pharmaceutically acceptable salt thereof can be capable of crossing the blood retina barrier (BRB) upon systemic, preferably, oral, administration to a subject.

[0029] In some embodiments, the non-retinoid pharmacochaperone can be capable of reversibly binding to rod opsin.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figs. l(A-B) illustrate molecular docking of the compounds to the opsin’s orthostcric site. (A) Two compounds JC3 and JC4 were identified in silico. The structures of bovine rod opsin (PDB ID: 3CAP) with the best binding poses of these compounds docked to the orthosteric binding pocket and close-ups of the binding site are shown. The ribbons in the rod opsin structure are shown in grey. The compounds are shown in purple. Trp265 is shown as green sticks. (B) Two-dimensional representation of the low energy structures obtained from molecular docking simulations. The interactions between the compound and the residues in the opsin’s polypeptide chain are shown. The structures obtained from molecular docking simulations (A) and the two-dimensional representations (B) were visualized with the Biovia Discovery Studio Visualizer 17.2.0 visualizer.

[0031] Figs. 2(A-E) illustrate plots showing binding of JC3 and JC4 to rod opsin, their effects on pigment regeneration, stability and signaling. (A) The binding of the identified compounds was determined by quenching the opsin’s intrinsic Trp fluorescence. Compounds were added to opsin membranes at different concentrations (100-1500 nM) 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 7.02 software. The Ka values of each compound were calculated and averaged from triplicates. These values ± standards deviation (S. D.s) are shown in the figure. (B) The UV-visible spectra of isoRho regenerated with 5 p M 9-cis-retinal (9cR) after treatment of opsin membranes with the JC3 and JC4 compounds at 10 uM and 100 pM concentrations. (C) The half-life (ti / 2) of isoRho regenerated with 5 pM 9cR and upon the treatment of opsin membranes with the new compounds at 1, 10 and 100 pM concentrations. (D) The effect of the JC3 and JC4 on the rod opsin stability. The temperature of melting (Tm) was determined for the opsin membrane in the presence of the JC compounds at different concentrations (0.0001-100 pM) by using a fluorescent probe BFC. The samples were incubated in the step-wise 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 7.02 software. The Tm for each compound is presented as a function of compound concentration and the values obtained at 0.1 pM compound concentration are shown in the figure. Error bars represent standard deviation (S. D.). Each measurement was repeated three times. Statistical analysis was performed with the one-way ANOVA and Turkey post hoc tests. The statistically differentchanges (P) are indicated in the figure. NS, not statistically significant. (E) The half-time (T1 / 2) of isoRho decay at 55 °C. isoRho was regenerated with 5 pM 9cR upon the treatment of opsin membranes with JC3 or JC4 compounds at 1, 10 and 100 pM concentrations.

[0032] Figs. 3(A-D) illustrate plots showing the effect of JC3 and JC4 on the Gt activation rates. (A) The rod opsin membranes were incubated with a 10 pM compound prior to pigment regeneration with 5 pM 9-cis-retinal (9cR). The activity of illuminated isoRho was recorded by monitoring changes in the Trp fluorescence at 345 nm upon the addition of 10 pM GTPyS. These changes related to the dissociation of Gtawere plotted as a function of time. The representative plot is shown. The excitation and emission wavelengths were 295 nm and 345 nm, respectively. (B) The initial rates and error bars (S. D.) calculated for each condition plotted are shown. Each measurement was performed three times and the experiment was repeated. (C and D). The effect of JC3 and JC4 on the function of WT and P23H isoRho in cultured cells, respectively. In cultured cells, Rho can signal through Gi signaling. Changes in the levels of cAMP upon light stimulation were monitored in the NIH-3T3 cells stably expressing WT (C) or P23H rod opsin (D). These cells were regenerated with 5 pM 9-cis-retinal (9cR) or incubated with JC3 or JC4 prior to regeneration. Levels of cAMP obtained in the illuminated cells were compared to the levels in cells kept in the dark. The cells were treated with the compound for 16 h before isoRho regeneration for 2 h. Cells treated with 9cR only and non- treated (NT) cells were used as controls. Each condition was performed in triplicate and the experiment was repeated. The one-way ANOVA and Turkey post hoc tests were used for the statistical analysis. The statistically different changes (P) are indicated in the figure.

[0033] Figs. 4(A-E) illustrate plots showing effect of JC3 and JC4 on opsin membrane targeting. (A) Cytotoxicity of JC3 and JC4 compounds. Toxicity of the identified compounds was tested in four cell lines, the NIH-3T3, HEK-293 cells, photoreceptor-derived 661W cells, and retinal pigment epithelium (RPE)-derived ARPE19 cells by MTT assay. The cells were treated with the compounds at 0.001-100 pM concentration for 24 h. The results are shown as a percentage of viable cells compared to non-treated control cells. (B) The fluorescence images of the NIH-3T3 cells stably expressing P23H rod opsin treated with the JC compounds at a final concentration of 10 pM or 5 pM 9-cis-retinal (9cR) for 16 h. Each condition was performed in triplicate and the experiment was repeated. The next day, cells were immunostained with the anti-Rho monoclonal antibody recognizing the N-tcrminalepitope of this receptor and the Alexa-Fluor 594-conjugated anti-mouse secondary antibody (orange) to detect the cell surface expression. The nuclei of the cells were labeled with DAPI (blue). The images were taken with a high-content imaging operetta microscope at 20x magnification. Scale bar, 50 pm. (C) Quantification of the cell surface fluorescence intensity. Statistical analysis was performed with the one-way ANOVA and Turkey post hoc tests. The statistically different changes (P) are indicated in the figure. (D) Immunoblot showing the effect of two compounds JC3 and JC4 on the expression level of WT and P23H rod opsin in the NIH-3T3 cells stably expressing these receptors. Total cell extracts (50 pg) were loaded and separated using SDS-PAGE gel, followed by transfer to polyvinyl difluoride membrane (PVDF). Rod opsin was detected with the 1D4 anti-Rho antibody detecting the C-terminal epitope. GAPDH was detected with an anti-GAPDH antibody and used as a loading control. PNGaseF-treated samples were deglycosylated for 1 h at room temperature prior to loading onto the gel. The experiment was repeated three times. Representative immunoblots are shown. (E) Quantification of band intensities of mature and immature P23H rod opsin in non-treated cells, treated with 9-crf-retinal and two JC compounds in the blots shown in D.

[0034] Figs. 5(A-B) illustrate images showing the effect of JC3 and JC4 on membrane expression of RP- linked rod opsin mutants. Deep mutational scanning was used to examine the chaperone effect of JC3 and JC4 on multiple RP-linked rod opsin mutants. The change in the plasma membrane levels of these variants upon treatment with a 10 pM compound was quantified and compared to the treatment with 5 pM 9-czs-retinal (9cR). (A) The cell surface immunostaining intensities for a collection of RP variants bearing individual amino acid substitutions examined in HEK-293 cells were depicted as heatmaps. The values represent the average from two biological replicates, and color bars indicate the scale of the observed effects under each condition. Red indicates an increased and blue reduced plasma membrane expression under each condition. (B) The Ca of mutated side chains are rendered as spheres in the context of the three-dimensional structure of Rho (PDB ID: 1U19). Spheres are colored according to the average change in the plasma membrane expression in the presence of JC3 and JC4. The I I -c / .v-rclinal chromophore is shown as green sticks to visualize the chromophore binding region.

[0035] Figs. 6(A-F) illustrate images and plots showing the protective effect of JC3 and JC4 against retinal degeneration triggered by bright light. The health of the retina was inspected by optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO)in vivo imaging, and histologically in Abca4- / -Rdh8- / -mice. Retinal function was examined by ERG. (A) The representative OCT images of the mouse eye after the indicated treatment and exposure to light. ONL, outer nuclear layer; 1NL, inner nuclear layer. (#) indicates a disorganized photoreceptor layer in vehicle-treated control mice. Scale bar, 100 pm. (B) Retinal sections stained with hematoxylin and eosin (H& E) prepared from eyes collected from mice either unexposed to light or exposed to bright light after the indicated treatment. (#) indicates a disorganized photoreceptor layer in vehicle-treated control mice. Scale bar, 50 pm. (C) Quantification of the ONL thickness measured at 0.5 mm from the optic nerve head (ONH) (n = 5 mice per treatment group). Error bars indicate standard deviation (S. D.). The statistically significant changes (P) in the ONL thickness observed between dark-adapted and vehicle-treated, exposed to light mice, and between compound-treated and the vehicle-treated group are indicated in the figure. No significant difference in the ONL thickness was observed between mice kept in the dark and those treated with the JC3 and JC4 compounds. (D) The representative SLO images. Autofluorescence (AF) spots were detected only in the retina of DMSO-treated mice injured with bright light. AF spots were not detected in mice kept in the dark or treated with the compounds before illumination. Scale bar, 1 mm. (E) Quantification of AF spots was performed in n = 5 mice per treatment group. Error bars indicate S. D. The changes in the number of AF spots after the treatment with JC3 and JC4 compared to vehicle-treated mice were statistically significant (P < 0.001). No significant difference was observed between mice kept in the dark and those exposed to light after treatment with the JC3 and JC4 compounds. (F) The effect of JC3 and JC4 on retinal function in mice injured with bright light (n = 4 for dark adapted mice, n = 7 for vehicle-treated mice, and n = 6 for JC3 and JC4-trcatcd mice). The statistically significant changes (P < 0.05) in the ERG responses obtained upon treatment with JC3 and JC4 are shown with asterisks. Statistical significance was calculated with the two-way ANOVA and post hoc Turkey’s tests.

[0036] Figs. 7(A-F) illustrate images and plots showing protective effect of JC3 and JC4 against retinal degeneration in RP. The health of the retina was inspected by optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) in vivo imaging, and histologically in RhoP23H / +mice. Retinal function was examined by the ERG. (A) The treatment strategy with JC3 and JC4 compounds is shown. (B) The representative OCT images of mouse eyes after the indicated treatment. ONL, outer nuclear layer; INL, innernuclear layer. Scale bar, 100 m. (C) Retinal sections stained with hematoxylin and eosin (H& E). Scale bar, 50 pm. (D) The labeling of the retina cryosections with 1D4 anti-Rho antibody and peanut agglutinin to detect rods and cones, respectively. (E) Quantification of the ONL thickness measured at 0.25, 0.5, 0.75, 1.0, and 1.25 mm from the optic nerve head (ONH) (n = 7-11 mice per treatment group). Error bars indicate standard deviation (S. D.). The statistically significant changes (P) in the ONL thickness observed between vehicle-treated and compound-treated mice are indicated in the figure with asterisks. (F) The effect of.103 and JC4 on retinal function in RhoF23H / +mice (n = 7-8 mice per treatment group). The statistically significant changes (P < 0.05) in the ERG responses obtained upon treatment with JC3 and JC4 are shown with asterisks. Statistical significance was calculated with the two-way ANOVA and post hoc Turkey’s tests.

[0037] Figs. 8(A-F) illustrate plots showing the detection of JC3 and JC4 in mouse eyes by LC-MS analysis and their effects on the visual cycle. (A) The HPLC elution profile of JC3 and JC4. The chromatogram represents ion intensity for m / z = 314.2.2 [M + H]+corresponding to JC3 and 298.2 [M + H]+for JC4. (B) The fragmentation patterns of JC3 and JC4 (MS / MS). (C) A standard curve was generated using different concentrations of one compound in the presence of 100 picomoles of the other compound (each compound was used as the internal standard for the other). The correlation for the signal intensity for each combination was determined. The linear regression was obtained from these values, and the concentration of the compounds in the eye was calculated. (D-E) The amount of JC3 and JC4 in the mouse eyes was determined at different time points, and their half-lives were calculated. Mice (n = 4) were used per each data. (F) Effect of JC3 and JC4 compounds on the visual cycle. Six-week-old WT C57BL / 6 mice were i.p. injected with 100 mg / kg of JC3, JC4, or vehicle 30 min before exposure to 10,000 lux light for 7 min. Then, these mice were placed in the dark. Eyes were collected from euthanized mice at 0, 2, or 24 h after illumination, followed by the extraction of the retinyl-oximes and their separation by HPLC. The amount of 11 -cA-retinyl-oxime is presented as a percentage of 11-cz.s-retinyl-oxime detected in vehicle-treated dark-adapted mice and plotted as a function of time after exposure to light illumination.

[0038] Figs. 9(A-C) illustrate images showing the structural basis for variant-specific pharmacochaperone effects. Bound state human WT rod opsin models, highlighted with interacting sites. (A) (R)-JC3 (orange sticks) and (S)-JC4 (green sticks) docked models. (B)(S)-JC3 (green sticks) and (R)-JC4 (orange sticks) docked models. (C) Ligand interface energies in REU of top 10% models for JC3- and JC4-bound variants.

[0039] Eigs. 10(A-C) illustrate an image and plots showing the effect of JC3 and JC4 on opsin plasma membrane expression in 661W cells stably expression rod opsin. (A) The fluorescence images of cells treated with the JC compounds at a final concentration of 10 pM or 5 pM 9-cw-retinal (9cR) labeled with the anti-Rho antibody recognizing the N-terminal epitope of this receptor and the Alexa-Fluor 594-conjugated anti- mouse secondary antibody (red) to detect the cell surface expression. The nuclei of the cells were labeled with DAPI (blue). Scale bar, 10 pm. (B) Immunoblot showing the effect of JC3 and JC4 on the expression level of WT and P23H rod opsin in 661W cells stably expressing these receptors. Total cell extracts (25 pg) were loaded and separated using SDS-PAGE gel, followed by transfer to polyvinyl difluoride membrane (PVDF). Rod opsin was detected with the 1D4 anti- Rho antibody detecting the C-terminal epitope. GAPDH was detected with an anti-GAPDH antibody and used as a loading control. PNGaseF-treated samples were deglycosylated for 1 h at room temperature prior to loading onto the gel. The experiment was repeated three times. Representative immunoblots are shown. (C) Quantification of band intensities of mature and immature P23H rod opsin in non-treated cells, treated with 9-cis-retinal and two JC compounds.

[0040] Figs. 1 l(A-F) illustrate image and plots showing the effect of JC3 and JC4 on retina health in homozygous RhoP23IPP 3Hmice. (A) Labeling of rod and cone photoreceptors in retina cryosections prepared from eyes of RhoP23H / F23Hmice treated with JC compounds or vehicle collected at P21. The expression of rod opsin was detected with 1D4 anti-Rho antibody (red) and cone opsin with PNA (green). Nuclei arc stained with DAPI (blue). The retina center and periphery are shown. Scale bar, 25 pm. (B) The number of nuclei rows in the ONL counted in the retina center and periphery. (C) The RT-qPCR gene expression levels of Rho and M cone opsin. (D) The immunoblot analyses of the Rho and M cone opsin protein expression. (E) Quantification of the Rho and M cone opsin protein expression normalized to GAPDH. (F) Retinal function was examined by the ERG. The measurements were performed in n = 5 mice per treatment group. Error bars indicate standard deviation (S. D.). The statistically significant changes (P) observed between vehicle-treated and compound-treated mice are indicated in the figure with asterisks. Statistical significance was calculated with the one-way ANOVA and post hoc Turkey’s tests.

[0041] Figs. 12(A-F) illustrate plots and images showing the toxicity of JC3 and JC4 in vivo. The effect of chronic or acute administration of JC3 and JC4 on body weight was examined in six-week-old WT C57BL / 6 mice. (A and B) Body weight examined in male and female mice (n = 5 each / group) treated with JC3 or JC4 at 10 mg / kg or vehicle delivered via i.p. injection every other day for 2 weeks. (C) Body weight examined every other day upon acute administration via i.p. injection of JC3 or JC4 at 100 mg / kg, or vehicle. (D- F) The effect of JC3 and JC4 on retinal function and structure examined in WT C57BL / 6 mouse eye treated at 10 mg / kg for 2 weeks every other day. The control mice were treated with a vehicle. The scotopic and photopic ERG responses were measured in n = 5 mice per treatment group. The representative in vivo OCT images of the retina. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 100 pm. (F) Quantification of the ONL thickness measured at 0.5 mm from the optic nerve head (ONH). The measurements were performed in n = 5 mice per treatment group. Error bars indicate standard deviation (S. D.). No significant differences in the ERG responses and the ONL thickness were observed between the treatment groups.

