A non-retinoid antagonist of retinol-binding protein 4 rescues phenotype in a model of Stargardt disease without inhibiting the visual cycle.

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the Abca4-/- mouse model of increased lipofuscinogenesis significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis, and this inhibition correlated with a partial reduction in visual cycle retinoids such as retinaldehydes serving as bisretinoid precursors. BPN-14136 administration at doses inducing maximal serum RBP4 reduction did not produce changes in the rate of the visual cycle, consistent with minimal changes in dark adaptation. Abca4-/- mice exhibited dysregulation of the complement system in the retina, and BPN-14136 administration normalized the retinal levels of proinflammatory complement cascade components such as complement factors D and H, C-reactive protein, and C3. We conclude that BPN-14136 has several beneficial characteristics, combining inhibition of bisretinoid synthesis and reduction in retinaldehydes with normalization of the retinal complement system. BPN-14136, or a similar compound, may be a promising drug candidate to manage Stargardt disease and dry AMD.

A novel RPE65 inhibitor CU239 suppresses visual cycle and prevents retinal degeneration.

The retinoid visual cycle is an ocular retinoid metabolism specifically dedicated to support vertebrate vision. The visual cycle serves not only to generate light-sensitive visual chromophore 11-cis-retinal, but also to clear toxic byproducts of normal visual cycle (i.e. all-trans-retinal and its condensation products) from the retina, ensuring both the visual function and the retinal health. Unfortunately, various conditions including genetic predisposition, environment and aging may attribute to a functional decline of the all-trans-retinal clearance. To combat all-trans-retinal mediated retinal degeneration, we sought to slow down the retinoid influx from the RPE by inhibiting the visual cycle with a small molecule. The present study describes identification of CU239, a novel non-retinoid inhibitor of RPE65, a key enzyme in the visual cycle. Our data demonstrated that CU239 selectively inhibited isomerase activity of RPE65, with IC50 of 6 µM. Further, our results indicated that CU239 inhibited RPE65 via competition with its substrate all-trans-retinyl ester. Mice with systemic injection of CU239 exhibited delayed chromophore regeneration after light bleach, and conferred a partial protection of the retina against injury from high intensity light. Taken together, CU239 is a potent visual cycle modulator and may have a therapeutic potential for retinal degeneration.

Pharmacological inhibition of lipofuscin accumulation in the retina as a therapeutic strategy for dry AMD treatment.

Age-related macular degeneration (AMD) is the leading cause of blindness in the western world. There is no FDA-approved treatment for the most prevalent dry (atrophic) form of AMD. Photoreceptor degeneration in dry AMD is triggered by abnormalities in the retinal pigment epithelium (RPE). It has been suggested that excessive accumulation of fluorescent lipofuscin pigment in the RPE represents an important pathogenic factor in etiology and progression of dry AMD. Cytotoxic lipofuscin bisretinoids, such as A2E, are formed in the retina in a non-enzymatic way from visual cycle retinoids. Inhibition of toxic bisretinoid production in the retina seems to be a sound treatment strategy for dry AMD. In this review we discuss the following classes of pharmacological treatments inhibiting lipofuscin bisretinoid formation in the retina: direct inhibitors of key visual cycle enzymes, RBP4 antagonists, primary amine-containing aldehyde traps, and deuterated analogs of vitamin A.

The novel visual cycle inhibitor (±)-RPE65-61 protects retinal photoreceptors from light-induced degeneration.

