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Fallopian tubal epithelium is a candidate for the origin of high-grade serous ovarian cancer. Transferrin-containing follicular fluid and/or retrograde menstrual blood are possible risk factors for carcinogenesis. Accumulation of DNA double-strand breaks (DNA-DSBs) in the fallopian tubal epithelium is considered to play an important role in the development of cancer. However, the mechanisms by which DNA-DSBs accumulate have not yet been fully elucidated. The hydroxyl radical, which is produced in a Fenton reaction catalyzed by an iron ion, serves as a potent DNA-DSB-inducing molecule, raising the potential of an iron ion transporter of transferrin in the formation of DNA-DSBs. We studied the potential involvement of transferrin in DNA damage and the development of ovarian cancer. Treatment with transferrin facilitated the formation of histone 2AX phosphorylated at Serine 139 (?H2AX), which is known as a DNA-DSB marker, in human fallopian tube secretory epithelial cells and A2780 ovarian cancer cells. Knockdown of transferrin receptor 1 (TfR1), but not transferrin receptor 2, suppressed the transferrin uptake and consequent formation of ?H2AX. As hydroxyl radicals in reactive oxygen species (ROS) are involved in DNA-DSBs, the formation of ROS was determined. Treatment with TfR1-specific small interference RNAs significantly diminished transferrin-induced formation of ROS. Moreover, TfR1-dependent uptake of transferrin was revealed to augment the formation of DNA-DSBs in the presence of hydrogen peroxide, which served as a substrate for the Fenton reaction. An ex vivostudy with murine fallopian tubes further demonstrated that transferrin treatment introduced DNA-DSBs in the fallopian tubal epithelium. Collectively, these data suggested that the transferrin-TfR1 axis accounts for the induction of DNA-DSBs that potentially lead to DNA damage/genome instability. These findings also suggested that exposure to transferrin initiates and promotes the development of ovarian cancer by aiding the accumulation of DNA-DSBs in the fallopian tubal epithelium.

Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2ß-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2ß and affect its compartmentalization, thereby suppressing PI3KC2ß activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2ß knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2ß in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2ß-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.

Twenty 5-alkyl-2-thiopyrimidine nucleosides were newly synthesized and examined for antiviral activities against herpes simplex virus (HSV), varicella-zoster virus (VZV) and human cytomegalovirus (HCMV). In this study, 2′-deoxy-5- alkyl-2-thiocytidine analogues had lower 50% effective concentration (EC50) values against HSV- 1, and 2/-deoxy-5-alkyl-2-thiouridine analogues showed lower EC50against VZV than their congeners of arabinoside form. Among the compounds examined, 2′-deoxy-5-ethyl and 5-propyl- 2-thiocytidine (TN-53 and TN-54) were most potent and selective anti-HSV compounds. Their EC50s were 0.04 and 0.15 μM, and selectivity indexes were more than 7215 and 1849, respectively. On the other hand, 2′-deoxy-5-propyl-2-thiouridine (TN-51), 5-bromovinyl-2-thiouracil arabinoside (TN-65) and 5-styryl-2-thiouracil arabinoside (TN-67) were most potent and selective anti-VZV compounds. Their EC50s were 3.1, 3.8 and 2.6 μM for CaQu strain of VZV, respectively, and 2.1 to 3.0 times lower than that of acyclovir. All 2-thiopyrimidine nucleoside analogues did not show antiviral activities against thymidine kinase (TK) negative strains of HSV-1 and VZV. Only three 2-thiocytosine arabinoside compounds showed marginal anti-CMV activities (EC50s were 57–159 μM). All of the five alkyl-2-thio-pyrimidine nucleoside analogues examined were not cytotoxic to human lymphoblastoid cells (RPMI8226) and human embryonic fibroblast cells (MRC-5) at 240 μM (100 pg/ml) or more. Regarding the structure-activity relationship of 5-alkyl-2-thiopyrimidine nucleoside analogues, the following remarks will be noted. Elongation of 5-alkyl chain (methyl to ethyl) of 2-thiocytosine in both deoxyribosyl and arabinosyl nucleosides increased anti-HSV-1 activity but not anti-VZV activity. Furthermore, elongation of the same chain (ethyl to propyl) of 2-thiodeoxyuridine increased anti-VZV activity whereas it did not in the case of 2-thiouracil arabinosides.

4‘-C-Ethynyl-β-d-arabino- and 4‘-C-ethynyl-2‘-deoxy-β-d-ribo-pentofuranosylpyrimidine and -purine nucleosides were synthesized and evaluated for their in vitro anti-HIV activity. The key intermediate, 4-C-ethynyl- or 4-C-triethylsilylethynyl-d-ribo-pentofuranose, was prepared from d-glucose and glycosidated with various pyrimidine or purine bases. The arabino pyrimidine derivatives were prepared from the corresponding ribo derivatives via O²,2‘-anhydro nucleosides. The 2‘-deoxy-ribo derivatives were synthesized by radical reduction of 2‘-bromo or 2‘- phenoxythiocarbonyloxy nucleosides. Among these 4‘-C-ethynyl nucleosides, seven analogues proved to be potent against HIV-1 in vitro with EC50 values ranging from 0.0003 to 0.03 μM. These compounds also exerted activity against clinical and multi-dideoxy-nucleoside-resistant HIV-1 strains with comparable EC50 values. Three such 4‘-C-ethynyl-2‘-deoxypurine analogues including 4‘-C-ethynyl-2‘-deoxyadenosine and 4‘-C-ethynyl-2,6-diamino-2‘-deoxypurine were less cytotoxic [selectivity indices (SIs):  975−2733] than three 4‘-C-ethynyl-2‘-deoxycytidine analogues (SIs:  63−363). 4‘-C-Ethynyl-5-fluoro-2‘-deoxycytidine was least toxic (SI:  >3333) and potent against all HIV strains tested.

A series of 4‘α-C-branched-chain pyrimidine nucleosides was synthesized from 2‘-deoxycytidine or uridine. In the 2‘-deoxycytidine series, the substituent at the 4‘α-position affected cytotoxicity against L1210 mouse leukemic cells in vitro in the order Me (23) > CN (22) > C&tbd1;CH (21) > CH&dbd;CH2 (19) > Et (24) > CH&dbd;CHCl (20). However, uridine and cytidine derivatives with ethynyl and cyano groups at the 4‘α-position did not show any cytotoxicity. The antiviral activities of these nucleosides against HSV-1, HSV-2, and HIV-1 in vitro were also examined. Compounds 22 and 23 showed antiviral activities against HSV-1 and HSV-2 without showing significant toxicity to the host cells (MRC-5 cells). Although almost all of the nucleosides showed anti-HIV-1 activities, they were also cytotoxic to the host cells (MT-4).

Twenty 2-thiopyrimidine nucleoside analogues were synthesized and examined for inhibitory activity against herpes simplex virus (HSV) type 1 and 2, varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and thymidine kinase-deficient HSV (HSV-TK) replication in vitro. 2-thiouracil (thymine) arabinoside, 2′-deoxy-2-thiouridine (or 2-thiothymidine) and their 5-halogenated derivatives showed anti-HSV activity in both RPMI8226 (human B-lymphoblastoid cells) and MRC-5 (human embryo lung cells). 2′-deoxy-5-halogenated-2-thiocytidines were also inhibitory against HSV, whereas 2-thiocytosine arabinoside and its derivatives were not inhibitory against HSV replication, except 5-bromo and 5-iodo congeners (TN-31, TN-32). Substitution of the halogen atom at the 5-position of the pyrimidine rings to an atom with a higher molecular weight increased anti-HSV and VZV activities, except for the anti-HSV activity of 2-thiouracil arabinosides. 2′-deoxy-5-methyl-, and 2′-deoxy-5-iodo-2-thiouridines (TN-17, TN-44) showed the most potent anti-HSV activity, and 2′-deoxy-5-chloro- and 2′-deoxy-5-bromo-2-thiocytidines were potent inhibitors of VZV replication. However, none of the compounds inhibited HCMV and HSV-TK−replication. TN-31 and TN-32 were shown to inhibited HCMV and HSV-TK−as well as HSV and VZV replication. The cytotoxicity of the 2-thiopyrimidine nucleoside analogues was less than that of the 2-oxy-congeners of the compounds (5-iodo-2′-deoxyuridine, 5-iodo-2′-deoxycytidine, thymine arabinoside and cytosine arabinoside). The selectivity index of 2′-deoxy-5-iodo-2-thiouridine (TN-44) was higher than that of 5-iodo-deoxyuridine. TN-17 and TN-44 were not cytotoxic to resting or stimulated human peripheral blood mononuclear cells at 400 μ, although TN-32 was cytotoxic at a concentration of 20 μ.

We have examined novel benzylhydroxylamine derivatives for their inhibitory effects on the replication of human immunodeficiency virus (HIV) in cell cultures. Among the series, O-(2-chloro-6-fluo-robenzyl)hydroxylamine (RD6-Y664) was found to be the most potent inhibitor of HIV-1. The EC50for HIV-1 strain IIIB was 1.6 μg/ml with a selectivity index greater than 38 in MT-4 cells. It also inhibited the replication of other HIV strains including a nonnucleoside reverse transcriptase (RT) inhibitor-resistant mutant, a nucleoside RT inhibitor-resistant mutant and HIV-2, in acutely infected cells. However, the compound did not affect HIV-1 production in chronically infected cells. A time-of-addition experiment and detection of proviral DNA synthesis suggested that RD6-Y664 targeted an early step of the viral replication cycle, presumably a process prior to reverse transcription. In fact, an assay for HIV-1 RT revealed that the compound did not suppress enzyme activity. Furthermore, RD6-Y664 did not show any inhibition of gp120–CD4 interaction, or binding of anti-CXCR4 antibody to CXCR4.

Sixty polyoxometalates were examined for anti-influenza A virus (FluV-A) activity in vitro. Two of the most potent and least cytotoxic compounds, PM-504 K9H5(Ge2Ti6W18O77)16H2O] and PM-523 (iPrNH3)6H [PTi2W10O38(O2)9H2O2] were selected for further studies. Examination of the antiviral effects of PM-504 and PM-523 against other human ortho- and paramyxoviruses revealed that both compounds had broad spectrum antimyxovirus activities. From a time of addition study and FACS analysis for influenza A virus infected cells, the compounds were found not to inhibit binding of virus to MDCK cells. However, these compounds inhibited haemolysis of chicken erythrocytes by virus and also inhibited fluorescence dequenching of octadecylrhodamine B-labelled virus after binding to cells. This indicates that these polyoxometalates inhibited fusion of the virus envelope to the cellular membrane.

Polyoxometalates have been shown to inhibit the replication of retro-, toga-, paramyxo- and herpesviruses. The primary mechanism of the anti-human immunodeficiency virus type 1 (HIV-1) action of polyoxometalates seems to be inhibition of binding of virus to cells and inhibition of syncytium formation. Since myxoviruses and HIV-1 are known to interact with the cytoplasmic membrane by adsorption and penetration of virus and by fusion of infected and uninfected cells, 25 polyoxometalates were examined for anti-ortho-, anti-paramyxovirus and anti-HIV-1 activity in vitro. Of the 25 compounds evaluated, 24 showed antiviral effects against influenza virus A, 11 showed activity against respiratory syncytial virus, six showed activity against measles virus, and 23 were considered effective against HIV-1 at a lower concentration than that producing cytotoxicity. Four polyoxotungstates which had potent inhibitory effects were examined for inhibitory effects against additional ortho- and paramyxoviruses, and proved to have a broad spectrum of antimyxoviral activity. HS-058, the Keggin sandwich compound K10Fe4(H2O)2(PW9O34)2·nH2O, was inhibitory against influenza viruses A and B, respiratory syncytial virus, measles virus, and parainfluenza virus 2, with median effective concentrations of 1.4, 21.8, 7.4, 0.8 and 0.32 μ,M, respectively. However, HS-058 had no effect on parainfluenza virus 3 or mumps virus. The median cytotoxic concentration of HS-058 for Madin-Darby canine kidney (MDCK) and HEp-2 cells was more than 200 μM and that for HMV-2 and Vero cells was about 50 μM. When HS-058 was added at different times after influenza A and respiratory syncytial virus infection, it inhibited binding of the latter but not of the former to cells. However, at higher concentrations, HS-054 and HS-058 inhibited haemolysis of chick erythrocytes by influenza virus and syncytium formation involving respiratory syncytial virus-infected cells and uninfected cells. Four times the median effective antiviral concentration of HS-058 completely inhibited the growth of influenza virus A in MDCK cells when compound was added before virus adsorption. Furthermore, when HS-058 was added after virus adsorption, it inhibited the yield of virus in MDCK cells infected at low but not at high multiplicity of infection.

Among thirteen newly synthesized pyridobenzoazole derivatives which have been examined for anti-myxovirus and antiherpesvirus activities, three benzimidazoles emerged as potent anti-orthomyxo- or paramyxovirus compounds. 4-Cyano-2-benzamide-1-oxo-1,5-dihydropyrido[1,2a]benzimidazole (CBO-PB) showed broad antiviral activities against paramyxo-and orthomyxoviruses with EC50of 0.1–2.0 μg ml−1, and 2-cyano-1-amino derivatives of CBO-PB (CCI-PB) were inhibitory to paramyxoviruses at 1.4–8.5 (μg ml−1by a plaque reduction method. The third compound, 2-ethoxycarbonyl derivatives of CCI-PB was inhibitory only to respiratory syncytial virus (RSV) at 15–28 μg ml−1. Selectivity indexes of these 3 compounds for RSV in HeLa cells were 60, 86, and >13, respectively. All three compounds inhibited syncytium formation of RSV and Parainfluenzavirus (PFLUV) type 3 at comparable concentrations with EC50for plaque formation. They inhibited antigen production of RSV and PFLUV at the concentrations that were 4 to 20-fold higher than those needed for plaque reduction, but they did not inhibit adsorption of virus to cells at all. All three compounds inhibited the growth of RSV in HeLa cells at 4-fold higher concentrations than their EC50for plaque reduction.

Mizoribine (4-carbamoyl-1-β-D-ribofuranosylimidazo-lelium-5-olate), EICAR (5-ethynyl-1-β-D-ribofuranosylimi-dazole-4-carboxamide), mycophenolic acid and ribavirin are antiviral agents targeted for inosine monophosphate (IMP) dehydrogenase. These compounds have been examined for their activities against orthomyxoviruses [influenza viruses (FluV)] and paramyxoviruses [parainfluenza viruses (PFIuV), mumps virus, measles virus (MLSV) and respiratory syncytial virus (RSV)] in vitro. Mizoribine was 1- to 9-fold more active than ribavirin against RSV, PFIuV and MLSV. EICAR and mycophenolic acid showed higher potency than mizoribine and ribavirin against all myxoviruses examined. None of the four compounds examined proved cytotoxic to stationary host cells (HeLa, Vero and MDCK) at a concentration of 200 μg ml−1or more. On the other hand, EICAR and mycophenolic acid were toxic to rapidly growing cells at concentrations of 2.2-9 and 0.1-1.1 μg ml−1, respectively. Mizoribine and ribavirin showed cytotoxicity to the growing cells at higher concentrations (12-51 μg ml−1). The antiviral activities of mizoribine against FluV and RSV were reversed by 25-100 μm of each of guanosine and guanosine monophosphate (GMP). The antiviral activity of ribavirin against FluV was reversed by 25 μg of each of guanosine and GMP, while its activity against RSV was reversed by ≥ 100 μm of each of these compounds. Neither xanthosine nor xanthosine monophosphate (XMP) reversed the antiviral effects of mizoribine and ribavirin at concentrations of 300 μM. Concentrations 9 times higher than the median effective doses (EC50) of mizoribine and ribavirin inhibited the growth of RSV in HeLa cells as determined in an assay of infectious virus yield. Mizoribin should be further pursued as a candidate drug for the treatment of ortho- and paramyxovirus infections.

Bis-naphthalenedisulphonic acid derivatives with a biphenyl spacer, 4,4′-[4,4′-biphenyldiylbis(sulphonyl-amino)]bis(5-hydroxy-2,7-naphthalenedisulphonic acid) and 3,3′-[4,4′-biphenyldiylbis(sulphonyl-amino)]bis(1,5-naphthalenedisulphonic acid), have previously been reported as potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in cell culture. These compounds have also proved inhibitory to syncytium formation in cocultures of MOLT-4 cells with HIV-1-infected HUT-78 cells. They also inhibit the binding of HIV-1 virions to MT-4 cells as determined by a flow cytometric (FACS) method. Further studies on their mechanism of action by the FACS have revealed that the compounds inhibit the binding of anti-gp120 monoclonal antibody to the viral envelope glycoprotein gp120. Binding of OKT4A/Leu3a monoclonal antibody to the cellular CD4 receptor is not affected by the compounds. These results suggest that the anti-HIV-1 activity of the naphthalenedisulphonic acid derivatives can be attributed to inhibition of the gp120-CD4 interaction through binding of the compounds to the viral gp120 antigen.

We established a haemadsorption-based colorimetric assay system, which may be used to screen a large number of anti-influenza compounds. Madin Darby canine kidney (MDCK) cells infected with influenza virus were cultured in the presence of test compounds and after 3 days of infection guinea pig erythrocytes (GPE) were added to infected MDCK cells. After 4 washings of the cells the adsorbed GPE were lysed with distilled water, and peroxidase activities contained in GPE were measured using o-phenylendia-mine and H2O2as substrates. The peroxidase activity that was read as optical density of oxidized o-phenylendiamine (quinoid form) correlated well with the dose of virus and day of infection in MDCK cells. Fifty per cent inhibitory concentration (IC50) of 1-(β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin) and adamantanamine hydrochloride (amantadine) against influenza virus A/H3N2/lshikawa and influenza B/Singapore, the IC50s of ribavirin to influenza A and B were 0.5 μg ml−1to 1.0 μg ml−1respectively. On the other hand, the IC50of amantadine for influenza A was 1 μg ml−1but was more than 100 μg ml−1for influenza B virus. By this method, ribavirin, 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide (EICAR), 3-(β-D-ribofuranosyl)-4-hydroxypyrazole-5-carboxamide (pyrazofurin) have shown inhibitory effects for virus replication at lower concentrations than their cytotoxic doses. On the other hand carbocyclic citidine (carbodine) was cytotoxic at the concentration of virus inhibition. Ribavirin and pyrazofurin showed one tenth or less EC50for influenza A and B viruses by the haemadsorption-based colorimetric assay compared with the ECso by TCID50method. It is possible to estimate the inhibitory effect of antiviral compounds against influenza viruses rapidly and quantitatively by this colorimetric assay system.

A natural sulphated mucopolysaccharide (OKU40), extracted from a marine plant Dinoflagellata, and an artificial sulphated polysaccharide (OKU41), prepared from a marine Pseudomonas, displayed antiviral activities against several enveloped viruses. OKU40 and OKU41 were found to be homogenous in electrophoresis and sedimation velocity and had a molecular weight of 8.0 × 1065.0 × 105respectively. The sulphation rate of OKU40 and OKU41 was 8.9% and 5.4%, respectively. Each OKU40 and OKU41 inhibited the cytopathic effect of human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and zidovudineresistant HIV-1 in MT-4 cells at similar concentrations to those of dextran sulphate (molecular weight: 5000) (50% inhibitory concentrations: 0.86-1.95 μg mL−1), whereas these compounds did not affect the growth and viability of mock-infected MT-4 cells at concentrations up to 500 μg mL−1. These compounds proved inhibitory not only to HIV-1 and HIV-2 but also to other enveloped viruses, i.e. herpes simplex virus type 1, influenza virus A and B, respiratory syncytial virus and measles virus. OKU40 and OKU41 suppressed syncytium formation induced by cocultivation of MOLT-4/IIIband MOLT-4 cells at concentrations higher than 20 μg mL−1. Although OKU41 inhibited the binding of HIV-1 to the host cells and the binding of anti-gp120 monoclonal antibody to HIV-1 gp120, OKU40 did not inhibit these bindings, suggesting that the mechanism of anti-HIV activity of OKU40 and OKU41 may be primarily due to the inhibition of virus-cell fusion and viral adsorption to the host cells, respectively. Furthermore, these compounds did not inhibit to the blood coagulation process at a concentration that was significantly inhibitory to HIV replication. The compounds appear to have an interesting potential as virucidal agents.

In this study, the anti-herpetic activities of novel 2′-methyl nucleoside analogues which were substituted at the 5 position of the pyrimidine with a halogen were investigated. The 2′-fluoro-5-iodo-aracytosine (FIAC) congeners (2′S)-2′-deoxy-2′-C-methylcytidine which were substituted with Br or I at the 5 position (SMBC or SMIC); and 2′-fluoro-5-iodo-arauridine (FIAU) congeners (2′S)-2′-deoxy-2′-C-methyluridine which were substituted with Br or I at the 5 position (SMBU or SMIU), proved to have potent antiviral activities against herpes simplex virus type-1 (HSV-1) and varicella-zoster virus (VZV) but not against herpes simplex virus type-2 (HSV-2). SMIU has a higher selective index against HSV-1 than FIAU, and both SMIC and SMIU showed higher inhibitory effects against VZV replication than aciclovir. The four effective compounds were not inhibitory to a thymidine kinase (TK)-negative HSV-1 strain, and this result indicates that phosphorylation of the compounds by HSV or VZV-TK is necessary for the activation of these compounds.

Several dextrin sulphate derivatives were prepared and investigated for their activity against human immunodeficiency virus type 1 (HIV-1) in vitro.These compounds have proved to be potent and selective inhibitors of HIV-1. One of the compounds, termed FG-752 [molecular weight (MW) 3000], was the most active, and its 50% antiviral effective concentration was 2.1 μg ml−1(0.7 μm) in MT-4 cells. No toxicity for the cells was observed at a concentration of 500 μg ml−1. The compounds were also inhibitory to HIV-1-induced giant cell (syncytium) formation. These results suggest that dextrin sulphate may be useful for the chemotherapy of HIV-1 infections.

Salcomine, N,N'-bis(salicylidene)ethylene diaminocobalt (II), and its derivatives were evaluated for their ability to inhibit selectively human cytomegalovirus (HCMV) proteinase activity. The 50% inhibitory concentration (IC50) of salcomine was 1.4 μM for HCMV proteinase, but >200 μM for three other serine proteinases (trypsin, >250 μM; chymotrypsin, 206 μM; and elastase, >250 μM). Two salcomine derivatives also inhibited HCMV proteinase with IC50values under 2 μM. Studies of the structure–activity relationship of salcomine-related compounds showed that the phenyl moiety and the spacer moiety (distance betweenthe two amines) were instrumental in the inhibition of HCMV proteinase. Moreover, salcomine inhibited the growth of laboratory strain AD169 and three clinical isolates at a 50% effective concentration (EC50) range of 1.92–2.89 μM. These results show that salcomine derivatives are potent and selective inhibitors of HCMV proteinase and HCMV replication in cell culture. Salcomine derivatives appear to be worth pursuing as candidate drugs for the chemotherapy of HCMV infection.

Podophyllotoxin and its derivatives were examined for inhibitory effects on the replication of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), including acyclovir-resistant virus and clinical isolates. Deoxypodophyllotoxin (RD4–6266) proved to be a highly potent and selective inhibitor of all HSV strains in MRC-5 cells. EC50values of RD4–6283 (in which the methylenedioxy ring A is modified) for HSV-1 and -2 were inferior to those of deoxypodophyllotoxin. However, podorhizol (RD4–6277) and 5′-methoxy-podorhizol (RD4–6276), in which ring C is absent, did notinhibit HSV replication. Moreover, RD4–6266 also inhibited the production of infectious virus particles of HSV-1 KOS strain and HSV-2 G strain. In contrast, none of the podophyllotoxin derivatives were found to have an antiviral effect against influenza A virus, respiratory syncytial virus or human cytomegalovirus in doses not toxic to the cells.

Background: Two predominant mite species, Dermatophagoides farinae and Dermatophagoides pteronyssinus, are responsible for the immediate hypersensitivity reaction. Although a large number of antigens and allergens was detected in a whole mite culture extract, only the major allergens of Der I and II groups have been extensively studied. Much information on other important allergens remains to be accumulated. Methods: A new allergen complementary DNA (cDNA) clone was isolated from a D. farinae protein cDNA library. The expression product of the cDNA and native tropomyosin from D. farinae were purified, characterized, and evaluated. Results: An amino acid sequence deduced from the cDNA clone revealed significant homology with tropomyosins conserved in a wide range of animals. The amino acid sequences of two fragments obtained by degradation of the purified protein with cyanogen bromide coincided completely with the corresponding portions of the deduced amino acid sequence. The native tropomyosin reacted with specific IgE in the 31 sera tested at a high frequency (80.6%), comparable to that of Der f I (90.3%) and Der f II (74.2%). Conclusions: The cloned cDNA encodes tropomyosin. The high binding response of specific IgE antibodies to recombinant and native tropomyosins demonstrates that mite tropomyosin is an additional important allergen in house dust mite. (J Allergy Clin Immunol 1995;96:74-83.)

We studied the effects of eight protease inhibitors on the multicycle replications of various orthomyxoviruses and paramyxoviruses. Among the compounds, nafamostat mesilate, camostat mesilate, gabexate mesilate, and aprotinin, which are widely used in the treatment of pancreatitis, inhibited influenza virus A and B replication at concentrations that were significantly lower than their cytotoxic thresholds in vitro. None of the protease inhibitors had activity against respiratory syncytial virus, measles virus, or parainfluenza virus type 3 at the highest concentrations tested. Camostat mesilate was found to be the most selective inhibitor. Its 50% effective concentration for influenza virus A replication was 2.2 micrograms/ml, and the selectivity index, which was based on the ratio of the 50% inhibitory concentration for host cell proliferation to the 50% effective concentration for influenza virus A replication, was 680. When the in ovo antiviral activity of the compounds was tested by using chicken embryos, camostat mesilate at a dose of 10 micrograms/g markedly reduced the hemagglutinin titers of influenza viruses A and B.

5-Ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and 6'-(R)-6'-C-methylneplanocin A (TJ13025) are two novel antiviral agents which are targeted against IMP dehydrogenase and S-adenosylhomocysteine hydrolase, respectively. These compounds have been examined for their activities against various strains of orthomyxoviruses (influenza virus) and paramyxoviruses (parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus) in vitro. EICAR was 10- to 59-fold more active than ribavirin and TJ13025 was 32- to 330-fold more active than ribavirin against parainfluenza virus types (2 and 3), mumps virus, and measles virus. EICAR was also more active than ribavirin against respiratory syncytial virus and influenza virus, whereas TJ13025 was virtually inactive against these viruses. The 50% virus-inhibitory concentrations of EICAR and TJ13025 were generally within the 0.1- to 1-microgram/ml range. Although the compounds did not prove cytotoxic to stationary host cells (HeLa, Vero, MDCK, and LLCMK2) at a concentration of 200 micrograms/ml, concentrations of 4 to 13 micrograms/ml inhibited the growth of dividing cells. EICAR and TJ13025 should be further pursued as candidate drugs for the treatment of ortho- and paramyxovirus infections.

Duck hepatitis B virus (DHBV) replication in primary duck hepatocytes was monitored by examining the synthesis of both DHBV DNA and DHBV core antigen. Several nucleoside analogs which were previously shown to inhibit the replication of DNA viruses (i.e., herpesviruses) and retroviruses were examined for their inhibitory effects on the synthesis of DHBV core antigen in primary duck hepatocytes. (S)-9-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine, 2',3'-dideoxyadenosine, and 2',3'-dideoxycytidine inhibited DHBV core antigen synthesis at concentrations that were significantly lower than those found to be toxic to the primary hepatocytes. Of all the compounds tested, (S)-HPMPA showed the lowest 50% effective concentration (0.5 micrograms/ml). The selectivity index or ratio of the 50% cytotoxic concentration to the 50% effective concentration of (S)-HPMPA was greater than 300. (S)-HPMPA not only inhibited DHBV core antigen but also DHBV DNA synthesis in DHBV-infected hepatocytes.

