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1.  A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity 
Journal of the American Chemical Society  2010;133(1):10.1021/ja108258d.
We developed a novel method to spatiotemporally control activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV-irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.
doi:10.1021/ja108258d
PMCID: PMC3850177  PMID: 21142151
2.  Moving molecules by light; Spatio-temporal manipulation of small GTPase activity at subcellular level and on timescale of seconds in living cells 
Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing.
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.
doi:10.3791/3794
PMCID: PMC3460574  PMID: 22433289
Small GTPase; rapamycin; caged compound; spatiotemporal control; heterodimerization; FKBP; FRB; light irradiation
3.  Oxysterol-Binding Protein Family I Is the Target of Minor Enviroxime-Like Compounds 
Journal of Virology  2013;87(8):4252-4260.
Enviroxime is an antipicornavirus compound that targets host phosphatidylinositol 4-kinase III beta (PI4KB) activity for its antipicornavirus activity. To date, several antipoliovirus (PV) compounds similar to enviroxime that are associated with a common resistance mutation in viral protein 3A (a G5318A [3A-Ala70Thr] mutation in PV) have been identified. Most of these compounds have a direct inhibitory effect on PI4KB activity, as well as enviroxime (designated major enviroxime-like compounds). However, one of the compounds, AN-12-H5, showed no inhibitory effect on PI4KB and was considered to belong to another group of enviroxime-like compounds (designated minor enviroxime-like compounds). In the present study, we performed a small interfering RNA (siRNA) sensitization assay targeting PI4KB-related genes and identified oxysterol-binding protein (OSBP) as a target of minor enviroxime-like compounds. Knockdown of OSBP and OSBP2 increased the anti-PV activities of AN-12-H5 and a newly identified minor enviroxime-like compound, T-00127-HEV2, and also to T-00127-HEV1 to a minor extent, in the cells. A ligand of OSBP, 25-hydroxycholesterol (25-HC), acted as a minor enviroxime-like compound. Minor enviroxime-like compounds induced relocalization of OSBP to the Golgi apparatus in cells. Treatment of the cells with major or minor enviroxime-like compounds suppressed the expression of genes (HMGCS1 and SQLE) in the SREBP/SCAP regulatory pathway and diminished endogenous phosphatidylinositol 4-phosphate (PI4P) at the Golgi apparatus. Our results suggested that minor enviroxime-like compounds are phenotypically identical to 25-HC and that major and minor enviroxime-like compounds suppress the production and/or accumulation of PI4P in PV-infected cells by targeting PI4KB and OSBP family I activities, respectively.
doi:10.1128/JVI.03546-12
PMCID: PMC3624399  PMID: 23365445
4.  Activatable Optical Imaging with a Silica-Rhodamine Based Near Infrared (SiR700) Fluorophore: A comparison with cyanine based dyes 
Bioconjugate chemistry  2011;22(12):2531-2538.
Optical imaging is emerging as an important tool to visualize tumors. However, there are many potential choices among the available fluorophores. Optical imaging probes that emit in the visible range can image superficial tumors with high quantum yields, however, if deeper imaging is needed then near infrared (NIR) fluorophores are necessary. Most commercially available NIR fluorophores are cyanine based and are prone to non-specific binding and relatively limited photostability. Silica-containing rhodamine (SiR) fluorophores represent a new class of NIR fluorophores, which permit photoactivation via H-dimer formation as well as demonstrate improved photostability. This permits higher tumor-to-background ratios (TBRs) to be achieved over longer periods of time. Here, we compared an avidin conjugated with SiR700 (Av-SiR700) to similar compounds based on cyanine dyes (Av-Cy5.5 and Av-Alexa Fluor 680) in a mouse tumor model of ovarian cancer metastasis. We found that the Av-SiR700 probe demonstrated superior quenching enabling activation after binding-internalization to the target cell. As a result, Av-SiR700 had higher TBRs compared to Av-Cy5.5, and better biostability compared to Av-Alexa Fluor 680.
