Membrane-Permeant, Environment-Sensitive Dyes Generate Biosensors within Living Cells.

Dyes with environment-sensitive fluorescence have proven useful to study the spatiotemporal dynamics of protein activity in living cells. When attached to proteins, their fluorescence can reflect protein conformational changes, post-translational modifications, or protein interactions. However, the utility of such dye-protein conjugates has been limited because it is difficult to load them into cells. They usually must be introduced using techniques that perturb cell physiology, limit throughput, or generate fluorescent vesicles (e.g., electroporation, microinjection, or membrane transduction peptides). Here we circumvent these problems by modifying a proven, environment-sensitive biosensor fluorophore so that it can pass through cell membranes without staining intracellular compartments and can be attached to proteins within living cells using unnatural amino acid (UAA) mutagenesis. Reactive groups were incorporated for attachment to UAAs or small molecules (mero166, azide; mero167, alkyne; mero76, carboxylic acid). These dyes are bright and fluoresce at long wavelengths (reaching ε = 100 000 M-1 cm-1, ϕ = 0.24, with excitation 565 nm and emission 594 nm). The utility of mero166 was demonstrated by in-cell labeling of a UAA to generate a biosensor for the small GTPase Cdc42. In addition, conjugation of mero166 to a small molecule produced a membrane-permeable probe that reported the localization of the DNA methyltransferase G9a in cells. This approach provides a strategy to access biosensors for many targets and to more practically harness the varied environmental sensitivities of synthetic dyes.

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, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 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 αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 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-ß 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.

Chlorophyll-Inspired Red-Region Fluorophores: Building Block Synthesis and Studies in Aqueous Media.

Fluorophores that absorb and emit in the red spectral region (600-700 nm) are of great interest in photochemistry and photomedicine. Eight new target chlorins (and 19 new chlorins altogether)-analogues of chlorophyll-of different polarities have been designed and synthesized for various applications; seven of the chlorins are equipped with a bioconjugatable tether. Hydrophobic or amphiphilic chlorins in a non-polar organic solvent (toluene), polar organic solvent (DMF), and aqueous or aqueous micellar media show a sharp emission band in the red region and modest fluorescence quantum yield (Φf = 0.2-0.3). A Poisson analysis implies most micelles are empty and few contain >1 chlorin. Water-soluble chlorins each bearing three PEG (oligoethyleneglycol) groups exhibit narrow emission bands (full-width-at-half maximum <25 nm). The lifetime of the lowest singlet excited state and the corresponding yields and rate constants for depopulation pathways (fluorescence, intersystem crossing, internal conversion) are generally little affected by the PEG groups or dissolution in aqueous or organic media. A set of chlorin-avidin conjugates revealed a 2-fold increase in Φf with increased average chlorin/avidin ratio (2.3-12). In summary, the chlorins of various polarities described herein are well suited as red-emitting fluorophores for applications in aqueous or organic media.

Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation.

Biosensors that report endogenous protein activity in vivo can be based on environment-sensing fluorescent dyes. The dyes can be attached to reagents that bind selectively to a specific conformation of the targeted protein, such that binding leads to a fluorescence change. Dyes that are sufficiently bright for use at low, nonperturbing intracellular concentrations typically undergo changes in intensity rather than the shifts in excitation or emission maxima that would enable precise quantitation through ratiometric imaging. We report here mero199, an environment-sensing dye that undergoes a 33 nm solvent-dependent shift in excitation. The dye was used to generate a ratiometric biosensor of Cdc42 (CRIB199) without the need for additional fluorophores. CRIB199 was used in the same cell with a FRET sensor of Rac1 activation to simultaneously observe Cdc42 and Rac1 activity in cellular protrusions, indicating that Rac1 but not Cdc42 activity was reduced during tail retraction, and specific protrusions had reduced Cdc42 activity. A novel program (EdgeProps) used to correlate localized activation with cell edge dynamics indicated that Rac1 was specifically reduced during retraction.

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 methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a K(d) of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds 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 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.

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.

Live-cell biosensors based on the fluorescence lifetime of environment-sensing dyes.

In this work, we examine the use of environment-sensitive fluorescent dyes in fluorescence lifetime imaging microscopy (FLIM) biosensors. We screened merocyanine dyes to find an optimal combination of environment-induced lifetime changes, photostability, and brightness at wavelengths suitable for live-cell imaging. FLIM was used to monitor a biosensor reporting conformational changes of endogenous Cdc42 in living cells. The ability to quantify activity using phasor analysis of a single fluorophore (e.g., rather than ratio imaging) eliminated potential artifacts. We leveraged these properties to determine specific concentrations of activated Cdc42 across the cell.

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.

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 EZH2(Y641N) 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 analogue of UNC1999 with potency >1,000-fold lower than that of 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.

Development and screening of water-soluble analogues of progesterone and allopregnanolone in models of brain injury.

Preclinical and clinical research findings have revealed that the hormone progesterone, when acutely administered, can dramatically reduce cerebral edema, inflammation, tissue necrosis, and programmed cell death following traumatic brain injury (TBI). The poor aqueous solubility of progesterone, however, limits its potential use as a therapeutic. Several chemically novel analogues of progesterone and its natural metabolite allopregnanolone have been synthesized and screened using both in vitro and whole animal models of TBI. The new derivatives demonstrated greatly improved solubility and select compounds have shown equivalent effectiveness to progesterone in reducing cerebral edema after TBI.

Stereoselective synthesis of quaternary center bearing azetines and their beta-amino acid derivatives.

We describe here the use of a stable, four-membered azetine heterocycle for the preparation of highly substituted beta-amino acid derivatives. Imidazolidinone chiral auxiliaries were found to eliminate a competitive reaction pathway that had been present under previously reported conditions for azetine synthesis. The ephedrine derived imidazolidin-2-one 21 was allowed to react as its chlorotitanium enolate with O-methyl or -benzyl oximes under optimized conditions to gain improved access to azetines at the gram scale. The azetines were further found to undergo alkylation with complete diastereocontrol, affording the creation of a quaternary center. Subsequent ring opening with benzoyl chloride and auxiliary cleavage provided the corresponding beta2,2,3-amino carbonyl derivatives in good yields.