ABSTRACT
Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.
Subject(s)
Disease Models, Animal , Drug Repositioning , Retinitis Pigmentosa , Animals , Mice , Dogs , Retinitis Pigmentosa/drug therapy , Retinitis Pigmentosa/genetics , Mutation , Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Mice, Knockout , Leber Congenital Amaurosis/drug therapy , Leber Congenital Amaurosis/genetics , Bromocriptine/pharmacology , Bromocriptine/therapeutic use , cis-trans-Isomerases/genetics , cis-trans-Isomerases/metabolism , Humans , Drug Therapy, Combination , Mice, Inbred C57BL , Retinal Cone Photoreceptor Cells/drug effects , Retinal Cone Photoreceptor Cells/metabolism , Retinal Cone Photoreceptor Cells/pathology , Female , Cyclic AMP/metabolism , Retinal Degeneration/drug therapy , Retinal Degeneration/genetics , Male , Calcium/metabolismABSTRACT
Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.
Subject(s)
Carrier Proteins , Mice, Knockout , Retinal Cone Photoreceptor Cells , Retinal Pigment Epithelium , Animals , Mice , Carrier Proteins/metabolism , Carrier Proteins/genetics , Ependymoglial Cells/metabolism , Mice, Inbred C57BL , Retinal Cone Photoreceptor Cells/metabolism , Retinal Pigment Epithelium/metabolism , Retinal Pigments/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Male , FemaleABSTRACT
Recycling of all-trans-retinal to 11-cis-retinal through the visual cycle is a fundamental metabolic pathway in the eye. A potent retinoid isomerase (RPE65) inhibitor, (R)-emixustat, has been developed and tested in several clinical trials; however, it has not received regulatory approval for use in any specific retinopathy. Rapid clearance of this drug presents challenges to maintaining concentrations in eyes within a therapeutic window. To address this pharmacokinetic inadequacy, we rationally designed and synthesized a series of emixustat derivatives with strategically placed fluorine and deuterium atoms to slow down the key metabolic transformations known for emixustat. Crystal structures and quantum chemical analysis of RPE65 in complex with the most potent emixustat derivatives revealed the structural and electronic bases for how fluoro substituents can be favorably accommodated within the active site pocket of RPE65. We found a close (â¼3.0 Å) F-π interaction that is predicted to contribute â¼2.4 kcal/mol to the overall binding energy.
Subject(s)
Eye/metabolism , Phenyl Ethers/pharmacokinetics , Propanolamines/pharmacokinetics , Amine Oxidase (Copper-Containing)/metabolism , Animals , Cattle , Cell Adhesion Molecules/metabolism , Crystallography, X-Ray , Deuterium/chemistry , Drug Design , Fluorine/chemistry , Halogenation , Mice , Molecular Structure , Phenyl Ethers/chemical synthesis , Phenyl Ethers/metabolism , Propanolamines/chemical synthesis , Propanolamines/metabolism , Protein Binding , Structure-Activity Relationship , cis-trans-Isomerases/metabolismABSTRACT
The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for cotranslational expression of CreERT2. Cre+/- mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/- mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre+/- mice. These data indicate that Rpe65CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE.