Timothy M. Dore

Imaging Dynamic Redox Changes in Mammalian Cells with Green Fluorescent Protein Indicators

Changes in the redox equilibrium of cells influence a host of cell functions. Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator. Ratios of fluorescence from excitation at 400 and 490 nm indicate the extent of oxidation and thus the redox potential while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 (GFP with mutations C48S, S147C, and Q204C) and roGFP2 (the same plus S65T) with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H2O2-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid.

Bhc‐cNMPs as either Water‐Soluble or Membrane‐Permeant Photoreleasable Cyclic Nucleotides for both One‐ and Two‐Photon Excitation

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6‐bromo‐7‐hydroxycoumarin‐4‐ylmethyl esters of cNMP (Bhc‐cNMP) and their acetyl derivatives (Bhc‐cNMP/Ac) as new caged second messengers. Irradiation of Bhc‐cNMPs quantitatively produced the parent cNMPs with one‐photon uncaging efficiencies (Φε) of up to one order of magnitude better than those of 2‐nitrophenethyl (NPE) cNMPs. In addition, two‐photon induced photochemical release of cNMP from Bhc‐cNMPs (7 and 8) can be observed with the two‐photon uncaging action cross‐sections (δu) of up to 2.28 GM (1 GM=10−50 cm4 s photon−1), which is the largest value among those of the reported Bhc‐caged compounds. The wavelength dependence of the δu values of 7 revealed that the peak wavelength was twice that of the one‐photon absorption maximum. Bhc‐cNMPs showed practically useful water solubility (nearly 500 μM), whereas 7‐acetylated derivatives (Bhc‐cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP‐mediated transduction channels in newt olfactory receptor cells and cAMP‐mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

Unraveling the Mechanism of the Photodeprotection Reaction of 8-Bromo- and 8-Chloro-7-hydroxyquinoline Caged Acetates

Abstract Photoremovable protecting groups (PPGs) when conjugated to biological effectors forming “caged compounds” are a powerful means to regulate the action of physiologically active messengers in vivo through 1-photon excitation (1PE) and 2-photon excitation (2PE). Understanding the photodeprotection mechanism is important for their physiological use. We compared the quantum efficiencies and product outcomes in different solvent and pH conditions for the photolysis reactions of (8-chloro-7-hydroxyquinolin-2-yl)methyl acetate (CHQ-OAc) and (8-bromo-7-hydroxyquinolin-2-yl)methyl acetate (BHQ-OAc), representatives of the quinoline class of phototriggers for biological use, and conducted nanosecond time-resolved spectroscopic studies using transient emission (ns-EM), transient absorption (ns-TA), transient resonance Raman (ns-TR2), and time-resolved resonance Raman (ns-TR3) spectroscopies. The results indicate differences in the photochemical mechanisms and product outcomes, and reveal that the triplet excited state is most likely on the pathway to the product and that dehalogenation competes with release of acetate from BHQ-OAc, but not CHQ-OAc. A high fluorescence quantum yield and a more efficient excited-state proton transfer (ESPT) in CHQ-OAc compared to BHQ-OAc explain the lower quantum efficiency of CHQ-OAc relative to BHQ-OAc.

Left-right patterning in Xenopus conjoined twin embryos requires serotonin signaling and gap junctions.

A number of processes operating during the first cell cleavages enable the left-right (LR) axis to be consistently oriented during Xenopus laevis development. Prior work showed that secondary organizers induced in frog embryos after cleavage stages (i.e. conjoined twins arising from ectopic induced primary axes) correctly pattern their own LR axis only when a primary (early) organizer is also present. This instructive effect confirms the unique LR patterning functions that occur during early embryogenesis, but leaves open the question: which mechanisms that operate during early stages are also involved in the orientation of later-induced organizers? We sought to distinguish the two phases of LR patterning in secondary organizers (LR patterning of the primary twin and the later transfer of this information to the secondary twin) by perturbing only the latter process. Here, we used reagents that do not affect primary LR patterning at the time secondary organizers form to inhibit each of 4 mechanisms in the induced twin. Using pharmacological, molecular-genetic, and photo-chemical tools, we show that serotonergic and gap-junctional signaling, but not proton or potassium flows, are required for the secondary organizer to appropriately pattern its LR axis in a multicellular context. We also show that consistently-asymmetric gene expression begins prior to ciliary flow. Together, our data highlight the importance of physiological signaling in the propagation of cleavage-derived LR orientation to multicellular cell fields.

