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%).

Evidence for methionine-sulfoxide-reductase gene transfer from Alphaproteobacteria to the transcriptionally active (macro)nucleus of the ciliate, Euplotes raikovi.

BACKGROUND: Deleterious phenomena of protein oxidation affect every aerobic organism and methionine residues are their elective targets. The reduction of methionine sulfoxides back to methionines is catalyzed by methionine-sulfoxide reductases (Msrs), enzymes which are particularly active in microorganisms because of their unique nature of individual cells directly exposed to environmental oxidation. RESULTS: From the transcriptionally active somatic genome of a common free-living marine protist ciliate, Euplotes raikovi, we cloned multiple gene isoforms encoding Msr of type A (MsrA) committed to repair methionine-S-sulfoxides. One of these isoforms, in addition to including a MsrA-specific nucleotide sequence, included also a sequence specific for a Msr of type B (MsrB) committed to repair methionine-R-sulfoxides. Analyzed for its structural relationships with MsrA and MsrB coding sequences of other organisms, the coding region of this gene (named msrAB) showed much more significant relationships with Msr gene coding sequences of Rhodobacterales and Rhizobiales (Alphaproteobacteria), than of other eukaryotic organisms. CONCLUSIONS: Based on the fact that the msrAB gene is delimited by Euplotes-specific regulatory 5' and 3' regions and telomeric C4A4/G4T4 repeats, it was concluded that E. raikovi inherited the coding region of this gene through a phenomenon of horizontal gene transfer from species of Alphaproteobacteria with which it coexists in nature and on which it likely feeds.

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.

Methionine sulfoxide reduction in ciliates: characterization of the ready-to-use methionine sulfoxide-R-reductase genes in Euplotes.

Genes encoding the enzyme methionine sulfoxide reductase type B, specific to the reduction of the oxidized methionine-R form, were characterized from the expressed (macronuclear) genome of two ecologically separate marine species of Euplotes, i.e. temperate water E. raikovi and polar water E. nobilii. Both species were found to contain a single msrB gene with a very simple structural organization encoding a protein of 127 (E. raikovi) or 126 (E. nobilii) amino acid residues that belongs to the group of zinc-containing enzymes. Both msrB genes are constitutively expressed, suggesting that the MsrB enzyme plays an essential role in repairing oxidative damages that appear to be primarily caused by physiological cell aging in E. raikovi and by interactions with an O(2) saturated environment in E. nobilii.

The water-borne protein signals (pheromones) of the Antarctic ciliated protozoan Euplotes nobilii: structure of the gene coding for the En-6 pheromone.

The marine Antarctic ciliate, Euplotes nobilii, secretes a family of water-borne signal proteins, denoted as pheromones, which control vegetative proliferation and mating in the cell. Based on the knowledge of the amino acid sequences of a set of these pheromones isolated from the culture supernatant of wild-type strains, we designed probes to identify their encoding genes in the cell somatic nucleus (macronucleus). The full-length gene of the pheromone En-6 was determined and found to contain an open-reading frame specific for the synthesis of the En-6 cytoplasmic precursor (pre-pro-En-6), which requires 2 proteolytic cleavages to remove the signal peptide (pre) and the prosegment before secretion of the mature protein. In contrast to the sequence variability that distinguishes the secreted pheromones, the pre- and pro-sequences appear to be tightly conserved and useful for the construction of probes to clone every other E. nobilii pheromone gene. Potential intron sequences in the coding region of the En-6 gene imply the synthesis of more En-6 isoforms.