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Despite the fact that enteroviruses are implicated in a variety of human diseases, there is no approved therapy for the treatment of enteroviral infections. Here, a series of 2,6-dihalophenyl-substituted 1H,3H-thiazolo[3,4-a]benzimidazoles with anti-enterovirus activity is reported. The compounds elicit potent activity against coxsackievirus A9, echovirus 9 and 11 and all six strains of coxsackievirus B. A structure-activity relationship analysis revealed that the presence of substituents at position 6 of the tricyclic system positively influences the antiviral activity, whereas substitutions at position 7 are less favorable. In particular a 6-trifluoromethyl substitution leads to a substantial improvement of the antiviral activity as compared to the unsubstituted structure. Furthermore, an additional introduction of a 2-Cl, 6-F substitution on the phenyl at C-1 results in a further increase of the antiviral activity. Hence, 1-(2-chloro-6-fluorophenyl)-6-trifluoromethyl-1H,3H-thiazolo[3,4-a]benzimidazole results in a dose-dependent inhibition of viral replication with a 50% effective concentration (EC50) of 0.41 microg/ml without any detectable cytotoxicity at the highest concentration (100 microg/ml) tested.

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC(50)) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78+/-21 microM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 microM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.

Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells including genes of viral origin. To evaluate the therapeutic efficacy of siRNA against the hepatitis B virus (HBV), we studied the effect of transfection of the HBV-inducible cell lines HepAD38 and HepAD79 with siRNA specific for the core gene of the HBV genome. HepAD38 cells produce wild-type HBV, whereas HepAD79 cells produce the lamivudine resistant YMDD variant. Transfection of HepAD38 cells with either 1.6 or 4 microg/ml siRNA resulted in a profound inhibition (72% and 98%, respectively) of viral replication (as assessed by real-time quantitative PCR). The inhibitory effect was corroborated by a marked reduction of HBV core protein synthesis in induced HepAD38 cells. In HepAD79 cells, transfected with 1.6 or 4 microg/ml HBV-specific siRNA, virus production was reduced by 75% and 89%, respectively.

We overexpressed and purified from Escherichia coli the dihydrofolate reductase (DHFR) of the gammaherpesviruses human herpesvirus 8 (HHV-8), herpesvirus saimiri (HVS), and rhesus rhadinovirus (RRV). All three enzymes proved catalytically active. The K(m) value of HHV-8 DHFR for dihydrofolate (DHF) was 2.02+/-0.44 microM, that of HVS DHFR was 4.31+/-0.56 microM, and that of RRV DHFR is 7.09+/-0.11 microM. These values are approximately 5-15-fold higher than the K(m) value reported for the human DHFR. The K(m) value of HHV-8 DHFR for NADPH was 1.31+/-0.23 microM, that of HVS DHFR was 3.78+/-0.61 microM, and that of RRV DHFR was 7.47+/-0.59 microM. These values are similar or slightly higher than the corresponding K(m) value of the human enzyme. Methotrexate, aminopterin, trimethoprim, pyrimethamine, and N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), all well-known folate antagonists, inhibited the DHFR activity of the three gammaherpesviruses competitively with respect to DHF but proved markedly less inhibitory to the viral than towards the human enzyme.

A novel membrane-soluble prodrug of the 5'-monophosphate derivative of 3TC containing a phenyl group and the methyl ester of L-alanine linked to the phosphorus through a phosphoramidate bond with the primary amino moiety (designated Cf 1109) was prepared. The 3TC prodrug proved less potent an inhibitor of HIV-1 and HIV-2 replication in CEM cell cultures than 3TC, but lost only 20-fold antiviral potency in 2'-deoxycytidine kinase-deficient CEM/dCK- cells compared with a more than 2,000-fold decrease of activity of 3TC. In contrast, 3TC and Cf 1109 proved equally highly effective in inhibiting HBV release in supernatants of HBV-transfected Hep G2 2.2.15 cell cultures (50% effective concentration approximately 0.02 microM). Both compounds easily selected for highly resistant HIV-1 strains at a comparable speed of breakthrough. The mutant viruses contained an 184-Ile and/or 184-Val amino acid change in their reverse transcriptase. Our data are suggestive for a relatively poor delivery of 3TC-MP in the intact CEM cells but a remarkably high delivery of 3TC and/or 3TC-MP in the intact Hep G2 2.2.15 cells.

To develop an animal model for the therapy of AIDS with human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors, we recently constructed a hybrid simian immunodeficiency virus (SIV)/HIV-1 in which the RT gene of SIV was replaced by the RT gene of HIV-1. This chimaeric virus, designated RT-SHIV, was found to be markedly sensitive to the inhibitory effects of both nucleoside (ddN) and non-nucleoside RT inhibitors (NNRTIs). In contrast, SIV was inhibited only by ddNs (i.e., 3TC and AZT), but not NNRTIs. When RT-SHIV was grown in the presence of 3TC, nevirapine, TSAO-m3T or the thiocarboxanilide UC-42 drug-resistant mutant virus strains emerged in cell culture as rapid as for HIV-1(IIIB). The antiviral sensitivity/resistance spectrum of the mutant RT-SHIV strains against NNRTIs and ddNs, and the nature of the mutations that appeared in their RT were similar to those of the mutant HIV-1 strains that were selected under identical experimental conditions. Infection of macaques with RT-SHIV may be a useful tool for studying the mechanism of NNRTI-resistance development and the therapy of NNRTI-resistant viruses in an animal model.

Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase (RT) containing lysine (Lys) instead of glutamic acid (Glu) at position 138 proved fully resistant to the inhibitory effect of TSAO derivatives, but retained marked sensitivity to all other HIV-1-specific inhibitors investigated. In contrast, 181 Tyr-->Cys mutated RT lost sensitivity to all HIV-1-specific inhibitors. There was a close correlation between the sensitivity/resistance pattern of HIV-1-specific inhibitors against mutated (138 Glu-->Lys) recombinant HIV-1 RT and mutant virus strains selected for resistance against TSAO-m3T in cell culture and proven to contain the 138-Lys mutation as the sole mutation within the amino acid 50-270 region of their RT.

Ribavirin and EICAR are two antiviral agents that share a similar antiviral activity spectrum and are targeted at inosine 5'-monophosphate (IMP) dehydrogenase. Neither ribavirin nor EICAR inhibit the replication of human immunodeficiency virus (HIV) in peripheral blood lymphocyte cells (PBL) at subtoxic concentrations. However, both compounds markedly potentiate the anti-HIV activity of 2',3'-dideoxyinosine (DDI) in PBL cells without a marked increase of toxicity. Both the increased IMP levels and the decreased guanine nucleotide levels caused by ribavirin and EICAR may be responsible for their potentiating effect on the anti-HIV activity of DDI.

A number of acyclic nucleoside phosphonate analogues, including 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and its 2,6-diaminopurine derivative PMEDAP, (R,S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine [(R,S)-FPMPA] and its 2,6-diaminopurine derivative (R,S)-FPMPDAP were evaluated for their inhibitory effects on HIV-1 replication in two natural human cell systems, i.e. peripheral blood lymphocytes (PBL) and freshly prepared monocyte/macrophages (M/M). All compounds were potent inhibitors of HIV-1 replication in PBL [50% effective concentration (EC50): 0.94-3.9 microM] and M/M (EC50: 0.022-0.95 microM). In particular, (R,S)-FPMPA and (R,S)-FPMPDAP showed a greater antiviral selectivity than PMEA and PMEDAP due to the virtual lack of toxicity of the former compounds in these cell systems. Also, the antiviral selectivity of the acyclic nucleoside phosphonate analogues was much higher in M/M than in the human T-cell lines MT-4, ATH8 and CEM.