We examined the inhibitory effect of 20 antiviral compounds, including ribavirin, on the replication of respiratory syncytial virus in HeLa and HEp-2 cell cultures. Of the compounds studied, pyrazofurin and 3-deazaguanine emerged as more potent inhibitors of respiratory syncytial virus than ribavirin. Based on their inhibitory effect on the cytopathogenicity of respiratory syncytial virus in HeLa cells, the average 50% effective dose of pyrazofurin and 3-deazaguanine for eight strains was 0.07 and 1.65 micrograms/ml, respectively; that of ribavirin was 5.82 micrograms/ml. The cytotoxicity of these compounds for HeLa cells was examined by monitoring the incorporation of radiolabeled uridine into cellular RNA. The selectivity indexes of pyrazofurin and 3-deazaguanine exceeded that of ribavirin by 70- and 11-fold, respectively. Pyrazofurin, 3-deazaguanine, and ribavirin inhibited both viral antigen expression and syncytium formation in HeLa cell cultures, as assessed by an indirect immunofluorescence assay. In these assays, pyrazofurin and 3-deazaguanine again proved more potent than ribavirin. 2,5-Diamidinoindole and carbodine were less potent than ribavirin. Various other compounds, i.e., 3-adenin-9-yl-2-hydroxypropanoic acid isobutyl ester, 3-deazauridine, 3'-C-methyluridine, 5'-deoxy-5-fluorouridine, 5-cyanoimidazole-4-carboxamide, and its ribofuranosyl derivative, did not inhibit the cytopathic effect of the Long strain of respiratory syncytial virus at concentrations greater than or equal to 125 micrograms/ml. Tubercidin, 5-chlorotubercidin, xylotubercidin, neplanocin A, thiosemicarbazone R, and 3-methylquercetine were too toxic to HeLa cells for their inhibitory effects on respiratory syncytial virus to be examined.

By using an assay system based on a human hepatoblastoma cell line (HB611) that continuously synthesizes hepatitis B virus (HBV) DNA, 56 acyclic nucleoside phosphonate analogues were examined for their inhibitory effects on HBV DNA synthesis. The following compounds were found to inhibit HBV DNA synthesis at concentrations that were significantly lower than their minimum cytotoxic concentrations; 9-(2-phosphonylmethoxyethyl)adenine (PMEA), 9-(2-phosphonylmethoxyethyl) guanine(PMEG), 9-(2-phosphonylmethoxyethyl) guanine ethyl ester (PMEGEE), 9 - (2 - phosphonylmethoxyethyl) - 1 - deazaadenine (PMEC1A), 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), 9-(3-isopropoxy-2-phosphonylmethoxypropyl)adenine (IPPMPA), 9-(RS)-(2-phosphonylmethoxypropyl)adenine (PMPA) and 9-(3-hydroxy-2-phosphonylmethoxypropyl)-2, 6-diaminopurine (HPMPDAP). The most selective compounds (with indexes greater than 100) were PMEDAP, PMEA, IPPMPA, and PMPA. Acyclic pyrimidine nucleoside phosphonate analogues did not prove markedly selective as anti-HBV agents. Diphosphoryl derivatives of some acyclic purine nucleoside phos-phonates (i.e. PMEA, PMEDAP, HPMPA) were prepared. They proved inhibitory to HBV DNA polymerase but not cellular DNA polymerase α.

This study evaluated the inhibitory effects of 2,4,6-trihydroxy,3-nitro-benzamide (THNB) derivatives against hepatitis C virus (HCV). protease and other human serine proteases. The inhibitory efficacy was tested with a reversed-phase HPLC assay system using a NS3-NS4A fusion protein as the HCV protease and a synthetic peptide substrate that mimics the NS5A-5B junction. Twelve THNB derivatives showed more than 50% inhibition at 100 μg mL-1. The most potent derivative was RD3-4082, with 50% inhibition at a concentration of 2.3 μg mL-1; this concentration was lower than those of other protease inhibitors reported previously. The most selective derivative was RD2-4039, with 50% inhibition at a concentration of 32.8 μg mL-1, a lower concentration than those against other serine proteases (chymotrypsin, trypsin, plasmin and elastase). These results suggest that the RD2-4039 skeleton is an important structure for inhibitory activity against the HCV protease NS3-NS4A.

Ingenol 3,5,20-triacetate has recently been identified as a highly potent and selective inhibitor of HIV replication in vitro. To evaluate the potential of ingenol derivatives as anti-HIV agents, several ingenol derivatives have been synthesized and investigated for their anti-HIV activities, structure-activity relationships, and possible mechanisms of action. Among the ingenol derivatives, 13-hydroxyingenol-3-(2,3-dimethylbutanoate)-13-dodecanoate (RD4-2138) proved to be a highly potent and selective inhibitor of HIV replication. Its 50% effective concentration for viral replication in MT-4 cells was 0.07-0.5 nM depending on viral strains, including HIV-2. This concentration was approximately 105-fold lower than its cytotoxic threshold. RD4-2138 was also inhibitory to the syncytium formation induced by cocultivation of Molt-4 cells with Molt-4/IIIBcells (Molt-4 cells chronically infected with HIV-1). Some correlation was observed with the ingenol derivatives between their inhibitory effects on HTLV-IIIBreplication and surface CD4 expression in MT-4 cells, suggesting that the mechanism of inhibition is in part attributed to the inhibition of virus adsorption through down-regulation of CD4 molecules in the host cells. However, such correlation was not identified between the inhibition of HTLV-IIIB and the activation of protein kinase C. Thus, they might have a potential as effective anti-HIV agents when toxicity in vivocould be elucidated.

