Crystal structure reveals the binding mode and selectivity of a photoswitchable ligand for the adenosine A2A receptor.

Rational synthetic expansion of photoresponsive ligands is important for photopharmacological studies. Adenosine A2A receptor (A2AR) is stimulated by adenosine and related in Parkinson's disease and other diseases. Here, we report the crystal structure of the A2AR in complex with the novel photoresponsive ligand photoNECA (blue) at 3.34 Å resolution. PhotoNECA (blue) was designed for this structural study and the cell-based assay showed a photoresponsive and receptor selective characteristics of photoNECA (blue) for A2AR. The crystal structure explains the binding mode, photoresponsive mechanism and receptor selectivity of photoNECA (blue). Our study would promote not only the rational design of photoresponsive ligands but also dynamic structural studies of A2AR.

[Next-Generation Chemogenetics for Elucidating Higher Brain Functions].

To understand higher brain function, we need to understand the cellular function in a cell-type-specific manner. In recent decades, cell manipulation techniques termed chemogenetics (e.g., DREADD) have enabled cell-type-specific control of nerve activity in vivo. These are powerful for elucidating brain function in live animals. However, artificially-designed receptors evoke unnatural cellular signals in these methods; thus, they may not reflect physiological responses. We have recently focused on "molecular-targeted chemogenetics," which allows the cell-type specific regulation of target endogenous receptors. This review describes our current results toward "molecular-targeted chemogenetics" along with the recent progress in cell manipulation techniques.

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue.

Direct activation of cell-surface receptors is highly desirable for elucidating their physiological roles. A potential approach for cell-type-specific activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most cases. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was activated directly by palladium complexes. A palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum. Moreover, cell-type-specific activation of mGlu1 was demonstrated successfully using adeno-associated viruses in mice, which shows the potential utility of this chemogenetics for clarifying the physiological roles of mGlu1 in a cell-type-specific manner.

Chemogenetics of cell surface receptors: beyond genetic and pharmacological approaches.

Cell surface receptors transmit extracellular information into cells. Spatiotemporal regulation of receptor signaling is crucial for cellular functions, and dysregulation of signaling causes various diseases. Thus, it is highly desired to control receptor functions with high spatial and/or temporal resolution. Conventionally, genetic engineering or chemical ligands have been used to control receptor functions in cells. As the alternative, chemogenetics has been proposed, in which target proteins are genetically engineered to interact with a designed chemical partner with high selectivity. The engineered receptor dissects the function of one receptor member among a highly homologous receptor family in a cell-specific manner. Notably, some chemogenetic strategies have been used to reveal the receptor signaling of target cells in living animals. In this review, we summarize the developing chemogenetic methods of transmembrane receptors for cell-specific regulation of receptor signaling. We also discuss the prospects of chemogenetics for clinical applications.

Tethering-based chemogenetic approaches for the modulation of protein function in live cells.

Proteins are the workhorse molecules performing various tasks to sustain life. To investigate the roles of a protein under physiological conditions, the rapid modulation of the protein with high specificity in a living system would be ideal, but achieving this is often challenging. To address this challenge, researchers have developed chemogenetic strategies for the rapid and selective modulation of protein function in live cells. Here, the target protein is modified genetically to become sensitive to a designer molecule that otherwise has no effect on other cellular biomolecules. One powerful chemogenetic strategy is to introduce a tethering point into the target protein, allowing covalent or non-covalent attachment of the designer molecule. In this tutorial review, we focus on tethering-based chemogenetic approaches for modulating protein function in live cells. We first describe genetic, optogenetic and chemical means to study protein function. These means lay the basis for the chemogenetic concept, which is explained in detail. The next section gives an overview, including advantages and limitations, of tethering tactics that have been employed for modulating cellular protein function. The third section provides examples of the modulation of cell-surface proteins using tethering-based chemogenetics through non-covalent tethering and covalent tethering for irreversible modulation or functional switching. The fourth section presents intracellular examples. The last section summarizes key considerations in implementing tethering-based chemogenetics and shows perspectives highlighting future directions and other applications of this burgeoning research field.

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking.

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N-methyl-D-aspartate-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

Orthogonal Activation of Metabotropic Glutamate Receptor Using Coordination Chemogenetics.

