Ratiometric Fluorescent Sensors Illuminate Cellular Magnesium Imbalance in a Model of Acetaminophen-Induced Liver Injury.

Magnesium(II) plays catalytic, structural, regulatory, and signaling roles in living organisms. Abnormal levels of this metal have been associated with numerous pathologies, including cardiovascular disease, diabetes, metabolic syndrome, immunodeficiency, cancer, and, most recently, liver pathologies affecting humans. The role of Mg2+ in the pathophysiology of liver disease, however, has been occluded by concomitant changes in concentration of interfering divalent cations, such as Ca2+, which complicates the interpretation of experiments conducted with existing molecular Mg2+ indicators. Herein, we introduce a new quinoline-based fluorescent sensor, MagZet1, that displays a shift in its excitation and emission wavelengths, affording ratiometric detection of cellular Mg2+ by both fluorescence microscopy and flow cytometry. The new sensor binds the target metal with a submillimolar dissociation constant─well suited for detection of changes in free Mg2+ in cells─and displays a 10-fold selectivity against Ca2+. Furthermore, the fluorescence ratio is insensitive to changes in pH in the physiological range, providing an overall superior performance over existing indicators. We provide insights into the metal selectivity profile of the new sensor based on computational modeling, and we apply it to shed light on a decrease in cytosolic free Mg2+ and altered expression of metal transporters in cellular models of drug-induced liver injury caused by acetaminophen overdose.

Lanthanide-based luminescent probes for biological magnesium: accessing polyphosphate-bound Mg2.

Biomolecule-bound Mg2+ species, particularly polyphosphate complexes, represent a large and dynamic fraction of the total cellular magnesium that is essential for cellular function but remains invisible to most indicators. Here we report a new family of Eu(III)-based indicators, the MagQEu family, functionalized with a 4-oxo-4H-quinolizine-3-carboxylic acid metal recognition group/sensitization antenna for turn-on, luminescence-based detection of biologically relevant Mg2+ species.

Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage.

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury.

Intracellular Localization Studies of the Luminescent Analogue of an Anticancer Ruthenium Iminophosphorane with High Efficacy in a Triple-Negative Breast Cancer Mouse Model.

The potential of ruthenium(II) compounds as an alternative to platinum-based clinical anticancer agents has been unveiled after extensive research for over 2 decades. As opposed to cisplatin, ruthenium(II) compounds have distinct mechanisms of action that do not rely solely on interactions with DNA. In a previous report from our group, we described the synthesis, characterization, and biological evaluation of a cationic, water-soluble, organometallic ruthenium(II) iminophosphorane (IM) complex of p-cymene, ([(η6-p-cymene)Ru{(Ph3P═N-CO-2N-C5H4)-κ-N,O}Cl]Cl (1 or Ru-IM), that was found to be highly cytotoxic against a panel of cell lines resistant to cisplatin, including triple-negative breast cancer (TNBC) MDA-MB-231, through canonical or caspase-dependent apoptosis. Studies on a MDA-MB-231 xenograft mice model (after 28 days of treatment) afforded an excellent tumor reduction of 56%, with almost negligible systemic toxicity, and a favored ruthenium tumor accumulation compared to other organs. 1 is known to only interact weakly with DNA, but its intracellular distribution and ultimate targets remain unknown. To gain insight on potential mechanisms for this highly efficacious ruthenium compound, we have developed two luminescent analogues containing the BOPIPY fluorophore (or a modification) in the IM scaffold with the general structure of [(η6-p-cymene)Ru{(BODIPY-Ph2P═N-CO-2-NC5H4)-κ-N,O}Cl]Cl {BODIPY-Ph2P = 8-[(4-diphenylphosphino)phenyl]-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene (3a) and 4,4-difluoro-8-[4-[[2-[4-(diphenylphosphino)benzamido]ethyl]carbamoyl]phenyl]-1,3,5,7-tetramethyl,2,6-diethyl-4-bora-3a,4a-diaza-s-indacene (3b)}. We report on the synthesis, characterization, lipophilicity, stability, luminescence properties, and cell viability studies in the TNBC cell line MDA-MB-231, nonmalignant breast cells (MCF10a), and lung fibroblasts (IMR-90) of the new compounds. The ruthenium derivative 3b was studied by fluorescence confocal microscopy. These studies point to a preferential accumulation of the compound in the endoplasmic reticulum, mitochondria, and lysosomes. Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis also confirms a greater ruthenium accumulation in the cytoplasmic fraction, including endoplasmic reticulum and lysosomes, and a smaller percentage of accumulation in mitochondria and the nucleus. ICP-OES analysis of the parent compound 1 indicates that it accumulates preferentially in the mitochondria and cytoplasm. Subsequent experiments in 1-treated MDA-MB-231 cells demonstrate significant reactive oxygen species generation.

