Choroidal neovascularization(CNV) is the most severe complication in Age-related macular degeneration(AMD) and the most common cause of irreversible blindness in the elderly in developed world. The aim of this study was to identify the effect of transforming growth factor-β(TGF-β) and Smad2/3-VEGF/TNF-α signaling on CNV angiopoiesis, and to explore TGF-β inhibitors on the development of CNV in a CNV mouse model. Fundus fluorescein angiography(FFA) was used to evaluate the laser-induced CNV formation. The histology of CNV lesions stained with hematoxylin-eosin(HE) was obtained. The immunofluorescent staining was performed to determine TGF-β protein expression. The expressions of TGF-β, phosphorylated Smad2/3, VEGF and TNF-α were determined by using Western blot analysis. The CNV areas were analyzed by using fluorescein stain on RPE/choroid-sclera flat mounts. We found the levels of TGF-β protein expression increasingly reached the peak till 3rd week during the CNV development. The protein levels of VEGF and TNF-α also increased significantly in CNV mice, which were inhibited by a synthetic TGF-β inhibitor LY2157299 or a natural TGF-β inhibitor Decorin. The phosphorylated Smad2/3 levels increased significantly in CNV mice, but this response was profoundly suppressed by the TGF-β inhibitors. Here we have demonstrated that TGF-β/Smad signaling plays an important role in Laser-induced CNV formation through down-regulation of VEGF and TNF-α expressions, suggesting TGF-β inhibitors may provide an alternative to traditional methods in wet AMD treatment.

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A series of novel, synthetic compounds containing lipids linked to a phosphate-containing acyclic backbone are shown to have similar biological properties to lipopolysaccharide (LPS). These compounds showed intrinsic agonistic properties when tested for their ability to stimulate tumor necrosis factor-␣ in human whole blood and interleukin-6 in U373 human glioblastoma cells without added LPS coreceptor CD14. The presence of the LPS antagonist E5564 completely blocked responses, suggesting that the novel compounds and LPS share a common mechanism of cell activation. Stereoselectivity of the molecules was observed in vitro; compounds with an R,R,R,R-configuration were strongly agonistic, whereas compounds with an R,S,S,R-configuration were much weaker in their activity on human whole blood and U373 cells. We also tested the effect of the compounds in cells transfected with the LPS receptor Tolllike receptor 4 (TLR4), with similar results, further supporting a shared mechanism with LPS. This was confirmed in vivo where the agonists failed to elicit cytokine responses in C3H/HeJ mice lacking TLR4 signaling. Because LPS-like molecules enhance immune responses, the compounds were mixed with tetanus toxoid and administered to mice in an immunization protocol to test for adjuvant activity. They enhanced the generation of specific antibodies against tetanus toxoid. Our results indicate that these unique compounds behave as agonists of TLR4, resulting in responses similar to those elicited by LPS. They display adjuvant activity in vivo and may be useful for the development of vaccine therapies.

The measurement of receptor occupancy (RO) using positron emission tomography (PET) has been instrumental in guiding discovery and development of CNS directed therapeutics. We and others have investigated muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs) for the treatment of symptoms associated with neuropsychiatric disorders. In this article, we describe the synthesis, in vitro, and in vivo characterization of a series of central pyridinerelated M4 PAMs that can be conveniently radiolabeled with carbon-11 as PET tracers for the in vivo imaging of an allosteric binding site of the M4 receptor. We first demonstrated its feasibility by mapping the receptor distribution in mouse brain and confirming that a lead molecule 1 binds selectively to the receptor only in the presence of the orthosteric agonist carbachol. Through a competitive binding affinity assay and a number of physiochemical properties filters, several related compounds were identified as candidates for in vivo evaluation. These candidates were then radiolabeled with 11 C and studied in vivo in rhesus monkeys. This research eventually led to the discovery of the clinical radiotracer candidate [ 11 C]MK-6884.

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A route to enantioenriched merrilactone A was found via intermediate 3. A degradation pathway in which meso compound 1 is transformed into key intermediate 2 in four steps occurs with full regiocontrol and promising enantiocontrol. A key feature of this reaction sequence is the desymmetrization of 1 through an intramolecular asymmetric ring‐opening reaction.

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Using the hydrated adenosine intermediate (6R)-6-amino-1, 6-dihydro-6-hydroxy-9-(beta-D-ribofuranosyl)purine (2) produced by adenosine deaminase (ADA, EC 3.5.4.4) as a starting point, the active site probe and inhibitor platform 5-(formylamino)imidazole riboside (FAIRs, 4) was designed by removal of the-C6(OH)(NH2)-molecular fragment of 2 generated by the early events of the enzyme-catalyzed hydrolysis. FAIRs was synthesized directly from the sodium salt of 5-amino-1-(beta-D-ribofuranosyl)imidazole-4-carboxylic acid (CAIR) along a reaction sequence involving a tandem N-formylation/decarboxylation that may have a mechanistic connection to the Escherichia coli purE-catalyzed constitutional isomerization of N5-CAIR to CAIR. The physical and spectral properties of FAIRs were elucidated, its X-ray crystal and NMR solution structures were determined, and its interaction with ADA was investigated. Crystalline FAIRs exists solely as the Z-formamide rotamer and exhibits many of the same intramolecular hydrogen bonding events known to contribute to the association of Ado to ADA. In water and various organic solvents, however, FAIRs exists as NMR-distinct, slowly interconverting Z and E rotamers. This truncated enzymatic tetrahedral intermediate analog was determined to be a competitive inhibitor of ADA with an apparent Ki binding constant of 40 microM, a value quite close to that (33 microM) of the natural substrate's K(m). The actual species selected for binding by ADA, though, is likely the minor hydroxyimino prototropic form of Z-FAIRs possessing a far lower true Ki value. As the structural features of FAIRs appear well-suited to support its use as a template for constructing active site probes of both ADA and AIR carboxylases, a variety of carbohydrate-protected versions of FAIRs suitable for facile aglycon elaborations were synthesized. The N3-alkylation, N3-borane complexation, and C4-iodination of some of these were investigated in order to assess physicochemical properties that may assist in the elucidation of mechanisms for the AIR carboxylases. The survey of these properties taken together with a reasonable mechanism for the model CAIRs-->FAIRs synthetic transformation is interpreted to support a mechanism for the purE-catalyzed N5-CAIR-->CAIR biosynthetic one that involves a carboxylative sp3-rehybridization of the imidazole C4 atom rather than one possessing a dipole-stabilized C4 sp2 carbanionic intermediate.