[0042] Fig. 13 illustrates images showing JC3 and JC4 enantiomer docking to selected rod opsin variants. Binding pockets of JC3- and JC4- bound human rod opsin homology model and variants. The mutated amino acids are shown in magenta.

[0043] Fig. 14 illustrates plots showing enantiomer assignment for JC3 compound. The isolated enantiomers of JC3 were assigned by the comparison of computed and measured circular dichroism spectra. The CD spectra of isomer 1 (solid blue) and isomer 2 (dashed blue) were recorded in acetonitrile, and their molar ellipticities are plotted against the wavelength. For the sake of comparison, the electronic structures of the R (solid red) and S (dashed red) enantiomers of JC3 were modeled using density functional theory in order to simulate their CD spectra. The computed change in ellipticity for each enantiomer is plotted against the wavelength for the sake of comparison for the lowest energy conformer, which corresponds to the major peak in each experimental structure. The coincidence between the major peaks suggests isomer 1 is R and isomer 2 is S.

[0044] Figs. 15(A-B) illustrate plots and graph showing the binding properties of JC3 and JC4 enantiomer. (A) The binding of JC3 and JC4 enantiomers to rod opsin was determined by an intrinsic Trp fluorescence quenching assay. Compounds were added to opsin membranes at different concentrations (62.5 - 1000 nM) and changes in Trpfluorescence intensity at 330 nm were recorded and plotted as a function of the compound concentration. The binding curves were fitted using PRISM GraphPad 7.02 software. The Kd values of each compound were calculated and averaged from triplicates. These values ± standard deviation (S. D.) are shown in the figure. (B) The change in the plasma membrane levels of WT and selected rod opsin variants upon treatment with a 10 pM JC3 and JC4 enantiomer mix and enantiopures was quantified and compared to the treatment with vehicle.

[0045] Figs. 16(A-B) illustrate plots showing the effect of JC3 and JC4 on the conformational stability of rod opsin variants. The molecular dynamic (MD) simulations were carried out for the JC3- and JC4- bound human rod opsin homology model and variants. A, the root-mean- square fluctuation (RMSF). B, the root- mean-square deviation (RMSD).

[0046] Fig. 17 illustrates images showing the effect of JC3 and JC4 enantiomers on the protein structure network (PSN) in P23H rod opsin. The specific residue interaction clusters are shown in the protein structures and specified in the tables. A protein structure network (PSN) analysis (3) carried out based on MD simulations identified clusters of stabilizing interactions within the native rod opsin structure. The binding of either JC3 or JC4 increased the number of interacting residues within the native clusters or generated additional clusters of interacting residues. Thus, JC compounds generally appear to stabilize the native fold by improving internal residue-residue interactions that form in the context of the RP variants.

[0047] Fig. 18 illustrates images showing the effect of JC3 and JC4 enantiomers on the protein structure network (PSN) in G51R rod opsin. The specific residue interaction clusters are shown in the protein structures and specified in the tables. A protein structure network (PSN) analysis (3) carried out based on MD simulations identified clusters of stabilizing interactions within the native rod opsin structure. The binding of cither JC3 or JC4 increased the number of interacting residues within the native clusters or generated additional clusters of interacting residues. Thus, JC compounds generally appear to stabilize the native fold by improving internal residue-residue interactions that form in the context of the RP variants.DETAILED DESCRIPTION

[0048] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0049] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0050] The terms "comprise," "comprising," "include," "including," "have," and "having" are used in the inclusive, open sense, meaning that additional elements may be included. The terms "such as", "e.g.", as used herein are non-limiting and are for illustrative purposes only. " Including" and "including but not limited to" are used interchangeably.

[0051] The term "or" as used herein should be understood to mean "and / or", unless the context clearly indicates otherwise.

[0052] The term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "about" or "approximately" refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0053] It will be noted that the structure of some of the compounds of the application include asymmetric (chiral) carbon or sulfur atoms. It is to be understood accordingly that the isomers arising from such asymmetry are included herein, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. The compounds of this application may exist in stereoisomeric form, therefore can be produced as individual stereoisomers or as mixtures.

[0054] The term "isomerism" means compounds that have identical molecular formulae but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of one another are termed "diastereoisomers", and stereoisomers that are non-superimposable mirror images are termed "enantiomers", or sometimes optical isomers. A carbon atom bonded to four nonidentical substituents is termed a "chiral center" whereas a sulfur bound to three or four different substituents, e.g., sulfoxides or sulfinimides, is likewise termed a “chiral center”.

[0055] The term "chiral isomer" means a compound with at least one chiral center. It has two enantiomeric forms of opposite chirality and may exist either as an individual enantiomer or as a mixture of enantiomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a "racemic mixture". A compound that has more than one chiral center has 2n- 1 enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as either an individual diastereomer or as a mixture of diastereomers, termed a "diastereomeric mixture". When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Alternatively, when one or more chiral centers are present, a stereoisomer may be characterized as (+) or (-). Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964, 41, 116).

[0056] The term "geometric Isomers" means the diastereomers that owe their existence to hindered rotation about double bonds. These configurations are differentiated in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold-Prelog rules. Further, the structures and other compounds discussed in this application include all atropic isomers thereof

[0057] The term "atropic isomers" are a type of stereoisomer in which the atoms of two isomers arc arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques, it has been possible to separate mixtures of two atropic isomers in select cases.

[0058] The terms "crystal polymorphs" or "polymorphs" or "crystal forms" means crystal structures in which a compound (or salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability andsolubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.

[0059] The term "derivative" refers to compounds that have a common core structure, and are substituted with various groups as described herein.

[0060] The term "bioisostere" refers to a compound resulting from the exchange of an atom or of a group of atoms with another, broadly similar, atom or group of atoms. The objective of a bioisosteric replacement is to create a new compound with similar biological properties to the parent compound. The bioisosteric replacement may be physicochemically or topologically based. Examples of carboxylic acid bioisosteres include acyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).

[0061] The phrases "parenteral administration" and "administered parenterally" are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-aiticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0062] The term "treating" is art-recognized and includes inhibiting a disease, disorder or condition in a subject, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected.

[0063] The term "preventing" is art-recognized and includes stopping a disease, disorder or condition from occurring in a subject, which may be predisposed to the disease, disorder and / or condition but has not yet been diagnosed as having it. Preventing a condition related to a disease includes stopping the condition from occurring after the disease has been diagnosed but before the condition has been diagnosed.

[0064] The term "pharmaceutical composition" refers to a formulation containing the disclosed compounds in a form suitable for administration to a subject. In a preferred embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unitdosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed compound or salts thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, inhalational, and the like. Dosage forms for the topical or transdermal administration of a compound described herein includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, nebulized compounds, and inhalants. In a preferred embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[0065] The term "flash dose" refers to compound formulations that are rapidly dispersing dosage forms.

[0066] The phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, the term includes compositions, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0067] The phrase "pharmaceutically acceptable carrier" is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient. In certain embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0068] The compounds of the application are capable of further forming salts. All of these forms are also contemplated herein.

[0069] " Pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. For example, the salt can be an acid addition salt. One embodiment of an acid addition salt is a hydrochloride salt. The pharmaceutically acceptable salts can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile being preferred. Fists of salts are found in Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0070] The compounds described herein can also be prepared as esters, for example pharmaceutically acceptable esters. For example, a carboxylic acid function group in a compound can be converted to its corresponding ester, e.g., a methyl, ethyl, or other ester. Also, an alcohol group in a compound can be converted to its corresponding ester, e.g., an acetate, propionate, or other ester.

[0071] The compounds described herein can also be prepared as prodrugs, for example pharmaceutically acceptable prodrugs. The terms "pro-drug" and "prodrug" are used interchangeably herein and refer to any compound, which releases an active parent drug in vivo. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals e.g., solubility, bioavailability, manufacturing, etc.) the compounds can be delivered in prodrug form. Thus, the compounds described herein are intended to cover prodrugs of the presently claimed compounds, methods of delivering the same and compositions containingthe same. " Prodrugs" are intended to include any covalently bonded earners that release an active parent drug in vivo when such prodrug is administered to a subject. Prodrugs are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include compounds wherein a hydroxy, amino, sulfhydryl, carboxy, or carbonyl group is bonded to any group that may be cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl, free carboxy or free carbonyl group, respectively.

[0072] Examples of prodrugs include, but are not limited to, esters (e.g., acetate, dialkylaminoacetates, formates, phosphates, sulfates, and benzoate derivatives) and carbamates (e.g., N, N-dimethylaminocarbonyl) of hydroxy functional groups, ester groups (e.g., ethyl esters, morpholinoethanol esters) of carboxyl functional groups, N-acyl derivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases and enaminones of amino functional groups, oximes, acetals, ketals and enol esters of ketone and aldehyde functional groups in compounds of Formula I, and the like, See Bundegaard, H. " Design of Prodrugs" pl-92, Elesevier, New York-Oxford (1985).

[0073] Additionally, the salts of the compounds described herein, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Nonlimiting examples of hydrates include monohydrates, dihydrates, etc. Nonlimiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0074] The term "solvates" means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate, when the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one of the substances in which the water retains its molecular state as H2O, such combination being able to form one or more hydrate.

[0075] The compounds, salts and prodrugs described herein can exist in several tautomeric forms, including the enol and imine form, and the keto and enamine form and geometric isomers and mixtures thereof. Tautomers exist as mixtures of a tautomeric set in solution. In solid form, usually one tautomer predominates. Even though one tautomer may be described, the present application includes all tautomers of the present compounds. A tautomer is one of two or more structural isomers that exist in equilibrium and arc readilyconverted from one isomeric form to another. This reaction results in the formal migration of a hydrogen atom accompanied by a switch of adjacent conjugated double bonds. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that are interconvertable by tautomerizations is called tautomerism.

[0076] Of the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism a simultaneous shift of electrons and a hydrogen atom occurs.

[0077] Tautomerizations can be catalyzed by: Base: 1. deprotonation; 2. formation of a delocalized anion (e.g., an enolate); 3. protonation at a different position of the anion; Acid: 1. protonation; 2. formation of a delocalized cation; 3. deprotonation at a different position adjacent to the cation.

[0078] The term "analog" refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.

[0079] A "patient," "subject," or "host" to be treated by the subject method may mean either a human or non-human animal, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term docs not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder.

[0080] The terms "prophylactic” or “therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic(i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0081] The terms "therapeutic agent", "drug", "medicament" and "bioactive substance" are art-recognized and include molecules and other agents that are biologically, physiologically, or pharmacologically active substances that act locally or systemically in a patient or subject to treat a disease or condition. The terms include without limitation pharmaceutically acceptable salts thereof and prodrugs. Such agents may be acidic, basic, or salts; they may be neutral molecules, polar molecules, or molecular complexes capable of hydrogen bonding; they may be prodrugs in the form of ethers, esters, amides and the like that are biologically activated when administered into a patient or subject.

[0082] The phrase "therapeutically effective amount" or “pharmaceutically effective amount” is an art-recognized term. In certain embodiments, the term refers to an amount of a therapeutic agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain a target of a particular therapeutic regimen. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In certain embodiments, a therapeutically effective amount of a therapeutic agent for in vivo use will likely depend on a number of factors, including: the rate of release of an agent from a polymer matrix, which will depend in part on the chemical and physical characteristics of the polymer; the identity of the agent; the mode and method of administration; and any other materials incorporated in the polymer matrix in addition to the agent.

[0083] The term " ED50" is art-recognized. In certain embodiments, ED50 means the dose of a drug, which produces 50% of its maximum response or effect, or alternatively, the dose, which produces a pre-determined response in 50% of test subjects or preparations. The term " LD50" is art-recognized. In certain embodiments, LD50 means the dose of a drug, which is lethal in 50% of test subjects. The term "therapeutic index" is an art-recognized term, which refers to the therapeutic index of a drug, defined as LD50 / ED50.

[0084] The terms " IC50," or “half maximal inhibitory concentration” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc.

[0085] With respect to any chemical compounds, the present application is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include C-13 and C-14.

[0086] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent can be bonded to any atom in the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent can be bonded via any atom in such substituent. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0087] When an atom or a chemical moiety is followed by a subscripted numeric range (e.g., Ci-Cg), it is meant to encompass each number within the range as well as all intermediate ranges. For example, " Ci-Ce alkyl" is meant to include alkyl groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5. and 5-6 carbons.

[0088] The term "alkyl" is intended to include both branched (e.g., isopropyl, tert-butyl, isobutyl), straight-chain e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), and cycloalkyl (e.g., alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cyclohcptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. Such aliphatic hydrocarbon groups have a specified number of carbon atoms. For example, Ci-Ce alkyl is intended to include Ci, C2, C3, C4, C5, and Ce alkyl groups. As used herein, "lower alkyl" refers to alkyl groups having from 1 to 6 carbon atoms in the backbone of the carbon chain. " Alkyl" further includes alkyl groups that have oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more hydrocarbon backbone carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has six or fewer carbon atoms in its backbone (e.g., Ci-Ce for straight chain, C3-C6 for branched chain), for example four or fewer. Likewise, certain cycloalkyls have from three to eight carbon atoms in their ring structure, such as five or six carbons in the ring structure.

[0089] The term "alkenyl" refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. Generally, although again not necessarily, alkenyl groups can contain 2 to about 18 carbon atoms, and more particularly 2 to 12 carbon atoms. The term "lower alkenyl" refers to an alkenyl group of 2 to 6 carbon atoms, and the specific term "cycloalkenyl" intends a cyclic alkenyl group, preferably having 5 to 8 carbon atoms. The term "substituted alkenyl" refers to alkenyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to alkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkenyl" and "lower alkenyl" include linear, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0090] The term "alkynyl" refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups can contain 2 to about 18 carbon atoms, and more particularly can contain 2 to 12 carbon atoms. The term "lower alkynyl" intends an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to alkynyl substituted with one or more substituent groups, and the terms"heteroatom-containing alkynyl" and "heteroalkynyl" refer to alkynyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkynyl" and "lower alkynyl" include linear, branched, unsubstituted, substituted, and / or heteroatomcontaining alkynyl and lower alkynyl, respectively.

[0091] The terms "alkyl", "alkenyl", and "alkynyl" are intended to include moieties which are diradicals, i.e., having two points of attachment. A nonlimiting example of such an alkyl moiety that is a diradical is -CH2CH2-, i.e., a C2 alkyl group that is covalently bonded via each terminal carbon atom to the remainder of the molecule.

[0092] The term "alkoxy" refers to an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group may be represented as -O-alkyl where alkyl is as defined above. A "lower alkoxy" group intends an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. Preferred substituents identified as " Ci-Ce alkoxy" or "lower alkoxy" herein contain 1 to 3carbon atoms, and particularly preferred such substituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

[0093] The term "aryl" refers to an aromatic substituent containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Aryl groups can contain 5 to 20 carbon atoms, and particularly preferred aryl groups can contain 5 to 14 carbon atoms. Examples of aryl groups include benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, the term "aryl" includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles", "heterocycles," "heteroaryls" or "heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diaryl amino, and al kylaryl amino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and urcido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylatc, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl). If not otherwise indicated, the term "aryl" includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0094] The term "alkaryl" refers to an aryl group with an alkyl substituent, and the term "aralkyl" refers to an alkyl group with an aryl substituent, wherein "aryl" and "alkyl" are as defined above. Exemplary aralkyl groups contain 6 to 24 carbon atoms, and particularlypreferred aralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groups include, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl,4-benzylcyclohexylmethyl, and the like. Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl, 3-ethyl-cyclopenta-l,4-diene, and the like.

