Reactivity of substituted 4-Pyridones in normal and inverse demand Diels-Alder cycloaddition

The synthesis of the decahydroquinoline ring system has been explored extensively due to its appearance within many biologically active natural alkaloids. New compounds containing such a moiety are being discovered on an ongoing basis. The present work has sought to develop an alternative synthetic pathway to this system through the use of Diels-Alder [4 + 2] cycloaddition chemistry. The initial focus of this research was on the development of an activated dienophile derived from 4-pyridone. Two such compounds were eventually synthesized, N-tosyl-3- formyl-5,6-dihydropyridin-4-one, and N-t-butoxycarbonyl-3-formyl-5,6-dihydropyridin- 4-one. The reactivity of these compounds with several different dienes was then investigated in an effort to obtain the desired decahydroquinoline cycloadduct. Under inverse electron demand Diels-Alder conditions, the synthesized compounds reacted with ethyl vinyl ether to consistently produce pyran containing bicyclic adducts under mild conditions. The products contained a mixture of isomers in varying ratio relative to the nitrogen substituent present. Non-activated dienes did not react with the dienophiles under normal demand conditions even with the addition of lewis acid catalysts. However, cycloadducts were formed through reaction with the highly activated diene 1-dimethylamino-3-tertbutyldimethylsiloxy- 1,3-butadiene. The products did not resemble the target decahydroquinoline system but instead appeared to be the result of reaction with the formyl group substituent, acting as a heterodienophile, to yield a dihydropyranone ring bridged to C-3 of the original pyridone derived dienophile

Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbβ3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbβ3 activation, but it activates αIIbβ3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbβ3 is tightly regulated by the topology of β3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin β3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-β TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs

Environment-Sensing Merocyanine Dyes for Live Cell Imaging Applications

Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye mero60 showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 in vitro. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells

A novel fluorescent sensor protein for detecting changes in airway surface liquid glucose concentration.

Both lung disease and elevation of blood glucose are associated with increased glucose concentration (from 0.4 to ~4.0 mM) in the airway surface liquid (ASL). This perturbation of ASL glucose makes the airway more susceptible to infection by respiratory pathogens. ASL is minute (~1 μl/cm(2)) and the measurement of glucose concentration in the small volume ASL is extremely difficult. Therefore, we sought to develop a fluorescent biosensor with sufficient sensitivity to determine glucose concentrations in ASL in situ. We coupled a range of environmentally sensitive fluorophores to mutated forms of a glucose/galactose-binding protein (GBP) including H152C and H152C/A213R and determined their equilibrium binding properties. Of these, GBP H152C/A213R-BADAN (Kd 0.86 ± 0.01 mM, Fmax/F0 3.6) was optimal for glucose sensing and in ASL increased fluorescence when basolateral glucose concentration was raised from 1 to 20 mM. Moreover, interpolation of the data showed that the glucose concentration in ASL was increased, with results similar to that using glucose oxidase analysis. The fluorescence of GBP H152C/A213R-BADAN in native ASL from human airway epithelial cultures in situ was significantly increased over time when basolateral glucose was increased from 5 to 20 mM. Overall our data indicate that this GBP is a useful tool to monitor glucose homoeostasis in the lung

An Orally Bioavailable Chemical Probe of the Lysine Methyltransferases EZH2 and EZH1

EZH2 or EZH1 is the catalytic subunit of the polycomb repressive complex 2 that catalyzes methylation of histone H3 lysine 27 (H3K27). The trimethylation of H3K27 (H3K27me3) is a transcriptionally repressive post-translational modification. Overexpression of EZH2 and hypertrimethylation of H3K27 have been implicated in a number of cancers. Several selective inhibitors of EZH2 have been reported recently. Herein we disclose UNC1999, the first orally bioavailable inhibitor that has high in vitro potency for wild-type and mutant EZH2 as well as EZH1, a closely related H3K27 methyltransferase that shares 96% sequence identity with EZH2 in their respective catalytic domains. UNC1999 was highly selective for EZH2 and EZH1 over a broad range of epigenetic and non-epigenetic targets, competitive with the cofactor SAM, and non-competitive with the peptide substrate. This inhibitor potently reduced H3K27me3 levels in cells and selectively killed diffused large B cell lymphoma cell lines harboring the EZH2Y641N mutant. Importantly, UNC1999 was orally bioavailable in mice, making this inhibitor a valuable tool for investigating the role of EZH2 and EZH1 in chronic animal studies. We also designed and synthesized UNC2400, a close analog of UNC1999 with >1,000-fold lower potency than UNC1999 as a negative control for cell-based studies. Finally, we created a biotin-tagged UNC1999 (UNC2399) which enriched EZH2 in pull-down studies, and a UNC1999 – dye conjugate (UNC2239) for co-localization studies with EZH2 in live cells. Taken together, these compounds represent a set of useful tools for the biomedical community to investigate the role of EZH2 and EZH1 in health and disease

Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyl-lysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides, and is greater than 50-fold selective versus other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a novel 2:2 polyvalent mode of interaction. In cells, UNC1215 is non-toxic and binds directly to L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. Finally, UNC1215 demonstrates a novel Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis

Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbβ3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbβ3 activation, but it activates αIIbβ3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbβ3 is tightly regulated by the topology of β3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin β3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-β TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs

Environment-Sensing Merocyanine Dyes for Live Cell Imaging Applications

Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications, as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye <b>mero60</b> showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 <i>in vitro</i>. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells

Environment-Sensing Merocyanine Dyes for Live Cell Imaging Applications

Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications, as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye <b>mero60</b> showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 <i>in vitro</i>. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells