Mamoru Haratake

Characterisation of radioiodinated flavonoid derivatives for SPECT imaging of cerebral prion deposits

Prion diseases are fatal neurodegenerative diseases characterised by deposition of amyloid plaques containing abnormal prion protein aggregates (PrPSc). This study aimed to evaluate the potential of radioiodinated flavonoid derivatives for single photon emission computed tomography (SPECT) imaging of PrPSc. In vitro binding assays using recombinant mouse PrP (rMoPrP) aggregates revealed that the 4-dimethylamino-substituted styrylchromone derivative (SC-NMe2) had higher in vitro binding affinity (Kdu2009=u200924.5u2009nM) and capacity (Bmaxu2009=u200936.3u2009pmol/nmol protein) than three other flavonoid derivatives (flavone, chalcone, and aurone). Fluorescent imaging using brain sections from mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice demonstrated that SC-NMe2 clearly labelled PrPSc-positive prion deposits in the mice brain. Two methoxy SC derivatives, SC-OMe and SC-(OMe)2, also showed high binding affinity for rMoPrP aggregates with Ki values of 20.8 and 26.6u2009nM, respectively. In vitro fluorescence and autoradiography experiments demonstrated high accumulation of [125I]SC-OMe and [125I]SC-(OMe)2 in prion deposit-rich regions of the mBSE-infected mouse brain. SPECT/computed tomography (CT) imaging and ex vivo autoradiography demonstrated that [123I]SC-OMe showed consistent brain distribution with the presence of PrPSc deposits in the mBSE-infected mice brain. In conclusion, [123I]SC-OMe appears a promising SPECT radioligand for monitoring prion deposit levels in the living brain.

Amyloid formation characteristics of GNNQQNY from yeast prion protein Sup35 and its seeding with heterogeneous polypeptides

Sup35 is a prion-like protein from yeast and shares the ability to transmit its aberrant fold and to aggregate into amyloid fibrils. 7GNNQQNY13 from the prion-determining domain of Sup35 was reported to form an amyloid. We first investigated the self-aggregation transition behavior of GNNQQNY to the β-sheet amyloid state under various conditions. Mechanical stirring using a magnetic bar resulted in accelerated aggregation of the GNNQQNY. The aggregation rate of GNNQQNY was also dependent on its concentration; the higher the GNNQQNY concentration, the faster the aggregation. Circular dichroism and Fourier transform-infrared spectral data indicated the formation of the β-sheet structure in the GNNQQNY aggregates. The fluorescence experiments using an amyloid-specific thioflavin T also demonstrated that the GNNQQNY aggregates formed the amyloid structures. The amyloid structure of the GNNQQNY aggregates served as seeds for the elongation of the monomeric GNNQQNY in the solution state. We further studied the ability of the GNNQQNY amyloid fibrils to act as seeds for the elongation of the amyloid-forming monomeric proteins (albumin, lysozyme and insulin). The cross-seeding experiments suggested that the GNNQQNY aggregate could possibly promote the amyloid fibril formation of heterogeneous insulin. The inverse monomeric GNNQQNY would have a binding capacity for the heterogeneous already-formed amyloid-β fibrils on a mice brain section. These basic data could be informative for elucidating the pathogenic and/or propagation mechanisms of prion agents and developing effective therapeutics and/or diagnosis for prion diseases.

A Comprehensive Analysis of Selenium-Binding Proteins in the Brain Using Its Reactive Metabolite

The intracellular metabolism of selenium in the brain currently remains unknown, although the antioxidant activity of this element is widely acknowledged to be important in maintaining brain functions. In this study, a comprehensive method for identifying the selenium-binding proteins using PenSSeSPen as a model of the selenium metabolite, selenotrisulfide (RSSeSR, STS), was applied to a complex cell lysate generated from the rat brain. Most of the selenium from L-penicillamine selenotrisulfide (PenSSeSPen) was captured by the cytosolic protein thiols in the form of STS through the thiol-exchange reaction (R-SH+PenSSeSPen→R-SSeSPen+PenSH). The cytosolic protein species, which reacted with the PenSSeSPen mainly had a molecular mass of less than 20u2009kDa. A thiol-containing protein at m/z 15155 in the brain cell lysate was identified as the cystatin-12 precursor (CST12) from a rat protein database search and a tryptic fragmentation experiment. CST12 belongs to the cysteine proteinase inhibitors of the cystatin superfamily that are of interest in mechanisms regulating the protein turnover and polypeptide production in the central nervous system and other tissues. Consequently, CST12 is suggested to be one of the cytosolic proteins responsible for the selenium metabolism in the brain.