The antiherpetic agent 9-beta-D-arabinofuranosyladenine (araA) needs to be phosphorylated to its 5'-triphosphate to be effective as an inhibitor of herpes simplex virus replication. Adenosine kinase and deoxycytidine kinase are assumed to convert araA to its 5'-monophosphate. We now found that araAMP is converted to its 5'-triphosphate through a direct pyrophosphate transfer from 5-phosphoribosyl-1-pyrophosphate (PRPP) by PRPP synthetase. The efficiency of phosphorylation of araAMP to araATP is about 5% of that of AMP, as estimated from their Vmax/Km ratios for PRPP synthetase. AraATP is converted to araAMP by PRPP synthetase at a 4-fold higher Km but similar Vmax as ATP.

The antiviral drug, 9-(2-phosphonylmethoxyethyl) adenine (PMEA) was linked to a synthetic and neutral polymer bearing mannosyl residues to allow its internalization by macrophages via membrane lectins. PMEA bound to the mannosylated polymer was more efficient in vitro than free PMEA in preventing lysis of human macrophages by herpes virus.

Tumor necrosis factor alpha (TNF-alpha) completely reverses the activity of azidothymidine (AZT) against human immunodeficiency virus type 1 (HIV-1) in MOLT-4 cell cultures. The 50% effective concentration of AZT, required to protect MOLT-4 cells against the cytopathic effect of HIV-1, increased from 5.8 nM in the absence of TNF-alpha to greater than 125 microM in the presence of TNF-alpha (100 U/ml). TNF-alpha also antagonized the anti-HIV-1 activity of dideoxycytidine but did not markedly affect the anti-HIV-1 activity of dextran sulfate. The intracellular phosphorylation pattern of AZT was not changed upon the presence of TNF-alpha.

3'-Azido-2',3'-dideoxyguanosine (AzddGuo) is a potent and selective inhibitor of human immunodeficiency virus (HIV) in vitro. AzddGuo completely inhibits HIV-induced cytopathogenicity and viral antigen expression in MT-4 cells at a concentration of 5.0 microM. Its 50% effective dose for inhibiting HIV-induced cytopathogenicity is 1.4 microM, as compared to 6.4 microM for 2',3'-dideoxyadenosine (ddAdo). Thus, AzddGuo is approximately 4.6-fold more potent as an anti-HIV agent than ddAdo, one of the most promising compounds for the treatment of AIDS. However, AzddGuo is about 4.7 times more cytotoxic than ddAdo, so that its selectivity index, as based on the ratio of the 50% cytotoxic dose to the 50% antiviral effective dose, is almost the same as that of ddAdo (136 and 139, respectively).

Aurintricarboxylic acid, an anionic triphenylmethane dye, and Evans Blue, an anionic compound structurally related to suramin, are, like suramin itself, inhibitors of human T-cell lymphotropic virus type III (HTLV-III)/-lymphadenopathy-associated virus (LAV) in vitro. These compounds may be targeted, at least in part, at the HTLV-III/LAV reverse transcriptase. The lack of any appreciable cytostatic action of aurintricarboxylic acid, Evans Blue and suramin against several murine and human cell lines, their inability to inhibit cellular DNA, RNA and protein synthesis, and their high lethal dose-50 (greater than or equal to 0.340 g/kg) for NMRI mice point to the selectivity of the compounds as inhibitors of HTLV-III/LAV.

For a series of adenosine analogues a close correlation (r = 0.986) was found between their antiviral potency (against vesicular stomatitis virus) and their inhibitory effects (Ki/Km) on S-adenosylhomocysteine (AdoHcy) hydrolase; thus, in order of increasing inhibitory potency for both virus replication and AdoHcy hydrolase activity: (S)-9-(2,3-dihydroxypropyl)adenine less than (RS)-3-adenin-9-yl-2-hydroxypropanoic acid (isobutyl ester) less than carbocyclic 3-deazaadenosine less than neplanocin A. Our findings point to AdoHcy hydrolase as the target for the broad-spectrum antiviral activity of these adenosine analogues.