The inhibitory effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) 5'-triphosphate on varicella zoster virus (VZV) DNA polymerase was studied using the parent strain (TK+-VZV) and the mutant strain (TK--VZV). The mutant strain was deficient in thymidine kinase (TK)-inducing activity and resistant to BVDU. In the absence of BVDU, TK--VZV and TK+-VZV induced an equivalent level of viral DNA polymerase activity in human embryo fibroblasts. In the presence of 5 microM BVDU, TK--VZV still induced viral DNA polymerase activity, whereas TK+-VZV failed to do so. BVDU 5'-triphosphate (BVDUTP) was considerably more inhibitory to the TK+- and TK--VZV DNA polymerases than to the cellular DNA polymerases. There were no significant differences in the affinity for dTTP as substrate and the sensitivity to BVDUTP as inhibitor between the TK+- and TK--VZV DNA polymerases. The Km value for dTTP and the Ki value for BVDUTP of the VZV DNA polymerases were 1.43 microM and 0.55 microM, respectively. The inhibitory effect of BVDUTP to VZV DNA polymerase was competitive with respect to the natural substrate.

Summary A long period of clinical latency before development of symptoms is characteristic of human immunodeficiency virus type 1 (HIV-1) infection. OM10.1, a promyelocyte cell line latently infected with HIV-1, has been developed as a model for studying the mechanism of viral latency and the activation of virus expression. We found that this latently infected cell line with heat shock at 42°C for 2 h resulted in a high level of HIV-1 production without addition of any cytokines. The mechanism of activation was analyzed by using anti-TNF-a antibody and various inhibitors. Although the TNF-a level in culture supernatants was below the sensitivity of an ELISA assay system, addition of anti-TNF-a antibody in culture medium could partially suppress the heat shock induced HIV-1 production. Staurosporine (PKC inhibitor), pentoxifylline (NF-?B inhibitor), and Ro5-3335 (HIV-1 Tat inhibitor) also inhibited significantly the heat shock induced virus activation. In particular, staurosporine achieved approximately 90% inhibition of the HIV-1 antigen expression in heat shock-treated OM10.1 at a non-toxic concentration. Although the mechanism of HIV-1 activation with heat shock has not been fully elucidated yet, it is presumed PKC plays an important role in HIV-1 activation. Thus, the present observations will provide a further insight into the pathogenesis of HIV-1 infections.

Sulfated polysaccharides (i.e., dextran sulfate) and sulfated polymers (i.e., sulfated polyvinylalcohol and sulfated copolymers of acrylic acid with vinylalcohol) were found to be potent and selective inhibitors of the replication of respiratory syncytial virus (RSV) and influenza virus type A (influenza A virus) but not of other myxoviruses (parainfluenza 3, measles, and influenza B viruses). The compounds were also inhibitory to human immunodeficiency virus type 1 (HIV-1) and HIV-2 and simian immunodeficiency virus but not simian AIDS-related virus. The mode of antiviral action of the sulfated polysaccharides and polymers can be attributed to an inhibition of virus binding to the cells (HIV-1), inhibition of virus-cell fusion (influenza A virus), or inhibition of both virus-cell binding and fusion (RSV). The fact that the sulfated polysaccharides and polymers are inhibitory to some myxoviruses and retroviruses but not to others seems to depend on the composition of the amino acid sequences of the viral envelope glycoproteins that are involved in virus-cell binding and fusion. All myxoviruses and retroviruses that are sensitive to the sulfated polysaccharides and polymers share a tripeptide segment (Phe-Leu-Gly). This tripeptide segment may be involved either directly (as a target sequence) or indirectly in the inhibitory effects of the compounds on virus-cell binding and fusion.

The in vitro susceptibility of eight strains of varicella-zoster virus (VZV) to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was examined in human embryonic fibroblasts by the following techniques: inhibition of focus formation by either cell-free VZV (4-day assay) or cell-associated VZV (2-day assay), inhibition of viral antigen formation (2-day assay), and inhibition of viral cytopathogenicity (15-day assay). The 50% inhibitory dose (ID50) of BVDU ranged from 0.001 microgram/ml (2-day assay) to 0.01 microgram/ml (15-day assay). BVDU appeared highly selective in its anti-VZV activity since even at concentrations as high as 100 micrograms/ml, BVDU did not markedly affect the viability of the host cells. The ID50 of BVDU for VZV was comparable to that of IVDU (E-5-(2-iodovinyl)-2'-deoxyuridine). Both drugs inhibited the replication of VZV at a much lower concentration than did other antiviral compounds such as iododeoxyuridine, ethyldeoxyuridine, arabinosylcytosine, arabinosyladenine, phosphonoacetic acid, iododeoxycytidine, and acycloguanosine. BVDU and IVDU were virtually inactive against a thymidine kinase-deficient VZV mutant, suggesting that phosphorylation by the viral enzyme is responsible, at least in part, for the selective anti-VZV activity of the compounds.

We examined eight isonucleoside analogues, which have variable bases at the 2' position of deoxyribose, for anti-herpes virus activities in vitro. Six of the eight compounds showed anti-herpes activity.

A novel 6-substituted acyclouridine derivative, 5-ethyl-1-ethoxymethyl-6-(phenylthio)uracil (E-EPU), has recently proved to be a highly potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. Combinations of 3'-azido-2',3'-dideoxythymidine (AZT) and E-EPU synergistically inhibit the replication of HIV-1 in MT-4 cells, whereas the cytotoxic effects of AZT and E-EPU on mock-infected MT-4 cells are not enhanced by the drug combination. Synergistic inhibition of HIV-1 replication has also been observed in peripheral blood lymphocytes. These results indicate that the combination of AZT and E-EPU should be further pursued in the treatment of AIDS.

