Kenji Sudo

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.

A sensitive assay system screening antiviral compounds against herpes simplex virus type 1 and type 2

A highly sensitive and accurate assay system was developed for in vitro evaluation of anti-herpes simplex virus (anti-HSV) agents using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and human embryonic lung fibroblast (MRC-5) cells. This assay system was found to be highly sensitive for both HSV-1 and -2. Confluent MRC-5 cells were infected with either HSV-1 KOS strain or HSV-2 G strain of 25 TCID50 in the presence of various concentrations of test compounds. The optical density of formazan was used to determine cell viability. The EC50 values of acyclovir and several other anti-HSV agents were found to be similar to those obtained by the plaque reduction method. These results indicate that this MTT assay is useful for screening anti-HSV-1 and -2 agents.

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.

Use of lactate dehydrogenase to evaluate the anti-viral activity against influenza A virus

The detection of lactate dehydrogenase (LDH) can be used to evaluate efficiently anti-influenza A virus agents. LDH levels in the virus-infected Madin-Darby canine kidney cell cultures were significantly higher than in controls, were in proportion to the degree of virus infection, and corresponded to a decrease in mitochondrial dehydrogenase activity as assayed using a tetrazolium colorimetric assay (MTT method). The EC50 value and cytotoxicity of ribavirin, 3-deazaguanine, pyrazofurin, and carbodine against influenza A virus as measured by the LDH detection method was equivalent to that derived by the MTT method.

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.

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.

Effect of ASP2151, a Herpesvirus Helicase-Primase Inhibitor, in a Guinea Pig Model of Genital Herpes

ASP2151 is a herpesvirus helicase-primase inhibitor with antiviral activity against varicella zoster virus and herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Here, we examined the potency and efficacy of ASP2151 against HSV in vitro and in vivo. We found that ASP2151 was more potent in inhibiting the replication of HSV-1 and HSV-2 in Vero cells in the plaque reduction assay and had greater anti-HSV activity in a guinea pig model of genital herpes than did acyclovir and valacyclovir (VACV), respectively. Oral ASP2151 given from the day of infection reduced peak and overall disease scores in a dose-dependent manner, resulting in complete prevention of symptoms at the dose of 30 mg/kg. The 50% effective dose (ED50) values for ASP2151 and VACV were 0.37 and 68 mg/kg, respectively, indicating that ASP2151 was 184-fold more potent than VACV. When ASP2151 was administered after the onset of symptoms, the disease course of genital herpes was suppressed more effectively than by VACV, with a significant reduction in disease score observed one day after starting ASP2151 at 30 mg/kg, whereas the therapeutic effect of VACV was only evident three days after treatment at the highest dose tested (300 mg/kg). This indicated that ASP2151 possesses a faster onset of action and wider therapeutic time window than VACV. Further, virus shedding from the genital mucosa was significantly reduced with ASP2151 at 10 and 30 mg/kg but not with VACV, even at 300 mg/kg. Taken together, our present findings demonstrated the superior potency and efficacy of ASP2151 against HSV.

Colorimetric Assay System for Screening Antiviral Compounds against Hepatitis B Virus

A highly sensitive, rapid, and accurate assay system was developed for the in vitro evaluation of anti‐hepatitis B virus (anti‐HBV) agents. Chronic HBV‐producing HB611 cells were used in combination with immunoaffinity purification, polymerase chain reaction (PCR), and hybrid capture detection. HB611 cells were incubated with putative anti‐HBV agents for 7 days in 96‐well microtiter plates. HBV was purified from HB611 cell culture media using immunoaffinity purification. The HBV DNA was extracted, amplified with PCR, and assayed using a hybrid capture colorimetric method. This assay provided quantitative detection of extracellular HBV DNA from 25 μl of cell culture media. Using the colorimetric method, we found that 50% effective concentration levels of several known anti‐HBV agents (HPMPA, PMEDAP, PMEA and others) were similar to those reported in studies using Southern blot analysis. These results demonstrate that this new and easily automated colorimetric assay system can be used for the rapid and accurate assessment of anti‐HBV compound selectivity.

Binding Modes of Two Novel Non-Nucleoside Reverse Transcriptase Inhibitors, YM-215389 and YM-228855, to HIV Type-1 Reverse Transcriptase

Background: YM-215389 and YM-228855 are thiazolidenebenzenesulfonamide (TBS) derivatives and novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) that inhibit not only wild-type, but also the K103N- and Y181C-substituted reverse transcriptase (RT) of HIV type-1 (HIV-1). Methods: To characterize the binding modes of the TBS derivatives in detail, the anti-HIV-1 activities of YM-215389 and YM-228855 against various NNRTI-resistant clones were examined. Docking studies with HIV-1 RT were also performed. Results: YM-215389, which effectively inhibits various NNRTI-resistant clones, interacted with L100, K103, V106 and Y318 through the benzene ring and with E138, V179, Y181, Y188 and W229 through the thiazole ring. A single amino acid substitution confers only moderate resistance to YM-215389; indeed, four amino acid substitutions (V106L, V108I, E138K and L214F) were necessary for high- level resistance. Although the activity of YM-228855, a derivative of YM-215389 that has two bulky and rigid cyano-moieties on the benzene ring, was 10x more potent against HIV-1 than YM-215389, its anti-HIV-1 activity was readily reduced with single substitutions as with Y181I and K103N. Conclusions: These results provide structural information for optimizing the TBS derivatives in an attempt to construct ideal NNRTIs that maintain anti-HIV-1 activity to various HIV-1 variants.

Colorimetric assay system for screening antiviral compounds against hepatitis B virus

A highly sensitive, rapid, and accurate assay system was developed for the in vitro evaluation of anti-hepatitis B virus (anti-HBV) agents. Chronic HBV-producing HB611 cells were used in combination with immunoaffinity purification, polymerase chain reaction (PCR), and hybrid capture detection. HB611 cells were incubated with putative anti-HBV agents for 7 days in 96-well microtiter plates. HBV was purified from HB611 cell culture media using immunoaffinity purification. The HBV DNA was extracted, amplified with PCR, and assayed using a hybrid capture colorimetric method. This assay provided quantitative detection of extracellular HBV DNA from 25 microliters of cell culture media. Using the colorimetric method, we found that 50% effective concentration levels of several known anti-HBV agents (HPMPA, PMEDAP, PMEA and others) were similar to those reported in studies using Southern blot analysis. These results demonstrate that this new and easily automated colorimetric assay system can be used for the rapid and accurate assessment of anti-HBV compound selectivity.