Hepatitis B virus evades the immune system by suppressing the NF-κB signaling pathway with DENND2A.

Overcoming hepatitis B virus (HBV) is a challenging problem because HBV deceives the host immune system. We have found that DENN domain-containing 2A (DENND2A) was essential for HBV maintenance, although its role remains unclear. In this study, we elucidate its function by screening a novel DENND2A-binding peptide, DENP4-3S. DENP4-3S exhibits homology to SAM and SH3 domain-containing protein 1 (SASH1), a scaffold protein involved in Toll-like receptor signaling that promotes proinflammatory cytokine production. We confirmed that DENND2A interacts with SASH1 specifically. Overexpression and knockdown experiments showed that overexpression of DENND2A suppressed the transcriptional activity of NF-κB, and the knockdown of DENND2A promoted it and the production of cytokines and interferons. Here, we constructed a fusion protein (10M-DEN3SN) consisting of an anti-asialoglycoprotein receptor antibody and DENP4-3S to deliver the peptide to hepatocytes specifically. 10M-DEN3SN inhibited the interaction between DENND2A and SASH1, and rescued SASH1 trapped by DENND2A, leading to the upregulation of NF-κB and its downstream signaling. In addition, 10M-DEN3SN suppressed HBV proliferation in PXB chimeric mice. These results with the DENND2A-binding peptide delivered into hepatocytes suggested the involvement of DENND2A, SASH, and NF-κB signaling pathway in the HBV infection and onset of hepatitis. In conclusion, this study indicates that HBV utilizes DENND2A and SASH1 to evade the immune system.IMPORTANCEHepatitis B virus (HBV) is a serious liver infection with no established cure, causing an abnormal host immune response. Here, we identified a novel peptide that interacts with DENN domain-containing 2A (DENND2A), a host factor essential for HBV maintenance. The resulting peptide showed sequence homology, revealing an interaction between DENND2A and the immune system regulator SASH1. This study suggests that DENND2A contributes to HBV infection by suppressing the cellular immune system by inhibiting SASH1. The DENND2A-binding peptide, incorporated into our hepatocyte-specific peptide delivery system, inhibited the DENND2A-SASH1 interaction and promoted the production of cytokines and interferons in cultured hepatocytes. As a consequence, the peptide suppressed HBV proliferation in humanized mice. We report new insights into the role of DENND2A and SASH1 in HBV maintenance and highlight the importance of the immune system.

Functional involvement of endothelial lipase in hepatitis B virus infection.

BACKGROUND: HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence. METHODS: The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane. RESULTS: We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells. CONCLUSIONS: LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity.

Hepatitis B Virus Utilizes a Retrograde Trafficking Route via the Trans-Golgi Network to Avoid Lysosomal Degradation.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified. METHODS: The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay. RESULTS: The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels. CONCLUSIONS: HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.

Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication.

Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.

A phenylphthalimide derivative, TC11, induces apoptosis by degrading MCL1 in multiple myeloma cells.

To date, the prognosis of multiple myeloma (MM) in patients harboring cytogenetic abnormalities (CA) involving t (4; 14) and deletion of chromosome 17 remains poor despite recent advances in drug development that include the use of immunomodulatory drugs (IMiDs) such as lenalidomide for MM. To address this issue, we have developed a novel phenylphthalimide derivative, TC11, that is structurally related to IMiDs. It remains unclear how TC11 induces apoptosis of MM cells with high-risk CA. Here, we show that TC11 does not induce degradation of CRBN's substrates, IKZF1/3 and CK1α, and induces apoptosis of CRBN-silenced MM; this effect was independent of the cereblon (CRBN) pathway, which is involved in the mechanism of action of IMiDs used for the treatment of MM. We also revealed that TC11, in contrast to existing IMiDs, induced degradation of MCL1 and activation of caspase-9. Furthermore, inhibition of CDK1 by CGP74514A prevented TC11-induced MCL1 degradation, caspase-9 activation, and the subsequent apoptotic cell death. We showed that ectopic MCL1 expression rescued apoptosis of MM. These observations suggest that TC11 induces apoptotic death caused by degradation of MCL1 during prolonged mitotic arrest. Therefore, our findings suggest that TC11 is a potential drug candidate for high-risk MM.

