Yasuaki Shimizu

ASP2151, a novel helicase-primase inhibitor, possesses antiviral activity against varicella-zoster virus and herpes simplex virus types 1 and 2

OBJECTIVES To evaluate and describe the anti-herpesvirus effect of ASP2151, amenamevir, a novel non-nucleoside oxadiazolylphenyl-containing herpesvirus helicase-primase complex inhibitor. METHODS The inhibitory effect of ASP2151 on enzymatic activities associated with a recombinant HSV-1 helicase-primase complex was assessed. To investigate the effect on viral DNA replication, we analysed viral DNA in cells infected with herpesviruses [herpes simplex virus (HSV), varicella-zoster virus (VZV) and human cytomegalovirus]. Sequencing analyses were conducted on an ASP2151-resistant VZV mutant. In vitro and in vivo antiviral activities were evaluated using a plaque reduction assay and an HSV-1-infected zosteriform-spread model in mice. RESULTS ASP2151 inhibited the single-stranded DNA-dependent ATPase, helicase and primase activities associated with the HSV-1 helicase-primase complex. Antiviral assays revealed that ASP2151, unlike other known HSV helicase-primase inhibitors, exerts equipotent activity against VZV, HSV-1 and HSV-2 through prevention of viral DNA replication. Further, the anti-VZV activity of ASP2151 (EC(50), 0.038-0.10 microM) was more potent against all strains tested than that of aciclovir (EC(50), 1.3-27 microM). ASP2151 was also active against aciclovir-resistant VZV. Amino acid substitutions were found in helicase and primase subunits of ASP2151-resistant VZV. In a mouse zosteriform-spread model, ASP2151 was orally active and inhibited disease progression more potently than valaciclovir. CONCLUSIONS ASP2151 is a novel herpes helicase-primase inhibitor that warrants further investigation for the potential treatment of both VZV and HSV infections.

Characterization of YM-58483/BTP2, a novel store-operated Ca2+ entry blocker, on T cell-mediated immune responses in vivo

YM-58483/BTP2 is a blocker of store-operated Ca2+ entry (SOCE), which regulates the activation of non-excitable cells such as lymphocytes. YM-58483 has been reported to inhibit cytokine production and proliferation in T cells, and to be useful as a probable medicinal candidate for treatment of bronchial asthma. The present study investigated the pharmacological profile and therapeutic potential of YM-58483 in relation to cell-mediated immune responses. In the mouse graft-versus-host disease (GVHD) model, YM-58483 (1-30 mg/kg, p.o.) and cyclosporine A (1-30 mg/kg, p.o.) inhibited donor anti-host cytotoxic T lymphocyte (CTL) activity and IFN-gamma production, and also reduced the number of donor T cells, especially donor CD8+ T cells, in the spleen. YM-58483 and cyclosporine A inhibited T cell proliferation in a one-way mixed lymphocyte reaction (MLR) with IC50 values of 330 and 12.7 nM, respectively. Additionally, YM-58483 (1-10 mg/kg, p.o.) and cyclosporine A (2, 10 mg/kg, p.o.) inhibited the sheep red blood cell (SRBC)-induced delayed type hypersensitivity (DTH) response. These results suggest that the inhibition of SOCE leads to the prevention of antigen-induced T cell responses, which participate in autoimmune diseases such as autoimmune hepatitis and rheumatoid arthritis.

Stabilization of androgen receptor protein is induced by agonist, not by antagonists ☆

The action of nuclear receptor ligands in target tissues is specified mainly by the expression levels of their cognate nuclear receptors. The expression levels of these receptors are controlled through transcriptional and post-transcriptional events. Among post-transcriptional events, the effect of ligand on nuclear receptor protein turnover still remains largely unknown. Therefore, we studied the effects of agonist and antagonists on the turnover of the human androgen receptor (hAR) protein in stably transformed Chinese hamster ovary cells expressing exogenous hAR. Western blot analysis showed that the most potent androgen, dihydrotestosterone (DHT), stabilizes hAR with the induction of the transactivation function of hAR. However, this androgen-induced stabilization of hAR protein was abrogated by well-known androgen antagonists, hydroxyflutamide and bicalutamide (BIC), with inhibition of the transactivation function of hAR. Thus, the present study suggests that androgen antagonists exert their effects through, at least in part, abrogating the agonist-induced stabilization of hAR protein as well as blocking the ligand-induced transactivation function of hAR.

Establishment of an in vitro assay system for screening hepatitis C virus protease inhibitors using high performance liquid chromatography.