doi:10.1021/bc2003617
PMCID: PMC3244508  PMID: 22034863
Near infrared; molecular imaging; cancer; activatable; cyanine; rhodamine
5.  Structural Basis for Specific Recognition of Substrates by Sapovirus Protease 
Sapovirus (SaV) protease catalyzes cleavage of the peptide bonds at six sites of a viral polyprotein for the viral replication and maturation. However, the mechanisms by which the protease recognizes the distinct sequences of the six cleavage sites remain poorly understood. Here we examined this issue by computational and experimental approaches. A structural modeling and docking study disclosed two small clefts on the SaV protease cavity that allow the stable and functional binding of substrates to the catalytic cavity via aromatic stacking and electrostatic interactions. An information entropy study and a site-directed mutagenesis study consistently suggested variability of the two clefts under functional constraints. Using this information, we identified three chemical compounds that had structural and spatial features resembling those of the substrate amino acid residues bound to the two clefts and that exhibited an inhibitory effect on SaV protease in vitro. These results suggest that the two clefts provide structural base points to realize the functional binding of various substrates.
doi:10.3389/fmicb.2012.00312
PMCID: PMC3433708  PMID: 22973264
sapovirus protease; substrate recognition; P1 and P4 amino acid residues; 3-D models; amino acid diversity; mutagenesis; 3-D pharmacophore; inhibitor screening
6.  Phosphatidylinositol 4-Kinase III Beta Is a Target of Enviroxime-Like Compounds for Antipoliovirus Activity▿ †  
Journal of Virology  2010;85(5):2364-2372.
Enviroxime is an antienterovirus compound that targets viral protein 3A and/or 3AB and suppresses a step in enterovirus replication by unknown mechanism. To date, four antienterovirus compounds, i.e., GW5074, Flt3 inhibitor II, TTP-8307, and AN-12-H5, are known to have similar mutations in the 3A protein-encoding region causing resistance to enviroxime (a G5318A [3A-Ala70Thr] mutation in poliovirus [PV]) and are considered enviroxime-like compounds. Recently, antienterovirus activity of a phosphatidylinositol 4-kinase III beta (PI4KB) inhibitor, PIK93, was reported, suggesting that PI4KB is an important host factor targetable by antienterovirus compounds (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we analyzed the inhibitory effects of previously identified enviroxime-like compounds (GW5074 and AN-12-H5) and a newly identified antienterovirus compound, T-00127-HEV1, on phosphoinositide (PI) kinases. We found that T-00127-HEV1 inhibited PI4KB activity with a higher specificity for than other PI kinases, in contrast to GW5074, which had a broad specificity for PI kinases. In contrast, AN-12-H5 showed no inhibitory effect on PI4KB activity and only moderate inhibitory effects on PI 3-kinase activity. Small interfering RNA (siRNA) screening targeting PI kinases identified PI4KB is a target of GW5074 and T-00127-HEV1, but not of AN-12-H5, for anti-PV activity. Interestingly, T-00127-HEV1 and GW5074 did not inhibit hepatitis C virus (HCV) replication, in contrast to a strong inhibitory effect of AN-12-H5. These results suggested that PI4KB is an enterovirus-specific host factor required for the replication process and targeted by some enviroxime-like compounds (T-00127-HEV1 and GW5074) and that enviroxime-like compounds may have targets other than PI kinases for their antiviral effect.
doi:10.1128/JVI.02249-10
PMCID: PMC3067798  PMID: 21177810
7.  Development of fluorescent probes for bioimaging applications 
Fluorescent probes, which allow visualization of cations such as Ca2+, Zn2+ etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group. The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems.
doi:10.2183/pjab.86.837
PMCID: PMC3037519  PMID: 20948177
probe; bioimaging; photoinduced electron transfer; fluorescence; spirocyclization
8.  Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes 
Nature medicine  2008;15(1):104-109.