Novel Antibody-Drug Conjugate with Anti-CD26 Humanized Monoclonal Antibody and Transcription Factor IIH (TFIIH) Inhibitor, Triptolide, Inhibits Tumor Growth via Impairing mRNA Synthesis.

Here, we report a novel antibody drug conjugate (ADC) with the humanized anti-CD26 monoclonal antibody YS110 and triptolide (TR-1). YS110 has an inhibitory activity against the CD26-positive tumor growth via the immunological and direct pathway, such as intra-nuclear transportation of CD26 and YS110, and suppressed transcription of RNA polymerase II (Pol II) subunit POLR2A. The ADC conjugated with YS110 and an antitumor compound triptolide (TR-1), which is an inhibitor for TFIIH, one of the general transcription factors for Pol II was developed. YS110 and triptolide were crosslinked by the heterobifunctional linker succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and designated Y-TR1. Antitumor efficacy of Y-TR1 against malignant mesothelioma and leukemia cell lines were assessed by the in vitro cell viability assay and in vivo assay using xenografted mouse models. Y-TR1 showed significant cytotoxicity against CD26-positive cell lines but not CD26-negative counterparts in a dose-dependent manner via suppression of mRNA synthesis by impairment of the Pol II activity. The tumors in xenografted mice administered Y-TR1 was smaller than that of the unconjugated YS110 treated mice without severe toxicity. In conclusion, the novel compound Y-TR1 showed antitumor properties against CD26-positive cancer cell lines both in vitro and in vivo without toxicity. The Y-TR1 is a unique antitumor ADC and functions against Pol II.

How Does the Recently Discovered Peptide MIP Exhibit Much Higher Binding Affinity than an Anticancer Protein p53 for an Oncoprotein MDM2?

An oncoprotein MDM2 binds to the extreme N-terminal peptide region of a tumor suppressor protein p53 (p53NTD) and inhibits its anticancer activity. We recently discovered a peptide named MIP which exhibits much higher binding affinity for MDM2 than p53NTD. Experiments showed that the binding free energy (BFE) of MDM2-MIP is lower than that of MDM2-p53NTD by approximately -4 kcal/mol. Here, we develop a theoretical method which is successful in reproducing this quantitative difference and elucidating its physical origins. It enables us to decompose the BFE into a variety of energetic and entropic components, evaluate their relative magnitudes, and identify the physical factors driving or opposing the binding. It should be applicable also to the assessment of differences among ligands in the binding affinity for a particular receptor, which is a central issue in modern chemistry. In the MDM2 case, the higher affinity of MIP is ascribed to a larger gain of translational, configurational entropy of water upon binding. This result is useful to the design of a peptide possessing even higher affinity for MDM2 as a reliable drug against a cancer.

Seasonality of i.v. immunoglobulin responsiveness in Kawasaki disease.

BACKGROUND: Evidence suggests that seasonal variation in the onset of Kawasaki disease (KD) exists worldwide. Whether a seasonal component to successful i.v. immunoglobulin (IVIG) therapy exists in KD-positive children, however, is unknown. We addressed this question by focusing on patients with primary onset KD who were non-responsive to IVIG treatment, in the large nationwide Japanese KD survey datasets from 2009 to 2016. METHODS: In these datasets, the IVIG therapy non-responders were defined as patients whose fever persisted ≥24 h or recurred ≤24 h after the end of the initial IVIG treatment (dosage, 2,000 mg/kg). Those who successfully responded to this treatment were defined as IVIG responders. The consecutive monthly trend of the proportion of IVIG non-responders was analyzed throughout the study period to investigate seasonal periodicity on Fourier analysis, and the monthly distributions of non-responders and responders were compared. RESULTS: From a total of 113 691 KD-positive patients, 15.7% were IVIG non-responders, and 61% were male. The proportion of non-responders increased across each calendar year with fluctuation, and Fourier analysis indicated seasonal periodicity. The seasonality effect differed between responders and non-responders, with the proportion of responders tending to increase in autumn through winter, while the non-responders showed a decreasing trend in autumn. The seasonality effect tended to differ by sex. CONCLUSIONS: The results indicate that the currently unknown etiological agents of KD might differ between IVIG responders and non-responders. In addition, immune reactivity against such agents possibly differs by sex in the IVIG non-responders.