The visual cycle refers to a series of biochemical reactions of retinoids in ocular tissues and supports the vision in vertebrates. The visual cycle regenerates visual pigments chromophore, 11-cis-retinal, and eliminates its toxic byproducts from the retina, supporting visual function and retinal neuron survival. Unfortunately, during the visual cycle, when 11-cis-retinal is being regenerated in the retina, toxic byproducts, such as all-trans-retinal and bis-retinoid is N-retinylidene-N-retinylethanolamine (A2E), are produced, which are proposed to contribute to the pathogenesis of the dry form of age-related macular degeneration (AMD). The primary biochemical defect in Stargardt disease (STGD1) is the accelerated synthesis of cytotoxic lipofuscin bisretinoids, such as A2E, in the retinal pigment epithelium (RPE) due to mutations in the ABCA4 gene. To prevent all-trans-retinal-and bisretinoid-mediated retinal degeneration, slowing down the retinoid flow by modulating the visual cycle with a small molecule has been proposed as a therapeutic strategy. The present study describes RPE65-61, a novel, non-retinoid compound, as an inhibitor of RPE65 (a key enzyme in the visual cycle), intended to modulate the excessive activity of the visual cycle to protect the retina from harm degenerative diseases. Our data demonstrated that (±)-RPE65-61 selectively inhibited retinoid isomerase activity of RPE65, with an IC50 of 80 nM. Furthermore, (±)-RPE65-61 inhibited RPE65 via an uncompetitive mechanism. Systemic administration of (±)-RPE65-61 in mice resulted in slower chromophore regeneration after light bleach, confirming in vivo target engagement and visual cycle modulation. Concomitant protection of the mouse retina from high-intensity light damage was also observed. Furthermore, RPE65-61 down-regulated the cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) pathway, decreased the inflammatory factor, and attenuated retinal apoptosis caused by light-induced retinal damage (LIRD), which led to the preservation of the retinal function. Taken together, (±)-RPE65-61 is a potent visual cycle modulator that may provide a neuroprotective therapeutic benefit for patients with STGD and AMD.

DPOFA, a Cl⁻/HCO3⁻ exchanger antagonist, stimulates fluid absorption across basolateral surface of the retinal pigment epithelium.

BACKGROUND: Retinal detachment is a disorder of the eye in which sensory retina separates from the retinal pigment epithelium (RPE) due to accumulation of fluid in subretinal space. Pharmacological stimulation of fluid reabsorption from subretinal space to choroid across the RPE has been suggested as a treatment strategy for retinal detachment. DPOFA, (R)-(+)-(5,6-dichloro 2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy]acetic acid, is an abandoned drug capable of inhibiting Cl⁻/HCO3⁻ exchanger activity. We hypothesized that DPOFA may increase fluid absorption across basolateral surface of the RPE. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA for six different transporters that may act as Cl⁻/HCO3⁻ exchangers was conducted in bovine and human RPE to confirm that RPE from two species expresses the same repertoire of Cl⁻/HCO3⁻ exchanger isoforms. The degree of amino acid homology between orthologous human and bovine RPE-specific isoforms was calculated after performing protein alignments. Transport of fluid across bovine RPE-choroid explants mounted in the Ussing chamber was used to assess the ability of DPOFA to modulate fluid absorption across the RPE. RESULTS: Using RT-PCR we showed that three isoforms (SLC4A2, SLC4A3, and SLC26A6) are strongly expressed in human and bovine RPE preparations. Amino acid comparisons conducted for RPE-specific isoforms support the use of bovine RPE-choroid explants as an adequate experimental system for assessing fluid absorption activity for DPOFA. Our data is consistent with the fact that DPOFA stimulates fluid absorption across the RPE in bovine RPE-choroid explants. CONCLUSIONS: DPOFA seems to stimulate transport of water across the RPE in bovine RPE-choroid explants. Additional experiments are required to establish dose-dependent effect of DPOFA on fluid absorption in the bovine RPE-choroid experimental system.

Identification of Transthyretin Tetramer Kinetic Stabilizers That Are Capable of Inhibiting the Retinol-Dependent Retinol Binding Protein 4-Transthyretin Interaction: Potential Novel Therapeutics for Macular Degeneration, Transthyretin Amyloidosis, and Their Common Age-Related Comorbidities.