The 2',3'-dideoxyriboside (ddDAPR), 2',3'-didehydro-2',3'-dideoxyriboside (ddeDAPR) and 3'-azido-2',3'-dideoxyriboside (AzddDAPR) of 2,6-diaminopurine have been previously recognized as potent inhibitors of human immunodeficiency virus replication. These compounds are also potent inhibitors of adenosine deaminase and inhibit the deamination of 9-beta-D-arabinofuranosyladenine (araA). ddDAPR, ddeDAPR and AzddDAPR markedly potentiate the antiviral activity of araA against herpes simplex virus type 1 (HSV-1), type 2 (HSV-2) and vaccinia virus (VV). When used at a concentration of 20 micrograms/ml, which had by itself no antiviral effect, ddDAPR, ddeDAPR and AzddDAPR increased the ability of araA to suppress HSV-1, HSV-2 and VV yield by several orders of magnitude. The maximum antiviral effect was obtained with the combinations of ddDAPR or ddeDAPR with araA concentrations of 1 and 10 micrograms/ml.

2',3'-Didehydro-2',3'-dideoxy-5-chlorocytidine (D4CC) is, in contrast with 2',3'-dideoxy-5-chlorocytidine (ddClCyd) and 2',3'-didehydro-2',3'-dideoxy-5-chlorouridine (D4CU), a potent and selective inhibitor of the replication of human immunodeficiency virus (HIV) types 1 and 2, simian immunodeficiency virus (SIV) and simian AIDS related virus (SRV). D4CC is a poor inhibitor of the phosphorylation of [5-3H]2'-deoxycytidine (dCyd) by partially purified MT-4 cell dCyd kinase (Ki: 612 microM). The findings that (i) D4CC has little, if any, affinity for MT-4 cell Cyd/dCyd deaminase, (ii) D4CU is not antivirally active and (iii) the antiretroviral action of D4CC can be reversed by dCyd, but not dThd, indicate that D4CC is antivirally active as its Cyd metabolite (D4CC 5'-triphosphate) and does not need to be deaminated (to the corresponding Urd metabolite) to exert its antiretroviral action.

The carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (C-BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (C-IVDU), in which the sugar moiety is replaced by a cyclopentane ring, are as efficient substrates for the herpes simplex type 1 (HSV-1)-encoded thymidine kinase (TK) as their parent compounds (BVDU and IVDU). This conclusion is based on the binding affinities (Ki) of BVDU, IVDU, C-BVDU and C-IVDU to the HSV-1 TK and on the phosphorylation rates (Km, Vmax) of (125I)IVDU and (125I)C-IVDU by the enzyme. The specific phosphorylation of C-BVDU and C-IVDU by the viral TK may explain why these compounds are highly selective inhibitors of HSV-1 replication.

The acyclic nucleotide analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] is a potent and selective inhibitor of African swine fever virus (ASFV) replication. Using the DNA-DNA hybridization technique with plasmid pRPEL-2 as probe, we have shown that (S)-HPMPA exerts a specific, dose-dependent, inhibitory effect on viral DNA synthesis. Also, (S)-HPMPA inhibits the production of late viral proteins, especially IP-73, in ASFV-infected MS and Vero cells. When evaluated under the same experimental conditions, phosphonoacetic acid (PAA) also caused an inhibition of viral DNA and late viral protein synthesis but only so at a concentration which was 10- to 20-fold higher than that required for (S)-HPMPA.

The 2',3'-dideoxyriboside of 2,6-diaminopurine(ddDAPR) is, like 2',3'-dideoxyadenosine (ddAdo), a potent and selective inhibitor of human immunodeficiency virus (HIV) in vitro. The ddDAPR compound inhibits HIV antigen expression and HIV-induced cytopathogenicity in MT4 cells at a 50% effective dose (ED50) of 2.5-3.6 microM, as compared to 3.1-6.4 microM for ddAdo. Both compounds are endowed with a high selectivity index: 112 for ddDAPR and 139 for ddAdo. The 2',3'-unsaturated derivatives of ddDAPR and ddAdo, i.e. ddeDAPR and ddeAdo, are considerably more cytotoxic and less effective against HIV than the parental compounds. Like ddAdo, ddDAPR is only weakly inhibitory to the proliferation and DNA and RNA synthesis of a series of human B-lymphoblast, T-lymphoblast and T-lymphocyte cell lines. In contrast to ddAdo, which is rapidly deaminated by beef intestine adenosine deaminase at an initial velocity (Vi) of 145 mumol/mg protein/min, ddDAPR and ddeDAPR are poor substrates for the enzyme (Vi: 8 and 0.7 mumol/mg protein/min, respectively), which further contributes to the potential of ddDAPR as a chemotherapeutic agent against AIDS.