Nationwide epidemiologic survey of Kawasaki disease in Japan, 2015-2016.

BACKGROUND: Approximately 50 years have passed since Kawasaki disease (KD) was first reported. The KD nationwide survey began in 1970. Although >360 000 cases have already been reported in Japan, the cause is still unknown. In Japan, the number of patients and incidence rate of KD has continued to increase. It is necessary to examine the trend of the occurrence in the surveillance of KD. METHODS: The nationwide survey of patient incidence in 2015 and 2016 was conducted in 2017, as the 24th nationwide survey of KD. A questionnaire was sent to pediatric departments in hospitals with >100 beds and specialized pediatric hospitals, and was responded to by the attending pediatricians. RESULTS: The total number of patients in 2 years was 31 595, and the sex ratio (male/female) was 1.34. The incidence rate (/100 000 children aged 0-4 years/year) was 330.2 (371.2 in boys, 287.3 in girls) in 2015, and 309.0 (343.2 in boys, 273.2 in girls) in 2016. The number of patients by month peaked in January. The age-specific incidence rate according to sex was highest in children between 9 and 11 months of age, after which the incidence rate gradually decreased with advancing age. CONCLUSIONS: We summarize the most recent nationwide survey of KD and consider the change in the epidemiologic picture.

The effects of early intravenous immunoglobulin therapy for Kawasaki disease: The 22nd nationwide survey in Japan.

BACKGROUND: Although intravenous immunoglobulin (IVIG) therapy is the standard therapy for Kawasaki disease (KD) to prevent coronary aneurysms including dilatations, it is unclear whether early IVIG therapy is more efficient in the acute stage of KD. METHODS: We conducted a cohort study using data from the 22nd nationwide survey of KD in Japan from January 2011 to December 2012. We excluded patients with recurrent KD and whose first admission day was later than seven days from the onset of symptoms. Finally, 20,933 patients with echocardiography assessment and IVIG therapy were divided into three groups according to the start of the IVIG therapy: 1) early: ≤4 days, 2) conventional: 5-7 days, and 3) late: 8-10 days. Then we investigated whether the early IVIG therapy prevented coronary dilatation or aneurysm after multiple adjustments for age, sex, total amount of IVIG, use of steroids, infliximab, other immunosuppressive agents, and plasma exchange. RESULTS: After multiple adjustments, conventional therapy had similar risks for coronary dilatation or aneurysm compared with early therapy (odds ratio [OR]:0.95; 95% confidence interval [CI], 0.78-1.16), whereas late therapy had a higher risk (OR:1.66; 95% CI, 1.03-2.68). Other risk factors for coronary dilatation or aneurysm were young male, older age, use of steroids, infliximab, other immunosuppressive agents, and a larger amount of total IVIG. CONCLUSIONS: Early IVIG therapy for KD did not reduce the risk for coronary dilatation or aneurysm compared with conventional therapy. It is recommended to start IVIG therapy within 7 days from the onset of symptoms.

Epidemiological observations of Kawasaki disease in Japan, 2013-2014.