[0095] The terms "heterocyclyl" or "heterocyclic group" include closed ring structures, e.g., 3- to 10-, or 4- to 7-membered rings, which include one or more heteroatoms." Heteroatom" includes atoms of any element other than carbon or hydrogen. Examples of heteroatoms include nitrogen, oxygen, sulfur and phosphorus.

[0096] Heterocyclyl groups can be saturated or unsaturated and include pyrrolidine, oxolane, thiolane, piperidine, piperazine, morpholine, lactones, lactams, such as azetidinones and pyrrolidinones, sultams, and sultones. Heterocyclic groups such as pyrrole and furan can have aromatic character. They include fused ring structures, such as quinoline and isoquinoline. Other examples of heterocyclic groups include pyridine and purine. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromcthyl, cyano, azido, heterocyclyl, or an aromatic or heteroaromatic moiety. Heterocyclic groups can also be substituted at one or more constituent atoms with, for example, a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, — CF3, or -CN, or the like.

[0097] The term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo." Counterion" is used to represent a small, negatively charged species such as fluoride, chloride, bromide, iodide, hydroxide, acetate, and sulfate.

[0098] The terms "substituted" as in "substituted alkyl," "substituted aryl," and the like, as alluded to in some of the aforementioned definitions, is meant that in the alkyl, aryl, orother moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, silyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), C2-C24 alkylcarbonato(-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(C0)-NH2), mono-(Ci-C24 alkyl)-substituted carbamoyl (-(CO)-NH(CI-C24alkyl)), di-(Ci-C4 alkyl) -substituted carbamoyl (-(CO)-N(CI-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NII-aryl), thiocarbamoyl (-(CS)-NIl2), carbamide (-NH-(C0)-NH2), cyano(-CN), isocyano (-N+C), cyanato (-O-CN), isocyanato (-ON+C_), isothiocyanate (-S-CN), azido (-N=N+=N‘), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(Ci-C24 alkyl)-substituted amino, mono- and di-(Cs-C2o aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH-(CO)-aryl), imino (-CR=NH where R=hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(-NO2), nitroso (-NO), sulfo (-SO2 -OH), sulfonato (-SO2-O ), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O )2), phosphinate (-P(O)(O )), phospho (-PO2), and phosphino (-PH2); and the hydrocarbyl moieties C1-C24 alkyl, C2-C24alkenyl, C2-C24 alkynyl, Cs-C2o aryl, Ce-C24 alkaryl, and Ce-C24 aralkyl.

[0099] In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.

[0100] When the term "substituted" appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl, alkenyl, and aryl" is to be interpreted as "substituted alkyl, substituted alkenyl, and substituted aryl." Analogously, when the term "hctcroatom-containing" appears prior to a list of possible heteroatom-containing groups, it is intended that the term apply to every member of that group. For example, the phrase "heteroatomcontaining alkyl, alkenyl, and aryl" is to be interpreted as "heteroatom-containing alkyl, substituted alkenyl, and substituted aryl.

[0101] " Optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.

[0102] The terms "stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation, and as appropriate, purification from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0103] The terms "free compound" is used herein to describe a compound in the unbound state.

[0104] Throughout the description, where compositions are described as having, including, or comprising, specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0105] The term "small molecule" is an art-recognized term. In certain embodiments, this term refers to a molecule, which has a molecular weight of less than about 2000 amu, or less than about 1000 amu, and even less than about 500 amu.

[0106] All percentages and ratios used herein, unless otherwise indicated, are by weight.

[0107] Embodiments described herein relate to non-retinoid compounds that act as pharmacochaperones to modulate opsin protein properties, and particularly to the use of the non-retinoid compounds in methods of treating retinal degeneration, such as retinal degeneration associated with inherited rhodopsin mutations in ocular tissue of a subject. It isbelieved that thermal- or photo-bleached rhodopsin mutations (e.g., P23H rhodopsin mutation) in the apo opsin form aggregates in situ, disrupting the disc organization of the rod outer segment (ROS), thereby compromising photoreceptor cell survival. The compounds described herein are small molecules that can act or behave as chaperones of rhodopsin and can have micromolar potency and efficacy equal or greater than d-c -relinal. By improving the internal protein-structure network, these rod opsin ligands also enhanced the plasma membrane expression of clinical retinitis pigmentosa (RP) variants, including the most prevalent P23H variant. The compounds described herein can potentially stabilize mutant rhodopsin in photoreceptor cells, and rescue the transport and glycosylation of unstable mutant opsin from the endoplasmic reticulum (ER) to the plasma membrane, thereby restoring rhodopsin homeostasis and preventing photoreceptor death related to retinal degeneration.

[0108] In some embodiments, a non-retinoid pharmacochaperone described herein can include a compound of formula (I):(I) or a pharmaceutically acceptable salt thereof; whereinthe dashed line is an optional bond;X is -N-, -C-, or -C(H)-;X1is -C(H)-, -C(H2)-, or -O-;X2is -C(R4)-. -C(R4)2-. or -S-;X3is absent, -N-, -N(H)-, -C(H)-, or -C(H2)-;R1is aryl, heterocyclyl, or heteroaryl, each of which is optionally substituted with one or more R5;Rlais phenyl or benzyl, each of which is optionally substituted with one or more R5;R4is H, halogen, alkyl, or haloalkyl;each R5is halogen, -CN, -N(R4)2, -OH, -O-(alkylcnc)-OH, -S(O)m( alkyl), - C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, alkoxy, haloalkyl, cycloalkyl, or heterocyclyl; and m is 0, 1, or 2.

[0109] In some embodiments, X is -N-, X1is -C(H2)-, X2is -S-, and X3is absent.

[0110] In other embodiments, X is -C-, X1is -O-, X2is -C(R4)2-, and X3is absent.

[0111] In some embodiments, X is -N-, X1is -C(H)-, X2is -C(R4)-. and X3is absent.

[00112] In other embodiments, X is -N-, X1is -C(H)-, X2is -C(R4)-, and X3is -N-.

[0113] In some embodiments, Rlais phenyl, which is optionally substituted with one or more R5.

[0114] In some embodiments, Rlais

[0115] In some embodiments, Rlais a benzyl, which is optionally substituted with one or more R5.R6

[0116] In other embodiments, R1is37, wherein R6and R7are each independently absent, halogen, -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m( alkyl), -C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, haloalkyl, cycloalkyl, or heterocyclyl; or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N, and wherein the heterocycle is optionally substituted with R8; and R8is halogen, alkyl, or alkoxy.

[0117] wherein R9is a halogen, C'-C6alkyl, C'-C6alkoxy, or -S(O)m(alkyl).

[0118] In other embodiments, R1is

[0119] In other embodiments, a non-retinoid pharmacochaperone described herein can include a compound of formula (II):x1-x2u< / ^RX1IR1a(II) or a pharmaceutically acceptable salt thereof; whereinthe dashed line is an optional bond;X is -N-, -C-, or -C(H)-;XIis -C(H)-, -C(H2)- or O-;X2is -C(R4)-, -C(R4)2-, or -S-;R1is aryl, heterocyclyl, or heteroaryl, each of which is optionally substituted with one or more R5;Rlais a phenyl or a benzyl, each of which is optionally substituted with one or more R5;R4is H, halogen, alkyl, or haloalkyl;each R5is halogen. -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m(alkyl). - C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, alkoxy, haloalkyl, cycloalkyl, or heterocyclyl; and m is 0, 1, or 2.

[0120] In some embodiments, X is -N-, X1is -C(H2)-, and X2is -S-.

[0121] In other embodiments, X is -C-, X1is -O-, and X2is -C(R4)2-.

[0122] In some embodiments, X is -N-, X1is -C(H)-, and X2is -C(R4)-.

[0123] In some embodiments, Rlais phenyl, which is optionally substituted with one or more R5.

[0124] In some embodiments, Rlais \,,°— / . or ’KZy.

[0125] In some embodiments, Rlais a benzyl, which is optionally substituted with one or more R5.R6

[0126] In other embodiments, R1is ' — ^ R, wherein R6and R7are each independently absent, halogen, -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m( alkyl),-C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, haloalkyl, cycloalkyl, or heterocyclyl; or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N, and wherein the heterocycle is optionally substituted with R8; and R8is halogen, alkyl, or alkoxy.

[0127] wherein R9is a halogen, C'-C6alkyl, C'-C6alkoxy, or -S(O)m( alkyl).

[0128] In other embodiments, R1is

[0129] In other embodiments, a non-retinoid pharmacochaperone described herein can include a compound of formula (III):y^X2R6°A, 'R3(III) or a pharmaceutically acceptable salt thereof; whereinthe dashed line is an optional bond;X2is -C(R4)-, -C(R4)2-, or -S-;R2and R3are each independently absent or halogen, alkyl, haloalkyl, or alkoxy;R4is H, halogen, alkyl, or haloalkyl;R6and R7are each independently absent, halogen, -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m( alkyl), -C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, haloalkyl, cycloalkyl, or heterocyclyl;or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N, and wherein the heterocycle is optionally substituted with R8;R8is halogen, alkyl, or alkoxy; andm is 0, 1, or 2.

[0130] In some embodiments, X2is S; R2and R3are each independently absent, a halogen, Ci-Ce alkyl, or Ci-Ce alkoxy; and R6and R7are each independently absent, halogen, Ci-Ce alkyl, or Ci-Ce alkoxy; or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N.

[0131] For example, the compound or the pharmaceutically acceptable salt thereof can include a compound selected from:acceptable salt thereof.

[0132] In other embodiments, X2is -C(R4)-; R2and R3are each independently absent, a halogen, C'I-O, alkyl, or Ci-Ce alkoxy; and R6and R7are each independently absent, halogen, Ci-Ce alkyl, or Ci-Ce alkoxy.

[0133] For example, the compound or the pharmaceutically acceptable salt thereof can include a compound selected from:pharmaceutically acceptable salt thereof.

[0134] In other embodiments, the compound or the pharmaceutically acceptable salt thereof can include a compound selected from:

[0135] In other embodiments, the compound or the pharmaceutically acceptable salt; or a pharmaceutically acceptable salt thereof.

[0136] In other embodiments, the compound or the pharmaceutically acceptable salt thereof can be selected from:an (S) enantiomer ofan (R) enantiomer ofan (S) enantiomer of salt thereof.

[0137] In some embodiments, the binding of a putative compound of formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof to rod opsin can be measured using a tryptophan quenching assay where human rod opsin is titrated with increasing concentration of ligands. The tryptophan excitation at 330 nm wavelength can be quenched with increasing concentration of the compound or pharmaceutically acceptable salt thereof to determine an equilibrium dissociation constant (Kd) of the compound or pharmaceutically acceptable salt thereof. In some embodiments, the compound or pharmaceutically acceptable salt thereof can have a Kd less than about 2500 nM, less than about 2000 nM, less than about 1500 nM, less than about 1000 nM, less than about 500 nM, less than about 450 nM, less than about 400 nM, less than about 350 nM, less than about 300 nM, less than about 250 nM, less than about 200 nM, or less than about 100 nM, for example, about 1 nM to about 250 nM, about 5 nM to about 200 nM, about 5 nM to about 175 nM, about 5 nM to about 150 nM, about 5 nM to about 125 nM, about 5 nM to about 100 nM, about 5 nM to about 75 nM, about 5 nM to about 50 nM, or about 5 nM to about 255 nM, including any range therebetween.

[0138] In other embodiments, the EC50 of a putative compound of formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof to effect trafficking or mistrafficking rodopsin mutant P23H in NIH-3T3 cells stably expressing this receptor can be measured. In some embodiments, the compound or pharmaceutically acceptable salt thereof can have an EC50 less than about less than about 1200 nM, less than about 1000 nM, less than about 500 nM, less than about 450 nM, less than about 400 nM, less than about 350 nM, less than about 300 nM, less than about 250 nM, less than about 200 nM, or less than about 100 nM, for example, about 1 nM to about 1100 nM, about 5 nM to about 1000 nM, about 5 nM to about 500 nM, about 5 nM to about 400 nM, about 5 nM to about 300 nM, about 5 nM to about 200 nM, or about 5 nM to about 100 nM, including any range therebetween.

[0139] In still other embodiments, to examine whether the enhanced membrane localization of P23H rod opsin arises from changes in the total expression or differences in maturation, western blotting can be used to compare the effects of a putative compound of formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof on the glycosylation state of rod opsin. Wild-type (WT) rod opsin expressed in NIH-3T3 cells predominantly bears higher weight, mature glycans and migrates at an apparent molecular weight of 55 kDa. By comparison, the predominant form of P23H rod opsin has an apparent molecular weight of 37 kDa, which reflects its impaired maturation within the secretory pathway. Treatment with 9-cw-retinal stabilized P23H rod opsin in a manner that resulted in the appearance of both higher weight mature P23H glycoforms and / or oligomers. This interpretation of the observed protein migration pattern is supported by the decreased apparent molecular weight of the P23H protein following treatment with the PNGaseF glycosidase. An increase in the relative abundance of the mature P23H glycoform can be observed in response to treatment with a putative compound of formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof. In some embodiments, a putative compound of formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof can increase glycosylation (the ratio of the mature to immature receptor) by at least about 10%, at least about 20%, least about 30%, least about 40%, least about 50%, least about 60%, least about 70%, least about 80%, least about 90%, least about 100%, least about 150%, at least about 200%, or more compare to a control devoid of such treatment.

[0140] In other embodiments, a putative compound of formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof, activity can be tested using the beta-galactosidase fragment complementation assay to measure and / or quantify the rescue of P23H opsin from ER to plasma membrane. Activity scores can be normalized with the effect from treatmentwith 5 |1M 9-c / .v-rctinal. In certain embodiments, effective compounds exhibit an efficacy higher than about 20%, about 30%, about 40%, about 50%, or more, compared to a control devoid of such treatment.

[0141] Compounds or pharmaceutically acceptable salts thereof described herein may be synthesized using standard synthetic techniques known to those of skill in the art or using methods known in the art in combination with methods described herein. In additions, solvents, temperatures and other reaction conditions presented herein may vary according to the practice and knowledge of those of skill in the art.

[0142] The starting material used for the synthesis of compounds described herein can be obtained from commercial sources, such as Aldrich Chemical Co. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.), or the starting materials can be synthesized. The compounds described herein, and other related compounds having different substituents can be synthesized using techniques and materials known to those of skill in the art, such as described, for example, in March, ADVANCED ORGANIC CHEMISTRY4thEd., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY4thEd., Vols. A and B (Plenum 2000, 2001), and Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS3rdEd., (Wiley 1999) (all of which are incorporated by reference in their entirety).

[0143] The compounds or pharmaceutically acceptable salts thereof described herein can be provided and administered in the form of pharmaceutical compositions for the in vivo administration and inhibition of photoreceptor cell death in a subject. The pharmaceutical compositions can be administered to any subject that can experience the beneficial effects of the compounds described herein. Foremost among such animals arc humans, although the present invention is not intended to be so limited.

[0144] The compounds or pharmaceutically acceptable salts thereof used in methods described herein can be administered to the subject to treat retinal degeneration (e.g., retinal degeneration associated with rhodopsin mutations) using standard delivery methods including, for example, ophthalmic, topical, parenteral, subcutaneous, intravenous, intraarticular, intrathecal, intramuscular, intraperitoneal, intradermal injections, or by transdermal, buccal, oromucosal, oral routes or via inhalation. The particular approach and dosage used for a particular subject depends on several factors including, for example, thegeneral health, weight, and age of the subject. Based on factors such as these, a medical practitioner can select an appropriate approach to treatment

[0145] “Treating” or “treatment” as used herein, refers to the reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, prevention of the occurrence of symptoms and / or their underlying cause, and improvement or remediation of disease. Such treatment need not necessarily completely ameliorate the disease. For example, treatment of a subject with retinal degeneration by administration of the compounds described herein can encompass inhibiting or causing regression of the disease. Further, such treatment can be used in conjunction with other traditional treatments for retinal degeneration known to those of skill in the art.

[0146] Treatment according to the method described herein can be altered, stopped, or re-initiated in a subject depending on the status of ocular disorder. Treatment can be carried out as intervals determined to be appropriate by those skilled in the art. For example, the administration can be carried out 1, 2, 3, or 4 times a day. In some embodiments, the compounds can be administered after induction of retinal degeneration has occurred.