Synthesis and evaluation of a radioiodinated 4,6-diaryl-3-cyano-2-pyridinone derivative as a survivin targeting SPECT probe for tumor imaging

Survivin is overexpressed in most of the cancerous tissues but not in terminally differentiated normal tissues, making it an attractive target for diagnosis and therapy of various types of cancers. In this study, we aimed to develop 4,6-diaryl-3-cyano-2-pyridinone (DCP) derivatives, as novel cancer imaging probes that target survivin. Chloro and iodo analogs of DCP (CDCP and IDCP, respectively) were successfully synthesized by using a previously unreported carbon monoxide-free procedure. IDCP exhibited a slightly higher binding affinity for recombinant human survivin (Kd=34 nM) than that of CDCP (Kd=44 nM). Fluorescence staining indicated that both CDCP and IDCP showed high signals in MDA-MB-231 cells with high levels of survivin expression. Significantly low fluorescent signals were observed in MCF-10A cells, which showed low levels of survivin expression. [(125)I]IDCP was synthesized for the application of IDCP to single photon emission computed tomography (SPECT) imaging. Quantitative in vitro binding of [(125)I]IDCP in cell cultures showed results consistent to those observed after fluorescent staining. In vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [(125)I]IDCP increased gradually with time and was 0.65% injected dose per gram (% ID/g) at 180 min. The maximum tumor/blood and tumor/muscle ratio at 60 min were 0.87 and 2.27, respectively, indicating inadequate [(125)I]IDCP accumulation in tumors necessary for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties of IDCP, this study demonstrates the feasibility of using the DCP backbone as a scaffold for the development of survivin-targeting tumor imaging probes.

Development of radioiodinated acridine derivatives for in vivo imaging of prion deposits in the brain.

Prion diseases are caused by deposition of abnormal prion protein aggregates (PrPSc) in the central nervous system. This study aimed to develop in vivo imaging probes that can detect cerebral PrPSc deposits. We synthesized several quinacrine-based acridine (AC) derivatives with 2,9-substitution and radioiodinated them. The AC derivatives were evaluated as prion-imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. The distribution of these compounds in mice was also evaluated. The 2-methoxy derivative [125I]2 exhibited the highest binding affinity for rMoPrP aggregates with an equilibrium dissociation constant (Kd) value of 43.4nM. Fluorescence imaging with 2 showed clear signals at the thioflavin T (ThT)-positive amyloid deposits in the mBSE-infected mouse brain. Although a discrepancy was observed between the in vitro binding of AC derivatives to the aggregates and in vivo distribution of these compounds in the brain and we failed to identify prospective prion-imaging probes in this study, the AC derivatives may be considered a useful scaffold for the development of in vivo imaging probes. Further chemical modification of these AC derivatives may discover clinically applicable prion imaging probes.

Development of a 68Ge/68Ga Generator System Using Polysaccharide Polymers and Its Application in PET Imaging of Tropical Infectious Diseases

Gallium-68 (68Ga) is a positron emitter for clinical positron emission tomography (PET) applications that can be produced by a 68Ge/68Ga generator without cyclotron. However, commercially available 68Ge/68Ga generator systems require multiple steps for the preparation of 68Ga radiopharmaceuticals and are sometimes plagued by metallic impurities in the 68Ga eluent. We developed a 68Ge/68Ga generator system using polysaccharide-based adsorbents and direct application of the generator-eluted 68Ga-citrate to PET imaging of tropical infectious diseases. N-Methylglucamine (MG) as a 68Ge-adsorbing unit (Sepha-MGs) was introduced to a series of Sephadex G-10, G-15, G-25, G-50, and G-75. In the batch method, over 97% of the 68Ge in the solution was adsorbed onto the Sepha-MG series within 15 min. In particular, 68Ge was effectively adsorbed on the Sepha(15)-MG packed columns and 70–80% of the 68Ga was eluted by 1 mL of 0.1 M trisodium citrate with low 68Ge contamination (<0.001%). The chemical form of the generator-eluted 68Ga solution was identified as 68Ga-citrate. In PET studies, affected regions in mice infected with Leishmania and severe fever with thrombocytopenia syndrome virus were clearly visualized using the 68Ga-citrate. Sepha-MGs are useful adsorbents for 68Ge/68Ga generator systems with high 68Ga elution efficiency and minimal 68Ge breakthrough. These results indicated that eluted 68Ga-citrate can be directly used for PET imaging of infectious sites in mice. This novel generator system may be useful for straightforward PET imaging of infection in clinical practice.