The hepatitis C virus (HCV) genome contains the code for a conserved, serine-type protease, called NS3, for the processing of the non-structural protein region of the viral polyproteins. Furthermore, a related protein, NS4A, is an effector or cofactor of NS3 protease activity in the cleavage of NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. To establish an in vitro assay system for the screening of those enzyme inhibitors that inhibit the protease NS3-4A, we prepared a maltose-binding protein-NS3-NS4A fusion protein and a synthetic peptide substrate that mimics the NS5A-5B junction. Cleavage of the synthetic peptide was analyzed by reversed-phase high performance liquid chromatography (HPLC). We showed that the enzymatic activity of the NS3-NS4A fusion protein was enhanced in comparison to the NS3 protein alone. The assay conditions for optimum NS3-4A protease activity were determined to be pH 7.6 and 37 degrees C. In addition, we evaluated several protease inhibitors using the same HPLC assay system. The activity of HCV protease NS3-4A was inhibited by 2714.4 microM diisopropyl fluorophosphate, 270.8 microM N-tosyl-L-lysyl chloromethyl ketone, and 825.5 microM chymostatin. The results of the present study indicated that the synthetic peptide substrate and HPLC assay system are suitable for studying HCV protease activity and may facilitate the development of anti-HCV therapeutic reagents.

Antinociceptive Effects of AS1892802, a Novel Rho Kinase Inhibitor, in Rat Models of Inflammatory and Noninflammatory Arthritis

Rho kinase (ROCK) is involved in various physiological functions, including cell motility, vasoconstriction, and neurite extension. Although a functional role of ROCK in nociception in the central nervous tissue has been reported in neuropathy, the peripheral function of this protein in hyperalgesia is not known. In this study, antinociceptive effects of AS1892802 [1-[(1S)-2-hydroxy-1-phenylethyl]-3-[4-(pyridin-4-yl)phenyl]urea], a novel and highly selective ROCK inhibitor, were investigated in two rat models of arthritis. Orally administered AS1892802 exhibited potent antinociceptive effect in both an adjuvant-induced arthritis (AIA) model (inflammatory arthritis model) and a monoiodoacetate-induced arthritis (MIA) model (noninflammatory arthritis model), with an ED50 of 0.15 mg/kg (MIA model). Fasudil, a ROCK inhibitor, and tramadol were also effective in both models; however, diclofenac was effective only in the AIA model. The onset of antinociceptive effect of AS1892802 was as fast as those of tramadol and diclofenac. AS1892802 did not induce gastric irritation or abnormal behavior. Because AS1892802 rarely penetrates the central nervous tissue and is also effective by intra-articular administration, it seemed to function peripherally. These results suggest that AS1892802 has an attractive analgesic profile for the treatment of severe osteoarthritis pain.

Structural Basis for the Interaction of CCR5 with a Small Molecule, Functionally Selective CCR5 Agonist

The chemokine receptor CCR5 is an attractive target for HIV-1 drug development, as individuals whose cells lack surface CCR5 expression are highly resistant to HIV-1 infection. CCR5 ligands, such as CCL5/RANTES, effectively inhibit HIV-1 infection by competing for binding opportunities to the CCR5 and inducing its internalization. However, the inherent proinflammatory activity of the chemotactic response of CCR5 ligands has limited their clinical use. In this study, we found that a novel small molecule, functionally selective CCR5 agonist, 2,2-dichloro-1-(triphenylphosphonio)vinyl formamide perchlorate (YM-370749), down-modulates CCR5 from the cell surface without inducing a chemotactic response and inhibits HIV-1 replication. In molecular docking studies of YM-370749 and a three-dimensional model of CCR5 based on the rhodopsin crystal structure as well as binding and functional studies using various CCR5 mutants, the amino acid residues necessary for interaction with YM-370749 were marked. These results provide a structural basis for understanding the activation mechanism of CCR5 and for designing functionally selective agonists as a novel class of anti-HIV-1 agents.

ASP3258, an orally active potent phosphodiesterase 4 inhibitor with low emetic activity.

We investigated the pharmacology of a novel phosphodiesterase (PDE) 4 inhibitor, ASP3258 (3-[4-(3-chlorophenyl)-1-ethyl-7-methyl-2-oxo-1,2-dihydro-1,8-naphthyridin-3-yl] propanoic acid), comparing its potency with that of the most advanced PDE4 inhibitors, roflumilast and cilomilast. PDE4 inhibition by ASP3258 (IC(50)=0.28nM) was as potent as that achieved with roflumilast. ASP3258 inhibited lipopolysaccharide-induced tumor necrosis factor (TNF)-α production in rat whole blood cells (IC(50)=8.8 nM) and rat alveolar macrophages (IC(50)=2.6 nM). Orally administered ASP3258, roflumilast, and cilomilast dose-dependently inhibited production of interleukin-4, TNF-α, and cysteinyl leukotrienes, as well as leukocyte infiltration in bronchoalveolar lavage fluid from the airways of ovalbumin-sensitized Brown Norway rats, and these compounds showed almost complete inhibition at doses of 3, 3, and 30 mg/kg, respectively. PDE4 inhibitors induce emesis by mimicking the pharmacological action of α(2)-adrenoceptor antagonist. However, orally administered roflumilast (3mg/kg) and cilomilast (10mg/kg), but not ASP3258 (3mg/kg), inhibited α(2)-adrenoceptor agonist-induced anesthesia in rats and induced emesis in ferrets. Although ASP3258 (3mg/kg) inhibited airway inflammation completely, it had no emetic activity. As such, this compound may be useful in treating airway inflammatory diseases such as asthma and COPD.