[0147] The treatment methods can include administering to the subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof described herein. Pharmaceutical compositions for use in the methods described herein can have a therapeutically effective amount of the compound or salts thereof in a dosage in the range of.01 to 1,000 mg / kg of body weight of the subject, and more preferably in the range of from about 10 to 100 mg / kg of body weight of the patient.

[0148] The overall dosage will be a therapeutically effective amount depending on several factors including the overall health of a subject, the subject’s disease state, severity of the condition, the observation of improvements and the formulation and route of administration of the selected agent(s). Determination of a therapeutically effective amount is within the capability of those skilled in the art. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject's condition.

[0149] The therapeutically effective amount of the compounds and salts thereof used in the methods can vary depending upon the manner of administration, the age and body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by those skilled in the art. In some embodiments, a therapeutically effective amount can be anamount (dose) effective in treating a subject, having, for example, retinal degeneration related disease or disorder (e.g. retinitis pigmentosa).

[0150] In some embodiments, the therapeutically effective amount of a compound described herein is the amount effective to: inhibit photoreceptor cell death in the subject; promote rod photoreceptor cell homeostasis in the subject; inhibit early ER associated protein degradation (ERAD) pathway in photoreceptor cells of the subject; mobilize the P23H opsin from the endoplasmic reticulum to the plasma membrane of photoreceptor cells; stabilize the P23H rod opsin mutant protein in a subject; and / or inhibit bright light-induced retinal degeneration in a Rdh8 / -Abca4 / -mouse.

[0151] Formulation of the pharmaceutical compositions for use in the modes of administration noted above (and others) are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. (also see, e.g., M. J. Rathbone, ed., Oral Mucosal Drug Delivery, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N. Y., U. S. A., 1996; M. J. Rathbone et al., eds., Modified- Release Drug Delivery Technology, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N. Y., U. S. A., 2003; Ghosh et al., eds., Dmg Delivery to the Oral Cavity, Dmgs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N. Y., U. S. A., 2005; and Mathiowitz et al., eds., Bioadhesive Dmg Delivery Systems, Dmgs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N. Y., U. S. A., 1999. Compounds or pharmaceutically acceptable salts thereof can be formulated into pharmaceutical compositions containing pharmaceutically acceptable non-toxic excipients and carriers. The excipients are all components present in the pharmaceutical formulation other than the active ingredient or ingredients. Suitable excipients and carriers can be composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects, or unwanted interactions with other medications. Suitable excipients and carriers are those, which are composed of materials that will not affect the bioavailability and performance of the agent. As generally used herein “excipient” includes, but is not limited to surfactants, emulsifiers, emulsion stabilizers, emollients, buffers, solvents, dyes, flavors, binders, fillers, lubricants, and preservatives. Suitable excipients include those generally known in the art such as the “Handbook of Pharmaceutical Excipients”, 4th Ed., Pharmaceutical Press, 2003.

[0152] In one example, a compound or pharmaceutically acceptable salt thereof described herein can be provided in an ophthalmic preparation that can be administered to the subject’s eye. The ophthalmic preparation can contain the compound or pharmaceutically acceptable salt thereof in a pharmaceutically acceptable solution, suspension or ointment. Some variations in concentration will necessarily occur, depending on the particular compound employed, the condition of the subject to be treated and the like, and the person responsible for treatment will determine the most suitable concentration for the individual subject. The ophthalmic preparation can be in the form of a sterile aqueous solution containing, if desired, additional ingredients, for example, preservatives, buffers, tonicity agents, antioxidants, stabilizers, nonionic wetting or clarifying agents, and viscosity increasing agents.

[0153] Subjects affected with or at risk of retinal degeneration, which are not readily accessible or suitable for ophthalmic (e.g. eye-drops) and / or topical administration, can be treated by a systemic approach, such as intravenous infusion. For example, the compound can be administered at a low dosage by continuous intravenous infusion or parenteral administration. In another example, in which a patient requires longer-term care, the compound can be administered intermittently (e.g., every 12-24 hours). In a variation of this approach, the initial or loading dose can be followed by maintenance doses that are less than, (e.g., half) the loading dose or by continuous infusion. The duration of such treatment can be determined by those having skill in the art, based on factors, for example, the severity of the condition and the observation of improvements.

[0154] When administering the compounds or pharmaceutically acceptable salts thereof described herein to the subject by intravenous infusion, devices and equipment(e.g., catheters, such as central or peripheral venous catheters, tubing, drip chambers, flashback bulbs, injection Y sites, stopcocks, and infusion bags) can be used that are compatible with the compound.

[0155] In some embodiments, the compounds or pharmaceutically acceptable salts thereof can be administered to a subject to treat retinal degeneration in a subject. Retinal degeneration, as contemplated for treatment by the methods described herein, can include but is not limited to retinal degenerations associated with disrupted rhodopsin homeostasis and inherited retinal degeneration associated with rhodopsin mutations. In some embodiments,retinal degeneration can be selected from the group consisting of Leber congenital amaurosis, Stargardt disease, and retinitis pigmentosa.

[0156] One particular aspect of the present invention contemplates the treatment of retinitis pigmentosa in a subject. Retinitis pigmentosa as contemplated for treatment by methods described herein, can include but is not limited to autosomal dominate retinitis pigmentosa associated with a P23H RHO mutation.

[0157] In one embodiment, a subject is diagnosed as having symptoms of retinal degeneration (such as impaired vision, night blindness, retinal detachment, light sensitivity, tunnel vision, and loss of peripheral vision to total loss of vision), and then a disclosed compound or pharmaceutically acceptable salt thereof is administered. In another embodiment, a subject may be identified as being at risk for developing retinal degeneration (risk factors may include family history or testing positive for a rhodopsin mutation), and then a disclosed compound or pharmaceutically acceptable salt thereof is administered. In another embodiment, a subject may have retinal degeneration in both eyes, and then a disclosed compound or pharmaceutically acceptable salt thereof is administered. In another embodiment, a subject may have retinal degeneration in one eye but not the other eye, and then a disclosed compound is administered to one or both eyes. In yet another embodiment, a subject may be diagnosed as having retinitis pigmentosa, and then a disclosed compound or pharmaceutically acceptable salt thereof is administered. In another embodiment, a subject can be diagnosed as having symptoms of other forms of retinal degeneration whose etiology involves a rhodopsin mutation (e.g., a P23H rod opsin mutation) in photoreceptor cells of a subject, and then the compound or pharmaceutically acceptable salt thereof is administered. In another embodiment, a subject may be identified as being at risk for developing other forms of retinal degeneration whose etiology involves a rhodopsin mutation in photoreceptor cells, and then the disclosed compound or pharmaceutically acceptable salt thereof is administered. In some embodiments, a compound or pharmaceutically acceptable salt thereof is administered prophylactically. In some embodiments, a subject has been diagnosed as having the disease before retinal damage is apparent. In some embodiments, a human subject may know that he or she is in need of the retinal generation treatment or prevention.

[0158] In some embodiments, a subject may be monitored for the extent of retinal degeneration. A subject may be monitored in a variety of ways, such as by eye examination, dilated eye examination, fundoscopic examination, visual acuity test, and / or biopsy.Monitoring can be performed at a variety of times. For example, a subject may be monitored after a compound is administered. The monitoring can occur, for example, one day, one week, two weeks, one month, two months, six months, one year, two years, five years, or any other time period after the first administration of a compound. A subject can be repeatedly monitored. In some embodiments, the dose of a compound may be altered in response to monitoring.

[0159] Other embodiments relate to a method of suppressing the pathogenic effects of misfolded rhodopsin variants in a subject in need thereof. The method can include administering to the subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof as described herein.

[0160] In some embodiments, the therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof is an amount required to inhibit photoreceptor cell death in the subject.

[0161] In other embodiments, the therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof is an amount effective to inhibit bright light-induced retinal degeneration in a Abca4- / -Rdh8- / -mouse.

[0162] In some embodiments, administration of the compound or the pharmaceutically acceptable salt thereof to the subject stabilizes P23H rod opsin mutant proteins.

[0163] In some embodiments, administration of the compound or the pharmaceutically acceptable salt thereof to the subject promotes rod photoreceptor cell homeostasis in the subject.

[0164] In some embodiments, administration of the compound or the pharmaceutically acceptable salt thereof to the subject mobilizes the P23H opsin from the endoplasmic reticulum to the plasma membrane of photoreceptor cells

[0165] In some embodiments, the compound or the pharmaceutically acceptable salt thereof can be used in a method of suppressing the pathogenic effects of misfolded rhodopsin variants.

[0166] In some embodiments, the compound or the pharmaceutically acceptable salt thereof can be used in a method of stabilizing unliganded or ligand- free opsin.

[0167] In some embodiments, the compound or the pharmaceutically acceptable salt thereof can be capable of crossing the blood retina barrier (BRB) upon systemic, preferably, oral, administration to a subject.

[0168] In some embodiments, the non-retinoid pharmacochaperone can be capable of reversibly binding to rod opsin.

[0169] Another strategy for treating a subject suffering from a retinal degeneration is to administer a therapeutically effective amount of a compound described herein along with a therapeutically effective amount of an additional compound that acts as a chaperone of rhodopsin and / or an anti-retinal degeneration agent or therapy. Examples of anti-retinal degeneration agents or therapies include but are not limited to supplements, such as vitamin A, DHA, and lutien, as well as optic prosthetic devices, gene therapy mechanisms and retinal sheet transplantations.

[0170] Therefore, in a further embodiment, the compounds described herein can be administered as part of a combination therapy with adjunctive therapies for treating retinal degeneration.

[0171] The phrase "combination therapy" embraces the administration of the compounds described herein and a therapeutic agent as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents. When administered as a combination, the compounds, which act as a chaperone of rhodopsin, and a therapeutic agent can be formulated as separate compositions. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).

[0172] Combination therapy" is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeuticagents may be administered orally or all therapeutic agents may be administered by intravenous injection. The sequence in which the therapeutic agents are administered is not narrowly critical. " Combination therapy" also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients (such as, but not limited to, a second and different therapeutic agent) and nondrug therapies (e.g., optic prosthetic devices and retinal sheets).

[0173] The invention is further illustrated by the following example, which is not intended to limit the scope of the claims.Example

[0174] Though several approaches have been previously employed to identify small molecules that rescue the expression of P23H and other misfolded Rho variants, most of these compounds have poor pharmacological properties or bioavailability, thus there are still no approved therapeutics for retinitis pigmentosa. Computational-aided screening approach offers an efficient means to identify novel small molecules that are capable of binding and stabilizing the folded opsin apoprotein. We recently employed these approaches to identify both a series of flavonoid and chromenone-containing compounds that bind to opsin’s orthosteric site and partially correct the expression of certain RHO variants. These compounds rescued the maturation and cellular trafficking of pathogenic mutants in cell culture and showed beneficial effects in vivo in the P23H Rho knock-in mouse model of RP. Though promising, many of the 100+ clinical Rho variants failed to respond to these compounds. Therefore, the discovery of pharmacological chaperones with favorable pharmacological properties that correct a wider array of RP variants is still needed. Such efforts may also provide avenues for the development of targeted, variant-specific combination therapies such as those that are currently offered for the treatment of cystic fibrosis. The extent to which various non-retinoid pharmacochaperones could potentially rescue distinct classes of misfolded RHO variants remains unclear.

[0175] In this example, we utilized virtual screening to identify alternative compounds that bind within the orthosteric site and stabilize the opsin protein. We then employed in vitro biochemical assays, cellular measurements, and animal model studies to validate the pharmacological utility of our top drug candidates. We identified two compounds that bind to rod opsin in vitro, enhance its stability, and restore its maturation and plasma membrane expression in a model cell line stably expressing the most common P23H Rho mutant. Usingdeep mutational scanning, we compared the effects of these compounds on the expression of 123 known clinically relevant RHO variants and showed that these compounds appear to be effective against multiple variants that could be effectively corrected by 9-czs-retinal, and a subset of variants that are distinct from those the most responsive to retinoids. Moreover, we identified several mutants that differentially respond to our two hit compounds. Structural models of selected variants bound to these compounds suggest that the variant-specific effects arise from subtle differences in the binding orientation. MD simulations and protein structure network (PSN) analysis showed that these compounds reduce structural fluctuations caused by mutations and improve internal residue-residue interactions shifting receptor conformation towards WT-like. Importantly, we showed that both hit compounds accumulate within the eyes of mice for several hours after their intraperitoneal (i.p.) administration. We validated the therapeutic potential of these compounds in two mouse models of retina degeneration, Abca4- / -Rdh8- / -mice, a model of acute light damage, and in an RP model, P23H Rho knock-in mice. Both compounds protected photoreceptors from cell death induced by light and the pathogenic P23H RHO mutation. Together, our findings suggest that the molecules identified herein represent promising compounds for the development of precision therapeutics for RP and other visual retinopathies.Materials and MethodsChemicals and Reagents

[0176] 4',6-Diamidino-2-phenyl-indole (DAPI) for nuclear staining was purchased from Life Technologies (Grand Island, NY). Dimethylsulfoxide (DMSO) was obtained from Sigma (St. Louis, MO). EDTA-free protease inhibitor cocktail tablets were purchased from Roche (Basel, Switzerland). 9-cis-retinal was purchased from Sigma. BODIPY FL L-cystine (BFC) was obtained from Thermofisher (Waltham, MA). Polyvinylidene difluoride (PVDF) membrane was obtained from Millipore (Burlington, MA). JC3 was purchased from ChemBridge (San Diego). JC4 was obtained from MolPort (Latvia). Larger amounts of JC3 and JC4 were custom-synthesized by Wuxi AppTech (China). JC3 and JC4 unassigned enantiomers also were purchased from Wuxi AppTech (China).List of AntibodiesAnti-GAPDH, mouse monoclonal, Abclonal, NO: AC002, dilution 1:10,000, Anti-mouse IgG, HRP conjugate, goat, Promega, No: W4021, dilution 1:10,000,Anti-rabbit IgG, HRP conjugated, goat, Promega, No: W4011, dilution 1:10,000, Anti-mouse IgG Alexa Fluor 555-conjugated, goat, Thermofisher, No: A28180, dilution 1:400.Anti-rabbit IgG Alexa Fluor 555-conjugated, goat, Thermofisher, No: A27039, dilution 1:400,Anti-rabbit IgG Alexa Fluor 594-conjugated, goat, Thermofisher, No: A11005, dilution 1:200,Dylight 550-labeled anti-HA, mouse, Thermofisher, No: 2-2.2.14, dilution 1:100, Virtual Screening

[0177] The computational screening of purchasable compounds available in the Zinc (http: / / zinc.docking.org) database was performed against the chromophore-binding site of rod opsin. The monomeric unit of the crystal structure of bovine rod opsin (PDB ID: 3CAP) was used for the molecular docking experiment. Water and other co- crystallized molecules were removed from the coordinate set, the hydrogen atoms were added, and partial charges were assigned to all atoms. The protein then was subjected to restrained molecular mechanics refinement with NAMD 2.12 software using the CHARMM22 force field. The coordinates for the ligand-binding pocket located in the orthosteric site were selected as reported in. Compounds then were docked within the orthosteric binding site using VINA / Vega 3.1.0.21 software, and the binding free energies were calculated in kcal / mol. The docking results were visualized using Biovia Discovery Studio Visualizer 17.2.0 software to assess binding poses within the orthosteric site and identify interactions within the binding pocket. The docking results obtained then were rescored by docking each compound ten times. Binding poses and interactions (location into the binding site, and number and type of interactions formed) were compared using Biovia Discovery Studio Visualizer 17.2.0 software.Pharmacokinetic Drug Properties

[0178] An analysis of physicochemical descriptors, parameters related to administration, distribution, metabolism, and elimination (ADMF), and drug-likeness of the compounds was carried out using SWISSADME tools (http: / / www.swissadme.ch). The results are presented in Table 1.Preparation of Opsin Membranes

[0179] Bovine retinas were used to isolate the rod outer segment (ROS) membranes and opsin membranes were prepared as previously described. Membranes were washed four times with a hypotonic buffer containing 5 mM HEPES (pH 7.5) and 1 mM EDTA then pelleted by centrifugation at 25,000xg for 25 min. The final membrane pellet was suspended in 10 mM sodium phosphate (pH 7.0) and 50 mM hydroxylamine at Rho concentrations of ~2 mg / ml. Membranes were then exposed to light with a 150 Watt bulb for 30 min at 0°C.Then, membranes were pelleted at 16,000xg for 10 min in the bench-top centrifuge. The membrane pellet was washed twice with 10 mM sodium phosphate (pH 7.0) and 2% BSA followed by four washes with 10 mM sodium phosphate (pH 7.0), and two washes with 20 mM BTP (pH 7.5) containing 100 mM NaCl. After each wash, the membranes were centrifuged at 16,000xg for 10 min at 4°C.