Synthesis of Nanovesicular Glutathione Peroxidase Mimics with a Selenenylsulfide-Bearing Lipid

In this article, we describe the development of a nanosized-glutathione peroxidase (GPx) mimic based on liposomes of which the amphiphilic selenenylsulfide derivative (R–Se–S–R′) was incorporated into a lipid membrane. A lipid membrane-compatible selenenylsulfide derivative, 1-oxo-headecyl-seleno-l-cysteine-methyl-Se-yl-S-l-penicillamine methyl ester (OHSeP), was synthesized. X-ray photoelectron spectroscopy revealed that the sulfur and selenium atoms of the OHSeP molecule formed a selenenylsulfide linkage. The use of OHSeP easily allowed the introduction of the seleno-l-cysteine (SeCys) moiety into the liposomal membranes by mixing with the phosphatidylcholines (PCs), which gave rise to the GPx-like catalytic activity because of the selenium atom in the SeCys moiety. The penicillamine moiety of the OHSeP molecule incorporated into the OHSeP/PC liposomes was thought to orient toward the outer water phase. The OHSeP/PC liposomes generated the GPx-like catalytic activity, which was ascribed to the SeCys moiety that was introduced into the PC-based liposomes. Consequently, the lipid/water interface of the liposomal membranes could possibly provide an effective colloidal platform for the development of water-soluble nanosized GPx mimics.

An effective method for profiling the selenium-binding proteins using its reactive metabolic intermediate

Currently, the intracellular reduction and/or transport of selenium still remain unknown. Certain reduced forms of selenium species are thought to be reactive with various endogenous molecules, particularly thiol-containing proteins. In this study, a profiling method for identifying the selenium-binding proteins using l-penicillamine selenotrisulfide (PenSSeSPen) as a model of the selenium metabolic intermediate was applied to the cell lysate generated from the rat liver. Several proteins with cysteine thiol were found to be reactive with PenSSeSPen through the thiol-exchange reaction by MALDI TOF–MS analysis. The most distinctive cysteine-containing protein at m/z 14,313 in the liver cell lysate was identified as the liver fatty acid-binding protein based on a rat protein database search and a tryptic fragmentation experiment. This methodology could be used for determining the selenium-binding proteins and/or selenium-interactive species and provide a better understanding of the selenium metabolism and utilization in biological systems.

In vitro assessment of bioavailability of selenium from a processed Japanese anchovy, Niboshi

Niboshi is a commonly used foodstuff that is processed from Japanese anchovy (Engraulis japonicus) in Japanese cuisine. It was previously demonstrated that Niboshi and its water extract contained highly bioavailable selenium for selenium deficient mice. In this study, we assessed the selenium bioavailability from the extract of the Niboshi, using cultured cells. The activity of selenium-dependent glutathione peroxidase (GPx) of rat dorsal ganglion cells and human cervical carcinoma cells incubated with selenium from the Niboshi extract was over 2 times of that of the extract-free control cells and comparable to that of cells incubated with selenious acid of the same selenium concentration. These results suggest that selenium from the Niboshi extract was utilized for synthesis of the selenoprotein. Such in vitro selenium bioavailability was consistent with our previous results of in vivo assessment in mice.

Selenoprotein L-inspired nano-vesicular peroxidase mimics based on amphiphilic diselenides

In this study, we developed selenoprotein L-inspired nano-vesicular peroxidase mimics based on amphiphilic diselenides. Selenocystine (SeCyst) was used as the starting material for the synthesis of four liposomal membrane-compatible diselenide derivatives (R-Se-Se-R) with two hydrophobic tails and a polar part. The diselenide derivatives were successfully incorporated into the phosphatidylcholine (PC)-based nano-vesicular scaffold. The results of the particle diameter and zeta-potential measurements suggested that the functional diselenide moiety was placed around the outer surface, not in the hydrophobic interior, of the liposomal membrane structures. The GPx-like catalytic activity of the diselenide/PC liposomes was determined by the conventional NADPH method using glutathione as the reducing substrate. For three peroxide substrates, i.e., hydrogen peroxide, organic tert-butyl hydroperoxide and cummen hydroperoxide, the cationic property-possessing diselenide derivatives in the PC-based liposomes resulted in a higher catalytic activity in comparison to electrically neutral and anionic derivatives. Overall, the diselenide derivatives at the surface of a liposomal colloidal scaffold could exert a GPx-like catalytic activity in physiological aqueous media.