BACKGROUND: The etiology of Kawasaki disease (KD) is unknown. In Japan, the number of patients and incidence rate of KD has increased continuously since its discovery. The aim of this report was to analyze the latest nationwide epidemiological survey of KD in Japan. METHODS: The 23rd nationwide survey of KD was conducted in 2015. To report on all patients diagnosed with KD in 2013 and 2014, a questionnaire was sent to hospitals with ≥100 beds containing pediatric departments, as well as specialized pediatric hospitals. RESULTS: The number of KD patients reported was 15 696 in 2013 and 15 979 in 2014, resulting in an annual incidence rate of 302.5 and 308.0 per 100 000 population aged 0-4 years, respectively. The number of patients and incidence rate of KD in 2014 were the highest ever recorded in Japan. The number of patients diagnosed per month peaked in January, and a gradual increase in summer was also observed. Eight patients died of KD in 2013 and 2014. CONCLUSIONS: The number of patients and incidence rate of KD in Japan continue to increase. Continued surveillance of epidemiological trends of KD is therefore required.

A novel phenylphthalimide derivative, pegylated TC11, improves pharmacokinetic properties and induces apoptosis of high-risk myeloma cells via G2/M cell-cycle arrest.

Despite the development of new drugs for multiple myeloma (MM), the prognosis of MM patients with high-risk cytogenetic abnormalities such as t (4; 14) and del17p remains poor. We reported that a novel phenylphthalimide derivative, TC11, induced apoptosis of MM cells in vitro and in vivo, and TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1). However, TC11 showed low water solubility and poor pharmacokinetic properties. Here we synthesized a water-soluble TC11-derivative, PEG(E)-TC11, in which HOEtO-TC11 is pegylated with PEG through an ester bond, and we examined its anti-myeloma activity. We observed that PEG(E)-TC11 and its hydrolyzed product, HOEtO-TC11, induced G2/M arrest and the apoptosis of MM cells. Intraperitoneal administration of PEG(E)-TC11 to xenografted mice revealed improved pharmacokinetic properties and significantly delayed tumor growth. TC11 and its derivatives did not bind to cereblon (CRBN), which is a responsible molecule for thalidomide-induced teratogenicity. These results suggest that PEG(E)-TC11 is a good candidate drug for treating high-risk MM.

Temporal and geographical clustering of Kawasaki disease in Japan: 2007-2012.

BACKGROUND: Since 1987, no study has reported the municipal-level geographical clustering of Kawasaki disease (KD) in Japan. Therefore, the aim of the present study was to identify the temporal and municipal-level geographical clustering of KD. METHODS: The annual incidence rates of KD for each municipality were calculated using nationwide data from 73 758 patients with KD (2007-2012). To determine whether temporal and municipal-level clustering existed, we calculated the correlations of the annual incidence rates for each municipality during the study years, and compared these rates with those of the adjacent municipalities. Spatial scanning analysis was used to identify the geographical clusters for each year, and the incidence rates in those clusters were compared with the rates in the surrounding region. RESULTS: The annual national incidence rate of KD, adjusted for the prefecture-specific response rate, was 322.45 patients per 100 000 children aged 0-4 years. The correlation between the annual incidence rates during 2 consecutive years was significantly positive (coefficients, 0.149-0.428). On spatial scanning analysis, the most likely clusters were in the Tokyo metropolitan area during 2007-2010 and 2012, and in Kumamoto prefecture during 2011. CONCLUSION: Kawasaki disease exhibits temporal and municipal-level clustering.

PURE mRNA display for in vitro selection of single-chain antibodies.

mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of the mRNA-protein conjugates was quite low. To establish an efficient platform for the PURE mRNA display of scFv, we performed affinity selection of a library of scFv antibodies with a C-terminal random sequence and obtained C-terminal sequences that increased the formation of mRNA-protein conjugates. We also identified unexpected common substitution mutations around the start codon of scFv antibodies, which were inferred to destabilize the mRNA secondary structure. This destabilization causes an increase in protein expression and the efficiency of the formation of mRNA-protein conjugates. We believe these improvements should make the PURE mRNA display more efficient for selecting antibodies for diagnostic and therapeutic applications.