Magnesium accumulation upon cyclin M4 silencing activates microsomal triglyceride transfer protein improving NASH.

BACKGROUND & AIMS: Perturbations of intracellular magnesium (Mg2+) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). METHODS: Serum Mg2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine® or conjugated to N-acetylgalactosamine. RESULTS: Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs. CONCLUSIONS: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH. LAY SUMMARY: Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein.

Fluorescence Quenching Effects of Tetrazines and Their Diels-Alder Products: Mechanistic Insight Toward Fluorogenic Efficiency.

Inverse electron demand Diels-Alder reactions between s-tetrazines and strained dienophiles have numerous applications in fluorescent labeling of biomolecules. Herein, we investigate the effect of the dienophile on the fluorescence enhancement obtained upon reaction with a tetrazine-quenched fluorophore and study the possible mechanisms of fluorescence quenching by both the tetrazine and its reaction products. The dihydropyridazine obtained from reaction with a strained cyclooctene shows a residual fluorescence quenching effect, greater than that exerted by the pyridazine arising from reaction with the analogous alkyne. Linear and ultrabroadband two-dimensional electronic spectroscopy experiments reveal that resonance energy transfer is the mechanism responsible for the fluorescence quenching effect of tetrazines, whereas a mechanism involving more intimate electronic coupling, likely photoinduced electron transfer, is responsible for the quenching effect of the dihydropyridazine. These studies uncover parameters that can be tuned to maximize fluorogenic efficiency in bioconjugation reactions and reveal that strained alkynes are better reaction partners for achieving maximum contrast ratio.

Advances in imaging of understudied ions in signaling: A focus on magnesium.

The study of metal ions in the context of cell signaling has historically focused mainly on Ca2+, the second messenger par excellence. But recent studies support an emerging paradigm in which other metals, including magnesium and d-block metals, play a role in signal transduction as well. Armed with the right indicators, fluorescence microscopy offers a unique combination of spatial and temporal resolution perfectly suited to reveal metal transients in real time, while also helping identify possible sources of ion mobilization and molecular targets. With a focus on Mg2+, we highlight recent advancements in the development of molecular indicators and imaging strategies for the study of metal ions in signaling. We discuss remaining conceptual and technical challenges in the field, and we illustrate through the case of Mg2+ how the study of nontraditional ions in signaling is inspiring technological developments applicable more broadly to the study of metals in biology.

Resolving the Fluorescence Quenching Mechanism of an Oxazine Dye Using Ultrabroadband Two-Dimensional Electronic Spectroscopy.

The design and optimization of fluorescent labels and fluorogenic probes rely heavily on their ability to distinguish among multiple competing fluorescence quenching mechanisms. Cresyl violet, a member of the 1,4-oxazine family of dyes, has generally been regarded as an exemplary fluorescent probe; however, recent ultrafast experiments revealed an excited-state decay kinetic of 1.2 ps, suggesting the presence of a transient photochemical state. Here, we present ultrabroadband two-dimensional electronic spectroscopy (2D ES) measurements of cresyl violet in the presence of the fluorescence quenching agent 3,6-di(2-hydroxyethyl)-1,2,4,5-tetrazine. The broad spectral bandwidth allows for the evaluation of multiple fluorescence quenching mechanisms such as exciton formation, photoinduced electron transfer, resonance energy transfer, and excited-state proton transfer. The 2D electronic spectra in the presence and absence of the quencher suggest that excited-state proton transfer drives the system's excited-state dynamics and leads to a cresyl violet tautomer involved in fluorescence quenching. The invocation of the tautomeric form of cresyl violet neatly resolves longstanding inconsistencies in the photophysics of oxazine dyes more generally. Although still under development, the application of ultrabroadband 2D ES to a molecular system represents a compelling demonstration of the technique's future role in the study of photochemical reaction mechanisms.