Dissociation of transthyretin (TTR) tetramers may lead to misfolding and aggregation of proamyloidogenic monomers, which underlies TTR amyloidosis (ATTR) pathophysiology. ATTR is a progressive disease resulting from the deposition of toxic fibrils in tissues that predominantly presents clinically as amyloid cardiomyopathy and peripheral polyneuropathy. Ligands that bind to and kinetically stabilize TTR tetramers prohibit their dissociation and may prevent ATTR onset. Drawing from clinically investigated AG10, we designed a constrained congener (14) that exhibits excellent TTR tetramer binding potency, prevents TTR aggregation in a gel-based assay, and possesses desirable pharmacokinetics in mice. Additionally, 14 significantly lowers murine serum retinol binding protein 4 (RBP4) levels despite a lack of binding at that protein's all-trans-retinol site. We hypothesize that kinetic stabilization of TTR tetramers via 14 is allosterically hindering all-trans-retinol-dependent RBP4-TTR tertiary complex formation and that the compound could present ancillary therapeutic utility for indications treated with RBP4 antagonists, such as macular degeneration.

Comparative pharmacokinetics and pharmacodynamics of the advanced Retinol-Binding Protein 4 antagonist in dog and cynomolgus monkey.

Accumulation of lipofuscin bisretinoids in the retina contributes to pathogenesis of macular degeneration. Retinol-Binding Protein 4 (RBP4) antagonists reduce serum retinol concentrations thus partially reducing retinol delivery to the retina which decreases bisretinoid synthesis. BPN-14136 is a novel RBP4 antagonist with good in vitro potency and selectivity and optimal rodent pharmacokinetic (PK) and pharmacodynamic (PD) characteristics. To select a non-rodent species for regulatory toxicology studies, we conducted PK and PD evaluation of BPN-14136 in dogs and non-human primates (NHP). PK properties were determined following oral and intravenous administration of BPN-14136 in beagle dogs and cynomolgus monkeys. Dynamics of plasma RBP4 reduction in response to compound administration was used as a PD marker. BPN-14136 exhibited favorable PK profile in both species. Dose-normalized exposure was significantly higher in NHP than in dog. Baseline concentrations of RBP4 were considerably lower in dog than in NHP, reflecting the atypical reliance of canids on non-RBP4 mechanisms of retinoid trafficking. Oral administration of BPN-14136 to NHP induced a strong 99% serum RBP4 reduction. Dynamics of RBP4 lowering in both species correlated with compound exposure. Despite adequate PK and PD characteristics of BPN-14136 in dog, reliance of canids on non-RBP4 mechanisms of retinoid trafficking precludes evaluation of on-target toxicities for RBP4 antagonists in this species. Strong RBP4 lowering combined with good PK attributes and high BPN-14136 exposure achieved in NHP, along with the biology of retinoid trafficking that is similar to that of humans, support the choice of NHP as a non-rodent safety species.

Discovery of Bispecific Antagonists of Retinol Binding Protein 4 That Stabilize Transthyretin Tetramers: Scaffolding Hopping, Optimization, and Preclinical Pharmacological Evaluation as a Potential Therapy for Two Common Age-Related Comorbidities.

Accumulation of cytotoxic lipofuscin bisretinoids may contribute to atrophic age-related macular degeneration (AMD) pathogenesis. Retinal bisretinoid synthesis depends on the influx of serum all-trans-retinol (1) delivered via a tertiary retinol binding protein 4 (RBP4)-transthyretin (TTR)-retinol complex. We previously identified selective RBP4 antagonists that dissociate circulating RBP4-TTR-retinol complexes, reduce serum RBP4 levels, and inhibit bisretinoid synthesis in models of enhanced retinal lipofuscinogenesis. However, the release of TTR by selective RBP4 antagonists may be associated with TTR tetramer destabilization and, potentially, TTR amyloid formation. We describe herein the identification of bispecific RBP4 antagonist-TTR tetramer kinetic stabilizers. Standout analogue (±)-44 possesses suitable potency for both targets, significantly lowers mouse plasma RBP4 levels, and prevents TTR aggregation in a gel-based assay. This new class of bispecific compounds may be especially important as a therapy for dry AMD patients who have another common age-related comorbidity, senile systemic amyloidosis, a nongenetic disease associated with wild-type TTR misfolding.

Design, Synthesis, and Preclinical Efficacy of Novel Nonretinoid Antagonists of Retinol-Binding Protein 4 in the Mouse Model of Hepatic Steatosis.