[0180] The concentration of opsin and Rho was determined after ROS membrane solubilization with 20 mM dodecyl-β-D-maltopyranoside (DDM) and pelleting insoluble material at 16,000xg for 15 min at 4°C using a UV-visible spectrophotometer (Cary 60, Varian, Palo Alto, CA). The absorption coefficients ε280nm=81,200 M-1cm-1for opsin, ε500nm=40,600 M-1cm-1for Rho, and ε485nm=43,600 M-1cm-1for isoRho were used to calculate the concentration.Detection of Compound Binding to Opsin

[0181] UV-visible spectroscopy - Washed ROS membranes suspended in the buffer consisting of 20 mM BTP (pH 7.5) and 100 mM NaCl at a final opsin concentration of 5 pM were incubated with 10 pM of each compound for 30 min at room temperature prior to incubation with 5 pM 9-cis-retinal for 15 min. For comparison, membranes were incubated only with 9-cis-retinal. Next, 20 mM DDM detergent was added to the membrane suspension. Following a 5 min incubation at room temperature, the samples were centrifuged at 16,000xg for 5 min at 4°C. The UV-visible spectra were measured.

[0182] Fluorescence spectroscopy - The intrinsic tryptophan fluorescence quenching was used to probe the binding of the compounds within the orthosteric binding pocket of rod opsin. Opsin membranes at 4.2 nM were solubilized in the buffer containing 20 mM BTP (pH 7.5), 100 mM NaCl and 1 mM DDM. Tryptophan fluorescence was monitored before and after incubation with compounds at 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, and 1.5 pM concentrations.The ligands were incubated with opsin for 2 min before the measurements. An FL 6500 Fluorescence Spectrometer was used to record the emission spectra 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 tryptophan fluorescence at 330 nm (AFZFo, where / IF is the difference between the initial Trp fluorescence (Fo) and fluorescence recorded upon addition of the compound) were plotted as a function of the ligand concentration. The ligand-binding curves were fitted and binding affinities (Ka) were calculated using GraphPad Prism 7.02 software. 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

[0183] ROS opsin membranes at 5 pM concentration were incubated with the compounds at 1, 10, and 100 pM concentrations for 30 min at room temperature prior to membrane solubilization with 20 mM DDM for 5 min at room temperature. Solubilized opsin was cleared by centrifugation at 16,000xg for 5 min at 4°C. 5 pM 9-cis-retinal was then added to the sample and UV-visible spectra were measured every 2 min for 60 min at 20°C. Each condition was repeated three times. The absorbance at 485 nm was plotted as a function of time and a time course of pigment regeneration was fitted to a second-order exponential decay to calculate the rates and the apparent half-lives of isorhodopsin (isoRho) regeneration.Thermal Stability

[0184] To compare their effect on pigment stability, isoRho’ s regenerated with 9-cis-retinal following treatment with JC3 and JC4 were incubated at 55°C in the dark. Their UV-visible spectra were then recorded every 2 min for 1 h. The absorbance at 485 nm was plotted as a function of time, and the half times (ti / 2) of the chromophore release were calculated by fitting the observed decay with a single exponential rate equation. All samples were measured in triplicate.

[0185] Thermal shift assays were used to examine the effect of JC3 and JC4 on the stability of unliganded opsin. The opsin membranes were suspended (20 pl) in 20 mM BTP, pH 7.5, and 100 mM NaCl at a concentration of 0.01 mg / ml and 20 pl were pipetted into a 96-well plate (Applied Biosystem). Indicated compounds were added to final concentrationsof 0.1, 1, 10, 100 nM, 1, and 10 pM and incubated for 1 h at 4°C. 5 pl of the BODIPY FL L-cystine (BFC) probe (Thermofisher) was then added to each well. Opsin membranes without treatment were included as a control. The plate was sealed with a ClearSeal film (HR4-521, Hampton Research) and incubated for 10 min on ice before the measurement. The changes in the sample fluorescence were measured with a StepOnePlus Real-Time PCR System (Applied Biosystems) and analyzed using the StepOne software version 2.3. The fluorescence in the SYBR, FAM, and ROX channels was recorded for each sample. Cycles included an initial cooling step at 4°C for 1 min then warmed by 1 °C and held at that temperature for 1 min until the sample reached 99.9°C. Multi-component data were then analyzed using GraphPad Prism 7.02 software. The melting temperatures (Tm) of bovine Rho and opsin within the membranes were 71.9 °C and 55.4 °C, respectively. Each condition was repeated in triplicate.Gt Activation Assay

[0186] The Gt protein was extracted and purified from ROS membranes isolated from a hundred dark-adapted bovine retinas. The effect of tested compounds on Rho function was tested by measuring the changes in the intrinsic tryptophan fluorescence of Gta. The opsin membranes at 50 nM concentration suspended in 20 mM BTP (pH 7.0) containing 120 mM NaCl and 1 mM Mg Cl 2 were incubated with a 10 uM compound for 30 min at room temperature prior to regeneration of the pigment with 5 pM 9-cis-retinal for 10 min at room temperature. Next, Gt was added to 500 nM concentration and the sample was illuminated for 1 min with a Fiber- Light illuminator (Dolan Jenner Industries Inc., Boxborough, MA) through a 480-520 nm band-pass wavelength filter (Chroma Technology Corporation, Bellows Falls, VT). Next, 10 pM GTPyS was added and the measurement was performed for 1200 s with an FL 6500 Fluorescence Spectrometer. Excitation and emission wavelengths were set at 300 nm and 345 nm, respectively. Gt activation rates were determined for the first 500 s.Cell Culture

[0187] The NIH-3T3, HEK-293, and ARPE19 cells were cultured in DMEM with 10% FBS (Hyclone, Logan, UT), and 1 unit / ml penicillin with 1 pg / ml streptomycin (Life Technologies) at 37°C under 5% CO2 according to the instructions from the ATCC Animal Cell Culture Guide. The 661W cells, murine photoreceptor-derived cells, were provided byDr. Muayyad Al-Ubaidi, University of Houston, and cultured in DMEM with 10% FBS (Hyclone, Logan, UT), and 1 unit / ml penicillin with 1 pg / ml streptomycin (Life Technologies) at 37 °C under 5% CO2 according to the received instructions.Cytotoxicity Assay

[0188] Cells were plated in the 96-well plate at a density of 3xl05cells / well and 24 h later were treated with different concentrations of the identified compounds for an additional 24 h. The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell proliferation assay (Sigma) was used to assess the effect of these compounds on cell viability. Non-trcatcd cells were used as a control. Cytotoxicity was determined by calculating the percentage of dead cells in each experimental condition. All experimental conditions were performed in triplicate and the experiments were repeated three times.Detection of Rod Opsin in the Cell Membrane and Signaling

[0189] The NIH-3T3 cells stably expressing rod opsin and GFP were plated in the 96-well plate at a density of 3x l(f cells / well. Compounds were added to a final concentration of 10 M for 6 h after plating and the cells were then incubated for 16 h. Cells treated with 5 pM 9-cis-retinal were included as a positive control. The next day, cells were fixed with 4% paraformaldehyde for 20 min at room temperature and then washed twice with phosphate buffer saline (PBS). Cells were then incubated with 10% normal goat serum in PBS for 1 h at 37°C prior to incubation with B6-30 anti-Rho antibody recognizing the receptor N-terminus for 2 h at 37°C. The cells were then washed three times with PBS prior to incubation with an anti-mouse antibody conjugated with Alexa Fluor 594 (Thermo Fisher Scientific) at 1:200 dilution for 1 h at room temperature. Cells were then washed with PBS three times and the nuclei were stained with DAPI following the manufacturer’s protocol. Finally, the plate was sealed with a ClearSeal film (HR4-521, Hampton Research) and used for cell imaging. Cells were imaged using an Operetta High Content Imager (Perkin Elmer Life Sciences). DAPI fluorescence was used to define nuclei and count cells. Bright-filed images and GFP fluorescence were used to define cell bodies. The plasma membrane fluorescence was defined within ± 5% of the cell border. In addition, photoreceptor-derived 661W cells stably expressing P23H rod opsin were used.cAMP Detection Assay

[0190] The NIH-3T3 cells stably expressing either W T or P23H rod opsin were plated in two 96- well plates at a density of 50,000 cells per well in 85 pl of DMEM medium containing 10% FBS and antibiotics. Cells were treated with compounds at different concentrations after 6 h and incubated for 16 h. The next day, 9-cw-retinal was added for 2 h in the dark to regenerate isoRho. Then, one plate was kept in the dark, while the second plate was exposed to bright (150 Watt) light for 15 min from a 10 cm distance. The cAMP-Glo™ assay (Promega) was used to detect the levels of accumulated cAMP following the manufacturer's protocol. The luminescence signal was recorded with a FlexStation 3 plate reader (Molecular Devices). A standard curve was prepared using cAMP provided by the kit and the percentage of cAMP was calculated for each condition. The values were expressed as a percentage, assuming the cAMP level detected in the non-treated cells as 100%.Mutational Scanning

[0191] We compared the effects of JC3 and JC4 compounds on the plasma membrane expression of 123 retinopathy variants in parallel using deep mutational scanning. Briefly, a mixed recombinant stable HEK-293T cell line in which each individual cell inducible expresses one of 123 known retinopathy variants was generated as previously described. An N-terminal hemagglutinin epitope tag was then used to mark the expressed Rho variants at the plasma membrane by surface immunostaining using a Dylight 550-labeled anti-HA antibody (ThermoFisher). Cells were then separated based on their relative surface immunostaining using fluorescence-activated cell sorting (FACS). Cellular isolates were then expanded prior to the extraction of the recombined genomic DNA from each fraction. Illumina sequencing was then used to characterize each cellular isolate by quantifying the relative abundance of a series of unique molecular identifiers associated with each variant. The relative surface immunostaining of each variant was then inferred from the sequencing data in the presence and absence of JC3, JC4, or 9-m-relinal. The analysis was repeated in triplicate for each condition.Structural Modeling and Ligand Docking

[0192] Comparative Modeling of Human Rod Opsin Receptor - To model human rod opsin in its ligand-free state, the crystal structure of bovine rod opsin (PDB ID: 3CAP) was used as a template (93% sequence identity). Comparative modeling was carried out withRosettaCM following a published protocol for G-protein coupled receptors. Sequence alignment was done with ClustalOmega using default settings. Membrane topology files are generated with TOPCONS and only OCTOPUS results were kept and converted to Rosetta readable span files using Rosetta built-in octopus2span.pl script. Coordinates of human rod opsin were then generated from aligned templates with Rosetta partial_thread application. The threaded models were hybridized and relaxed through Rosetta XML scripts. Since the template sequences are highly similar to human opsin sequence, hybridization created 2000 models and the relaxed models were ranked by total_score. The best-scoring model was selected for later docking studies.

[0193] Ligand Docking into Wild Type Homology Models - The ligand docking protocol applied here was modified from RosettaLigand standard docking protocol. Ligand conformers were generated with Biochemical Library (BCL) using default settings of BCL:: Conf. The retinal orthosteric binding site was chosen as the pocket for ligand docking. Low-resolution docking phase allowed to sample ligand binding modes within 5.0 A from the center of the pocket. High-resolution phase performed six cycles of flexible sampling of sidechain rotamer and ligand conformer. The structure was then relaxed with the ligand and protein non-neighbor ( 10.0 A or more away from the ligand) atoms fixed after low-resolution and high-resolution phases. Before the final minimization, another round of high-resolution docking phase was carried out to refine the binding mode. The RosettaLigand energy function was used. This protocol was repeated to generate 10,000 models for each enantiomer of JC3 and JC4. The models were sorted by interface delta score and the top 5% models were clustered into 3 groups based on root-mean-square deviation (RMSD). The model for further analysis were manually selected from top models of each cluster after visual inspection.

[0194] Ligand Docking into Mutation Variants - The top-scoring docked wild type models were mutated with Rosetta MutateResidue mover. Another phase of high-resolution docking was then applied to the mutated complex, followed by relaxation with fixed ligand atoms and final minimization. This protocol was repeated to generate 5000 models for each variant. The models were first sorted by total score and the top 10% models were then sorted by interface delta score. The top models were clustered into three groups based on RMSD and top models of each cluster were visually inspected.MD Simulations and PSN Analysis

[0195] Models of selected human opsin variants and compound complexes were used for MD simulations (Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2020. Maestro-Desmond Interoperability Tools, Schrodinger, New York, NY, 2020) (15, 33, 54-57). The protein-compound complexes were inserted in a membrane of POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) (54) at 300K, while the transmembrane region was obtained following the data of UniProt database (https: / / www.uniDrot.org / ). The complexes created were immersed in a box filled with water molecules using the simple point charge (SPC) scheme. The dimension of the solvent buffer was set to 10 A3. Counter ions (2 Na+) were added to neutralize charges and additional CT and Na+ions were added to obtain a final NaCl concentration of 150 mM. Energy minimization was carried out by 2000 steps using the steepest descent method with a threshold of 1.0 kcal / mol / A. Periodic boundary conditions were used and a cutoff of 9 A was established for van der Waals interactions and the Particle Mesh Ewald (PME) method with a tolerance of 10'9was used in the electrostatic part. The NPT simulations were realized at 300 K with the Nose-Hoover algorithm and the pressure was maintained at 1 bar with the Martyna-Tobias-Klein barostat. The OPLS3e force field was used in all runs. The simulation length was 1000 ns. The final states of the proteins were analyzed by the Protein Structure Network (PSN) using the software webPSN. This tool allows inferring the hubs, links, and communities between the residues of the protein.Stereochemistry of JC Compounds

[0196] Stable stereoisomers of JC3 were obtained with high purity, JC3-1 (98.5%) and JC3-2 (99.5%). However, only one JC4 enantiomer, JC4-1 (99.5%) was stable, while JC4-2 rapidly converted back to a racemic mixture (62.5% compound enrichment). To assign the stereochemistry of the JC3 isolates, we collected circular dichroism spectra for each compound at 1 pM in acetonitrile using a Jasco J- 1500 CD Spectrometer (Jasco, Oklahoma City, OK). To match these spectra to specific isomers, we then used the Gaussian 16 suite to model the electronic structure of each isomer and simulate their ECD spectra. The geometric optimization and frequency calculations were carried out with the 6-311 G(d,p) Pople basis set and wB97X-D functional. The ECD calculations were obtained by performing the TD-DFT calculation method on the geometrically optimized structures at the same level of theoryin acetonitrile using the SMD solvation model. The ECD calculations were obtained by performing the TD-DFT calculation method on the geometry -optimized structure at the same level of theory and spectra for individual conformers were Boltzmann weighted to obtain the final mixture ECD spectrum.Mouse Models

[0197] Six- week-old Abca4 ' Rdh8 ' mice (Research Resource Identifier, RRID: IMSR_JAX:030503, The Jackson Laboratory, Bar Harbor, ME) were used to test the protective effects of JC3 and JC4 compounds against acute light-induced retinal degeneration. Abca4~ / ~Rdh8~ / ~ mice were gcnotypcd to confirm that they do not carry the Rd8 mutation. These mice carry the Leu variation at amino acid 450 of retinal pigment epithelium 65 kDa protein (RPE65). Substitution of Leu to Met decreases sensitivity to light-induced photoreceptor degeneration. Heterozygous RhoP23H / +knock-in mice were used to evaluate the effectiveness of JC3 and JC4 in RP. To obtain heterozygous RhoP23H / +mice, RhoP23H / P23H(RRID: IMSR_JAX:017628, The Jackson Laboratory, Bar Harbor, ME) were crossed with WT C57BL / 6J mice (RRID: IMSR_JAX:000664, The Jackson Laboratory, Bar Harbor, ME). Compounds dissolved in 50% DMSO in PBS were administered to mice by intraperitoneal (i.p.) injection. Both male and female mice were used in all experiments. All mice were housed in the Animal Resource Center at the School of Medicine, Case Western Reserve University (CWRU), and maintained in a 12-hour light / dark cycle. All animal procedures and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at CWRU and conformed to recommendations of both the American Veterinary Medical Association Panel on Euthanasia and the Association for Research in Vision and Ophthalmology as well as the National Eye Institute Animal Care and Use Committee (NELASP 682). The protocol number is 2015-0124. Efforts were taken to minimize animal suffering.Animal Treatment

[0198] The Abca4 ' Rdh8 ' mice were dark-adapted 24 h before the treatment. The compounds at a concentration of 100 mg / kg body weight (b.w.) or DMSO vehicle were delivered to mice 30 min before the exposure to bright light. Mice pupils were dilated with 1% tropicamide and the retinal degeneration was initiated by illumination of mice with 10,000 lux light (150-W bulb, Hampton Bay; Home Depot, Atlanta, GA) for 30 min. Retinalstructures were visualized and analyzed in vivo by ultra-resolution spectral domain-optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) (Heidelberg Engineering, Franklin, MA). Retinal function was examined with electroretinography (ERG). Analyses were performed 7-10 days after light exposure. Mice were euthanized by cervical dislocation under deep anesthesia with a cocktail containing ketamine (20 mg / ml) and xylazine (1.75 mg / ml) at a dose of 4 pl / g b.w. Eyes were collected for preparing paraffin sections, which were used for staining with hematoxylin and eosin (H& E) or cryosections.

