Toshiyuki Hiramatsu

Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A

Dyrk1A (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A) is a serine/threonine kinase essential for brain development and function, and its excessive activity is considered a pathogenic factor in Down syndrome. The development of potent, selective inhibitors of Dyrk1A would help to elucidate the molecular mechanisms of normal and diseased brains, and may provide a new lead compound for molecular-targeted drug discovery. Here, we report a novel Dyrk1A inhibitor, INDY, a benzothiazole derivative showing a potent ATP-competitive inhibitory effect with IC(50) and K(i) values of 0.24 and 0.18 μM, respectively. X-ray crystallography of the Dyrk1A/INDY complex revealed the binding of INDY in the ATP pocket of the enzyme. INDY effectively reversed the aberrant tau-phosphorylation and rescued the repressed NFAT (nuclear factor of activated T cell) signalling induced by Dyrk1A overexpression. Importantly, proINDY, a prodrug of INDY, effectively recovered Xenopus embryos from head malformation induced by Dyrk1A overexpression, resulting in normally developed embryos and demonstrating the utility of proINDY in vivo.

Novel bifunctional probe for radioisotope-free photoaffinity labeling: compact structure comprised of photospecific ligand ligation and detectable tag anchoring units

A novel method for radioisotope-free photoaffinity labeling was developed, in which a bifunctional ligand is connected to a target protein by activation of a photoreactive group, such as an aromatic azido or 3-trifluoromethyl-3H-diazirin-3-yl group, and identification of the ligated product is achieved by anchoring of a detectable tag through the Staudinger-Bertozzi reaction with an alkyl azido moiety that survives photolysis. The chemical ground of this method was confirmed using model compounds with the bifunctional group under photoirradiation in the presence of trapping agents for reactive intermediates. The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase.

Dantrolene analogues revisited: general synthesis and specific functions capable of discriminating two kinds of Ca2+ release from sarcoplasmic reticulum of mouse skeletal muscle

The general synthesis of dantrolene analogues with various substituents on its phenyl ring has been developed via palladium-catalyzed cross-coupling reactions, the Stille or Suzuki reaction, as the key step. The effects of synthesized analogues have been evaluated by two kinds of Ca(2+) release modes from sarcoplasmic reticulum (SR) of mouse skeletal muscle fibers based on: (1) the measurement of twitch contraction caused by the physiological Ca(2+) release (PCR) of intact skeletal muscle and (2) the rate of Ca(2+)-induced Ca(2+) release (CICR) in saponin-treated skinned muscle fibers. Although dantrolene, a lead compound, inhibits both twitch contraction and CICR, some structurally modified analogues exhibit one or the other of these effects. The methoxy congener, GIF-0185, potently inhibits the twitch contraction without affecting the CICR, while GIF-0166 and GIF-0248, the ortho-nitro regioisomer and ortho, ortho-dinitro substituted analogues, respectively, doubly potentiate the CICR exclusively.

11C-Cetrozole: An Improved C-11C-Methylated PET Probe for Aromatase Imaging in the Brain

Aromatase (an enzyme that converts androgens to estrogens) in the brain is involved in neuroprotection, synaptic plasticity, and regulation of sexual and emotional behaviors. To investigate the physiologic and pathologic importance of aromatase in the brain, including in humans, we here report the development of a novel PET probe for aromatase, 11C-cetrozole, which allows noninvasive quantification of aromatase expression. Methods: 11C-cetrozole was synthesized by the C-11C-methylation method developed by our group. In vitro autoradiography of frozen sections and a binding study with rat brain homogenates were conducted to demonstrate the specific binding and the dissociation constant. PET studies with anesthetized rhesus monkeys were performed to analyze the dynamics in the brain. Results: In vitro and in vivo studies using 11C-cetrozole showed its superiority in brain aromatase imaging in terms of specificity and selectivity, compared with previously developed 11C-vorozole. PET studies showed that 11C-cetrozole had a higher signal-to-noise ratio, providing a sharper image than 11C-vorozole, because the radioactive metabolite of 11C-vorozole was taken up into the brain. High specific binding of 11C-cetrozole was observed in the amygdala and hypothalamus, and we also noted binding in the nucleus accumbens of rhesus monkeys for the first time. Conclusion: These results suggest that PET imaging with newly developed 11C-cetrozole is suitable for quantifying the expression of brain aromatase in vivo, possibly providing critical information regarding the functional roles of aromatase in human neurologic and emotional disorders.

