Takayuki Hamasaki

Modulation of innate and adaptive immunity by biodegradable nanoparticles.

Vaccine strategy needs efficient adjuvants to induce potent antigen-specific immune responses by targeting antigens to antigen presenting cells followed by their functional maturation. In this study, biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) were examined for their immunological activities in mice. Like lipopolysaccharide, gamma-PGA NPs strongly activated spleen dendritic cells (DCs) and induced their cytokine production and costimulatory molecule expression through the nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways. The immunization of mice with ovalbumin-carrying gamma-PGA NPs could induce the antigen-specific and long-lived effector and central memory CD8(+) T cells as well as antibody responses. Thus, gamma-PGA NPs have great potential as an efficient antigen carrier and strong adjuvant to DCs.

Highly Enhanced Expression of CD70 on Human T-Lymphotropic Virus Type 1-Carrying T-Cell Lines and Adult T-Cell Leukemia Cells

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). In Japan, the number of HTLV-1 carriers is estimated to be 1.2 million and more than 700 cases of ATL have been diagnosed every year. Considering the poor prognosis and lack of curative therapy of ATL, it seems mandatory to establish an effective strategy for the treatment of ATL. In this study, we attempted to identify the cell surface molecules that will become suitable targets of antibodies for anti-ATL therapy. The expression levels of approximately 40,000 host genes of three human T-cell lines carrying HTLV-1 genomes were analyzed by oligonucleotide microarray and compared with the expression levels of the genes in an HTLV-1-negative T-cell line. The HTLV-1-carrying T-cell lines used for experiments had totally different expression patterns of viral genome. Among the genes evaluated, the expression levels of 108 genes were found to be enhanced more than 10-fold in all of the T-cell lines examined and 11 of the 108 genes were considered to generate the proteins expressed on the cell surface. In particular, the CD70 gene was upregulated more than 1,000-fold and the enhanced expression of the CD70 molecule was confirmed by laser flow cytometry for various HTLV-1-carrying T-cell lines and primary CD4+ T cells isolated from acute-type ATL patients. Such expression was not observed for primary CD4+ T cells isolated from healthy donors. Since CD70 expression is strictly restricted in normal tissues, such as highly activated T and B cells, CD70 appears to be a potential target for effective antibody therapy against ATL.

Modulation of Gene Expression Related to Toll-Like Receptor Signaling in Dendritic Cells by Poly(γ-Glutamic Acid) Nanoparticles

ABSTRACT Poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) have previously been reported as an efficient antigen delivery system with adjuvant activity. In this study, the gene expression in murine bone marrow-derived dendritic cells (DCs) treated with γ-PGA NPs was examined by oligonucleotide microarray analysis and compared with that in cells treated with other adjuvants. The gene expression of proinflammatory chemokines, cytokines, and costimulatory molecules was upregulated considerably in DCs treated with γ-PGA NPs. The upregulation pattern was similar to that in DCs treated with lipopolysaccharide (LPS) but not to that in DCs treated with unparticulate γ-PGA. The activation of DCs by γ-PGA NPs was confirmed by real-time reverse transcriptase PCR (RT-PCR) analysis of genes related to Toll-like receptor (TLR) signaling. The effect of γ-PGA NPs on DCs was not annihilated by treatment with polymyxin B, an inhibitor of LPS. Furthermore, the immunization of mice with γ-PGA NPs carrying ovalbumin (OVA) as an antigen significantly induced antigen-specific CD8+ T cells and antigen-specific production of interleukin-2, tumor necrosis factor alpha, and gamma interferon from the cells. Such activities of γ-PGA NPs were more potent than those obtained with immunization with OVA plus aluminum hydroxide or OVA plus complete Freunds adjuvant. These results suggest that γ-PGA NPs induce a CD8+ T-cell response by activating innate immunity in a fashion different from that of LPS. Thus, γ-PGA NPs may be an attractive candidate to be developed further as a vaccine adjuvant.