Catalytic subunits of the phosphatase calcineurin interact with NF-κB-inducing kinase (NIK) and attenuate NIK-dependent gene expression.

Nuclear factor (NF)-κB-inducing kinase (NIK) is a serine/threonine kinase that activates NF-κB pathways, thereby regulating a wide variety of immune systems. Aberrant NIK activation causes tumor malignancy, suggesting a requirement for precise regulation of NIK activity. To explore novel interacting proteins of NIK, we performed in vitro virus screening and identified the catalytic subunit Aα isoform of serine/threonine phosphatase calcineurin (CnAα) as a novel NIK-interacting protein. The interaction of NIK with CnAα in living cells was confirmed by co-immunoprecipitation. Calcineurin catalytic subunit Aß isoform (CnAß) also bound to NIK. Experiments using domain deletion mutants suggested that CnAα and CnAß interact with both the kinase domain and C-terminal region of NIK. Moreover, the phosphatase domain of CnAα is responsible for the interaction with NIK. Intriguingly, we found that TRAF3, a critical regulator of NIK activity, also binds to CnAα and CnAß. Depletion of CnAα and CnAß significantly enhanced lymphotoxin-ß receptor (LtßR)-mediated expression of the NIK-dependent gene Spi-B and activation of RelA and RelB, suggesting that CnAα and CnAß attenuate NF-κB activation mediated by LtßR-NIK signaling. Overall, these findings suggest a possible role of CnAα and CnAß in modifying NIK functions.

Cardiac lesions and initial laboratory data in Kawasaki disease: a nationwide survey in Japan.

BACKGROUND: Cardiac lesions, such as coronary dilatation, aneurysms, narrowing, myocardial infarction, and valvular lesions, sometimes occur in Kawasaki disease, but most studies have only evaluated cardiac lesions in the later phase of the disease. This study was undertaken to clarify the related factors between cardiac lesions and laboratory data in the initial phase of Kawasaki disease. METHODS: We conducted a cross-sectional study using data for 26 691 patients from the 22nd nationwide survey of Kawasaki disease in Japan, the observation period of which was from January 2011 through December 2012. We excluded patients with recurrent Kawasaki disease and who were more than seven days from the start of symptoms at admission. We analyzed 23 155 cases (13 353 boys; mean age: 923 ± 734 days) with available laboratory data for white blood cell count, platelet count, serum albumin, and C-reactive protein (CRP). RESULTS: Cardiac lesions were detected in 984 cases (656 boys and 328 girls); lesions were classified as coronary dilatation (764 cases), coronary aneurysm (40), giant coronary aneurysm (6), coronary narrowing (3), and valvular lesions (204). The significant related factors of initial coronary dilatation were male sex (odds ratio [OR] 1.73), older age (OR per 100 days increase 1.03), higher platelet count (OR per 10 000 cells/µL increase 1.006), lower albumin (OR per 1 g/dL increase 0.66), and higher CRP (OR per 1 mg/dL increase 1.02). The factors related to coronary aneurysm were higher platelet count (OR 1.01) and lower albumin (OR 0.34). No factors were significantly related to giant coronary aneurysm. The related factors of valvular lesions were age (OR 0.98), and higher CRP (OR 1.05). CONCLUSIONS: Clinicians should consider male sex, older age, higher platelet count, lower albumin levels, and higher CRP levels when assessing risk of cardiac lesions in the initial phase of Kawasaki disease.

Descriptive epidemiology of Kawasaki disease in Japan, 2011-2012: from the results of the 22nd nationwide survey.