Highly Selective, Red Emitting BODIPY-Based Fluorescent Indicators for Intracellular Mg2+ Imaging.

Most fluorescent indicators for Mg2+ suffer from poor selectivity against other divalent cations, especially Ca2+, thus do not provide reliable information on cellular Mg2+ concentrations in processes in which such metals are involved. We report a new set of highly selective fluorescent indicators based on alkoxystyryl-functionalized BODIPY fluorophores decorated with a 4-oxo-4H-quinolizine-3-carboxylic acid metal binding moiety. The new sensors, MagQ1 and MagQ2, display absorption and emission maxima above 600 nm, with a 29-fold fluorescence enhancement and good quantum yields (Φ > 0.3) upon coordination of Mg2+ in aqueous buffer. Fluorescence response to Mg2+ is not affected by the presence of competing divalent cations typically present in the cellular milieu, and displays minimal pH dependence in the physiologically relevant range. The choice of alkoxy groups decorating the styryl BODIPY core does not influence the basic photophysical and metal binding properties of the compounds, but has a marked effect on their intracellular retention and thus in their applicability for detection of cellular Mg2+ by fluorescence imaging. In particular, we demonstrate the utility of a triethyleneglycol (TEG) functionalization tactic that endows MagQ2 with superior cellular retention in live cells by reducing active extrusion through organic anion transporters, which are thought to cause fast leakage of typical anionic dyes. With enhanced retention and excellent photophysical properties, MagQ2 can be applied in the detection of cellular Mg2+ influx without interference of high concentrations of Ca2+ akin to those involved in signaling.

Peptide-Based Fluorescent Probes for Deacetylase and Decrotonylase Activity: Toward a General Platform for Real-Time Detection of Lysine Deacylation.

Histone deacetylases regulate the acetylation levels of numerous proteins and play key roles in physiological processes and disease states. In addition to acetyl groups, deacetylases can remove other acyl modifications on lysines, the roles and regulation of which are far less understood. A peptide-based fluorescent probe for single-reagent, real-time detection of deacetylase activity that can be readily adapted for probing broader lysine deacylation, including decrotonylation, is reported. Following cleavage of the lysine modification, the probe undergoes rapid intramolecular imine formation that results in marked optical changes, thus enabling convenient detection of deacylase activity with good statistical Z' factors for both absorption and fluorescence modalities. The peptide-based design offers broader isozyme scope than that of small-molecule analogues, and is suitable for probing both metal- and nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylases. With an effective sirtuin activity assay in hand, it is demonstrated that iron chelation by Sirtinol, a commonly employed sirtuin inhibitor, results in an enhancement in the inhibitory activity of the compound that may affect its performance in vivo.

Tuning the Spectroscopic Properties of Ratiometric Fluorescent Metal Indicators: Experimental and Computational Studies on Mag-fura-2 and Analogues.

In this joint theoretical and experimental work, we investigate the properties of Mag-fura-2 and seven structurally related fluorescent sensors designed for the ratiometric detection of Mg2+ cations. The synthesis of three new compounds is described, and the absorption and emission spectra of all of the sensors in both their free and metal-bound forms are reported. A time-dependent density functional theory approach accounting for hydration effects using a hybrid implicit/explicit model is employed to calculate the absorption and fluorescence emission wavelengths, study the origins of the hypsochromic shift caused by metal binding for all of the sensors in this family, and investigate the auxochromic effects of various modifications of the "fura" core. The metal-free forms of the sensors are shown to undergo a strong intramolecular charge transfer upon light absorption, which is largely suppressed by metal complexation, resulting in predominantly locally excited states upon excitation of the metal complexes. Our computational protocol might aid in the design of new generations of fluorescent sensors with low-energy excitation and enhanced properties for ratiometric imaging of metal cations in biological samples.