Highly potent and selective inhibition of bovine viral diarrhea virus replication by γ-carboline derivatives

Several novel γ-carboline derivatives were identified as selective inhibitors of bovine viral diarrhea virus (BVDV) replication in cell cultures. Among them, 3,4,5-trimethyl-γ-carboline (SK3M4M5M) was the most active against BVDV (Nose strain) in MDBK cells, with a 50% effective concentration of 0.017±0.005μM and a selectivity index of 435. The compound inhibited viral RNA synthesis in a dose-dependent fashion. In a time of drug-addition experiment during a single viral replication cycle, SK3M4M5M lost its antiviral activity when first added at 8h or later after infection, which coincides with the onset of viral RNA synthesis. When selected γ-carboline derivatives, including SK3M4M5M, were examined for their inhibitory effect on the mutant strains resistant to some classes of nonnucleoside BVDV RNA-dependent RNA polymerase inhibitors, all of which target the top of the finger domain of the polymerase, the strains displayed cross-resistance to the γ-carboline derivatives. These results indicate that the γ-carboline derivatives may possibly target a hot spot of the RNA-dependent RNA polymerase. Although SK3M4M5M was highly active against BVDV, the compound proved inactive against hepatitis C virus (HCV) in HCV RNA replicon cells.

Identification of Novel Inhibitors of Human Immunodeficiency Virus Type 1 Replication by In Silico Screening Targeting Cyclin T1/Tat Interaction

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) transcription is essential for viral replication and the only step for viral genome amplification. Cyclin T1 (CycT1) interacts with HIV-1 Tat and transactivation-responsive (TAR) RNA, leading to the activation of viral transcription through the hyperphosphorylation of RNA polymerase II (RNAPII). Thus, the CycT1/Tat/TAR RNA interaction represents a novel target for inhibition of HIV-1 replication. In this study, we conducted in silico screening of compounds targeting the CycT1/Tat/TAR RNA complex and found that two structurally related compounds (C1 and C2) had high docking scores for a model of the complex. These compounds proved inhibitory to HIV-1 replication in tumor necrosis factor alpha-stimulated chronically infected cells. In addition, C3, a derivative of C1 and C2, was found to be a more potent inhibitor of HIV-1 replication in chronically infected cells. C3 also inhibited HIV-1 replication in acutely infected cells. The compound could suppress Tat-mediated HIV-1 long terminal repeat-driven gene expression and phosphorylation of RNAPII through inhibition of Tat binding to CycT1. Furthermore, the docking pose of C3 was defined by analyses for its in silico docking energy and in vitro antiviral activity, which indicates that C3 interacts with Tat-binding amino acids of CycT1. Thus, a series of compounds described herein are novel inhibitors of HIV-1 transcription through inhibition of CycT1/Tat interaction.

Discovery of tetrahydrotetramethylnaphthalene analogs as adult T-cell leukemia cell-selective proliferation inhibitors in a small chemical library constructed based on multi-template hypothesis

Adult T cell leukemia (ATL), caused by infection of human T-lymphotropic virus type 1 (HTLV-1), has a poor prognosis and curative therapy is unavailable, so it is important to find or design superior lead compounds for the drug treatment of ATL. We used our micro-reversed fragment-based drug design hypothesis and multi-template hypothesis to extract the tetrahydrotetramethylnaphthalene (TMN) skeleton from tamibarotene, a useful medicament for the treatment of acute promyelocytic leukemia (APL). Structural development of TMN yielded highly ATL cell-selective growth inhibitors, including 2-acetyl-3-hydroxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalene (6). Structure-activity relationship analysis suggests the existence of a specific target molecule for ATL cell-selective inhibition of proliferation through G2 arrest.

Potent and selective inhibition of hepatitis C virus replication by novel phenanthridinone derivatives.

A number of novel phenanthridinone derivatives were examined for their inhibitory effect on hepatitis C virus (HCV) replication in Huh-7 cells harboring self-replicating subgenomic viral RNA replicons with a luciferase reporter (LucNeo#2). The activity of compounds was further confirmed by inhibition of viral RNA copy number in different subgenomic and full-genomic replicon cells using real-time reverse transcription polymerase chain reaction. Among the compounds, 4-butyl-11-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-7-methoxy-[1,3]dioxolo[4,5-c]phenanthridin-5(4H)-one (HA-719) was found to be the most active with a 50% effective concentration of 0.063 ± 0.010 μM in LucNeo#2 cells. The compound did not show apparent cytotoxicity to the host cells at concentrations up to 40 μM. Western blot analysis demonstrated that HA-719 reduced the levels of NS3 and NS5A proteins in a dose-dependent fashion in the replicon cells. Interestingly, the phenanthridinone derivatives including HA-719 were less potent inhibitors of JFH1 strain (genobtype 2a HCV) in cell-free virus infection assay. Although biochemical assays revealed that HA-719 proved not to inhibit NS3 protease or NS5B RNA polymerase activity at the concentrations capable of inhibiting viral replication, their molecular target (mechanism of inhibition) remains unknown. Considering the fact that most of the anti-HCV agents currently approved or under clinical trials are protease and polymerase inhibitors, the phenanthridinone derivatives are worth pursuing for their mechanism of action and potential as novel anti-HCV agents.

