Hyang Yeon Lee

A Two-Photon Tracer for Glucose Uptake†

Glucose is the principal energy source essential for cell growth. Fast-growing cancer cells exhibit a high rate of glycolysis; hence, the rate of glucose uptake is faster in these cells, primarily due to overexpression or enhanced intracellular translocation of glucose transporters (GLUTs) and increased activity of mitochondria-bound hexokinases in the tumor. To monitor glucose metabolism in living systems, a variety of tracers, such as [F]-2-fluoro-2-deoxyglucose (FDG), 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2deoxy-d-glucose (2-NBDG; Scheme 1), and IR dye 800CW-2DG, have been developed. FDG is widely used in the in vivo analysis of glucose metabolism by positron emission tomography (PET), whereas 2-NBDG and IR dye 800CW-2DG are fluorescent probes that have been used for studying cellular metabolic functions involving GLUTs and in tumorimaging studies. Recently, we developed a new fluorescent probe, cyanine-3-linked O-1-glycosylated glucose (Cy3-Glc-a ; Scheme 1), which is a better glucose probe than 2-NBDG because it can be used without glucose starvation, produces a much brighter image, and can be applied for the screening of anticancer agents. In one-photon microscopy (OPM), the probes are excited with short-wavelength light ( 350–550 nm); this, however, limits their application in tissue imaging, owing to inherent problems such as shallow penetration depth (< 80 mm), interference by cellular autofluorescence, photobleaching, and photodamage. 11] To overcome these problems, it is crucial to use two-photon microscopy (TPM), which utilizes two near-infrared photons for excitation. TPM offers a number of advantages over OPM, including greater penetration depth (> 500 mm), localized excitation, and longer observation times. In particular, the extra penetration depth afforded by TPM is an essential element for application in tissue-imaging studies because the artifacts arising from surface preparation, such as damaged cells, can extend over 70 mm into the tissue interior. However, visualization of glucose uptake by live cells and tissues with two-photon (TP) tracers has not been reported so far. The requirements for a TP tracer to visualize glucose uptake include sufficient water solubility for staining cells and tissues, preferential uptake by cancer cells, a large TP crosssection for a bright TPM image, pH resistance, and high photostability. Our strategy was to link a-d-glucose with the fluorophore 2-acetyl-6-dimethylaminonaphthalene (acedan) through 3,6-dioxaoctane-1,8-diamine or a piperazine linkage (in AG1 and AG2, respectively; Scheme 1), so that the tracers are transported into the cells through the glucose-specific mechanism. Acedan is a polarity-sensitive fluorophore that has been successfully employed in the development of TP probes for the cell membrane, metal ions, and acidic vesicles. We now report that these tracers facilitate the visualization of glucose uptake in cancer cells and live tissues at a depth of 75–150 mm for more than 3000 s and can be used for screening anticancer agents. The preparation of AG1 and AG2 is shown in Scheme 2. 6-Acetyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene Scheme 1. Structures of fluorescent tracers AG1, AG2, 2-NBDG, and Cy3-Gly-a.

Development of Fluorescent Glucose Bioprobes and Their Application on Real‐Time and Quantitative Monitoring of Glucose Uptake in Living Cells

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases.

Crystal structure of Tpa1 from Saccharomyces cerevisiae, a component of the messenger ribonucleoprotein complex

Tpa1 (for termination and polyadenylation) from Saccharomyces cerevisiae is a component of a messenger ribonucleoprotein (mRNP) complex at the 3′ untranslated region of mRNAs. It comprises an N-terminal Fe(II)- and 2-oxoglutarate (2OG) dependent dioxygenase domain and a C-terminal domain. The N-terminal dioxygenase domain of a homologous Ofd1 protein from Schizosaccharomyces pombe was proposed to serve as an oxygen sensor that regulates the activity of the C-terminal degradation domain. Members of the Tpa1 family are also present in higher eukaryotes including humans. Here we report the crystal structure of S. cerevisiae Tpa1 as a representative member of the Tpa1 family. Structures have been determined as a binary complex with Fe(III) and as a ternary complex with Fe(III) and 2OG. The structures reveal that both domains of Tpa1 have the double-stranded β-helix fold and are similar to prolyl 4-hydroxylases. However, the binding of Fe(III) and 2OG is observed in the N-terminal domain only. We also show that Tpa1 binds to poly(rA), suggesting its direct interaction with mRNA in the mRNP complex. The structural and functional data reported in this study support a role of the Tpa1 family as a hydroxylase in the mRNP complex and as an oxygen sensor.

Pharmacophore-based strategy for the development of general and specific scFv biosensors for abused antibiotics.

We developed fluorescent biosensor systems that are either general or selective to fluoroquinolone antibiotics by using a single-chain variable-fragment (scFv) as a recognition element. The selectivity of these biosensors to fluoroquinolone antibiotics was rationally tuned through the structural modification on the pharmacophore of fluoroquinolone antibiotics and the subsequent selection of scFv receptor modules against these antibiotics-based antigens using phage display. The resulting A2 and F9 scFvs bound to their representative antigen with a moderate affinity (K(D) in micromolar range as determined by surface plasmon resonance). A2 is a specific binder for enrofloxacin and did not cross-react with other fluoroquinolone antibiotics including structurally similar ciprofloxacin, while F9 is a general fluoroquinolone binder that likely bound to the antigen at the common pyridone-carboxylic acid pharmacophore. These scFv-based receptors were successfully applied to the development of one-step fluorescent biosensor which can detect fluoroquinolone antibiotics at concentrations below the level suggested in animal drug application guidelines. The strategy described in this report can be applied to developing convenient field biosensors that can qualitatively detect overused/misused antibiotics in the livestock drinking water.

Small Molecule Microarray: Functional-Group Specific Immobilization of Small Molecules

Proteomic screening with small molecule microarrays can be a powerful tool in conjunction with various forward chemical genetics screening and high-throughput phenotype assays. Small molecule microarray screening can provide high quality information from the direct binding interaction between proteins of interest and a collection of small molecules in a high-throughput fashion. To realize this potential of small molecule microarray in the postgenomic era, the immobilization of small molecules on the surface of microscope glass slides has been a critical step, to apply small molecule library in protein screening assays and dissecting the protein network. In this chapter, we would like to focus on the protocol for the systematic immobilization of synthetic drug-like small molecules containing either specific functional handles or common functional groups.