BACKGROUND: The number of patients and incidence rate of Kawasaki disease (KD) are increasing in Japan. We have therefore characterized the latest epidemiological information on KD. METHODS: The 22nd nationwide survey of KD, which targeted patients diagnosed with KD in 2011 and 2012, was conducted in 2013 and included a total of 1983 departments and hospitals. In order to report on all patients with KD during the 2 survey years, we targeted hospitals of 100 beds or more with pediatric departments, or specialized pediatric hospitals. RESULTS: From a total of 1420 hospitals and departments (71.6% response rate), 26,691 KD patients were reported (12,774 in 2011 and 13,917 in 2012; 15,442 males and 11,249 females). The annual incidence rates were 243.1 per 100,000 population aged 0 to 4 years in 2011 and 264.8 in 2012. The number of cases of KD recorded in 2012 was the highest ever reported in Japan. The incidence rate of complete cases was also the highest ever reported in Japan and contributed to the increase in the rate of total cases in recent years. The number of patients diagnosed per month peaked in January, and additional peaks were noted during summer months, although these peaks were lower than those seen in winter. Age-specific incidence rate showed a monomodal distribution with a peak in the latter half of the year in which patients were born. CONCLUSIONS: The number of patients and the incidence rate of KD in Japan continue to increase. A similar trend has also been seen for patients with complete KD.

A novel phthalimide derivative, TC11, has preclinical effects on high-risk myeloma cells and osteoclasts.

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 µM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.

Structural basis for inhibition of the MDM2:p53 interaction by an optimized MDM2-binding peptide selected with mRNA display.

The oncoprotein MDM2 binds to tumor suppressor protein p53 and inhibits its anticancer activity, which leads to promotion of tumor cell growth and tumor survival. Abrogation of the p53:MDM2 interaction reportedly results in reactivation of the p53 pathway and inhibition of tumor cell proliferation. We recently performed rigorous selection of MDM2-binding peptides by means of mRNA display and identified an optimal 12-mer peptide (PRFWEYWLRLME), named MDM2 Inhibitory Peptide (MIP), which shows higher affinity for MDM2 (and also its homolog, MDMX) and higher tumor cell proliferation suppression activity than known peptides. Here we determined the NMR solution structure of a MIP-MDM2 fusion protein to elucidate the structural basis of the tight binding of MIP to MDM2. A region spanning from Phe3 to Met11 of MIP forms a single α-helix, which is longer than those of the other MDM2-binding peptides. MIP shares a conserved Phe3-Trp7-Leu10 triad, whose side chains are oriented towards and fit into the hydrophobic pockets of MDM2. Additionally, hydrophobic surface patches that surround the hydrophobic pockets of MDM2 are covered by solvent-exposed MIP residues, Trp4, Tyr6, and Met11. Their hydrophobic interactions extend the interface of the two molecules and contribute to the strong binding. The potential MDM2 inhibition activity observed for MIP turned out to originate from its enlarged binding interface. The structural information obtained in the present study provides a road map for the rational design of strong inhibitors of MDM2:p53 binding.

NESmapper: accurate prediction of leucine-rich nuclear export signals using activity-based profiles.

The nuclear export of proteins is regulated largely through the exportin/CRM1 pathway, which involves the specific recognition of leucine-rich nuclear export signals (NESs) in the cargo proteins, and modulates nuclear-cytoplasmic protein shuttling by antagonizing the nuclear import activity mediated by importins and the nuclear import signal (NLS). Although the prediction of NESs can help to define proteins that undergo regulated nuclear export, current methods of predicting NESs, including computational tools and consensus-sequence-based searches, have limited accuracy, especially in terms of their specificity. We found that each residue within an NES largely contributes independently and additively to the entire nuclear export activity. We created activity-based profiles of all classes of NESs with a comprehensive mutational analysis in mammalian cells. The profiles highlight a number of specific activity-affecting residues not only at the conserved hydrophobic positions but also in the linker and flanking regions. We then developed a computational tool, NESmapper, to predict NESs by using profiles that had been further optimized by training and combining the amino acid properties of the NES-flanking regions. This tool successfully reduced the considerable number of false positives, and the overall prediction accuracy was higher than that of other methods, including NESsential and Wregex. This profile-based prediction strategy is a reliable way to identify functional protein motifs. NESmapper is available at http://sourceforge.net/projects/nesmapper.