Photochemical Modulation of Ras-Mediated Signal Transduction Using Caged Farnesyltransferase Inhibitors: Activation by One- and Two-Photon Excitation

The creation of caged molecules involves the attachment of protecting groups to biologically active compounds such as ligands, substrates and drugs that can be removed under specific conditions. Photoremovable caging groups are the most common due to their ability to be removed with high spatial and temporal resolution. Here, the synthesis and photochemistry of a caged inhibitor of protein farnesyltransferase is described. The inhibitor, FTI, was caged by alkylation of a critical thiol group with a bromohydroxycoumarin (Bhc) moiety. While Bhc is well established as a protecting group for carboxylates and phosphates, it has not been extensively used to cage sulfhydryl groups. The resulting caged molecule, Bhc‐FTI, can be photolyzed with UV light to release the inhibitor that prevents Ras farnesylation, Ras membrane localization and downstream signaling. Finally, it is shown that Bhc‐FTI can be uncaged by two‐photon excitation to produce FTI at levels sufficient to inhibit Ras localization and alter cell morphology. Given the widespread involvement of Ras proteins in signal transduction pathways, this caged inhibitor should be useful in a plethora of studies.

8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells.

Ras converting enzyme 1 (Rce1) is an endoprotease that catalyzes processing of the C-terminus of Ras protein by removing -aaX from the CaaX motif. The activity of Rce1 is crucial for proper localization of Ras to the plasma membrane where it functions. Ras is responsible for transmitting signals related to cell proliferation, cell cycle progression, and apoptosis. The disregulation of these pathways due to constitutively active oncogenic Ras can ultimately lead to cancer. Ras, its effectors and regulators, and the enzymes that are involved in its maturation process are all targets for anti-cancer therapeutics. Key enzymes required for Ras maturation and localization are the farnesyltransferase (FTase), Rce1, and isoprenylcysteine carboxyl methyltransferase (ICMT). Among these proteins, the physiological role of Rce1 in regulating Ras and other CaaX proteins has not been fully explored. Small-molecule inhibitors of Rce1 could be useful as chemical biology tools to understand further the downstream impact of Rce1 on Ras function and serve as potential leads for cancer therapeutics. Structure-activity relationship (SAR) analysis of a previously reported Rce1 inhibitor, NSC1011, has been performed to generate a new library of Rce1 inhibitors. The new inhibitors caused a reduction in Rce1 in vitro activity, exhibited low cell toxicity, and induced mislocalization of EGFP-Ras from the plasma membrane in human colon carcinoma cells giving rise to a phenotype similar to that observed with siRNA knockdowns of Rce1 expression. Several of the new inhibitors were more effective at mislocalizing K-Ras compared to a potent farnesyltransferase inhibitor (FTI), which is significant because of the preponderance of K-Ras mutations in cancer.

Comparison of the Absorption, Emission, and Resonance Raman Spectra of 7-Hydroxyquinoline and 8-Bromo-7-Hydroxyquinoline Caged Acetate

To better understand the deprotection reaction of the new promising phototrigger compound BHQ-OAc (8-bromo-7-hydroxyquinoline acetate), we present a detailed comparison of the UV-vis absorption, resonance Raman, and fluorescence spectra of BHQ-OAc with its parent compound 7-hydroxyquinoline in different solvents. The steady-state absorption and resonance Raman spectra provide fundamental information about the structure, properties, and population distribution of the different prototropic forms present under the different solvent conditions examined. The species present in the excited states that emit strongly were detected by fluorescence spectra. It is shown that the ground-state tautomerization process of BHQ-OAc is disfavored compared with that of 7-HQ in aqueous solutions. The observation of the tautomeric form of BHQ-OAc in neutral aqueous solutions demonstrates the occurrence of the excited-state proton-transfer process, which would be a competing process for the deprotection reaction of BHQ-OAc in aqueous solutions.