YM-341619 suppresses the differentiation of spleen T cells into Th2 cells in vitro, eosinophilia, and airway hyperresponsiveness in rat allergic models

T helper (Th) 2 cells play a central role in the pathogenesis of allergic diseases such as allergic asthma, atopic dermatitis, and allergic rhinitis. We have found that YM-341619 hydrochloride, which suppressed IL-4-induced STAT6-dependent reporter gene expression, inhibited the differentiation of mouse spleen T cells into Th2 cells in vitro. YM-341619 suppressed the production of IL-4 and the expression of GATA-3 mRNA, a Th2 transcription factor, in T cells cultured with anti-CD3 antibody and anti-CD28 antibody in the presence of IL-4. In contrast, the production of IFN-gamma and the expression of T-bet mRNA, a Th1 transcription factor, in T cells cultured with anti-CD3 antibody in the presence of IL-12, were not effected by YM-341619. Orally administered YM-341619 (0.003-0.03 mg/kg) reduced the plasma IgE level of DNP-Ascaris-sensitized rats, but not the IgG(2a) level. YM-341619 suppressed IL-4 and IL-13 production in the splenocytes of these DNP-Ascaris-sensitized rats without augmenting IFN-gamma production. YM-341619 also dose-dependently suppressed eosinophil accumulation in the lung (0.003-3 mg/kg, p.o.) and airway hyperresponsiveness (0.3-3 mg/kg, p.o.) induced by repeated exposure to ovalbumin in ovalbumin-sensitized rats. These results suggest that YM-341619 has the ability to suppress allergen-induced Th2 responses by selectively inhibiting the differentiation of CD4(+) T cells into the Th2 subset.

High-Throughput Screening of Low Molecular Weight NS3-NS4A Protease Inhibitors Using a Fluorescence Resonance Energy Transfer Substrate

Hepatitis C virus (HCV) NS3-NS4A protease is an attractive target for anti-HCV agents because of its important role in replication. An optimized fluorescence resonance energy transfer (FRET) substrate for NS3-NS4A protease, based on the sequence of the NS5A-5B cleavage site, was designed and synthesized. High-throughput screening of in-house compound libraries was performed using a FRET substrate FS10 (MOCAc-DKIVPC-SMSYK-Dnp) and MBP-NS3-NS4A fusion protein. Several hit compounds were found, including YZ-9577 (2-oxido-1,2,5-oxadiazole-3,4-diyl) bis (phenylmethanone) with potent inhibitory activity (IC50=1.6 μM) and good selectivity against other human serine proteases.

Efficacy of drugs with different mechanisms of action in relieving spontaneous pain at rest and during movement in a rat model of osteoarthritis.

Patients with osteoarthritis (OA) suffer from joint pain aggravated by movement, which affect their quality of life. In the present study, a weight bearing paradigm for pain at rest and a gait paradigm for pain during movement were tested in rats with unilateral knee arthritis induced by an intra-articular injection of sodium monoiodoacetate (MIA). At week 3 after MIA (1mg/knee) injection, animals developed pain-associated, right-left imbalances of weight distribution (weight bearing) or foot print parameters (gait). Diclofenac, at doses up to 30 mg/kg orally (p.o.), did not have a significant effect on either paradigm. Morphine rectified the weight bearing and gait imbalances at 1 and 3mg/kg subcutaneously, respectively. The weak opioid and serotonin/norepinephrine reuptake inhibitor (SNRI) tramadol also significantly corrected the indices at 10mg/kg (weight bearing) and 100mg/kg p.o. (gait). The SNRI duloxetine at 30 mg/kg p.o. corrected the weight bearing imbalance but not gait imbalance. We assessed the effect of different drugs on pain-induced disturbances in weight distribution and gait in MIA-induced arthritic rats. Analgesic drugs, each with different mechanisms of action, were less effective in rectifying the imbalance in gait than that in weight distribution. The assessment of the effect of analgesics on not only rest pain but pain during movement is valuable for the comprehensive examination of their therapeutic efficacies in OA.