Beata Jastrzebska

Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinal degenerative disorders characterized by progressive photoreceptor loss and vision impairment, for which broadly applicable mutation-independent therapies remain limited. To examine the therapeutic potential of combined galanin receptor 3 (GALR3) inhibition and antioxidant therapy in a mutation-independent context, we utilized the rd10 mouse model of RP. We first evaluated the effects of individual treatments with the GALR3 antagonist SNAP-37889 and the antioxidant quercetin, followed by a combined treatment regimen to determine whether simultaneous targeting of neuroinflammatory and oxidative stress pathways provides enhanced retinal protection. Treatment efficacy was assessed using functional and morphological analyses, including electroretinography (ERG) to measure retinal function, optical coherence tomography (OCT) to evaluate retinal structure in vivo, and histological and immunohistochemical analyses to quantify photoreceptor survival and markers of retinal oxidative stress and inflammation. Although the expression levels of individual inflammatory and oxidative stress markers did not consistently exhibit additive responses, the combined treatment produced greater photoreceptor survival and preservation of photopic retinal function than either monotherapy alone. These findings support the hypothesis that simultaneous modulation of oxidative stress and neuroinflammation provides greater neuroprotective benefits, establish a foundation for the development of mutation-independent therapeutic strategies for RP, and identify GALR3 as a promising therapeutic target.

Rhodopsin misfolding underlies rhodopsin-linked retinitis pigmentosa, and small-molecule pharmacochaperones represent a promising therapeutic strategy. However, the mechanisms by which these compounds interact with and stabilize rhodopsin remain poorly understood. Here, we combine backbone amide hydrogen-deuterium exchange mass spectrometry (amide HDX-MS), histidine-specific HDX (His-HDX), protein structure network (PSN) analysis, molecular docking, and functional spectroscopy to define ligand-induced conformational signatures in this receptor elicited by three non-retinoid small molecules, quercetin, myricetin, and the chromenone CR5, and to compare them with those of the native chromophore 11- cis -retinal. Binding of 11- cis -retinal to ligand-free opsin establishes a benchmark orthosteric conformational signature, characterized by strong backbone HDX protection across TM4-TM7 and adjacent loops, suppression of EX1-like hydrogen-deuterium exchange kinetics at the N-terminal ends of TM1 and TM4, and reorganization of PSN hubs that stabilizes an inactive-state residue interaction network. All three non-retinoid ligands generate HDX footprints that closely track this retinal-induced pattern within the chromophore pocket, consistent with direct orthosteric engagement, but they confer weaker and ligand-specific stabilization. Among them, quercetin most closely reproduces the retinal-like backbone protection and His-HDX microenvironment changes, whereas myricetin and CR5 only partially recapitulate retinal-induced stabilization and redistribute conformational flexibility toward TM1 and intradiscal regions, without fully suppressing EX1-like gating. In addition, all three compounds induce weak cytoplasmic allosteric effects in retinal-bound rhodopsin, indicating secondary interactions in addition to a primary orthosteric mechanism. Together, these results provide the first residue-level experimental framework for understanding the differential pharmacochaperoning capacity of non-retinoid ligands and highlight key conformational principles for future optimization of opsin stabilizers.

Photoreceptors require a finely regulated balance of oxygen, nutrients, and waste removal to sustain visual function. In inherited retinopathies like rhodopsin (RHO)-associated retinitis pigmentosa (RP), disruption in retinal homeostasis leads to neurodegeneration. The most common mutation in RHO, P23H, causes protein misfolding, endoplasmic reticulum (ER) stress, and activation of inflammatory and oxidative stress pathways, ultimately leading to photoreceptor death. Upregulation of the NLRP3 inflammasome and NF-κB signaling in RHO mutant models, highlight inflammation as a key contributor to disease progression, yet targeted therapies remain limited. G protein-coupled receptor (GPCR) signaling is a crucial regulator of retinal homeostasis. We identified galanin receptor 3 (GALR3), a GPCR expressed in retinal cells, as a mediator of photoreceptor degeneration. In the Rho^P23H/+ mouse model, GALR3 expression was upregulated in response to the mutation-induced chronic stress. Both genetic ablation and pharmacological inhibition of GALR3 with the selective antagonist SNAP-37,889 attenuated photoreceptor loss and improved retinal survival. Mechanistically, GALR3 inhibition suppressed pro-inflammatory signaling, promoted anti-inflammatory responses, and activated antioxidant defense pathways. These findings reveal GALR3 as a critical mediator of inflammatory and oxidative stress responses in RHO P23H-associated RP, and its inhibition offers a promising therapeutic strategy to slow retinal degeneration and preserve vision in inherited retinopathies.