Cell-surface receptors play a pivotal role as transducers of extracellular input. Although different cell types express the same receptor, the physiological roles of the receptor are highly dependent on cell type. To understand each role, tactics for cell-specific activation of the target receptor are in high demand. Herein, we developed an orthogonal activation method targeting metabotropic glutamate receptor 1 (mGlu1), a G-protein coupled receptor. In this method, direct activation via coordination-based chemogenetics (dA-CBC) was adopted, where activation of mGlu1 was artificially induced by a protein conformational change in response to the coordination of a metal ion or metal-ion complex. Our structure-based protein design and screening approach identified mGlu1 mutants that were directly activated by the coordination of Cu2+ or Zn2+, in addition to our previous Pd-complex-sensitive mGlu1 mutant. Notably, the activation of the mutants was mutually orthogonal, resulting in cell-type selective activation in a model system using HEK293 cells.

Selective acetylcholinesterase inhibitors derived from muscle relaxant dantrolene.

Dantrolene, the only therapeutic agent for malignant hyperthermia, is known to have not only a muscle relaxant effect, but also a neuroprotective effect and Alzheimer's disease improving effect. Recently, it has been reported that dantrolene has a weak inhibitory effect on acetylcholinesterase (AChE), which is a therapeutic drug target for Alzheimer's disease. Thus, we focused on developing of AChE inhibitors with benzylpiperidine/piperazine moieties that are based on the dantrolene skeleton. Several derivatives showed an inhibitory activity. Among them, ortho-nitro derivative 8c showed the most potent inhibitory activity with the IC50 value of 34.2 nM. Furthermore, Lineweaver-Burk plot analysis indicated that 8c is AChE-selective inhibitor, which shows only a weak inhibitory effect on butyrylcholinesterase (BuChE) and a non-competitive inhibition.

Hybrid cell reactor system from Escherichia coli protoplast cells and arrayed lipid bilayer chamber device.

We developed a novel hybrid cell reactor system via functional fusion of single Escherichia coli protoplast cells, that are deficient in cell wall and expose plasma membrane, with arrayed lipid bilayer chambers on a device in order to incorporate the full set of cytosolic and membrane constituents into the artificial chambers. We investigated gene expression activity to represent the viability of the hybrid cell reactors: over 20% of hybrid cells showed gene expression activity from plasmid or mRNA. This suggests that the hybrid cell reactors retained fundamental activity of genetic information transduction. To expand the applicability of the hybrid cell reactors, we also developed the E. coli-in-E. coli cytoplasm system as an artificial parasitism system. Over 30% of encapsulated E. coli cells exhibited normal cell division, showing that hybrid cells can accommodate and cultivate living cells. This novel artificial cell reactor technology would enable unique approaches for synthetic cell researches such as reconstruction of living cell, artificial parasitism/symbiosis system, or physical simulation to test functionality of synthetic genome.

Miniaturization of thiol-organosilica nanoparticles induced by an anionic surfactant.

Thiol-organosilica nanoparticles are a promising nanomaterial for biomedical applications. The enhanced permeability and retention (EPR) effect is useful for tumor targeting within the biomedical applications of nanomaterials, and nanomaterials with a size of less than 200 nm exhibit the maximum EPR effect. However, the synthesis of thiol-organosilica nanoparticles with a diameter of less than 200 nm is not efficient for the yield using the present conventional synthetic methods. Herein, we report the development of an efficient synthetic method of thiol-organosilica nanoparticles with a diameter of less than 200 nm using an anionic surfactant and discuss its mechanism. Compared with the conventional synthetic methods, a greater than 10-fold miniaturization of thiol-organosilica nanoparticles and an approximately 40-fold increase in the production efficiency of small thiol-organosilica nanoparticles were achieved using the sodium dodecyl sulfate (SDS)-addition synthetic method or sodium dodecylbenzenesulfonate (SDBS)-addition synthetic method. This is the first report about the miniaturization of organosilica nanoparticles induced by an anionic surfactant. The SDS-addition synthetic method or SDBS-addition synthetic method will accelerate the biomedical applications of thiol-organosilica nanoparticles.

Combretastatin A4-ß-Galactosyl Conjugates for Ovarian Cancer Prodrug Monotherapy.