Retinol-binding protein 4 (RBP4) serves as a transporter for all- trans-retinol (1) in the blood, and it has been proposed to act as an adipokine. Elevated plasma levels of the protein have been linked to diabetes, obesity, cardiovascular diseases, and nonalcoholic fatty liver disease (NAFLD). Recently, adipocyte-specific overexpression of RBP4 was reported to cause hepatic steatosis in mice. We previously identified an orally bioavailable RBP4 antagonist that significantly lowered RBP4 serum levels in Abca4-/- knockout mice with concomitant normalization of complement system protein expression and reduction of bisretinoid formation within the retinal pigment epithelium. We describe herein the discovery of novel RBP4 antagonists 48 and 59, which reduce serum RBP4 levels by >80% in mice upon acute oral dosing. Furthermore, 59 demonstrated efficacy in the transgenic adi-hRBP4 murine model of hepatic steatosis, suggesting that RBP4 antagonists may also have therapeutic utility for the treatment of NAFLD.

Haploinsufficiency is not the key mechanism of pathogenesis in a heterozygous Elovl4 knockout mouse model of STGD3 disease.

PURPOSE: Autosomal dominant Stargardt-like (STGD3) disease results from mutations in the ELOVL4 gene (elongation of very-long-chain fatty acids). This study was undertaken to characterize a mouse model with a targeted deletion of Elovl4 and to explore the role of this gene in retinal/macular degeneration. METHODS: A construct targeted to exon 2 of the Elovl4 gene was used to suppress expression of the gene. Elovl4 homozygous pups were nonviable and were not available for study. Hence, the analysis was performed on heterozygous Elovl4(+/-) mice 16 to 22 month of age and littermate wild-type (WT) control mice of the same age. Characterization included examining gene message and protein levels, electroretinogram (ERG), retinal morphology and ultrastructure, and plasma and retinal fatty acid composition. RESULTS: Although the level of Elovl4 mRNA was reduced in Elovl4(+/-) retinas, only minimal morphologic abnormalities were found, and the retinal (ERG) function was essentially normal in Elovl4(+/-) retinas compared with the WT control retinas. Systemic fatty acid profiles of Elovl4(+/-) mice were unremarkable, although the concentration of several fatty acids was significantly lower in Elovl4(+/-) retinas, particularly the monounsaturated fatty acids. CONCLUSIONS: The detailed characterization of this animal model provides the first in vivo evidence that Elovl4 haploinsufficiency is not the underlying key disease mechanism in STGD3. The results are consistent with a dominant negative mechanism for the deletion mutation. The Elovl4 knockout mouse is one of three complementary animal models that will help elucidate the disease mechanism.

Development of the high throughput screening assay for identification of agonists of an orphan nuclear receptor.

Retina-specific nuclear receptor (RNR), also known as PNR and NR2E3, is an orphan nuclear receptor expressed exclusively in photoreceptor cells of the retina. Here we describe homogeneous cell-based resonance energy transfer assay for identification of RNR agonists using beta-lactamase as the reporter gene. Bacterial beta-lactamase reporter construct containing GAL4 response elements was randomly integrated into the genome with subsequent selection of responsive cell pools by fluorescence-activated cell sorting. Chimeric RNR (RNR hinge and ligand-binding domains fused to GAL4 DNA-binding domain) was stably transfected into mammalian Flp-In Chinese hamster ovary cells using Flp-mediated recombination into a single pre-integrated Flp recombination target site. Since no RNR ligand could be used as a control for monitoring the development of the RNR assay, we developed a parallel cell line with the functionally related well-characterized thyroid hormone nuclear receptor. This parallel thyroid hormone nuclear receptor system was used as a "guide" in optimizing the RNR assay for ultra-high-throughput screening in 3,456-well nanoplate format. The assay was successfully used to screen a large compound collection for RNR agonists. In this study we demonstrated the feasibility of developing and optimization of the high-throughput screening-compatible assay for the orphan nuclear receptor in the absence of its cognitive ligand.

