Masatoshi Yuri

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3′ UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

Sepantronium Bromide (YM155) induces disruption of the ILF3/p54nrb complex, which is required for survivin expression

YM155, a small-molecule survivin suppressant, specifically binds to the transcription factor ILF3, which regulates the expression of survivin[1]. In this experiment we have demonstrated that p54(nrb) binds to the survivin promoter and regulates survivin expression. p54(nrb) forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54(nrb) complex, which results in a different subcellular localization between ILF3 and p54(nrb). Thus, identification of molecular targets of YM155 in suppression of the survivin pathway, might lead to development of its use as a novel potential target in cancers.

Interleukin Enhancer-binding Factor 3/NF110 Is a Target of YM155, a Suppressant of Survivin

Survivin is responsible for cancer progression and drug resistance in many types of cancer. YM155 selectively suppresses the expression of survivin and induces apoptosis in cancer cells in vitro and in vivo. However, the mechanism underlying these effects of YM155 is unknown. Here, we show that a transcription factor, interleukin enhancer-binding factor 3 (ILF3)/NF110, is a direct binding target of YM155. The enhanced survivin promoter activity by overexpression of ILF3/NF110 was attenuated by YM155 in a concentration-dependent manner, suggesting that ILF3/NF110 is the physiological target through which YM155 mediates survivin suppression. The results also show that the unique C-terminal region of ILF3/NF110 is important for promoting survivin expression and for high affinity binding to YM155.

Absence of Increased α1-Microglobulin in IgA Nephropathy Proteinuria

To search for biomarkers of IgA nephropathy, protein profiles of urine samples from patients with IgA nephropathy and normal volunteers were compared using two-dimensional DIGE. Most of the 172 spots identified in the urine were serum proteins, and their amounts in IgA nephropathy urine were much higher than those in normal urine; this can be explained as proteinuria caused by glomerular dysfunction. However, only α1-microglobulin, also one of the major serum proteins, in IgA nephropathy urine was not higher in amount than that in normal urine. We confirmed using ELISA analysis that the amounts of transferrin and albumin in IgA nephropathy and diabetic nephropathy urine were much higher than those in normal urine, whereas the amount of α1-microglobulin in IgA nephropathy urine was not higher than that in normal urine and was much lower than that in diabetic nephropathy urine. Approximately 50% of α1-microglobulin forms a complex with IgA in serum. These results suggest that α1-microglobulin in IgA nephropathy urine is a characteristic protein and might be a biomarker for IgA nephropathy and that α1-microglobulin might have a relationship with IgA nephropathy pathology.

Abstract 1728: ASP8273, a novel mutant-selective irreversible EGFR inhibitor, inhibits growth of non-small cell lung cancer (NSCLC) cells with EGFR activating and T790M resistance mutations