Resonance Raman Characterization of Different Forms of Ground-State 8-Bromo-7-hydroxyquinoline Caged Acetate in Aqueous Solutions

The 8-bromo-7-hydroxyquinolinyl group (BHQ) is a derivative of 7-hydroxyquinoline (7-HQ) and BHQ molecules coexisting as different forms in aqueous solution. Absorption and resonance Raman spectroscopic methods were used to examine 8-bromo-7-hydroxyquinoline protected acetate (BHQ-OAc) in acetonitrile (MeCN), H(2)O/MeCN (60:40, v/v, pH 6 approximately 7), and NaOH-H(2)O/MeCN (60:40, v/v, pH 11 approximately 12) to obtain a better characterization of the forms of the ground-state species of BHQ-OAc in aqueous solutions and to examine their properties. The absorption spectra of BHQ-OAc in water show no absorption bands of the tautomeric species unlike the strong band at about 400 nm observed for the tautomeric form in 7-HQ aqueous solution. The resonance Raman spectra in conjunction with Raman spectra predicted from density functional theory (DFT) calculations reveal the observation of a double Raman band system characteristic of the neutral form (the nominal C=C ring stretching, C-N stretching, and O-H bending modes at 1564 and 1607 cm(-1)) and a single Raman band diagnostic of the enol-deprotonated anionic form (the nominal C=C ring, C-N, and C-O(-) stretching modes in the 1593 cm(-1) region). These results suggest that the neutral form of BHQ-OAc is the major species in neutral aqueous solution. There is a modest increase in the amount of the anionic form and a big decrease in the amount of the tautomeric form of the molecules for BHQ-OAc compared to 7-HQ in neutral aqueous solution. The presence of the 8-bromo group and/or competitive hydrogen bonding that hinder the formation and transfer process of a BHQ-OAc-water cyclic complex may be responsible for this large substituent effect.

Resonance Raman characterization of the different forms of ground-state 8-substituted 7-hydroxyquinoline caged acetate compounds in aqueous solutions.

To investigate the substituent effect on the distribution of the forms of the ground-state species of 8-substituted 7-hydroxyquinolines, ultraviolet-absorption and resonance Raman experiments were performed for 8-chloro-7-hydroxyquinoline (CHQ-OAc) and 8-cyano-7-hydroxyquinoline (CyHQ-OAc) in acetonitrile (MeCN), in NaOH-H(2)O/MeCN (60:40, v/v, pH 11-12), and in H(2)O/MeCN (60:40, v/v, pH 6-7) solutions, and these results were compared to those previously reported for the 8-bromo-7-hydroxyquinoline (BHQ-OAc) compound. Swapping a bromine atom in BHQ-OAc for a chlorine atom in CHQ-OAc causes the amount of the tautomeric species to become larger, although the neutral species is still the predominant species for both systems in water-rich solutions. The absorption spectra and the resonance Raman spectra of CyHQ-OAc suggest that, because of the strong electron-withdrawing nature of the cyano substituent, a measurable amount of the anionic species is present and the tautomeric species cannot be easily detected in water-rich solutions. The results reported here reveal large substituent effects on the distribution of the different forms of the XHQ-OAc compounds in largely aqueous solutions. The steric effect of the 8-substituted group and competitive hydrogen bonding between the 8-substituted group and water molecules hinders the formation of a cyclic BHQ-OAc-water complex, and the electron-withdrawing property of the 8-substituted group enhances the deprotonation of the phenol group while disfavoring the formation of the positively charged quinoline nitrogen. We briefly discuss the implications of the substituent effects for using these compounds as phototriggers.

Targeting Type 2 Diabetes with C-Glucosyl Dihydrochalcones as Selective Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitors: Synthesis and Biological Evaluation

Inhibiting glucose reabsorption by sodium glucose co-transporter proteins (SGLTs) in the kidneys is a relatively new strategy for treating type 2 diabetes. Selective inhibition of SGLT2 over SGLT1 is critical for minimizing adverse side effects associated with SGLT1 inhibition. A library of C-glucosyl dihydrochalcones and their dihydrochalcone and chalcone precursors was synthesized and tested as SGLT1/SGLT2 inhibitors using a cell-based fluorescence assay of glucose uptake. The most potent inhibitors of SGLT2 (IC50 = 9-23 nM) were considerably weaker inhibitors of SGLT1 (IC50 = 10-19 μM). They showed no effect on the sodium independent GLUT family of glucose transporters, and the most potent ones were not acutely toxic to cultured cells. The interaction of a C-glucosyl dihydrochalcone with a POPC membrane was modeled computationally, providing evidence that it is not a pan-assay interference compound. These results point toward the discovery of structures that are potent and highly selective inhibitors of SGLT2.