Bright, red emitting fluorescent sensor for intracellular imaging of Mg2+ .

Fluorescent sensors with low-energy excitation are in great demand for the study of cellular Mg2+ by fluorescence miscroscopy, but to date they remain scarce. Addressing this gap, we report herein a new set of molecular fluorescent sensors for the detection of Mg2+ based on an o-aminophenol-N,N,O-triacetic acid (APTRA) metal-recognition moiety combined with two different BODIPY fluorophores. The new sensors, MagB1 and MagB2, display absorption and emission maxima in the visible range and respond to Mg2+ in aqueous buffer with large fluorescence enhancements. MagB2, a red-emitting fluorescent indicator based on a styryl-BODIPY, displays superior metal selectivity and optical properties compared to its green emitting counterpart, MagB1. With an excellent 58-fold fluorescence turn-on and Mg2+ dissociation constant in tune with physiological concentrations of the cation (low millimolar range), MagB2 enables visualization of changes in intracellular levels of free Mg2+ in live cells with no significant interference from basal levels of Ca2+, the most common competitor.

Visualizing Compartmentalized Cellular Mg2+ on Demand with Small-Molecule Fluorescent Sensors.

The study of intracellular metal ion compartmentalization and trafficking involved in cellular processes demands sensors with controllable localization for the measurement of organelle-specific levels of cations with subcellular resolution. We introduce herein a new two-step strategy for in situ anchoring and activation of a fluorescent Mg2+ sensor within an organelle of choice, using a fast fluorogenic reaction between a tetrazine-functionalized pro-sensor, Mag-S-Tz, and a strained bicyclononyne conjugated to a genetically encoded HaloTag fusion protein of known cellular localization. Protein conjugation does not affect the metal-binding properties of the o-aminophenol-N,N,O-triacetic acid (APTRA)-based fluorescent indicator, which displays a dissociation constant Kd = 3.1 mM suitable for the detection of low millimolar concentrations of chelatable Mg2+ typical of the intracellular environment. We demonstrate the application of our sensing system for the ratiometric detection of Mg2+ in target organelles in HEK 293T cells, providing the first direct comparison of subcellular pools of the metal without interfering signal from other compartments. Activation of the fluorescence in situ through a fluorogenic conjugation step effectively constrains the fluorescence signal to the locale of interest, thus improving the spatial resolution in imaging applications and eliminating the need for washout of mislocalized sensor. The labeling strategy is fully compatible with live cell imaging, and provides a valuable tool for tracking changes in metal distribution that to date have been an unsolved mystery in magnesium biology.

Structural and spectroscopic insight into the metal binding properties of the o-aminophenol-N,N,O-triacetic acid (APTRA) chelator: implications for design of metal indicators.

The o-aminophenol-N,N,O-triacetic acid (APTRA) chelator is employed extensively as a metal-recognition moiety in fluorescent indicators for biological free Mg(2+), as well as in low-affinity indicators for the detection of high levels of cellular Ca(2+). Despite its widespread use in sensor design, the limited metal selectivity of this chelating moiety can lead to binding of competing cations that complicate the fluorescence-based detection of metals of interest in complex samples. Reported herein are the structural characterization of APTRA complexes with various biologically relevant cations, and the thermodynamic analysis of complex formation with Mg(2+), Ca(2+) and Zn(2+). Our results indicate that the low affinity of APTRA for Mg(2+), which makes it a suitable metal-recognition moiety for sensitive analysis of typical millimolar levels of this metal in cells, stems from a much higher enthalpic cost of Mg(2+) binding compared to that of other cations. The results are discussed in the context of indicator design, highlighting the aspects that may aid the future development of fluorescent sensors with enhanced metal selectivity profiles.