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: Reversible EGFR TKIs, gefitinib and erlotinib, have shown antitumor efficacy in NSCLC patients with activating mutations in EGFR kinase domain. But the clinical efficacy of these agents is limited by the development of acquired drug resistance, which is most commonly caused by T790M resistance mutation in EGFR. This mutation has been detected in approximately 50% to 60% of patients. The 2nd generation irreversible EGFR inhibitors inhibit EGFR with T790M, but their clinical efficacy to NSCLC patients with T790M appears to be limited due to severe adverse effects caused by concomitant WT EGFR inhibition. Therefore, an EGFR TKI which inhibits T790M mutant EGFR selectively with less activity against WT EGFR may be beneficial. Here we report ASP8273, a novel, small molecule EGFR TKI that inhibits the kinase activity of EGFR containing the activating and T790M resistance mutations with less activity against WT EGFR. METHODS: The inhibitory effect and the selectivity of ASP8273 were evaluated against mutant EGFR (L858R, del ex19, L858R/T790M and del ex19/T790M) and WT EGFR using in vitro enzymatic and cell-based assay. Binding mode of ASP8273 to EGFR was assessed by mass spectrometry. Antitumor activity of ASP8273 was evaluated in xenograft models using PC-9 (del ex19), HCC827 (del ex19), NCI-H1975 (L858R/T790M) and PC-9ER (Erlotinib Resistant)(del ex19/T790M) NSCLC cells. RESULTS: ASP8273 inhibited mutant EGFR containing del ex19 or L858R activating mutations as well as the T790M resistance mutation with lower IC50 values than WT EGFR. Mass spectrometry analysis revealed that ASP8273 is covalently bound to a mutant EGFR(L858R/T790M) via C797 in the kinase domain of EGFR. In NCI-H1975 cells, ASP8273 induced long-lasting inhibition of EGFR phosphorylation for 24 h after washout of compound. In assays using endogenously EGFR-dependent cells, ASP8273 inhibited the growth of PC-9(del ex19), HCC827(del ex19), NCI-H1975(del ex19/T790M) and PC-9ER(del ex19/T790M) with IC50 values of 8-33 nM, more potently than that of NCI-H1666(WT) with IC50 value of 230 nM. In mouse xenograft studies, ASP8273 induced tumor regression in NCI-H1975 (L858R/T790M), HCC827 (del ex19) and PC-9 (del ex19) xenograft models by repeated oral dosing in a dose-dependent manner. Dosing schedules did not affect the efficacy of ASP8273. In an NCI-H1975 xenograft model, complete regression of tumor was achieved after 14-days of ASP8273 treatment. Complete regression was maintained in 50% of mice more than 85 days after cessation of ASP8273 treatment. CONCLUSIONS: ASP8273 inhibits the growth of NSCLC cells with EGFR activating and T790M resistance mutations with evidence of tumor regression. Therefore, ASP8273 may show therapeutic efficacy in NSCLC patients with EGFR mutations. Clinical trials of ASP8273 in NSCLC patients are planned in the US/EU and Asia. Citation Format: Hideki Sakagami, Satoshi Konagai, Hiroko Yamamoto, Hiroaki Tanaka, Takahiro Matsuya, Masamichi Mori, Hiroyuki Koshio, Masatoshi Yuri, Masaaki Hirano, Sadao Kuromitsu. ASP8273, a novel mutant-selective irreversible EGFR inhibitor, inhibits growth of non-small cell lung cancer (NSCLC) cells with EGFR activating and T790M resistance mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2014-1728

Combination of MS Protein Identification and Bioassay of Chromatographic Fractions to Identify Biologically Active Substances from Complex Protein Sources

Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

Inhibition of c-Rel DNA binding is critical for the anti-inflammatory effects of novel PIKfyve inhibitor

Aberrant production of proinflammatory cytokines is linked to many autoimmune diseases, and their inhibition by small molecule compounds is considered beneficial. Here, we performed phenotypic screening in IFNγ/LPS-activated RAW264.7, mouse macrophage cells, and discovered AS2677131 and AS2795440 as novel and potent inhibitors of IL-12p40, a subunit of IL-23. Interestingly, these compounds exhibited unique pharmacological activities in their inhibition of the production of IL-12p40, IL-6 and IL-1β but not TNFα in activated macrophages or dendritic cells, and expression of IgM-induced MHC class II on B cells. To reveal these mechanisms, we synthesized two different activity probes which were structurally related to the AS compounds, and identified probe-specific binding proteins, including PIKfyve, a Class III PI kinase. The AS compounds inhibited PIKfyve activity and mimicked the properties of PIKfyve-deficient cells, eventually validating PIKfyve as target molecule. Regarding mechanism, AS2677131 regulated DNA binding activity of c-Rel on IL-12p40 and IL-1β promoter. As expected, a PIKfyve inhibitor prevented the development of arthritis in rats. Taken together, our findings of the novel and potent PIKfyve inhibitors AS2677131 and AS2795440 reveal the critical role of PIKfyve in proinflammatory cytokine production and B cell activation, and may indicate a potential new therapeutic option for treatment of inflammatory diseases.

Identification and relative quantitation of an orphan G-protein coupled receptor SREB2 (GPR85) protein in tissue using a linear ion trap mass spectrometer.

SREB2 (GPR85) is an orphan G-protein coupled receptor (GPCR) whose function is unknown. We previously prepared a SREB2-overexpressing transgenic mouse for functional analysis but were unable to confirm SREB2 protein expression level by immunochemical or biochemical methods. In this article, we report mass spectrometric identification and relative quantitative analysis of SREB2 in the forebrains of transgenic and wild type mice using nanoliquid chromatography coupled with a linear ion-trap mass spectrometer. By analyzing Chinese hamster ovary (CHO) cells overexpressing the SREB2 gene, we identified a proteotypic SREB2 peptide, GPTPPTLLGIR. Using a stable isotope-labeled analog as an authentic peptide for protein identification and as an internal control for relative quantitation, SREB2 was directly identified from the membrane fraction of forebrains from wild type and SREB2 transgenic mice. SREB2 protein expression level in the transgenic mouse was estimated to be 3-fold higher than that in the wild type littermate.

