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.

Beta-galactosidase-responsive synthetic biomarker for targeted tumor detection.

Tumor biomarkers are highly desirable for the screening of patients with a risk of tumor development and progression. Here, we report a beta-galactosidase (ß-gal)-responsive acetaminophen (ß-GR-APAP) as a synthetic plasma biomarker for targeted tumor detection. Tumor ß-gal labeling via the recognition of tumor-related antigen enabled the detection of a tumor using ß-GR-APAP.

Direct Monitoring of γ-Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized (13) C-Labeled Molecular Probe.

The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.

Design of a Hyperpolarized Molecular Probe for Detection of Aminopeptidase N Activity.

Aminopeptidase N (APN) is an important enzyme that is involved in tumor angiogenesis. Detection of APN activity can thus lead to early diagnosis and elucidation of tumor development. Although some molecular probes for APN have been developed, the detection of APN activity in opaque biological samples remains a challenge. To this end, we designed a hyperpolarized NMR probe [1-(13) C]Ala-NH2 which satisfies the prerequisites for APN detection, namely, sufficient retention of the hyperpolarized state, a high reactivity to APN, and an APN-induced chemical shift change. The [1-(13) C]Ala-NH2 probe allowed sensitive detection of APN activity using (13) C NMR spectroscopy.

In-cell covalent labeling of reactive His-tag fused proteins.

A new method for in-cell protein labeling was developed. This method employed a binding-induced nucleophilic reaction between the Cys-appended His-tag and the Ni(II)-NTA containing an α-chloroacetamide. Using this method, not only labeling of His-tag fused proteins but also the detection of a protein-protein interaction was achieved inside living cells.

Selective covalent labeling of tag-fused GPCR proteins on live cell surface with a synthetic probe for their functional analysis.

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions in living cells. In this paper, we report a covalent labeling method of tag-fused G-protein coupled receptor (GPCR) proteins expressing on cell surfaces utilizing small functional molecules. This method employs the selective and rapid reaction of a peptide tag and a molecular probe, which comprises the cysteine-containing short CA6D4x2 tag (CAAAAAADDDDGDDDD) and a tetranuclear Zn(II)-DpaTyr probe containing a reactive alpha-chloroacetyl moiety. The covalent labeling of tag-fused GPCRs such as bradykinin receptor (B2R) and acetylcholine receptor (m1AchR) selectively proceeded under physiological conditions during short incubation (10-30 min) with Zn(II)-DpaTyr probes bearing various functional groups. Labeling with fluorophore-appended Zn(II)-DpaTyr probes enabled visualization of the GPCRs on the surface of HEK293 cells by fluorescence. Labeling with the biotin-appended probe allowed introduction of a biotin unit into the GPCRs. This biotin label was utilized for fluorescence bioimaging studies and postlabeling blotting analysis of the labeled GPCRs by use of the specific biotin-streptavidin interaction. The utility of this labeling method was demonstrated in several function analyses of GPCRs, such as fluorescence visualization of the stimuli-responsive internalization of GPCRs and pH change in endosomes containing the internalized GPCRs.

FLAG-tag selective covalent protein labeling via a binding-induced acyl-transfer reaction.

A FLAG tag selective protein labeling method is newly developed in this study. Coupling of the selective binding between synthetic Ni-complex probe and FLAG tag with the acyl transfer reaction enables the site-selective covalent modification of FLAG peptide and FLAG-tag fused protein.

Turn-on fluorescence sensing of nucleoside polyphosphates using a xanthene-based Zn(II) complex chemosensor.

Fluorescence sensing with small molecular chemosensors is a versatile technique for elucidation of function of various biological substances. We now report a new fluorescent chemosensor for nucleoside polyphosphates such as ATP using metal-anion coordination chemistry. The chemosensor 1-2Zn(II) is comprised of the two sites of 2,2'-dipicolylamine (Dpa)-Zn(II) as the binding motifs and xanthene as a fluorescent sensing unit for nucleoside polyphosphates. The chemosensor 1-2Zn(II) selectively senses nucleoside polyphosphates with a large fluorescence enhancement (F/F(o) > 15) and strong binding affinity (K(app) approximately = 1 x 10(6) M(-1)), whereas no detectable fluorescence change was induced by monophosphate species and various other anions. The 'turn-on,' fluorescence of 1-2Zn(II) is based on a new mechanism, which involves the binding-induced recovery of the conjugated form of the xanthene ring from its nonfluorescent deconjugated state which was formed by an unprecedented nucleophilic attack of zinc-bound water. The selective and highly sensitive ability of 1-2Zn(II) to detect nucleoside polyphosphates enables its bioanalytical applications in fluorescence visualization of ATP particulate stores in living cells, demonstrating the potential utility of 1-2Zn(II).