Spectral Properties of Fluorogenic Thiophene-derived Triarylmethane Dyes.

Spectral properties and fluorogenic behaviors of five novel thiophene variants of malachite green (MG), termed MGTs, were determined. Appreciable changes as a function of homologation and substitution pattern, including absorption band positions and intensities and fluorescence quantum yields were observed. In particular, the shorter wavelength y-band absorption was found to shift over a nearly 200 nm range based on aryl group variation, allowing fine-tuning of the excitation wavelength for these dyes. In addition, the fluorescence intensity of some MGTs increased significantly (up to 4600-fold) when the dye was bound to a cognate protein partner, which is potentially useful for cell imaging studies.

Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif.

The DIR2s RNA aptamer, a second-generation, in-vitro selected binder to dimethylindole red (DIR), activates the fluorescence of cyanine dyes, DIR and oxazole thiazole blue (OTB), allowing detection of two well-resolved emission colors. Using Fab BL3-6 and its cognate hairpin as a crystallization module, we solved the crystal structures of both the apo and OTB-SO3 bound forms of DIR2s at 2.0 Å and 1.8 Å resolution, respectively. DIR2s adopts a compact, tuning fork-like architecture comprised of a helix and two short stem-loops oriented in parallel to create the ligand binding site through tertiary interactions. The OTB-SO3 fluorophore binds in a planar conformation to a claw-like structure formed by a purine base-triple, which provides a stacking platform for OTB-SO3, and an unpaired nucleotide, which partially caps the binding site from the top. The absence of a G-quartet or base tetrad makes the DIR2s aptamer unique among fluorogenic RNAs with known 3D structure.

A versatile optical tool for studying synaptic GABAA receptor trafficking.

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABAAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABAAR γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2pHFAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2pHFAP GABAARs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2pHFAP GABAARs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2pHFAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2pHFAP-MG dye approach reveals enhanced GABAAR turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor measurements. To our knowledge, this is the first application of the FAP-MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABAAR trafficking.

Fluoromodules Consisting of a Promiscuous RNA Aptamer and Red or Blue Fluorogenic Cyanine Dyes: Selection, Characterization, and Bioimaging.

An RNA aptamer selected for binding to the fluorogenic cyanine dye, dimethylindole red (DIR), also binds and activates another cyanine, oxazole thiazole blue (OTB), giving two well-resolved emission colors. The aptamer binds to each dye with submicromolar KD values, and the resulting fluoromodules exhibit fluorescence quantum yields ranging from 0.17 to 0.51 and excellent photostability. The aptamer was fused to a second aptamer previously selected for binding to the epidermal growth factor receptor (EGFR) to create a bifunctional aptamer that labels cell-surface EGFR on mammalian cells. The fluorescent color of the aptamer-labeled EGFR can be switched between blue and red in situ simply by exchanging the dye in the medium. The promiscuity of the aptamer can also be used to distinguish between cell-surface and internalized EGFR on the basis of the addition of red or blue fluorogen at different times.

Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness.

The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/ß-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H+-ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockade in vivo A LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior.

Longer Left Ventricular Electric Delay Reduces Mitral Regurgitation After Cardiac Resynchronization Therapy: Mechanistic Insights From the SMART-AV Study (SmartDelay Determined AV Optimization: A Comparison to Other AV Delay Methods Used in Cardiac Resynchronization Therapy).

BACKGROUND: Mitral regurgitation (MR) is associated with worse survival in those undergoing cardiac resynchronization therapy (CRT). Left ventricular (LV) lead position in CRT may ameliorate mechanisms of MR. We examine the association between a longer LV electric delay (QLV) at the LV stimulation site and MR reduction after CRT. METHODS AND RESULTS: QLV was assessed retrospectively in 426 patients enrolled in the SMART-AV study (SmartDelay Determined AV Optimization: A Comparison to Other AV Delay Methods Used in CRT). QLV was defined as the time from QRS onset to the first large peak of the LV electrogram. Linear regression and logistic regression were used to assess the association between baseline QLV and MR reduction at 6 months (absolute change in vena contracta width and odds of ≥1 grade reduction in MR). At baseline, there was no difference in MR grade, LV dyssynchrony, or LV volumes in those with QLV above versus below the median (95 ms). After multivariable adjustment, increasing QLV was an independent predictor of MR reduction at 6 months as reflected by an increased odds of MR response (odds ratio: 1.13 [1.03-1.25]/10 ms increase QLV; P=0.02) and a decrease in vena contracta width (P<0.001). At 3 months, longer QLV (≥median) was associated with significant decrease in LV end-systolic volume (ΔLV end-systolic volume -28.2±38.9 versus -4.9±33.8 mL, P<0.001). Adjustment for 3-month ΔLV end-systolic volume attenuated the association between QLV and 6-month MR reduction. CONCLUSIONS: In patients undergoing CRT, longer QLV was an independent predictor of MR reduction at 6 months and associated with interval 3-month LV reverse remodeling. These findings provide a mechanistic basis for using an electric-targeting LV lead strategy at the time of CRT implant.

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein.