Bicyclic [3.3.0]-Octahydrocyclopenta[c]pyrrolo Antagonists of Retinol Binding Protein 4: Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease.

Antagonists of retinol-binding protein 4 (RBP4) impede ocular uptake of serum all-trans retinol (1) and have been shown to reduce cytotoxic bisretinoid formation in the retinal pigment epithelium (RPE), which is associated with the pathogenesis of both dry age-related macular degeneration (AMD) and Stargardt disease. Thus, these agents show promise as a potential pharmacotherapy by which to stem further neurodegeneration and concomitant vision loss associated with geographic atrophy of the macula. We previously disclosed the discovery of a novel series of nonretinoid RBP4 antagonists, represented by bicyclic [3.3.0]-octahydrocyclopenta[c]pyrrolo analogue 4. We describe herein the utilization of a pyrimidine-4-carboxylic acid fragment as a suitable isostere for the anthranilic acid appendage of 4, which led to the discovery of standout antagonist 33. Analogue 33 possesses exquisite in vitro RBP4 binding affinity and favorable drug-like characteristics and was found to reduce circulating plasma RBP4 levels in vivo in a robust manner (>90%).

In vivo effect of mutant ELOVL4 on the expression and function of wild-type ELOVL4.

PURPOSE: Mutations in the elongation of very long chain fatty acids 4 (ELOVL4) gene cause human Stargardt's macular dystrophy 3 (STGD3), a juvenile onset dominant form of macular degeneration. To understand the role of the ELOVL4 protein in retinal function, several mouse models have been developed by using transgenic (TG), knock-in (Elovl4(+/mut)), and knockout (Elovl4(+/-)) approaches. Here we analyzed quantitatively the ELOVL4 protein and its enzymatic products (very long chain saturated fatty acid [VLC-FA] and VLC-polyunsaturated fatty acid [VLC-PUFA]) in the retinas of 8 to 10-week-old TG1(+), TG2(+), and Elovl4(+/mut) mice that harbor the mutant ELOVL4 and compared them to their wild-type littermates and Elovl4(+/-) that do not express the mutant protein. We also analyzed skin from these mice to gain insight into the pathogenesis resulting from the ELOVL4 mutation. METHODS: ELOVL4 protein localization in the retina was determined by immunohistochemistry. Levels of wild-type ELOVL4 protein in skin and retinas were determined by Western blotting. Total lipids from skin and retinas were measured by gas chromatography-mass spectrometry (GC-MS). Retinal glycerophosphatidylcholines (PC) were analyzed by tandem mass spectrometry. RESULTS: Immunohistochemical and Western analysis indicated that wild-type ELOVL4 protein was reduced in heterozygous Elovl4(+/mut) and Elovl4(+/-) retinas, but not in TG2(+) retinas. We found that VLC-FA was reduced by 50% in the skin of Elovl4(+/-) and by 60% to 65% in Elovl4(+/mut). We found VLC-PUFA levels at ∼ 50% in both the retinas, and wild-type levels of VLC-PUFA in TG2(+) retinas. CONCLUSIONS: We conclude that the presence of the mutant ELOVL4 does not affect the function of wild-type ELOVL4 in the fully developed 8- to 10-week-old retinas.

Design, synthesis, and evaluation of nonretinoid retinol binding protein 4 antagonists for the potential treatment of atrophic age-related macular degeneration and Stargardt disease.

Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4(-/-) mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%.

In pursuit of synthetic modulators for the orphan retina-specific nuclear receptor NR2E3.