THU0541 Anti-Inflammatory Activity of A Novel Small Molecule Inhibitor of Pikfvye, A Class III PI Kinase

Background Aberrant production of proinflammatory cytokines is associated with various chronic inflammatory diseases, and their functional inhibition is considered beneficial. We previously reported the discovery of novel small molecule inhibitors of PIKfyve, AS2677131 and AS2795440. These compounds were found as IL-12p40 inhibitors by phenotypic screening in IFNγ/LPS-activated mouse macrophage cell line RAW264.7, but their pharmacological properties and therapeutic potential in chronic inflammation have not been fully explored. Objectives The aim of this study was to characterize in vitro and in vivo anti-inflammatory activity of a novel PIKfyve inhibitor, AS2795440. Methods In vitro and in vivo TLR ligand-induced cytokine production assays were conducted in RAW264.7 cells, mouse perioneal macrophages, and rat acute inflammation model. Concentrations of IL-12p40, IL-6, and TNFα were measured in culture supernatants or plasma by ELISA. B cell receptor (BCR)-mediated B cell activation activity was examined by measuring MHC class II expression in whole blood from rats treated with AS2795440. To evaluate therapeutic potential in chronic inflammation, rat adjuvant-induced arthritis (AIA) was examined. AS2795440 was daily administered from the day after adjuvant injection. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc. Results AS2795440 inhibited the production of TLR ligand-induced proinflammatory cytokines, IL-12p40 and IL-6 but not TNFα, and expression of IgM-induced MHC class II on B cells, dose-dependently. In a rat AIA, AS2795440 (1-10 mg/kg) significantly suppressed the paw swelling and bone destruction, dose-dependently. Conclusions These results indicate that AS2795440 is a potent PIKfyve inhibitor, which has unique anti-inflammatory activity, such as inhibition of proinflammatory cytokine production and B cell activation, and has a potential therapeutic benefit in chronic inflammatory diseases. Disclosure of Interest : M. Terajima Employee of: Astellas Pharma Inc., Y. Kaneko-Kobayashi Employee of: Astellas Pharma Inc., H. Mizuhara Employee of: Astellas Pharma Inc., T. Ishikawa Employee of: Astellas Pharma Inc., E. Imamura Employee of: Astellas Pharma Inc., K. Hattori Employee of: Astellas Pharma Inc., N. Nakamura Employee of: Astellas Pharma Inc., M. Yuri Employee of: Astellas Pharma Inc., Y. Higashi Employee of: Astellas Pharma Inc., N. Seki Employee of: Astellas Pharma Inc. DOI 10.1136/annrheumdis-2014-eular.1310

Abstract 4756: YM155 suppresses survivin expression by disrupting the ILF3/p54nrb transcription factor complex

Survivin, a member of the anti-apoptosis proteins family, is highly expressed in all primary tumor types, and is responsible for cancer progression and drug resistance in many types of cancer. YM155, a small chemical compound, selectively suppressed the expression of survivin and induced apoptosis in cancer cells both in vitro and in vivo. However, the mechanisms underlying the suppression of survivin expression by YM155 are unknown. In order to identify the molecular targets and analyze the drug mechanism of action, affinity purification was performed using an active analogue of YM155. We identified interleukin enhancer-binding factor 3 (ILF3) as the binding target of YM155. From the complex analysis of ILF3 and survivin promoter sequence, we also found that ILF3 forms a complex with p54nrb transcription factor, and then binds to the survivin promoter. Overexpression of ILF3 enhanced survivin promoter activity, which was attenuated by YM155 in a concentration-dependent manner. Furthermore, the ILF3/p54nrb complex was disrupted by YM155, thus dispersing the components in the nucleus.In conclusion, our study suggests that binding to ILF3 by YM155 causes the dissociation of the ILF3/p54nrb complex, thus inhibits ILF3 dependent survivin expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4756. doi:1538-7445.AM2012-4756