Herein we report an injectable film by which antibodies can be localized in vivo. The system builds upon a bifunctional polypeptide consisting of a fluorogen-activating protein (FAP) and a ß-fibrillizing peptide (ßFP). The FAP domain generates fluorescence that reflects IgG binding sites conferred by Protein A/G (pAG) conjugated with the fluorogen malachite green (MG). A film is generated by mixing these proteins with molar excess of EAK16-II, a ßFP that forms ß-sheet fibrils at high salt concentrations. The IgG-binding, fluorogenic film can be injected in vivo through conventional needled syringes. Confocal microscopic images and dose-response titration experiments showed that loading of IgG into the film was mediated by pAG(MG) bound to the FAP. Release of IgG in vitro was significantly delayed by the bioaffinity mechanism; 26% of the IgG were released from films embedded with pAG(MG) after five days, compared to close to 90% in films without pAG(MG). Computational simulations indicated that the release rate of IgG is governed by positive cooperativity due to pAG(MG). When injected into the subcutaneous space of mouse footpads, film-embedded IgG were retained locally, with distribution through the lymphatics impeded. The ability to track IgG binding sites and distribution simultaneously will aid the optimization of local antibody delivery systems.

Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.

A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.

A Diet Rich in Medium-Chain Fatty Acids Improves Systolic Function and Alters the Lipidomic Profile in Patients With Type 2 Diabetes: A Pilot Study.

CONTEXT: Excessive cardiac long-chain fatty acid (LCFA) metabolism/storage causes cardiomyopathy in animal models of type 2 diabetes. Medium-chain fatty acids (MCFAs) are absorbed and oxidized efficiently. Data in animal models of diabetes suggest MCFAs may benefit the heart. OBJECTIVE: Our objective was to test the effects of an MCFA-rich diet vs an LCFA-rich diet on plasma lipids, cardiac steatosis, and function in patients with type 2 diabetes. DESIGN: This was a double-blind, randomized, 2-week matched-feeding study. SETTING: The study included ambulatory patients in the general community. PATIENTS: Sixteen patients, ages 37-65 years, with type 2 diabetes, an ejection fraction greater than 45%, and no other systemic disease were included. INTERVENTION: Fourteen days of a diet rich in MCFAs or LCFAs, containing 38% as fat in total, was undertaken. MAIN OUTCOME MEASURES: Cardiac steatosis and function were the main outcome measures, with lipidomic changes considered a secondary outcome. RESULTS: The relatively load-independent measure of cardiac contractility, S', improved in the MCFA group (P < .05). Weight-adjusted stroke volume and cardiac output decreased in the LCFA group (both P < .05). The MCFA, but not the LCFA, diet decreased several plasma sphingolipids, ceramide, and acylcarnitines implicated in diabetic cardiomyopathy, and changes in several sphingolipids correlated with improved fasting insulins. CONCLUSIONS: Although a diet high in MCFAs does not change cardiac steatosis, our findings suggest that the MCFA-rich diet alters the plasma lipidome and may benefit or at least not harm cardiac function and fasting insulin levels in humans with type 2 diabetes. Larger, long-term studies are needed to further evaluate these effects in less-controlled settings.

A rapid and affordable screening platform for membrane protein trafficking.

BACKGROUND: Membrane proteins regulate a diversity of physiological processes and are the most successful class of targets in drug discovery. However, the number of targets adequately explored in chemical space and the limited resources available for screening are significant problems shared by drug-discovery centers and small laboratories. Therefore, a low-cost and universally applicable screen for membrane protein trafficking was developed. RESULTS: This high-throughput screen (HTS), termed IRFAP-HTS, utilizes the recently described MarsCy1-fluorogen activating protein and the near-infrared and membrane impermeant fluorogen SCi1. The cell surface expression of MarsCy1 epitope-tagged receptors can be visualized by simple addition of SCi1. User-friendly, rapid, and quantitative detection occurs on a standard infrared western-blotting scanner. The reliability and robustness of IRFAP-HTS was validated by confirming human vasopressin-2 receptor and dopamine receptor-2 trafficking in response to agonist or antagonist. The IRFAP-HTS screen was deployed against the leucine-rich G protein-coupled receptor-5 (Lgr5). Lgr5 is expressed in stem cells, modulates Wnt/ß-catenin signaling, and is therefore a promising drug target. However, small molecule modulators have yet to be reported. The constitutive internalization of Lgr5 appears to be one primary mode through which its function is regulated. Therefore, IRFAP-HTS was utilized to screen 11,258 FDA-approved and drug-like small molecules for those that antagonize Lgr5 internalization. Glucocorticoids were found to potently increase Lgr5 expression at the plasma membrane. CONCLUSION: The IRFAP-HTS platform provides a versatile solution for screening more targets with fewer resources. Using only a standard western-blotting scanner, we were able to screen 5,000 compounds per hour in a robust and quantitative assay. Multi-purposing standardly available laboratory equipment eliminates the need for idiosyncratic and more expensive high-content imaging systems. The modular and user-friendly IRFAP-HTS is a significant departure from current screening platforms. Small laboratories will have unprecedented access to a robust and reliable screening platform and will no longer be limited by the esoteric nature of assay development, data acquisition, and post-screening analysis. The discovery of glucocorticoids as modulators for Lgr5 trafficking confirms that IRFAP-HTS can accelerate drug-discovery and drug-repurposing for even the most obscure targets.