PURPOSE: NR2E3 is an orphan nuclear receptor expressed exclusively in photoreceptor cells of the retina. NR2E3-specific modulators may prolong photoreceptor survival in patients with dry age-related macular degeneration and other forms of retinal degeneration. To definitively establish NR2E3 as a photoreceptor protection target, identification of small-molecule NR2E3 modulators and their testing in animal models of retinal degeneration are required. Development of the high-throughput screen (HTS)-compatible screen for small-molecule NR2E3 modulators is the first step toward this goal. METHODS: Purification protocol for isolation of the functionally competent soluble NR2E3 protein after its expression in the insect Sf9 cells was developed. The time-resolved fluorescence energy-transfer (TR-FRET) assay assessing agonist-sensitive interaction between apo-NR2E3 and transcriptional corepressor RetCOR was used for characterization of the previously reported putative NR2E3 agonist, Compound 11a, and to conduct the HTS for novel small-molecule NR2E3 modulators (direct and inverse agonists). A counterscreen TR-FRET assay that measures the affect of test compounds on PPARγ interaction with corepressor NCOR was used for assessing the specificity of compounds identified in the HTS. RESULTS: We developed the cell-free TR-FRET assay for small-molecule NR2E3 modulators, which is based on agonist-induced disruption of the interaction between GST-tagged apo-NR2E3 and MBP-tagged fragment of transcriptional corepressor RetCOR. Compound 11a, a putative NR2E3 agonist, did not affect the NR2E3-RetCOR interaction, as was established by its titration in the developed assay. The assay was miniaturized for an ultralow-volume 1,536-well format and automated into 3 simple pipetting steps. Consistent with excellent assay performance, the test runs established a Z'-score within the 0.6-0.8 range. Analysis of the mid-size National Institutes of Health collection of 315,001 structurally diverse drug-like compounds confirmed excellent assay performance, but did not reveal NR2E3-specific agonists or inverse agonists. CONCLUSIONS: A robust and reliable TR-FRET assay for small-molecule NR2E3-specific modulators suitable for the analysis of million compound-strong HTS libraries was developed. A previously described putative NR2E3 agonist, Compound 11a, is unlikely to represent a direct NR2E3 agonist. Application of the developed assay for screening of a more abundant and diverse compound collection be required for identification of synthetic NR2E3 ligands.

A1120, a nonretinoid RBP4 antagonist, inhibits formation of cytotoxic bisretinoids in the animal model of enhanced retinal lipofuscinogenesis.

PURPOSE: Excessive accumulation of lipofuscin is associated with pathogenesis of atrophic age-related macular degeneration (AMD) and Stargardt disease. Pharmacologic inhibition of the retinol-induced interaction of retinol-binding protein 4 (RBP4) with transthyretin (TTR) in the serum may decrease the uptake of serum retinol to the retina and reduce formation of lipofuscin bisretinoids. We evaluated in vitro and in vivo properties of the new nonretinoid RBP4 antagonist, A1120. METHODS: RBP4 binding potency, ability to antagonize RBP4-TTR interaction, and compound specificity were analyzed for A1120 and for the prototypic RBP4 antagonist fenretinide. A1120 ability to inhibit RPE65-mediated isomerohydrolase activity was assessed in the RPE microsomes. The in vivo effect of A1120 administration on serum RBP4, visual cycle retinoids, lipofuscin bisretinoids, and retinal visual function was evaluated using a combination of biochemical and electrophysiologic techniques. RESULTS: In comparison to fenretinide, A1120 did not act as a RARα agonist, while exhibiting superior in vitro potency in RBP4 binding and RBP4-TTR interaction assays. A1120 did not inhibit isomerohydrolase activity in the RPE microsomes. A1120 dosing in mice induced 75% reduction in serum RBP4, which correlated with reduction in visual cycle retinoids and ocular levels of lipofuscin fluorophores. A1120 dosing did not induce changes in kinetics of dark adaptation. CONCLUSIONS: A1120 significantly reduces accumulation of lipofuscin bisretinoids in the Abca4(-/-) animal model. This activity correlates with reduction in serum RBP4 and visual cycle retinoids confirming the mechanism of action for A1120. In contrast to fenretinide, A1120 does not act as a RARα agonist indicating a more favorable safety profile for this nonretinoid compound.

Dual role of Nr2e3 in photoreceptor development and maintenance.