Real-time detection of histone deacetylase activity with a small molecule fluorescent and spectrophotometric probe.

Histone deacetylases (HDACs) are central players in transcription regulation and important targets in cancer treatment. Activity assays are critical tools for the study of the function and regulation of these enzymes, as well as for the screening of potential inhibitors. We report a small-molecule probe for single-step, continuous detection of deacetylase activity based on an acetyl-lysine mimic functionalized with an amine-reactive fluorophore, designed to undergo rapid intramolecular imine formation upon deacetylation. The probe exhibits a bathochromic shift in the absorption spectrum and changes in fluorescence emission intensity that enable unprecedented real-time detection of HDAC activity of purified enzymes or in cell lysates, and offers a means to evaluate HDAC inhibitors via simple spectrophotometric or fluorescence readings without the need of additional reagents.

Enhanced ratiometric fluorescent indicators for magnesium based on azoles of the heavier chalcogens.

Red-shifted fluorescent indicators for magnesium were developed by incorporation of sulfur or selenium in the azole moiety of 'fura' fluorophores. Single atom replacement in the acceptor of these ITC probes affords longer excitation and emission wavelengths as well as greater separation between excitation bands, valuable for ratiometric intracellular Mg(2+) imaging.

Formation of ternary complexes with MgATP: effects on the detection of Mg2+ in biological samples by bidentate fluorescent sensors.

Fluorescent indicators based on ß-keto-acid bidentate coordination motifs display superior metal selectivity profiles compared to current o-aminophenol-N,N,O-triacetic acid (APTRA) based chelators for the study of biological magnesium. These low denticity chelators, however, may allow for the formation of ternary complexes with Mg(2+) and common ligands present in the cellular milieu. In this work, absorption, fluorescence, and NMR spectroscopy were employed to study the interaction of turn-on and ratiometric fluorescent indicators based on 4-oxo-4H-quinolizine-3-carboxylic acid with Mg(2+) and ATP, the most abundant chelator of biological magnesium, thus revealing the formation of ternary complexes under conditions relevant to fluorescence imaging. The formation of ternary species elicits comparable or greater optical changes than those attributed to the formation of binary complexes alone. Dissociation of the fluorescent indicators from both ternary and binary species have apparent equilibrium constants in the low millimolar range at pH 7 and 25 °C. These results suggest that these bidentate sensors are incapable of distinguishing between free Mg(2+) and MgATP based on ratio or intensity-based steady-state fluorescence measurements, thus posing challenges in the interpretation of results from fluorescence imaging of magnesium in nucleotide-rich biological samples.

Visualization of peroxynitrite-induced changes of labile Zn2+ in the endoplasmic reticulum with benzoresorufin-based fluorescent probes.

Zn(2+) plays essential roles in biology, and the homeostasis of Zn(2+) is tightly regulated in all cells. Subcellular distribution and trafficking of labile Zn(2+), and its inter-relation with reactive nitrogen species, are poorly understood due to the scarcity of appropriate imaging tools. We report a new family of red-emitting fluorescent sensors for labile Zn(2+), ZBR1-3, based on a benzoresorufin platform functionalized with dipicolylamine or picolylamine-derived metal binding groups. In combination, the pendant amines and fluorophore afford an [N3O] binding motif that resembles that of previously reported fluorescein-based sensors of the Zinpyr family, reproducing well their binding capabilities and yielding comparable Kd values in the sub-nanomolar and picomolar ranges. The ZBR sensors display up to 8.4-fold emission fluorescence enhancement upon Zn(2+) binding in the cuvette, with similar responses obtained in live cells using standard wide-field fluorescence microscopy imaging. The new sensors localize spontaneously in the endoplasmic reticulum (ER) of various tested cell lines, allowing for organelle-specific monitoring of zinc levels in live cells. Study of ER zinc levels in neural stem cells treated with a peroxynitrite generator, Sin-1, revealed an immediate decrease in labile Zn(2+) thus providing evidence for a direct connection between ER stress and ER Zn(2+) homeostasis.