[0199] RhoP23H / +mice (28) were used to determine the effects of the JC3 and JC4 on the progression of retinal degeneration in RP. Compounds at 10 mg / kg b.w. or vehicle were administered i.p. to mice starting at postnatal (P) day 21 (P21). A total of six injections were performed every other day at 3:00 p.m. Retinal morphology was visualized with the spectral domain optical coherence tomography (SD-OCT) and retinal function was examined with ERG. Before each procedure, mice were anesthetized with a cocktail containing ketamine (20 mg / ml) and xylazine (1.75 mg / ml) at a dose of 4 pl / g b.w. For histological and immunohistochemical examinations eyes were collected from mice euthanized by cervical dislocation under deep anesthesia.

[0200] In addition, RhoP23H2F23Hmice were treated with JC compounds at 10 mg / kg b.w. or vehicle every other day (3 injections) between P14 and P21.

[0201] To assess the potential toxicity of JC3 and JC4 WT C57BL / 6J mice were treated either with a single dose of these compounds (100 mg / kg) at P33 or 6 doses (10 mg / kg) administered every other day between P21 and P33. The body weight of these mice was monitored every other day for two weeks. Additionally, the ERG and SD-OCT measurements were performed at the end of prolonged compound administration.In vivo Imaging of Mouse Retina

[0202] The protective effects of the identified compounds against retinal damage induced by acute light in Abca4~ / ~Rdh8~ / ~ and inherited mutation in RhoP23H / +mice were evaluated by in vivo imaging with the scanning laser ophthalmoscopy (SD-OCT) (Bioptigen, Morrisville, NC). Before imaging, mice pupils were dilated with 1% tropicamide and mice were anesthetized by i.p. injection of a cocktail containing ketamine (20 mg / ml) and xylazine (1.75 mg / ml) at a dose of 4 pl / g bw. The a-scan / b-scan ratio was set at 1200 lines. The SD-OCT retinal images were obtained by scanning at 0 and 90 degrees in the b-mode. Five image frames were captured and averaged. The changes in the retinas of mice exposed tobright light and control mice were determined by measuring the outer nuclear layer (ONL) thickness at 0.5 mm from the optic nerve head (ONH). Values of the ONL thickness were plotted using means and standard deviation. Each experimental group contained at least n = 6 mice.

[0203] The in vivo whole-fundus imaging of mouse retinas in Abca4zRdh8zmice was performed with the SLO (Heidelberg Engineering, Franklin, MA). After the SD-OCT imaging, mice immediately were subjected to the SLO imaging. Images were collected in the auto-fluorescence mode. The number of autofluorescence spots (AF) detected was counted, and the data were analyzed to determine the statistical significance. Each experimental group contained at least n = 5 mice.Retina Histology

[0204] After the in vivo imaging, mouse eyes (n = 6 per group) were collected from euthanized mice and fixed in 0.5% glutaraldehyde in 2% paraformaldehyde in PBS for 24 h at room temperature on a rocking platform. Then, the fixation solution was changed to 1% PFA for 48 h at room temperature. Eyes were embedded in paraffin and sectioned (5 pm thick) followed by their staining with H& E. Histological slides were imaged with a ZEISS Axio Scan. Zl slide scanner (Carl Zeiss Microscopy GmBH, Jena, Germany). The data were processed using Zeiss-Zen 3.2 software (blue edition).Retinal Function

[0205] The effects of JC3 and JC4 on retinal function in Abca4zRdh8zmice injured with bright light, RhoP23H / +and RhoP23H / P23Hmice, and WT C57BL / 6J mice were examined by using electroretinography (ERG). Before ERG measurements, mice were anesthetized with a cocktail of 20 mg / ml ketamine and 1.75 mg / ml xylazine at 4 pl / g b.w., and pupils were dilated with 1% tropicamide. Scotopic and photopic ERGs were recorded for both eyes of each mouse using a Celeris rodent ERG system and Espion Dyagnosys software Version 6 (Dyagnosys, LLC, Lowell, MA). The ERG data were processed for each condition and presented as mean and standard deviation (S. D.) for both a-wave and b-wave amplitudes. Each experimental group contained at least n = 5 mice.Detection, Quantification, and Pharmacokinetics of JC3 and JC4 in Mouse Eves

[0206] High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) was used to examine the eye targeting of JC3 and JC4 compounds.Compounds were administered i.p. to 5-week-old C57BL / 6. T mice. Eyes were collected from euthanized mice at 0.5, 2, 4, 8, and 16 h after compound administration (n = 4 mice / time point). Two eyes from each treatment group were pooled together and homogenized on ice in 1 ml of a solution containing acetonitrile / methanol / water at 50:40:10 in the presence of 100 pmol of an internal standard (JC3 or JC4, respectively). These homogenates were mixed with 1000 pl of hexane and agitated for 2 min followed by centrifugation at 2,200xg for 5 minutes. The hydrophobic phase was discarded, while the polar phase was collected and dried in a Savant Speedvac concentrator (ThermoFisher Scientific). The compounds were dissolved in 300 pl of acetonitrile, and 100 pl was injected into an HPLC system. The compounds were separated on the X-Bridge BEII C4 column 2.1 x 50 mm (Waters) using a linear gradient of acetonitrile in water (30% - 100%) for 15 min at a flow rate of 0.3 ml / min. MS-based detection and quantification of the compounds were performed with an LTQ linear ion trap mass spectrometer (ThermoFisher Scientific) equipped with an electrospray ionization interface operated in the positive ionization mode. Each compound was used to determine the ionization parameters and to tune the instrument. The compounds were detected in the selected reaction-monitoring mode using the following ion transition 314.2 — 238.2 for JC3 and JC4298.2 — > 269.8 for JC4. A calibration curve was determined for each compound by calculating the relationship between the areas for ion intensity peaks corresponding to each compound versus their molar ratios in a range of 10 - 1000 pmol.Regeneration of 11 -cA-Retinal in the Mouse Eye

[0207] The effects of JC3 and JC4 on the recovery of I 1 -m-relinal after photobleaching were evaluated in 4-6-week-old dark-adapted WT C57BL / 6J mice. Mice were i.p. injected with a single dose of JC3, JC4, or vehicle. Pupils of these mice were dilated by 1% tropicamide after 30 min prior to their exposure to 10,000 lux white light for 10 min. Mice were then reared in the dark room and euthanized at 0, 2, or 24 h post illumination. Eyes were enucleated and stored in the dark at -80°C. To quantify 11 -cis-retinal, eyes were homogenized in 1 ml of PBS: methanol (1:1, v / v) supplemented with 40 mM hydroxylamine and incubated for 20 min at room temperature in the dark. Retinoids were extracted twice with 2 ml of hexane. The mixture was centrifuged at 3,220xg for 5 min at 4 °C to separate the hexanes from the aqueous layer. From the top hexane layer, 1.8 ml was transferred to a glass vial. These samples were then dried in a Savant speed- vac concentrator (Thermofisher, Waltham, Massachusetts, USA) and re-dissolved in 300 pl ofhexane, which was then analyzed by normal phase analytical HPLC column (Zorbax SIL 5 pm, 4.6x250 mm) with mobile phase hexane / ethyl acetate at a flow rate of 1.4 ml / min. The signals at 325 nm and 360 nm were collected. Three mice were included per time point. Each eye (n = 6) was analyzed individually.Statistical Analyses

[0208] Compound cytotoxicity, thermal shift assay, and cAMP quantification experiments were performed in triplicate and repeated. Each compound was tested at different concentrations. Each assay included positive and negative controls. The opsinligand binding, pigment regeneration, thermal stability, and Gt activation assays were performed three times. The effect of each compound was shown in a dose-dependent or time-dependent manner. The parameters derived from these measurements were shown as an average and statistical deviation (S. D.). One or two-way ANOVA with Turkey’s post hoc tests were used for hypothesis testing. All statistical calculations were performed using the Prism GraphPad 7.02 software. Typel error tolerance for the experiments was established at 5%. P values < 0.05 were considered statistically significant. A different person than the experimenter performed the analysis.ResultsIn silico discovery of new non-retinoid ligands of rod opsin

[0209] We recently found that dietary flavonoids and unrelated chromenone-containing compounds bind within the orthosteric site of rod opsin and act as pharmacological chaperones for RP-linked mutants. To expand on the growing list of Rho pharmacophores, we carried out a virtual screen of a library of non-retinoid small molecules commercially available within the Zinc database (http: / / zinc.docking.ofg). We performed pharmacophorebased screening using as a molecular descriptor the rod opsin ligand pharmacophores that we recently discovered as well as those of other previously reported non-retinoid opsin ligands. Comparative docking of these compounds within the crystal structure of unliganded bovine rod opsin (PDB ID: 3CAP) revealed several compounds with predicted binding free energies the same or lower relative to that of quercetin (-9.3 kcal / mol). From this subset of compounds, we selected two commercially available compounds that we will refer to hereafter as JC3 and JC4 (Table 1 and Table 2). These compounds form a network of favorable interactions in proximity to K296 residue within the orthosteric site of rod opsinwith binding free energies of -9.5 kcal / mol, and -9.7 kcal / mol, respectively (Fig. 1A). Both compounds formed direct interactions with various residues that are known to stabilize the natural 11 -cA-retinal chromophore within the binding pocket including Alai 17, Ala292, Tyrl91, and Glul81 (Fig. IB). The most favorable orientations of these ligands also placed them near Trp265 and Tyr268, similarly to 11-cA-retinal.Table 1 - Chemical compound characterizationOpsin Binding PubChemCompound Structure Chemical name Free Energy ID(kcal / mol) 2-( 1,3 -benzodioxol- 5-yl)-3-(4- JC3 00^ 2860307 -9.5methylpheny 1) -1,3- thiazolidin-4-one\ w— 02-(4-chlorophenyl)- oJC4 I TX 3 -methyl- 1 -(4- 12006040 -9.7 methylphenyl)-2H- 0 pyrrol-5-oneCl

[0210] JC3 and JC4 were also chosen for experimental characterization due to their favorable drug-like properties according to the analysis of their absorption, distribution, metabolism, and elimination (ADME) profiles, which were determined using SWISSADME tools (http: / / www.swissadme.ch). These tools score compounds based on their predicted lipophilicity, hydrophilicity, solubility, absorption in the gastrointestinal (GI) tract, and permeability to the blood-brain barrier (BBB). The main ADME parameters are presented in Table 2. None of the selected compounds violated Lipinski’s rule of five. The JC3 and JC4 compounds are moderately soluble and have predicted high absorption in the gastrointestinal tract. Importantly these results suggest that JC3 and JC4 should be able to cross the BBB, and thus likely the blood-retina barrier (BRB). These properties are critical considerations for the discovery of orally bioavailable small molecules that may correct the folding of Rho within the eye.Table 2 - ADME ParametersMW | ESOL: AU Gf BEB Lipisisfci: PAINS | SjsPwPe: Mokx'Hie•! Log S: Oass Log SI: k-kxfeastdvSB.37 j <38: -457 High | ¥es!> 3.3:, sohihk'I | MctfemSyK4 2i>7.7§ ■ -4.49: •4 J 2 High | Yes o i n | 2,94 i i sosabfeImpact of JC3 and JC4 on pigment regeneration and stability in vitro

[0211] To confirm that JC3 and JC4 bind within the orthosteric binding pocket of rod opsin, we used a previously established Trp fluorescence quenching assay to track ligand binding. Incubation of purified rod opsin membranes (ROS) with increasing concentrations of these compounds resulted in a progressive increase in the quenching of the intrinsic Trp fluorescence of opsin at 330 nm (Fig. 2A). This change results from the conformational rearrangement of Trp265 within the ligand-binding pocket upon ligand binding. Fits of the observed quenching data with a single-site binding model suggest equilibrium dissociation constant (Ka) values for JC3 and JC4 of 175 ± 20 nM and 98.5 ± 33 nM, respectively.Consistent with in silico docking scores, these findings indicate that both molecules bind within the orthosteric pocket and that JC4 binds with higher affinity relative to JC3.

[0212] Next, we examined the effect of JC3 and JC4 binding on the ability of rod opsin to bind 9-ci.v-relinal and regenerate its isochromophore. The binding of 9-cz.y-retinal results in the appearance of an absorption peak at 485 nm, a well-known spectroscopic signature for the formation of a Schiff base between the retinal and Lys296 in the protein backbone. As expected, pretreatment of opsin membranes with JC3 and JC4 did not generate a pigment. However, pre-saturation of the orthosteric pocket with these compounds partially decreased the binding efficiency of the retinal chromophore following a 10 min incubation with 9-cis-retinal (Fig. 2B). This inhibition is concentration-dependent and more pronounced at 100 pM relative to 10 pM. Pre-saturation with JC3 and JC4 also slowed the regeneration kinetics, the regeneration half-time of isorhodopsin (isoRho) increased from 3.9 ± 0.6 min to 8.3 ± 0.9 min and 8.4 ± 1.4 min in the presence of 100 pM IC3 and 100 pM IC4, respectively (Fig. 2C). Together, these observations confirm that JC3 and JC4 compete with retinal for the binding of the rod opsin orthosteric site, but do not inhibit the Schiff base formation between the retinal and the receptor.