It is a long-term goal of cancer diagnosis to develop tumor-imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from non-target tissues is critical. Here, we achieve highly specific in vivo cancer visualization by employing a newly-designed targeted “activatable” fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH-activatable probes based on the BODIPY fluorophore were synthesized, and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex and in vivo imaging of HER2-positive lung cancer cells in mice were performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific cell-surface-targeting molecules that result in cellular internalization.
doi:10.1038/nm.1854
PMCID: PMC2790281  PMID: 19029979
9.  Bioimaging Probes for Reactive Oxygen Species and Reactive Nitrogen Species 
Reactive oxygen species (ROS) play key roles in many pathogenic processes, including carcinogenesis, inflammation, ischemia-reperfusion injury and signal transduction. Also, reactive nitrogen species (RNS) cause various biological events such as neurodegenerative disorders. Sensitive and specific detection methods for ROS and RNS in biological samples should be useful for elucidation of biological events both in vitro and in vivo. Fluorescent probes based on small organic molecules have become indispensable tools in modern biology because they provide dynamic information concerning the localization and quantity of biological molecules of interest, without the need of genetic engineering of the sample. In this review, we recount some recent achievements in the field of small molecular fluorescent probes. First, the probes for nitric oxide and peroxynitrite as RNS are introduced and the probes of hydroxyl radical, hydrogen peroxide, hypochlorous and singlet oxygen as ROS are discussed, based on the fluorescence off/on switching mechanisms including photoinduced electron transfer and spirocyclization processes, and with some applications for in vitro and in vivo systems.
doi:10.3164/jcbn.R09-66
PMCID: PMC2735621  PMID: 19794917
bioimaging probe; reactive oxygen species; reactive nitrogen species; fluorescence; photoinduced electron transfer
10.  An enzymatically activated fluorescence probe for targeted tumor imaging 
β-Galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TG-βGal (J. Am. Chem. Soc., 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-βGal, provides a dramatic fluorescence enhancement upon reaction with β-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-βGal, after confirming that tumors in the model could be labeled with avidin-β-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-βGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 μm. This strategy has potential clinical application, for example in video-assisted laparoscopic tumor resection.
doi:10.1021/ja067710a
PMCID: PMC2555972  PMID: 17352471
11.  Mossy fiber Zn2+ spillover modulates heterosynaptic N-methyl-d-aspartate receptor activity in hippocampal CA3 circuits 
The Journal of Cell Biology  2002;158(2):215-220.
Although Zn2+ is contained in large amounts in the synaptic terminals of hippocampal mossy fibers (MFs), its physiological role in synaptic transmission is poorly understood. By using the newly developed high-sensitivity Zn2+ indicator ZnAF-2, the spatiotemporal dynamics of Zn2+ was monitored in rat hippocampal slices. When high-frequency stimulation was delivered to the MFs, the concentration of extracellular Zn2+ was immediately elevated in the stratum lucidum, followed by a mild increase in the stratum radiatum adjacent to the stratum lucidum, but not in the distal area of stratum radiatum. The Zn2+ increase was insensitive to a non–N-methyl-d-aspartate (NMDA) receptor antagonist but was efficiently attenuated by tetrodotoxin or Ca2+-free medium, suggesting that Zn2+ is released by MF synaptic terminals in an activity-dependent manner, and thereafter diffuses extracellularly into the neighboring stratum radiatum. Electrophysiological analyses revealed that NMDA receptor–mediated synaptic responses in CA3 proximal stratum radiatum were inhibited in the immediate aftermath of MF activation and that this inhibition was no longer observed in the presence of a Zn2+-chelating agent. Thus, Zn2+ serves as a spatiotemporal mediator in imprinting the history of MF activity in contiguous hippocampal networks. We predict herein a novel form of metaplasticity, i.e., an experience-dependent non-Hebbian modulation of synaptic plasticity.
doi:10.1083/jcb.200204066
PMCID: PMC2173116  PMID: 12119362
zinc; mossy fiber; hippocampus; synaptic plasticity; indicator

Results 1-11 (11)