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

Rhodopsin, the prototypical Class A G protein-coupled receptor (GPCR) and visual pigment of rod photoreceptors, has long served as a structural and mechanistic model for GPCR biology. Mutations in rhodopsin are the leading cause of autosomal dominant retinitis pigmentosa (adRP), making this receptor a critical therapeutic target. In this review, we summarize the chemical, structural, and biophysical features of small-molecule modulators of this receptor, spanning both classical retinoid analogs and emerging non-retinoid scaffolds. These ligands reveal recurrent binding modes within the orthosteric chromophore pocket as well as peripheral allosteric and bitopic sites, where they mediate folding, rescue trafficking, photocycle modulation, and mutant stabilization. We organize ligand performance into a three-tier framework linking binding affinity, cellular rescue potency, and stability gains. Chemotypes in tier 2, which show sub-micromolar to low-micromolar activity with broad mutant coverage, emerge as promising candidates for optimization into next-generation scaffolds. Across scaffolds, a recurring minimal pharmacophore is evident by a contiguous hydrophobic π-surface anchored in the β-ionone region, coupled with a strategically oriented polar handle that modulates the Lys296/Glu113 microenvironment, offering tractable design vectors for non-retinoid chemotypes. Beyond the chromophore binding pocket, we highlight opportunities to exploit extracellular loop epitopes, cytoplasmic microswitch clefts, dimer/membrane interfaces, and ion co-binding sites to engineer safer, state-biased control with fewer photochemical liabilities. By integrating rhodopsin photobiophysics with environment-aware, multi-state medicinal chemistry, and by addressing current translational challenges in drug delivery, this review outlines a rational framework for advancing rhodopsin-targeted therapeutics toward clinically credible interventions for RP and related retinal degenerations.

Pathogenic mutations that cause rhodopsin misfolding lead to a spectrum of currently untreatable blinding diseases collectively termed retinitis pigmentosa. Small molecules to correct rhodopsin misfolding are therefore urgently needed. In this study, we utilized virtual screening to search for drug-like molecules that bind to the orthosteric site of rod opsin and improve its folding and trafficking. We identified and validated the biological effects of 2 non-retinoid compounds with favorable pharmacological properties that cross the blood–retina barrier. These compounds reversibly bind to unliganded rod opsin, each with a K_d comparable to 9-cis-retinal and improve opsin stability. By improving the internal protein structure network (PSN), these rod opsin ligands also enhanced the plasma membrane expression of total 36 of 123 tested clinical RP variants, including the most prevalent P23H variant. Importantly, these compounds protected retinas against light-induced degeneration in mice vulnerable to bright light injury and prolonged survival of photoreceptors in a retinitis pigmentosa mouse model for rod opsin misfolding.

Retinitis pigmentosa (RP) is a hereditary disease characterized by progressive vision loss ultimately leading to blindness. This condition is initiated by mutations in genes expressed in retinal cells, resulting in the degeneration of rod photoreceptors, which is subsequently followed by the loss of cone photoreceptors. Mutations in various genes expressed in the retina are associated with RP. Among them, mutations in the rhodopsin gene (RHO) are the most common cause of this condition. Due to the involvement of numerous genes and multiple mutations in a single gene, RP is a highly heterogeneous disease making the development of effective treatments particularly challenging. The progression of this disease involves complex cellular responses to restore cellular homeostasis, including the unfolded protein response (UPR) signaling, autophagy, and various cell death pathways. These mechanisms, however, often fail to prevent photoreceptor cell degradation and instead contribute to cell death under certain conditions. Current research focuses on the pharmacological modulation of the components of these pathways and the direct stabilization of mutated receptors as potential treatment strategies. Despite these efforts, the intricate interplay between these mechanisms and the diverse causative mutations involved has hindered the development of effective treatments. Advancing our understanding of the interactions between photoreceptor cell death mechanisms and the specific genetic mutations driving RP is critical to accelerate the discovery and development of therapeutic strategies for this currently incurable disease.