[0213] The binding of pharmacochaperones typically enhances the stability of natively folded proteins. To determine whether the binding of these compounds stabilizes opsin, we incubated opsin ROS membranes with each JC compound and then compared the melting temperature (Tm) of the bound protein using a thermal shift assay. The Tmof opsin increased slightly in the presence of 0.1 ja M JC3 (56.5 ± 0.3 °C) or 0.1 pM JC4 (57.5 ± 0.7°C) relative to untreated opsin under these conditions (55.4 ± 0.4 °C), (Fig. 2D). As expected, this stabilization by JC3 or JC4 was not observed if the pigment was first regenerated by retinal, which would inhibit JC3 and JC4 binding. IsoRho and the pigments regenerated in the presence of JC3 and JC4 all exhibited comparable kinetic stability at 55°C, which indicates that the binding of these compounds does not impair the stability of the mature pigments (Fig. 2E).Pharmacological activity of JC3 and JC4

[0214] To examine the effect of JC3 and JC4 compounds on the function of the visual receptor, we measured their effects on the receptor-mediated photoactivation of G-protein transducin (Gt). Briefly, we incubated opsin ROS membranes that were preincubated with JC3 or JC4 followed by the regeneration with 9-cz.v-retinal, with purified Gt protein, and then used Tip fluorescence to monitor changes in Trp fluorescence of Gtaoccurring due to the exchange of GTPyS and the dissociation of the receptor-Gtcomplex upon photoactivation (Fig. 3A and B). The initial rates of Gt activation decreased slightly relative to the untreated control (kimtM= 5.1 ± 0.3 xlO’3s’1) when opsin membranes were preincubated with 10 uM JC3 (kmtia\= 4.4 ± 0.3 xlO’3s’1) or JC4 (fen,ftl7i= 4.3 ± 0.4 xlO’3s’1). To determine whether this result coincides with the inhibition of cellular signaling, we evaluated the effect of JC3 and JC4 on signaling in situ in cultured NIH-3T3 cells heterologously expressing WT rod opsin and compared it to P23H rod opsin mutant. In the absence of Gt Rho signals through Gi, resulting in a decrease in cellular levels of cAMP. Thus, we examined the level of cAMP in cells treated with the JC compounds followed by regeneration of isoRho and compared it with cells incubated with 9-cis-retinal only, either after their exposure to light or kept in the dark (Fig. 3C and D). Upon light stimulation, in the cells regenerated with 9-cis-retinal the cAMP concentration lowered to -50% of the level present in the non-regenerated cells in both, cells expressing WT and P23H mutant receptors. However, in the cells preincubated with JC3 or JC4, the decrease in cAMP level was much lower and its concentration reached about 80-90% as compared to non-regenerated cells, indicating a partial antagonistic effect ofJC3 and JC4 compounds. In addition, cells that were kept in the dark upon treatment with JC3 and JC4 showed a slight increase in the cAMP levels as compared to non-treated cells. Although, the reason for such a result is unclear it could be related to the silencing of constitutive opsin activity by the JC compounds. These results indicate that JC3 and JC4 compounds act as new rod opsin ligands with partial antagonist activity. Altogether, our computational and in vitro receptor binding analyses, along with the functional assays demonstrate that JC3 and JC4 could modulate Rho signaling through direct interaction with rod opsin protein.Impact of JC3 and JC4 on the plasma membrane expression of P23H rod opsin

[0215] The P23H mutation in Rho causes misfolding, ER retention, and enhanced degradation in a manner that causes degeneration of the retina. The proper cellular trafficking of Rho is critical for its function. Thus, we examined the effect of 1C3 and JC4 on membrane trafficking of P23H rod opsin in the NIH-3T3 cells stably expressing this receptor. Neither compound causes appreciable cellular toxicity at 10 pM concentration in the four cell lines tested, which included NIH-3T3 and HEK-293 cells as well as the retina-derived cell lines 661W and ARPE19 (Fig. 4A). Interestingly, treatment with IC3 and JC4 at 10 pM for 16 h enhanced the surface immunostaining of P23H rod opsin relative to untreated cells, with JC4 having a slightly higher efficacy (Fig. 4B and C). Importantly, the effect generated by JC4 was comparable to that of 9-cis-retinal. These findings demonstrate that JC compounds improve the plasma membrane expression of the most common RP-linked rod opsin mutant in the absence of a retinal chromophore.

[0216] To examine whether the enhanced membrane localization of P23H rod opsin arises from changes in the total expression or differences in maturation, we utilized western blotting to compare the effects of these compounds on the glycosylation state of rod opsin. Wild-type (WT) rod opsin expressed in the NIH-3T3 cells predominantly bears higher weight, mature glycans and migrates at an apparent molecular weight of 55 kDa (Fig. 4D). By comparison, the predominant form of P23H rod opsin has an apparent molecular weight of 37 kDa, which reflects its impaired maturation within the secretory pathway (Fig. 4D). Treatment with 9-m-relinal stabilized P23H rod opsin in a manner that resulted in the appearance of both higher weight mature P23H glycoforms and / or oligomers. This interpretation of the observed protein migration pattern is supported by the decreased apparent molecular weight of the P23H protein following treatment with the PNGaseFglycosidase. An increase in the relative abundance of the mature P23H glycoform was also observed in response to treatment with JC4 and, to a lesser extent, upon treatment with JC3 (the ratio of the mature to immature receptor increased to ~1.3 ± 0.2 and 1.6 ± 0.2 fold in the presence of JC3 and JC4, respectively) (Fig. 4E). Similar results showing an increase in the plasma membrane localization of P23H rod opsin upon treatment with the JC compounds were obtained in the photoreceptor-derived 661W cells stably expressing this mutant receptor. Interestingly, the ratio of mature to immature receptors was slightly greater in these cells and reached 1.5 ± 0.2 fold in the presence of JC3 and 3.0 ± 0.5 fold in the presence of JC4 (Fig. 10). Together, these observations show that JC3 and JC4 enhance the maturation of the nascent P23H rod opsin protein within the ER in a manner that ultimately increases its plasma membrane expression. In addition, these findings stimulated a focused analysis of the effects of the JC3 and JC4 compounds on the processing of various RP mutants.Survey of the mutation-specific effects of JC3 and JC4

[0217] Though several molecules that partially correct the misfolding of P23H have been previously reported, the effects of these compounds vary tremendously in their effects against the full spectrum of pathogenic RHO variants. To survey the effects of JC3 and JC4 against the large spectrum of known clinically relevant variants, we utilized deep mutational scanning to quantitatively compare their impacts on the plasma membrane expression of 123 clinical variants in HEK-293T cells as previously described (8, 16). Treatment of cells with JC3 and JC4 increased the expression of 32 and 26 of the 123 tested rod opsin variants, respectively, by at least 5% (Fig. 5A and B). Importantly, both compounds corrected membrane trafficking of the most prevalent RHO variant P23H. Though these compounds generally enhance the expression of a common subset of these variants, the magnitude of their responses varied considerably. For instance, M39R, L47R, L57R, R135L, A164E, Pl 80 A, D190N, and AC264 exhibited larger gains in expression in the presence of JC3 relative to JC4. In contrast, A169P, H211R, and S297R preferentially responded to JC4 over JC3. Notably, many of the destabilizing mutations that can be partially corrected by these compounds are located within the N-terminus or within the extracellular loop (ECL) 2 region that forms the plug stabilizing the retinal within the binding pocket. Other sensitive mutations are located in the proximity of the retinal within the orthosteric site. A few residues are located within transmembrane helix (TM) 1 and are possibly important for receptor-receptor interactions required for proper folding and trafficking. Though the effectsof these compounds are modest relative to that of the 9-cis-retinal isochromophore for some mutants, certain variants do appear to be more sensitive to the JC compounds. P23H, P23A, Q28H, Q28R, Q184P, C185R are more sensitive to JC4 while G89D exhibits sensitivity to JC3 but does not respond to 9-ci.v-rclinal. Together, these results revealed the scope of the mutation-specific effects of JC3 and JC4 across the spectrum of known RP-related rod opsin mutants. These results suggest that effective pharmacochaperones for Rho misfolding will eventually need to be targeted to a specific subset of clinical variants to ensure the success of the treatment.Impacts of JC3 and JC4 on retinal degeneration

[0218] To validate the therapeutic potential of JC3 and JC4 in vivo, we examined their effectiveness in two mouse models of retina degeneration Abca4 / 'Rdh8i / ' and RhoP23H / +knock-in mice. Accumulation of ligand-free opsin after photobleaching in the retina of Abca4 / 'Rdh8 / ' mice accelerates the degeneration of their photoreceptors as a result of constitutive signaling activation. Retinal health is compromised in these mice due to delayed clearance of all-tram-retinal photoproducts and / or regeneration of functional Rho. Given that JC3 and JC4 stabilize opsin in vitro, we assessed whether treatment with these compounds protects their retina from light insult. Abca4 / Rdh8 / ' mice were treated with an i.p. injection of 100 mg / kg body weight (b.w.) of JC3 or JC430 min prior to a 30 min exposure to 10,000 lux light. For comparison, we included control mice that were either withheld from treatment and kept in the dark or treated with a vehicle and subjected to the same illumination. The effects of each compound on both retinal morphology and visual function were then examined 7 days later. In vivo retina imaging (spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO)) and histological analysis showed that, while vehicle-treated mice exhibited a thinning of their outer nuclear layer (ONL), the retina of the JC3 and JC4-treated mice closely resembled those of the non-treated dark-adapted mice (Fig. 6A-C). In addition, the exposure of Abca4~pRdh8'pmice to bright light activates the migration of microglial cells to the retina to clear dying photoreceptors as evidenced by the appearance of autofluorescence (AF) spots.Though vehicle-treated mice exhibited an increase in the number of AF spots, this pathology was not detected in mice treated with either JC3 or JC4 (Fig. 6D and E). We next examined retinal function by measuring the electroretinography (ERG) responses, which provide a measure of retina activity in response to a light stimulus. Both dark- adapted ERG responsesof rod photoreceptors and light-adapted ERG responses of cone photoreceptors were much smaller for mice treated with a vehicle and exposed to bright light relative to mice that were not subjected to illumination (Fig. 6F). However, treatment with either JC3 or JC4 preserved normal retina function. By comparison, ERG responses for the treated mice closely resembled those of unexposed control mice (Fig. 6F). Together, our results indicate that JC3 and JC4 protect the retina of Abca^'RdhS'2' mice from retinal degeneration caused by bright light injury.

[0219] We next examined whether JC3 and JC4 could attenuate or slow down retina degeneration in heterozygous RhoP23H / +knock-in mice. These mice feature many hallmarks of human RP. The substitution of Pro23 to I lis in Rho is the most common mutation responsible for Rho-linked RP. We, therefore, injected RhoP23H / +mice with 10 mg / kg of JC3 or JC4 every other day from postnatal (P) day 21 (P21) until P33 followed by the established earlier protocol, and then used in vivo SD-OCT imaging, histological analyses, and immunostaining to assess the physiological effects of these treatments as shown in (Fig. 7A). Treatment with these compounds increased the thickness of the ONL in the retina relative to those of the vehicle-treated control mice, which indicates that JC3 and JC4 enhance the survival of photoreceptors (Fig. 7B-E). Indeed, the detection of rod cells with an anti-Rho antibody and cone photoreceptors with peanut agglutinin (PNA) confirmed that both JC3 and, to a lesser extent JC4, slowed the progression of photoreceptor cell death in RhoP23H / +mice (Fig. 7D). The ERG responses of mice treated with JC3 and JC4 exhibited enhanced amplitudes of both dark- adapted and light- adapted responses. However, treatment with JC4 resulted in slightly less pronounced ERG responses (Fig. 7F). Together these findings suggest that JC3 and JC4 potentially protect the retina through distinct mechanisms, and the effective dose for JC4 may require additional optimization. Differences in the efficacy of these compounds are likely to arise, in part, from differences in their bioavailability and / or pharmacokinetics (Fig. 8). Nevertheless, our results indicate that both JC3 and JC4 are good lead compound candidates for the development of next-generation more effective pharmacochaperones that stabilize the rod opsin protein, enhance photoreceptor survival, and ultimately slow down the progression of Rho-related RP.

[0220] To ensure that advantageous effects of JC3 and JC4 on retina health observed in the heterozygous RhoP23H / +mice result from the direct modulation of the rod opsin mutant we performed treatment of the homozygous RhoP23H / P23Hmice with these compounds followingthe earlier established protocol. These mice were administered with JC3 or JC4 every other day starting at P14 and were evaluated at P21. As shown in Fig. 11 this treatment resulted in improved retina morphology evidenced by an increased number of nuclei rows within the ONL layer as compared to the vehicle -treated control mice in both the retina center and periphery (Fig. 1 IB). The labeling of rod and cone photoreceptors while hardly detectable in the vehicle-treated mice was greatly enhanced in JC3 and JC4-treated mice (Fig. 11A). The expression of Rho and M cone opsin was barely detectable in the RhoP23H / P23Hmice, however, treatment with JC3 and JC4 resulted in increased both gene and protein expression levels of these receptors, indicating that JC compounds enhance the stability of the mutant Rho, slowing down degeneration of rod photoreceptors, and consequently cone photoreceptors (Fig. 11C-E). Enhanced survival of photoreceptor cells in RhoP23" / P23" mice treated with JC compounds was confirmed by increased amplitudes of both scotopic and photopic ERG responses as compared to the vehicle-treated mice (Fig. 1 IF). This data strongly indicates that the beneficial effects of JC3 and JC4 are related to their pharmacochaperone properties correcting the misfolded Rho mutant and shifting its properties towards WT-like.Bioavailability and toxicity of JC3 and JC4

[0221] Previous pharmacochaperones for the rod opsin protein have failed to advance to clinical studies due to their toxicity and poor bioavailability. To determine whether JC3 and JC4 indeed cross the BRB and persist in the eye in their active forms, we searched for these compounds in biological specimens from treated mice using reverse-phase liquid chromatography coupled with mass spectrometry (LC-MS). C57BL / 6J WT mice were treated with a single i.p. injection of each compound prior to harvesting their eyes at various time points. Internal JC3 or JC4 standards were then added to the eye homogenates from mice treated with the opposite compound prior to extraction. An MS signal for the JC3 standard was observed at m / z = 314.2 [M + H]+along with an MS / MS fragmentation peak at m / z = 238.2 [M + H]+. The MS signal of the JC4 standard was observed at m / z = 298.2 [M + II]+with a corresponding MS / MS fragmentation product at m / z = 269.8 [M + II]+. Both JC3 and JC4 were detected in the samples extracted from mouse eyes as unmodified compounds (Fig. 8A-C). Quantification based on the ion intensities for internal standards revealed 521.1 ± 120.3 pmols of JC3 accumulated in the eye within 30 min of the injection. By comparison, only 28.2 ± 3.2 pmols of JC4 accumulated within the eye during this time. These compounds were cleared from the eye with a calculated half-life of 2.5 h for JC3 and 1.7 h for JC4(Fig. 8D-E). Together, these results indicate that both JC3 and JC4 can cross the BRB and reach the eye, similarly to other small molecules with retinal protective activity.

[0222] To determine whether the JC compounds are well tolerated in mice, we tested their overall systemic toxicity and their effects on the retinoid (visual) cycle in the eye. The WT mice treated with JC3 or JC4 did not show a decrease in body weight either under acute (at 100 mg / kg) or chronic (at 10 mg / kg) doses administered over the course of two weeks (Fig. 12A-C). We also observed no apparent changes in behavior after the chronic administration of JC3 or JC4. A decrease in the exploratory activity observed after the acute administration of JC3 reversed after 2 h. Moreover, these mice treated with JC3 or JC4 for two weeks did not exhibit any significant changes in the ERG responses or the ONL thickness in comparison to the vehicle-treated mice, indicating that JC3 and JC4 do not exhibit detrimental effects on the overall retina function and morphology (Fig. 12D-F). Additionally, we did not detect any changes in the levels of regenerated 11 -cA-retinal at 2 h and 24 h post-illumination among mice treated with JC3 or JC4 compared to the vehicle-treated mice (Fig. 8F). These findings suggest that these compounds do not inhibit the overall regeneration of the visual chromophore. Together, our results suggest these compounds are well tolerated and accumulate within the eyes of mice without compromising their visual health.Structural basis for variant-specific pharmacochaperone effects

[0223] Emerging evidence suggests the variant-specific effects of pharmacochaperones arise from differences in their binding energies and the structural context of their binding sites. Indeed, we observed prominent differences in the response of rod opsin to JC3 and JC4 even though they share a similar pharmacological scaffold, bind with comparable affinity, and compete for the same orthosteric pocket. To evaluate the structural basis for the stabilization generated by these compounds, we employed computational modeling to identify specific interactions that form in the bound state. For this purpose, we used RosettaCM to generate structural models of the human rod opsin apoprotein using the crystallographic structures of the bovine rod opsin apoprotein (93% sequence identity). We then used RosettaLigand to dock each enantiomer of JC3 and JC4 into the orthosteric pocket of the homology model. The lowest energy pose of (R)-JC3 appears similar to that of (S)-JC4 (Fig. 9A). The methylbenzyl group in (R)-JC3 and the chlorobenzyl group in (S)-. TC4 are predicted to have ^-stacking with W265 and Y268. Polar interactions are predictedbetween the benzodioxole in (R)-JC3 and K296 residue, as well as between the carbonyl in (S)-JC4 and Y191 (Fig. 9A). Likewise, similar low energy poses were also determined for (S )- JC3 and (R)-JC4. The carbonyls in both molecules were predicted to have polar interactions with Y191 and Y268. The methylbenzyl group in (R)-. TC4 may also be involved in 71-stacking with Y191, while (S)-JC3 is slightly shifted and excluded from the interaction (Fig. 9B). These putative differences in the coordination of these two compounds may factor into their biological activity.

