Katsumi Doh-ura

Anti-prion activities and drug-like potential of functionalized quinacrine analogs with basic phenyl residues at the 9-amino position.

In this paper, we report the synthesis and cell-based anti-prion activity of quinacrine analogs derived by replacing the basic alkyl side chain of quinacrine with 4-(4-methylpiperazin-I-yl)phenyl, (1-benzylpiperidin-4-yl) and their structural variants. Several promising analogs were found that have a more favorable anti-prion profile than quinacrine in terms of potency and activity across different prion-infected murine cell models. They also exhibited greater binding affinities for a human prion protein fragment (hPrP(121-231)) than quinacrine, and had permeabilities on the PAMPA-BBB assay that fall within the range of CNS permeant candidates. When evaluated on bidirectional assays on a Pgp overexpressing cell line, one analog was less susceptible to Pgp efflux activity compared to quinacrine. Taken together, the results point to an important role for the substituted 9-amino side chain attached to the acridine, tetrahydroacridine and quinoline scaffolds. The nature of this side chain influenced cell-based potency, PAMPA permeability and binding affinity to hPrP(121-231).

Protease-resistant PrP and PrP oligomers in the brain in human prion diseases after intraventricular pentosan polysulfate infusion

Intraventricular infusion of pentosan polysulfate (PPS) as a treatment for various human prion diseases has been applied in Japan. To evaluate the influence of PPS treatment we performed pathological examination and biochemical analyses of PrP molecules in autopsied brains treated with PPS (one case of sporadic Creutzfeldt‐Jakob disease (sCJD, case 1), two cases of dura mater graft‐associated CJD (dCJD, cases 2 and 4), and one case of Gerstmann‐Sträussler‐Scheinker disease (GSS, case 3). Six cases of sCJD without PPS treatment were examined for comparison. Protease‐resistant PrP (PrPres) in the frontal lobe was evaluated by Western blotting after proteinase K digestion. Further, the degree of polymerization of PrP molecules was examined by the size‐exclusion gel chromatography assay. PPS infusions were started 3–10 months after disease onset, but the treatment did not achieve any clinical improvements. Postmortem examinations of the treated cases revealed symmetrical brain lesions, including neuronal loss, spongiform change and gliosis. Noteworthy was GFAP in the cortical astrocytes reduced in all treated cases despite astrogliosis. Immunohistochemistry for PrP revealed abnormal synaptic deposits in all treated cases and further plaque‐type PrP deposition in case 3 of GSS and case 4 of dCJD. Western blotting showed relatively low ratios of PrPres in case 2 of dCJD and case 3 of GSS, while in the treated sCJD (case 1), the ratio of PrPres was comparable with untreated cases. The indices of oligomeric PrP were reduced in one sCJD (case 1) and one dCJD (case 2). Although intraventricular PPS infusion might modify the accumulation of PrP oligomers in the brains of patients with prion diseases, the therapeutic effects are still uncertain.

Less protease-resistant PrP in a patient with sporadic CJD treated with intraventricular pentosan polysulphate.

Terada T, Tsuboi Y, Obi T, Doh‐ura K, Murayama S, Kitamoto T, Yamada T, Mizoguchi K. Less protease‐resistant PrP in a patient with sporadic CJD treated with intraventricular pentosan polysulphate.u2028Acta Neurol Scand: 2010: 121: 127–130.u2028© 2009 The Authors Journal compilation

Binding and Selectivity of the Marine Toxin Neodysiherbaine A and Its Synthetic Analogues to GluK1 and GluK2 Kainate Receptors.