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging.

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule-based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter-tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.

Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation.

The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration.

Spectral fine tuning of cyanine dyes: electron donor-acceptor substituted analogues of thiazole orange.

The introduction of electron donor and acceptor groups at strategic locations on a fluorogenic cyanine dye allows fine-tuning of the absorption and emission spectra while preserving the ability of the dye to bind to biomolecular hosts such as double-stranded DNA and a single-chain antibody fragment originally selected for binding to the parent unsubstituted dye, thiazole orange (TO). The observed spectral shifts are consistent with calculated HOMO-LUMO energy gaps and reflect electron density localization on the quinoline half of TO in the LUMO. A dye bearing donating methoxy and withdrawing trifluoromethyl groups on the benzothiazole and quinoline rings, respectively, shifts the absorption spectrum to sufficiently longer wavelengths to allow excitation at green wavelengths as opposed to the parent dye, which is optimally excited in the blue.

Type 2 diabetes, obesity, and sex difference affect the fate of glucose in the human heart.

Type 2 diabetes, obesity, and sex difference affect myocardial glucose uptake and utilization. However, their effect on the intramyocellular fate of glucose in humans has been unknown. How the heart uses glucose is important, because it affects energy production and oxygen efficiency, which in turn affect heart function and adaptability. We hypothesized that type 2 diabetes, sex difference, and obesity affect myocardial glucose oxidation, glycolysis, and glycogen production. In a first-in-human study, we measured intramyocardiocellular glucose metabolism from time-activity curves generated from previously obtained positron emission tomography scans of 110 subjects in 3 groups: nonobese, obese, and diabetes. Group and sex difference interacted in the prediction of all glucose uptake, utilization, and metabolism rates. Group independently predicted fractional glucose uptake and its components: glycolysis, glycogen deposition, and glucose oxidation rates. Sex difference predicted glycolysis rates. However, there were fewer differences in glucose metabolism between diabetic patients and others when plasma glucose levels were included in the modeling. The potentially detrimental effects of obesity and diabetes on myocardial glucose metabolism are more pronounced in men than women. This sex difference dimorphism needs to be taken into account in the design, trials, and application of metabolic modulator therapy. Slightly higher plasma glucose levels improve depressed glucose oxidation and glycogen deposition rates in diabetic patients.

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules.

Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nanomolar KD values for their respective proteins, >95% energy transfer efficiency and high fluorescence quantum yields.

Computational and experimental characterization of a fluorescent dye for detection of potassium ion concentration.

The fluorescence of the SKC-513 ((E)-N-(9-(4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl)-6-(butyl(3-sulfopropyl)amino)-3H-xanthen-3-ylidene)-N-(3-sulfopropyl)butan-1-aminium) dye is shown experimentally to have high sensitivity to binding of the K(+) ion. Computations are used to explore the potential origins of this sensitivity and to make some suggestions regarding structural improvements. In the absence of K(+), excitation is to two nearly degenerate states, a neutral (N) excited state with a high oscillator strength, and a charge-transfer (CT) state with a lower oscillator strength. Binding of K(+) destabilizes the CT state, raising its energy far above the N state. The increase in fluorescence quantum yield upon binding of K(+) is attributed to the increased energy of the CT state suppressing a nonradiative pathway mediated by the CT state. The near degeneracy of the N and CT excited states can be understood by considering SKC-513 as a reduced symmetry version of a parent molecule with 3-fold symmetry. Computations show that acceptor-donor substituents can be used to alter the relative energies of the N and CT state, whereas a methylene spacer between the heterocycle and phenylene groups can be used to increase the coupling between these states. These modifications provide synthetic handles with which to optimize the dye for K(+) detection.

A bifunctional converter: fluorescein quenching scFv/fluorogen activating protein for photostability and improved signal to noise in fluorescence experiments.

Monoclonal antibodies are one of the most useful and ubiquitous affinity reagents used in the biological sciences. Immunostaining of fixed and live cells for microscopy or cytometry measurements frequently employs fluorescently labeled antibodies, in particular fluorescein-labeled antibodies. This dye emits light at a wavelength overlapping with cellular autofluorescence, making it difficult to measure antibody binding to proteins of relatively low copy number or in cells of high green autofluorescence. A number of high affinity fluorescein binding antibodies and antibody domains have been developed that quench the dye's fluorescence. Using a fluorescein-binding recombinant antibody domain genetically fused to a fluorogen activating protein (FAP), we demonstrate a molecular converter capable of binding and quenching fluorescein, while binding and activating a fluorogenic triarylmethane dye. This reagent converts fluorescein conjugates to far-red fluorescent probes, where cellular autofluorescence is low, improving signal-to-background of cell-based antibody binding measurements by ∼7-fold. Microscopy experiments show colocalization of both fluorescein and MG fluorescence. This dual affinity fluorescein-quenching-FAP can also be used to convert fluorescein to the red fluorescing MG fluorogen on biological molecules other than antibodies.