Chemotherapy for ovarian cancer often causes severe side effects. As candidates for combretastatin A4 (CA4) prodrug for ovarian cancer prodrug monotherapy (PMT), we designed and synthesized two ß-galactose-conjugated CA4s (CA4-ßGals), CA4-ßGal-1 and CA4-ßGal-2. CA4 was liberated from CA4-ßGals by ß-galactosidase, an enzyme more strongly expressed in ovarian cancer cells than normal cells. CA4-ßGal-2, which has a self-immolative benzyl linker between CA4 and the ß-galactose moiety, was more cytotoxic to ovarian cancer cell lines than CA4-ßGal-1 without a linker. Therefore, CA4-ßGal-2 can serve as a platform for the design and manufacture of prodrugs for ovarian cancer PMT.

Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution.

The LacZ gene, which encodes Escherichia coli ß-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic ß-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.

PAMAM Dendron Lipid Assemblies That Undergo Structural Transition in Response to Weakly Acidic pH and Their Cytoplasmic Delivery Capability.

Dendron lipids designed to consist of amine-terminated polyamidoamine G1 dendron and two octadecyl chains were used for the preparation of pH-responsive molecular assemblies having phase structures that are changed through their dynamic molecular shape. The dendron lipid contains two primary amines and two tertiary amines in the dendron moiety, changing its charged state in the pH region between pH 10 and pH 4. The assemblies were shown to take a vesicle structure at neutral and alkaline pHs, but their structure changed to a micelle-like structure below pH 6.4. Because this pH region corresponds to one in which tertiary amines of the dendron lipid became protonated, protonation of tertiary amines in addition to primary amines in the dendron moiety might affect its dynamic molecular shape, resulting in a sharp pH response of the assemblies. The assemblies tended to form aggregates when taking on a vesicle form with a gel phase, but incorporation of a poly(ethylene glycol)-lipid provided dendron lipid vesicles with both sharp pH response and high colloidal stability. The poly(ethylene glycol)-incorporated dendron lipid vesicles tightly retained ovalbumin molecules in their internal aqueous space but released them almost completely at pH 6.0. In addition, the vesicles were shown to achieve efficient ovalbumin delivery into cytosol of DC2.4 cells (mouse dendritic cell line) after internalization through endocytosis.

Phenylboronic acid-based (19)F MRI probe for the detection and imaging of hydrogen peroxide utilizing its large chemical-shift change.

Herein, we report on a new (19)F MRI probe for the detection and imaging of H2O2. Our designed 2-fluorophenylboronic acid-based (19)F probe promptly reacted with H2O2 to produce 2-fluorophenol via boronic acid oxidation. The accompanying (19)F chemical-shift change reached 31 ppm under our experimental conditions. Such a large chemical-shift change allowed for the imaging of H2O2 by (19)F chemical-shift-selective MRI.

An adhesive (19)F MRI chemical probe allows signal off-to-on-type molecular sensing in a biological environment.

We report a new strategy for designing a signal off-to-on-type (19)F MRI chemical probe that operates in biological environments. The present strategy is based on the control of adherence of a (19)F MRI chemical probe to certain blood proteins, accompanied by a change in transverse relaxation time of (19)F nuclei.

A platform for designing hyperpolarized magnetic resonance chemical probes.

Hyperpolarization is a highly promising technique for improving the sensitivity of magnetic resonance chemical probes. Here we report [(15)N, D(9)]trimethylphenylammonium as a platform for designing a variety of hyperpolarized magnetic resonance chemical probes. The platform structure shows a remarkably long (15)N spin-lattice relaxation value (816 s, 14.1 T) for retaining its hyperpolarized spin state. The extended lifetime enables the detection of the hyperpolarized (15)N signal of the platform for several tens of minutes and thus overcomes the intrinsic short analysis time of hyperpolarized probes. Versatility of the platform is demonstrated by applying it to three types of hyperpolarized chemical probes: one each for sensing calcium ions, reactive oxygen species (hydrogen peroxide) and enzyme activity (carboxyl esterase). All of the designed probes achieve high sensitivity with rapid reactions and chemical shift changes, which are sufficient to allow sensitive and real-time monitoring of target molecules by (15)N magnetic resonance.

Atom arrangement strategy for designing a turn-on 1H magnetic resonance probe: a dual activatable probe for multimodal detection of hypochlorite.

The first dual activatable hypochlorite ((-)OCl)-sensing probe was developed, based on a new proof-of-concept design involving signal-activatable (1)H chemical probes using the triple-resonance NMR technique. The probe enabled fluorescence-(1)H MR dual turn-on detection of (-)OCl in solution and in crude tissue extracts.