Dysiherbaine (DH) and neodysiherbaine A (NDH) selectively bind and activate two kainate-type ionotropic glutamate receptors, GluK1 and GluK2. The ligand-binding domains of human GluK1 and GluK2 were crystallized as bound forms with a series of DH analogues including DH, NDH, 8-deoxy-NDH, 9-deoxy-NDH and 8,9-dideoxy-NDH (MSVIII-19), isolated from natural sources or prepared by total synthesis. Since the DH analogues exhibit a wide range of binding affinities and agonist efficacies, it follows that the detailed analysis of crystal structure would provide us with a significant opportunity to elucidate structural factors responsible for selective binding and some aspects of gating efficacy. We found that differences in three amino acids (Thr503, Ser706 and Ser726 in GluK1 and Ala487, Asn690 and Thr710 in GluK2) in the ligand-binding pocket generate differences in the binding modes of NDH to GluK1 and GluK2. Furthermore, deletion of the C(9) hydroxy group in NDH alters the ligand conformation such that it is no longer suited for binding to the GluK1 ligand-binding pocket. In GluK2, NDH pushes and rotates the side chain of Asn690 (substituted for Ser706 in GluK1) and disrupts an interdomain hydrogen bond with Glu409. The present data support the idea that receptor selectivities of DH analogues resulted from the differences in the binding modes of the ligands in GluK1/GluK2 and the steric repulsion of Asn690 in GluK2. All ligands, regardless of agonist efficacy, induced full domain closure. Consequently, ligand efficacy and domain closure did not directly coincide with DH analogues and the kainate receptors.

Semisynthesis of a Protein with Cholesterol at the C-Terminal, Targeted to the Cell Membrane of Live Cells

Various proteins are modified post-translationally to localize them at the cell membrane. Among them, hedgehog-family proteins are modified by cholesterol at the C-terminal. In this study, green fluorescent protein (GFP) modified with cholesterol (GFP-Chol) at the C-terminal was prepared semisynthetically and investigated. This semi-synthesis was performed using the following native chemical ligation: GFP-Cα-thioester was prepared using the intein-mediated thioester exchange reaction and was ligated to Cys-NH-diethylene glycol-NHCO-cholesterol in the presence of a detergent. After removal of the detergent, the GFP-Chol was applied to mouse live cells. Confocal laser fluorescent microscopy confirmed localization of GFP-Chol at the cell membrane. The findings suggest that modifying proteins with cholesterol at the C-terminal is useful for targeting the proteins to the cell membrane of live cells.

Efficacy and mechanism of a glycoside compound inhibiting abnormal prion protein formation in prion-infected cells: implications of interferon and phosphodiesterase 4D interacting protein

ABSTRACT A new type of antiprion compound, Gly-9, was found to inhibit abnormal prion protein formation in prion-infected neuroblastoma cells, in a prion strain-independent manner, when the cells were treated for more than 1 day. It reduced the intracellular prion protein level and significantly modified mRNA expression levels of genes of two types: interferon-stimulated genes were downregulated after more than 2 days of treatment, and the phosphodiesterase 4D-interacting protein gene, a gene involved in microtubule growth, was upregulated after more than 1 day of treatment. A supplement of interferon given to the cells partly restored the abnormal prion protein level but did not alter the normal prion protein level. This interferon action was independent of the Janus activated kinase-signal transducer and activator of transcription signaling pathway. Therefore, the changes in interferon-stimulated genes might be a secondary effect of Gly-9 treatment. However, gene knockdown of phosphodiesterase 4D-interacting protein restored or increased both the abnormal prion protein level and the normal prion protein level, without transcriptional alteration of the prion protein gene. It also altered the localization of abnormal prion protein accumulation in the cells, indicating that phosphodiesterase 4D-interacting protein might affect prion protein levels by altering the trafficking of prion protein-containing structures. Interferon and phosphodiesterase 4D-interacting protein had no direct mutual link, demonstrating that they regulate abnormal prion protein levels independently. Although the in vivo efficacy of Gly-9 was limited, the findings for Gly-9 provide insights into the regulation of abnormal prion protein in cells and suggest new targets for antiprion compounds. IMPORTANCE This report describes our study of the efficacy and potential mechanism underlying the antiprion action of a new antiprion compound with a glycoside structure in prion-infected cells, as well as the efficacy of the compound in prion-infected animals. The study revealed involvements of two factors in the compounds mechanism of action: interferon and a microtubule nucleation activator, phosphodiesterase 4D-interacting protein. In particular, phosphodiesterase 4D-interacting protein was suggested to be important in regulating the trafficking or fusion of prion protein-containing vesicles or structures in cells. The findings of the study are expected to be useful not only for the elucidation of cellular regulatory mechanisms of prion protein but also for the implication of new targets for therapeutic development.