[0224] To determine how these compounds stabilize specific variants, we used these WT models to generate bound-state ensembles of two variants that exhibit preferential stabilization by JC3 (G51R & D190N) and two that are preferentially stabilized by JC4 (P23H and Q184P). The docked poses and interactions predicted in the WT models were preserved in all variants. These mutations have modest effects on the shape and / or volume of the ligand-binding pocket, except for (S)-. TC4 bound G51R where the deeper pocket is closed (Fig. 13). The pockets of (R)-JC3 bound models and (S)-JC4 bound models are elongated in a manner that is perpendicular to the plane of the membrane, while those for (S)-JC3 and (R)-JC4 are more compact. For all four variants, the R enantiomer of JC3 forms more stabilizing contacts than the S enantiomer (Fig. 9C). To test this prediction, we assigned the isolated JC3 enantiomers (Fig. 14) and then measured their binding affinities (Fig. 15A). Consistent with modeling results, (R)-JC3 bound to rod opsin with a higher affinity (Ka = 66.7±1.3 nM) relative to (S)-JC3 (Kd = 333.7 ± 23 nM). Moreover, only the R enantiomer was able to enhance the plasma membrane expression of G51R and D190N rod opsins in HEK293T cells (Fig. 15B). Thus, we conclude that the R enantiomer of JC3 is the most potent. In contrast, the interface energies formed by the two JC4 enantiomers arc comparable for each variant except for G51R, where the compounds achieve a distinct pose (Fig. 9C and Fig. 13). Indeed, the isolated JC4 enantiomers appear to bind with similar affinity (Kd values of 91.6 ± 2.1 nM and 109.6 ± 6.1 nM determined for the two unassigned enantiomers). Across all four variants, (R)-JC3 and (S)-. TC4 have comparable predicted binding energies, however, (R)-JC4 binds with the lowest energy overall. Mutation- specific variations in the energies are generally modest and do not coincide with the selectivity of the variants for one compound over another. Though the similarity of the interaction energies is expected given the comparable binding affinities of these compounds (Fig. 2A), the modest differences between the bound-state structures of these variants suggest that their divergentpharmacological responses may instead arise from the ability of these compounds to suppress the distinct classes of conformational defects caused by these mutations.

[0225] To evaluate the manner in which J C compounds alter the dynamics of pathogenic rod opsin variants, we utilized molecular dynamics (MD) simulations to compare the conformational fluctuations of their bound states over the course of 1000 ns in an explicit bilayer solvent. The unliganded opsin of the common P23H variant exhibits enhanced flexibility within its N-terminal loop relative to WT, while G51R opsin undergoes dynamic fluctuations in TM1 (Fig. 16A). MD simulations of these variants bound to (R)-JC3, (S)-JC3, (R)-. TC4, and (S)-JC4 showed dampened conformational fluctuations, which suggests that the binding of these compounds partially restores the native structural dynamics of these variants (Fig. 16A). All four compounds remained bound to the orthosteric site over the entire course of the simulations (Fig. 16B). Based on these simulations, we carried out a protein structure network (PSN) analysis, which identified six clusters of stabilizing interactions within the native rod opsin structure. The binding of either JC3 or JC4 increased the number of interacting residues within the native clusters or generated additional clusters of interacting residues (Figs. 17 and 18). Although we observed minimal differences between the enantiomers in these simulations, generally JC compounds appear to stabilize the native fold by improving internal residue-residue interactions that form in the context of the RP variants.

[0226] Proper biogenesis, folding, and stability of rod opsin are required for the correct routing of this receptor to the ROS disc membranes. Mutations in the RHO gene cause structural defects in the receptor protein, which can lead to incorrect folding and defective binding of the native chromophore 11 -cA-rctinal due to weakening internal interactions critical for the intrinsic stability of opsin. These changes typically result in the retention and degradation of the mutant receptor in the endoplasmic reticulum (ER). In P23H Rho mice, the mutant receptor escapes ER quality control and is transported to the ROS, but ultimately compromises the integrity of its membrane discs in a manner that causes adRP. This pathology could potentially be rescued using pharmacological chaperones, which may include its native retinal chromophore or its analogs that restore opsin stability. However, stabilization and folding correction of misfolded opsin variants with retinoids can only occur under dark conditions due to the photo-sensitivity of the Schiff base bond formed between the retinal and the opsin’s Lys296. The utility of retinoids is also undermined by the fact thatthese compounds and their photoproducts are toxic, which limits their therapeutic potential. For these reasons, the use of commercially available and commonly used in in vitro assays 9-cM-relinal is not desirable, and thus the development of novel non-retinoid folding correctors that are insensitive to light provides a clear advantage in ongoing efforts to develop pharmacochaperones for the rod opsin protein. In fact, small molecule correctors of protein folding have been proven effective in other protein misfolding diseases.

[0227] Flavonoids and other unrelated small molecules are capable of stabilizing the opsin protein and acting as pharmacochaperones under certain conditions. Nevertheless, these compounds exhibit limited bioavailability and appear only to partially correct the misfolding of certain RP variants. In this Example, we described two new pharmacochaperones, JC3 and JC4, which bind reversibly to ligand-free opsin and modulate opsin properties without regulation by light. These molecules increased the stability of opsin and substantially improved the maturation and expression of the heterologously expressed P23H rod opsin mutant. Thermal stability assay indicated that JC3 and JC4 did not provide additional stabilization in the presence of 9-cw-retinal, which suggests they likely bind to the same orthosteric binding pocket. Indeed, the binding of JC compounds partially antagonized the coupling of the 9-cz.v-retinal isochromophore, slowing down the rate of pigment regeneration without inhibition of isoRho formation. This decelerated regeneration of the visual receptor could potentially be beneficial under pathological conditions in which the clearance of cytotoxic all-trans-retinal photoproducts is impaired, which is known to occur in Stargardt disease and age-related macular degeneration (AMD). These excessive concentrations of all- / ran.v-relinal induce a cellular stress response within the retina, which has detrimental consequences for photoreceptors. Slower formation of the functional Rho pigment could also attenuate the production and release of all-zra / z -relinal upon illumination. Indeed, we showed that JC3 and JC4 prevented photoreceptor death that occurs in response to bright light injury in an Abca4- / -Rdh8- / -mouse model of Stargardt disease, likely due to their stabilization of opsin and their ability to antagonize opsin constitutive activation under these conditions, which is known to accelerate degenerative processes in the retina.

[0228] In addition to their photosensitivity and limited bioavailability, previously described rod opsin pharmacochaperones generally appear to only rescue partially a subset of known retinopathy variants. Using deep mutational scanning, we showed that in total, JC3 and JC4 measurably enhanced the plasma membrane expression of 36 of 123 heterologouslyexpressed RP-related rod opsin mutants. Notably, 22 of these mutants appear to be sensitive to both compounds. Interestingly, treatment with JC3 enhanced the plasma membrane expression of eight distinct mutants, while JC4 appears to be uniquely effective towards four other distinct rod opsin mutants. Although JC3 and JC4 restore the expression of fewer mutants than 9-c / .v-relinal under these conditions, we suspect this limitation could potentially be overcome by modifications that enhance the affinity of these compounds for the rod opsin protein. Regardless of whether these compounds could be modified to correct the expression of a broader array of mutants, we noted that several of these mutants exhibit a response to JC compounds that is comparable in magnitude to the effect of 9-cis-retinal or even greater. Together, the observed differences in the response of this panel of mutants to retinoids, chromenones, and JC compounds suggest that, regardless of the specific details of the chemical scaffold, this class of molecules may eventually need to be targeted to specific patient genotypes in clinical trials. The development of a diverse portfolio of rod opsin pharmacochaperones may therefore be beneficial for the development of precision therapeutics, as has proven successful for CF.

[0229] While the in vitro studies described herein provide insights into the mechanistic effects of these compounds, the phenotypic effects of JC3 and JC4 in Abca4⁻ / ⁻Rdh8⁻ / ⁻ and RhoP23H / +mice provide compelling evidence of the therapeutic potential of these compounds. Importantly, treatment with either compound improved the overall retina health and function in these mice by prolonging the survival of their photoreceptors. The beneficial effect of JC3 and JC4 on the photoreceptor survival in the homozygous RhoP23H / P23Hmice showing similar outcomes as reported previously for treatment with flavonoids additionally confirms the pharmacochapcronc potential of these compounds for RP-linkcd rod opsin mutant. We noted that, although JC4 appeared to be most effective in vitro, JC3 proved more effective in vivo, especially in RhoP23H / +mice, likely due to its longer half-life in the eye. Furthermore, neither of these compounds caused significant toxicity in mice nor had adverse effects on retina morphology or function in the WT mice. Based on these considerations, the two compounds described herein offer considerable translational potential for the development of precision therapeutics for RP and other visual retinopathies.

[0230] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art arc intended to be covered by the appended claims. All references,publications, and patents cited in the present application are herein incorporated by reference in their entirety.

Claims

Having described the invention, we claim:

1. A compound of formula (I):IR1a(I) or a pharmaceutically acceptable salt thereof for use as a non-retinoid pharmacochaperone;whereinthe dashed line is an optional bond;Xis -N-, -C-, or -C(H)-;X1is -C(H)-, -C(H2)-, or -O-:X2is -C(R4)-, -C(R4)2-, or -S-;X3is absent, -N-, -N(H)-, -C(H)-, or -C(H2)-;R1is aryl, heterocyclyl, or heteroaryl, each of which is optionally substituted with one or more R5;Rlais phenyl or benzyl, each of which is optionally substituted with one or more RR4is H, halogen, alkyl, or haloalkyl;each R5is halogen, -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m( alkyl), - C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, alkoxy, haloalkyl, cycloalkyl, or heterocyclyl; and m is 0, 1, or 2.

2. The compound or the pharmaceutically acceptable salt thereof of claim 1, comprising a compound of formula (II):x1-x2U'Z^RX1IR1a(II) or a pharmaceutically acceptable salt thereof for use as a non-retinoid pharmacochaperone;whereinthe dashed line is an optional bond;X is -N-, -C-, or -C(H)-;X1is -C(H)-, -C(H2)-, or -O-;X2is -C(R4)-, -C(R4)2-, or -S-;R1is aryl, heterocyclyl, or heteroaryl, each of which is optionally substituted with one or more R5;Rlais a phenyl or a benzyl, each of which is optionally substituted with one or moreR4is H, halogen, alkyl, or haloalkyl;each R5is halogen, -CN, -N(R4)2, -OH, -O-(alkylene)-OH, -S(O)m( alkyl), -C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, alkoxy, haloalkyl, cycloalkyl, or heterocyclyl; and m is 0, 1, or 2.

3. The compound or the pharmaceutically acceptable salt thereof of claim 1 or claim 2, wherein Rlais a phenyl, which is optionally substituted with one or more R5.

4. The compound or the pharmaceutically acceptable salt thereof of any of R6ACclaims 1 to 3, wherein R1is ' — 'JR7,R6and R7are each independently absent, halogen, -CN, -N(R4)2, -OH, -O- (alkylene)-OH, -S(O)m( alkyl), -C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, haloalkyl, cycloalkyl, or heterocyclyl;or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N, and wherein the heterocycle is optionally substituted with R8; and R8is halogen, alkyl, or alkoxy.

5. The compound or the pharmaceutically acceptable salt thereof of any ofR9is a halogen, C'-C6alkyl, or C ’-C6alkoxy.

6. The compound or the pharmaceutically acceptable salt thereof of any of claims 1 to 5, comprising a compound of formula (III):(III) or a pharmaceutically acceptable salt thereof; whereinthe dashed line is an optional bond;X2is -C(R4)-, -C(R4)2-, or -S-;R2and R3are each independently absent or halogen, alkyl, haloalkyl, or alkoxy;R4is H, halogen, alkyl, or haloalkyl;R6and R7are each independently absent, halogen, -CN, -N(R4)2, -OH, -O- (alkylene)-OH, -S(O)m( alkyl), -C(O)(alkyl), -C(O)-(cycloalkyl), alkyl, haloalkyl, cycloalkyl, or heterocyclyl;or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N, and wherein the heterocycle is optionally substituted with R8;R8is halogen, alkyl, or alkoxy; andm is 0, 1, or 2.

7. The compound or the pharmaceutically acceptable salt thereof of claim 6, whereinX2is S;R2and R3are each independently absent, a halogen, Ci-Ce alkyl, or Ci-Ce alkoxy; andR6and R7are each independently absent, halogen, Ci-Ce alkyl, or Ci-Ce alkoxy; or alternatively, R6and R7together with the atom to which they are attached can form a 4- to 7-membered heterocycle, optionally containing an additional heteroatom selected from O, S, or N.

8. The compound or the pharmaceutically acceptable salt thereof of claim 7, comprising a compound selected from:or a pharmaceutically9. The compound or the pharmaceutically acceptable salt thereof of claim 6, whereinX2is -C(R4)-;R2and R3are each independently absent, a halogen, Ci-Ce alkyl, or Ci-Ce alkoxy; andR6and R7are each independently absent, halogen, Ci-Ce alkyl, or Ci-Ce alkoxy.

10. The compound or the pharmaceutically acceptable salt thereof of claim 9, comprising a compound selected from:

11. The compound or the pharmaceutically acceptable salt thereof of claim 1, selected from:

12. The compound or the pharmaceutically acceptable salt thereof of claim 1, selected from:so>oan (R) enantiomer ofsan (S) enantiomer ofan (R) enantiomer ofan (S) enantiomer of; or a pharmaceutically acceptable salt thereof.

13. A method of suppressing the pathogenic effects of misfolded rhodopsin variants in a subject in need thereof, the method comprising;administering to the subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof of any of claims 1 to 12.

14. A method of treating retinal degeneration in a subject in need thereof, the method comprising:administering to the subject a therapeutically effective amount of a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof of any of claims 1 to 1215. The method of claim 14, wherein the retinal degeneration is selected from the group consisting of Leber congenital amaurosis, Stargardt disease, and retinitis pigmentosa.

16. The method of claim 15, the retinitis pigmentosa comprising autosomal dominate retinitis pigmentosa associated with a P23H RHO mutation.

17. The method of claim 14, the therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof is an amount required to inhibit photoreceptor cell death in the subject.

18. The method of claim 14, the therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof is an amount effective to inhibit bright light-induced retinal degeneration in a Rdh8_ / Abca4 / -mouse.

19. The method of claim 14, wherein administration of the compound or the pharmaceutically acceptable salt thereof to the subject stabilizes P23H rod opsin mutant proteins.

20. The method of claim 14, wherein administration of the compound or the pharmaceutically acceptable salt thereof to the subject promotes rod photoreceptor cell homeostasis in the subject.

21. The method of claim 13, wherein administration of the compound or the pharmaceutically acceptable salt thereof to the subject mobilizes the P23H opsin from the endoplasmic reticulum to the plasma membrane of photoreceptor cells.

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