A series of novel compounds, 5-alkynyl-1-beta-D-ribofuranosylimidazole-4- carboxamides, have been identified as broad-spectrum antiviral agents. 5-Ethynyl-1-beta-D-ribofuranosylimidazole-4- carboxamide (EICAR), the most potent congener of the group, showed antiviral potency about 10- to 100-fold greater than that of ribavirin. Similar in spectrum to ribavirin, EICAR was particularly active (50% inhibitory concentration, 0.2 to 4 micrograms/ml) against poxviruses (vaccinia virus), togaviruses (Sindbis and Semliki forest viruses), arenaviruses (Junin and Tacaribe viruses), reoviruses (reovirus type 1), orthomyxoviruses (influenza A and B viruses), and paramyxoviruses (parainfluenza virus type 3, measles virus, subacute sclerosing panencephalitis virus, and respiratory syncytial virus). EICAR was also cytostatic for rapidly growing cells (50% inhibitory concentration, 0.2 to 0.9 microgram/ml). EICAR inhibited vaccinia virus tail lesion formation at doses that were not toxic to the host. EICAR is a candidate antiviral drug for the treatment of pox-, toga-, arena-, reo-, orthomyxo, and paramyxovirus infections.

By using an assay system based on a human hepatoblastoma cell line (HB611) that continuously synthesizes hepatitis B virus DNA, the following compounds were found to inhibit hepatitis B virus DNA synthesis at concentrations that were significantly lower than their minimum cytotoxic concentrations: 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, 9-(phosphonylmethoxyethyl)adenine, 2',3'-dideoxy-2',3'-didehydrocytidine, and 2',3'-dideoxycytidine. The most potent compound was PMEDAP (50% effective concentration, 0.02 micrograms/ml). The selective index, or ratio of the 50% cytotoxic concentration to 50% effective concentration, of PMEDAP was greater than 750.

We have developed a new sensitive enzyme immunoassay (EIA) and MTT (tetrazolium salt) assay for screening compounds against two variants of human herpesvirus 6 (HHV-6A, HHV-6B) and human herpesvirus 7 (HHV-7) and evaluated the anti-HHV-6 and HHV-7 activity of a series of anti-herpesvirus compounds and acyclic nucleoside phosphonate analogues. The results indicate that the pattern of activity of these compounds against these betaherpesviruses is similar to that for human cytomegalovirus (HCMV). The highest potency and selectivity against the two variants of HHV-6 and HHV-7 was demonstrated by S2242 (N7-isomer of 6-deoxy-ganciclovir). Also, ganciclovir (GCV), foscarnet, (phosphonoformic acid; PFA) and the acyclic nucleoside phosphonate analogues such as cidofovir (HPMPC) exhibited selective inhibitory activity against these viruses. Thymidine kinase (TK)-dependent drugs (acyclovir, ACV; brivudin, BVDU; and sorivudine, BVaraU) showed little, if any, activity. These results suggest a structural homology of the DNA polymerase and a lack of TK gene among these three betaherpesviruses (HHV-6, HHV-7 and HCMV). The finding that HHV-7 was highly sensitive to GCV also suggests that HHV-7 may have an HCMV-UL97-homologue gene for the phosphorylation of GCV. The present EIA method is more rapid and sensitive than the previously reported procedures and could be useful for the large-scale screening of compounds against HHV-6 and HHV-7.

The authors investigated the effect' of thymidylate synthetase (TS) on the antivariclllazoster virus (VZV) activity of (E)5-(2-bromovinyl)-2′-deoxyuridine (BVDU). TS catalyses the conversion of deoxyuridylate (dUMP) to thymidylate (dTMP) and is a key enzyme in pyrimidine biosynthesis, providing the only source of dTMP synthesized de novoin mammalian cells. VZV encodes a specialized viral form of TS. TS activity in cells infected with VZV (TIO-VZV and TK−-VZV) increased proportionally with focus formation. From kinetic analysis using the Michaelis-Menten equation, the authors determined a Kmvalue of 6.6μm for dUMP of TS induced in VZV-infected cells and a corresponding value from mock-infected cells of only 2.8μm. BVDU inhibited the induction of TS activity in TK+-VZV-infected cells at concentrations under ×10−3μm, but did not inhibit TS activity of TK−-VZV- or mock-infected cells at concentrations as high as 10μm. Inhibitory activity of BVDU against TS induced in TK+-VZV-infected cells appears to occur when BVDU is phosphorylated to BVDU monophosphate by viral pyrimidine kinase. These results suggest that the selective inhibitory action of BVDU on VZV replication depends on a specific interaction with both viral TK and TS. 5-Bromodeoxyuridine and 5-iododeoxyuridine inhibited TS activities induced in both VZV (TK+, TK−) and mock-infected cells. Other antiherpes compounds [i.e. 1-β-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil, 9- 2-hydroxyethoxymethyl)-guanine, arabinosyladenine, and others] did not inhibit TS activity in VZV-infected cells at concentrations Of 10μm.

When BALB/c mice (young and adult animals of both sexes) were infected intraperitoneally with 10(3) viable cells of Francisella tularensis (10(2) 50% lethal dose), all mice in these groups died on day 4. Reductions in thymus weights and in numbers of thymic cortex lymphocytes were observed in all the groups, but the decline was not so severe in the young females. Increases of plasma corticosterone in the adult males began 1 day after infection, but in the young females, the levels did not increase until day 3, the same days on which the respective thymus weights began to decline. Depletion of the thymus weights in the infected mice was prevented by adrenalectomy. The lymphocytes of the thymus (T)-dependent areas in peripheral lymphoid tissues in all groups were destroyed. By using an electron microscope, we found a large quantity of F. tularensis within the macrophages in the T-dependent areas but not in the thymus. The destruction of lymphocytes in the T-dependent areas was not prevented by adrenalectomy. Therefore, it was concluded that the weight reduction of the thymus is due to the stress of the F. tularensis infection. However, we think other mechanisms are responsible for the depression of lymphocytes in the T-dependent areas of peripheral lymphoid tissues.