Practical synthesis of precursor of [N-methyl-11C]vorozole, an efficient PET tracer targeting aromatase in the brain

A practical method to prepare precursor of [N-methyl-(11)C]vorozole ([(11)C]vorozole), an efficient positron emission tomography (PET) tracer for imaging aromatase in the living body, was established. Sufficient amount of the racemate including norvorozole, a demethylated vorozole derivative used as a precursor of [(11)C]vorozole, became available by means of high-yield eight-step synthesis. The enantiomers were separated by preparative HPLC using a chiral stationary phase column to give optically pure norvorozole and its enantiomer. From the latter, ent-[(11)C]vorozole, an enantiomer of [(11)C]vorozole, was prepared and used in the PET study for the first time, which was shown to bind very weakly to aromatase in rhesus monkey brain supporting the previous pharmacological results. The stable supply of norvorozole will facilitate further researches on aromatase in the living body including brain by the PET technique.

Detection of structural changes in a cofactor binding protein by using a wheat germ cell‐free protein synthesis system coupled with unnatural amino acid probing

A cell‐free protein synthesis system is a powerful tool with which unnatural amino acids can be introduced into polypeptide chains. Here, the authors describe unnatural amino acid probing in a wheat germ cell‐free translation system as a method for detecting the structural changes that occur in a cofactor binding protein on a conversion of the protein from an apo‐form to a holo‐form. The authors selected the FMN‐binding protein from Desulfovibrio vulgaris as a model protein. The apo‐form of the protein was synthesized efficiently in the absence of FMN. The purified apo‐form could be correctly converted to the holo‐form. Thus, the system could synthesize the active apo‐form. Gel filtration chromatography, analytical ultracentrifugation, and circular dichroism‐spectra studies suggested that the FMN‐binding site of the apo‐form is open as compared with the holo‐form. To confirm this idea, the unnatural amino acid probing was performed by incorporating 3‐azido‐L‐tyrosine at the Tyr35 residue in the FMN‐binding site. The authors optimized three steps in their system. The introduced 3‐azido‐L‐tyrosine residue was subjected to specific chemical modification by a fluorescein‐triarylphosphine derivative. The initial velocity of the apo‐form reaction was 20 fold faster than that of the holo‐form, demonstrating that the Tyr35 residue in the apo‐form is open to solvent. Proteins 2007.

3-Azidodifluoromethyl-3H-diazirin-3-yl group as an all-in-one functional group for radioisotope-free photoaffinity labeling.

The 3-azidodifluoromethyl-3H-diazirin-3-yl group was designed and synthesized as an all-in-one functional group for radioisotope-free photoaffinity labeling.

1‐Benzyl‐1,2,3,4‐tetrahydroisoquinoline binds with tubulin β, a substrate of parkin, and reduces its polyubiquitination

J. Neurochem. (2010) 114, 1291–1301.

Facile synthesis of diazido-functionalized biaryl compounds as radioisotope-free photoaffinity probes by Suzuki-Miyaura coupling.

Suzuki-Miyaura coupling of 3-azido-5-(azidomethyl)phenylboronic acid pinacol ester with various aryl bromides affords corresponding diazido-functionalized biaryl compounds in good yields. This approach provides an easy access to radioisotope-free photoaffinity probes possessing biaryl structure. By using this method, we prepared a novel diazido-functionalized dantrolene analog, which showed selective inhibitory effect on physiological Ca(2+) release (PCR) from sarcoplasmic reticulum (SR) in mouse skeletal muscle without affecting Ca(2+)-induced Ca(2+) release (CICR).

[125I]-N-[(3-azido-5-iodo)benzyl]dantrolene and [125I]-N-{[3-iodo-5-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl}dantrolene: Photoaffinity probes specific for the physiological Ca2+ release from sarcoplasmic reticulum of skeletal muscle

In order to capture and identify key molecules that regulate the release of Ca(2+) from the sarcoplasmic reticulum (SR) of skeletal muscle, we designed specific photoaffinity probes based on the structural modification of dantrolene. Thus, GIF-0082 and GIF-0276 possessing azido- and trifluoromethyldiazirinyl-benzyl groups, respectively, at the hydantoin moiety were found to have a highly selective inhibitory effect on physiological Ca(2+) release (PCR) without affecting Ca(2+)-induced Ca(2+) release (CICR). Successful realization of the sharp discrimination between PCR and CICR has led to the creation of [(125)I]GIF-0082 and [(125)I]GIF-0276, which were synthesized by substituting a stannyl group with (125)I in the corresponding phenylstannane precursors.