Synthesis of 1-benzyl-3-(3,5-dimethylbenzyl)uracil derivatives with potential anti-HIV activity

Background: Nine novel uracil analogues were synthesized and evaluated as inhibitors of HIV-1. Methods: Key structural modifications included replacement of the 6-chloro group of 1-benzyl-6-chloro-3-(3,5-dimethylbenzyl)uracil by other functional groups or N1-alkylation of 3-(3,5-dimethylbenzyl)-5-fluorouracil. Results: These compounds showed only micromolar potency against HIV-1 in MT-4, though two of them; 6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil were highly potent (half maximal effective concentration =0.067 and 0.069 μM) and selective (selectivity index =685 and 661), respectively. Structure–activity relationships among the newly synthesized uracil analogues suggest the importance of the H-bond formed between 6-amino group of 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil and amide group of HIV-1 reverse transcriptase. Conclusions: We discovered two 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl) uracils, (6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil) as novel anti-HIV agents. These compounds should be further pursued for their toxicity and pharmacokinetics in vivo as well as antiviral activity against non-nucleoside reverse transcriptase inhibitor-resistant strains.

Impact of Novel Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutations P119S and T165A on 4′-Ethynylthymidine Analog Resistance Profile

ABSTRACT 2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T), a derivative of stavudine (d4T), has potent activity against human immunodeficiency virus and is much less inhibitory to mitochondrial DNA synthesis and cell growth than its progenitor, d4T. 4′-Ed4T triphosphate was a better reverse transcriptase (RT) inhibitor than d4T triphosphate, due to the additional binding of the 4′-ethynyl group at a presumed hydrophobic pocket in the RT active site. Previous in vitro selection for 4′-Ed4T-resistant viral strains revealed M184V and P119S/T165A/M184V mutations on days 26 and 81, respectively; M184V and P119S/T165A/M184V conferred 3- and 130-fold resistance to 4′-Ed4T, respectively. We investigated the relative contributions of these mutations, engineered into the strain NL4-3 background, to drug resistance, RT activity, and viral growth. Viral variants with single RT mutations (P119S or T165A) did not show resistance to 4′-Ed4T; however, M184V and P119S/T165A/M184V conferred three- and fivefold resistance, respectively, compared with that of the wild-type virus. The P119S/M184V and T165A/M184V variants showed about fourfold resistance to 4′-Ed4T. The differences in the growth kinetics of the variants were not more than threefold. The purified RT of mutants with the P119S/M184V and T165A/M184V mutations were inhibited by 4′-Ed4TTP with 8- to 13-fold less efficiency than wild-type RT. M184V may be the primary resistance-associated mutation of 4′-Ed4T, and P119S and T165A are secondary mutations. On the basis of our findings and the results of structural modeling, a virus with a high degree of resistance to 4′-Ed4T (e.g., more than 50-fold resistance) will be difficult to develop. The previously observed 130-fold resistance of the virus with P119S/T165A/M184V to 4′-Ed4T may be partly due to mutations both in the RT sequence and outside the RT sequence.

Anti-human immunodeficiency virus type 1 activity of novel 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl)uracil derivatives.

ABSTRACT Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. In screening of chemical libraries, we found 6-azido-1-benzyl-3-(3,5-dimethylbenzyl)uracil (AzBBU) and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl)uracil (AmBBU) to be highly active and selective inhibitors of HIV-1 replication in vitro. To determine the resistance profiles of these compounds, we conducted a long-term culture of HIV-1-infected MT-4 cells with escalating concentrations of each compound. After serial passages of the infected cells, escape viruses were obtained, and they were more than 500-fold resistant to the uracil derivatives compared to the wild type. Sequence analysis was conducted for RT of the escape viruses at passages 12 and 24. The amino acid mutation Y181C in the polymerase domain of RT was detected for all escape viruses. Docking studies using the crystal structure of RT showed that AmBBU requires the amino acid residues Leu100, Val106, Tyr181, and Trp229 for exerting its inhibitory effect on HIV-1. Four additional amino acid changes (K451R, R461K, T468P, and D471N) were identified in the RNase H domain of RT; however, their precise role in the acquisition of resistance is still unclear. In conclusion, the initial mutation Y181C seems sufficient for the acquisition of resistance to the uracil derivatives AzBBU and AmBBU. Further studies are required to determine the precise role of each mutation in the acquisition of HIV-1 resistance.