Nr2e3 is a photoreceptor-specific nuclear receptor believed to play a role in photoreceptor development, differentiation, and survival. Much research has focused on the interaction of Nr2e3 with other transcription factors in determining the milieu of target gene expression in photoreceptors of the neonatal and adult retina. To investigate the downstream targets of Nr2e3 and thereby shed light on the functional pathways relevant to photoreceptor development and maintenance, expression profiling was performed on retinas from two different mouse knockout lines, one containing a targeted disruption of the Nr2e3 gene (Nr2e3 -/-), the other containing a spontaneous null allele of the Nr2e3 locus (rd7). Using whole genome microarrays, mRNA expression profiles of retinas from the two mutant strains were compared to those of wildtype C57BL/6 mice over a time course that ranged from postnatal day (p) 2 to 6months of age (p180). Additionally, expression profiling was performed on retinal explants treated with a putative NR2E3 agonist. The molecular profiling of Nr2e3 -/- and rd7/rd7 retinas identified 281 putative Nr2e3-dependent genes that were differentially expressed between wildtype and mutant retinas during at least one time point. Consistent with previous reports that Nr2e3 is necessary for the repression of cone-specific genes, increased expression of cone-specific genes was observed in the mutant samples, thereby providing proof-of-concept for the microarray screen. Further annotation of these data sets revealed ten predominant functional classes involved in the Nr2e3-mediated development and/or maintenance of photoreceptors. Interestingly, differences in the expression of Nr2e3-dependent genes exhibited two distinct temporal patterns. One group of genes showed a sustained difference in expression as compared to wildtype over the entire time course of the study, whereas a second group showed only transient differences which were largest around p10. Comparison of gene expression changes in Nr2e3 -/- and rd7/rd7 retinas with those uncovered by treating retinal explants with a putative NR2E3 agonist revealed four genes that were down-regulated in mutant retinas that lack Nr2e3 function but were up-regulated in agonist-treated explants. These results strongly suggest that the four genes may be direct targets of Nr2e3. Our identification of two sets of Nr2e3-regulated genes provides further evidence of a dual role for Nr2e3 in specification of photoreceptor fate during development as well as photoreceptor maintenance in the adult.

New therapeutic targets in atrophic age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. There is no effective treatment for the most prevalent atrophic (dry) form of AMD. Atrophic AMD is triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath the photoreceptor cells and normally provides critical metabolic support to these light-sensing cells. Secondary to RPE dysfunction, macular rods and cones degenerate leading to the irreversible loss of vision. Oxidative stress, formation of drusen, accumulation of lipofuscin, local inflammation and reactive gliosis represent the pathologic processes implicated in pathogenesis of atrophic AMD. This review discusses potential target areas for small-molecule and biologic intervention, which may lead to development of new therapeutic treatments for atrophic AMD.

Identification of potent agonists of photoreceptor-specific nuclear receptor (NR2E3) and preparation of a radioligand.

Agonists of NR2E3 (PNR, RNR) have been identified and optimized to EC(50)< 200 nM. A tritiated analogue of one agonist was prepared to aid in the development of a binding assay.

Preparation and characterization of calibration beads for sorting cells expressing a beta-lactamase gene reporter.

BACKGROUND: Modern drug discovery has been based on high-throughput screening using whole-cell assays. A prominent role has been assigned to the reporter gene technology based on a beta-lactamase and the fluorogenic substrate CCF2. Successful application of this technology requires fluorescence-activated cell sorting. We describe the preparation and characterization of calibration beads for sorting cells expressing the beta-lactamase gene using the CCF2 substrate. METHODS: To model Forster resonance energy transfer (FRET) between the coumarin donor and the fluorescein acceptor of the CCF2 reporting dye, we used activated polystyrene beads with primary amino groups. Donor and acceptor fluorophores were attached to the beads at different ratios via succinimidyl esters. The beads were characterized with a fluorescence plate reader and a flow cytometer. RESULTS: We prepared polystyrene beads with five different ratios of donor and acceptor fluorophores and beads that carried a donor or a receptor fluorophore alone. Fluorescence measurements demonstrated that the prepared beads well represent the FRET of CCF2 substrate. CONCLUSION: We have demonstrated that the prepared beads can be successfully used for the setup of fluorescence-activated cell sorting to sort cells with CCF2 reporter substrate and the beta-lactamase reporter gene.