Anti-prion activity of protein-bound polysaccharide K in prion-infected cells and animals

Protein-bound polysaccharide K (PSK) is a clinical immunotherapeutic agent that exhibits various biological activities, including anti-tumor and anti-microbial effects. In the present study, we report on the anti-prion activity of PSK. It inhibited the formation of protease-resistant abnormal prion protein in prion-infected cells without any apparent alterations in either the normal prion protein turnover or the autophagic function in the cells. Its anti-prion activity was predominantly composed of the high molecular weight component(s) of the protein portion of PSK. A single subcutaneous dose of PSK slightly but significantly prolonged the survival time of peritoneally prion-infected mice, but PSK-treated mice produced neutralizing antibodies against the anti-prion activity of PSK. These findings suggest that PSK is a new anti-prion substance that may be useful in elucidating the mechanism of prion replication, although the structure of the anti-prion component(s) of PSK requires further evaluation.

Autoantibody to glial fibrillary acidic protein in the sera of cattle with bovine spongiform encephalopathy

It is desirable to make the diagnosis in live cattle with bovine spongiform encephalopathy (BSE), and thus surrogate markers for the disease have been eagerly sought. Serum proteins from BSE cattle were analyzed by 2‐D Western blotting and TOF‐MS. Autoantibodies against proteins in cytoskeletal fractions prepared from normal bovine brains were found in the sera of BSE cattle. The protein recognized was identified to be glial fibrillary acidic protein (GFAP), which is expressed mainly in astrocytes in the brain. The antigen protein, GFAP, was also found in the sera of BSE cattle. The percentages of both positive sera in the autoantibody and GFAP were 44.0% for the BSE cattle, 0% for the healthy cattle, and 5.0% for the clinically suspected BSE‐negative cattle. A significant relationship between the presence of GFAP and the expression of its autoantibody in the serum was recognized in the BSE cattle. These findings suggest a leakage of GFAP into the peripheral blood during neurodegeneration associated with BSE, accompanied by the autoantibody production, and might be useful in understanding the pathogenesis and in developing a serological diagnosis of BSE in live cattle.

Amyloid-binding compounds and their anti-prion potency.

Prion diseases, or transmissible spongiform encephalopathies, are characterized by abnormal prion protein accumulation in the brain. Abnormal prion proteins, having properties of amyloids when extracted from the brain, are observed as amyloid plaque deposits in the brain in some prion diseases such as variant Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. This article reviews amyloid-binding compounds from the perspective of their usefulness for diagnosis and therapy of prion diseases. Styrylbenzoazole derivatives and phenylhydrazine derivatives are recently developed amyloid binding compounds that present benefits for prion-disease-related medicinal applications. For instance, styrylbenzoazole derivative BF-227, currently used as an amyloid imaging probe of positron emission tomography in Alzheimer disease, is useful also for the diagnosis of Gerstmann-Sträussler-Scheinker syndrome. A phenylhydrazine derivative, compB, has remarkable prophylactic effects on intracerebrally infected animals with certain prion strains, even when administered orally. These amyloid-binding compounds, however, are not applicable to prion strains or prion diseases of all types. For example, amyloid-binding compounds are ineffective for inhibiting prion strains such as 263K. They are not feasible for detecting abnormal prion protein accumulation in the brain for prion diseases having no amyloid plaques. To elucidate the limitations of amyloid-binding compounds, further investigation is necessary to clarify the binding mode of the compounds to abnormal prion protein structures at an atomic level.

A Single Subcutaneous Injection of Cellulose Ethers Administered Long before Infection Confers Sustained Protection against Prion Diseases in Rodents

Prion diseases are fatal, progressive, neurodegenerative diseases caused by prion accumulation in the brain and lymphoreticular system. Here we report that a single subcutaneous injection of cellulose ethers (CEs), which are commonly used as inactive ingredients in foods and pharmaceuticals, markedly prolonged the lives of mice and hamsters intracerebrally or intraperitoneally infected with the 263K hamster prion. CEs provided sustained protection even when a single injection was given as long as one year before infection. These effects were linked with persistent residues of CEs in various tissues. More effective CEs had less macrophage uptake ratios and hydrophobic modification of CEs abolished the effectiveness. CEs were significantly effective in other prion disease animal models; however, the effects were less remarkable than those observed in the 263K prion-infected animals. The genetic background of the animal model was suggested to influence the effects of CEs. CEs did not modify prion protein expression but inhibited abnormal prion protein formation in vitro and in prion-infected cells. Although the mechanism of CEs in vivo remains to be solved, these findings suggest that they aid in elucidating disease susceptibility and preventing prion diseases.