Fuchsin acid, an anionic dye, is a selective inhibitor of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in vitro. Its 50% effective dose for inhibition of HIV-1-induced cytopathogenicity in MT-4 cells and HIV-1 antigen expression in HUT-78 cells is 42 and 16 microM, respectively. These values are comparable to those of suramin, the first compound shown to be a selective inhibitor of HIV-1. However, fuchsin acid is less cytotoxic than suramin. The selectivity index of fuchsin acid, based on the ratio of the 50% cytotoxic dose to the 50% effective dose, is greater than 74 in MT-4 cells and greater than 39 in HUT-78 cells. Fuchsin acid is a much weaker inhibitor of HIV-1 reverse transcriptase than are suramin and aurintricarboxylic acid. Fuchsin acid does not interfere with the adsorption of HIV-1 particles to MT-4 cells even at concentrations that completely block HIV-1 replication in MT-4 cells. The mechanism of action of fuchsin acid remains subject of further study.

The 2',3'-dideoxyriboside of 2,6-diaminopurine (ddDAPR) and its 2',3'-didehydro derivative (ddeDAPR) are poor substrates for adenosine deaminase (ADA) but potent inhibitors of the enzyme. Their Km values for ADA are of the same order of magnitude as those of the natural adenosine (Ado) and 2'-deoxyadenosine (dAdo), but their Vmax values are 35-fold (ddDAPR) to 350-fold (ddeDAPR) lower than those of Ado and dAdo. The Ki/K values of ADA for ddeDAPR (as inhibitor) and Ado, 2',3'-dideoxyadenosine (ddAdo) and 9-beta-D-arabinofuranosyladenine (araA) as the substrates are 0.17, 0.05 and 0.06, respectively. ddDAPR is about 3-fold less potent as an inhibitor of ADA than ddeDAPR. The 2,6-diaminopurine derivatives ddeDAPR and ddDAPR [which is also a potent inhibitor of human immunodeficiency virus (HIV)], may hold great promise, from a chemotherapeutic viewpoint, in combination with other adenosine analogues such as ddAdo and araA, which have been recognized and/or being pursued as either anti-retrovirus or anti-herpesvirus agents.

2',3'-Dideoxythymidine (ddThd) and its 2',3'-unsaturated derivative 2',3'-dideoxythymidinene (ddeThd) are potent and selective inhibitors of human immunodeficiency virus (HIV) in vitro. When evaluated for their inhibitory effects on the cytopathogenicity of HIV in MT-4 cells, ddThd and ddeThd completely protected the cells against destruction by the virus at a concentration of 1 microM and 0.04 microM, respectively. In this aspect, ddeThd was about 5 times more potent than 2',3'-dideoxycytidine (ddCyd), one of the most potent and selective anti-HIV compounds now pursued for its therapeutic potential in the treatment of AIDS. ddThd and ddeThd also suppressed HIV antigen expression at 1 microM and 0.04 microM, respectively. Their selectivity indexes, as based on the ratio of the 50% cytotoxic dose to the 50% antiviral effective dose, were 120 (ddeThd) and greater than 625 (ddThd).

The effect of tumor necrosis factor (TNF) on the replication of human immunodeficiency virus type 1 (HIV-1) was investigated in several T4 lymphocyte cell lines. TNF markedly enhanced the cytopathogenicity of HIV-1, virion-associated reverse transcriptase (RT) activity in the cell culture supernatant, and viral antigen expression in MOLT-4 cells as early as 3 days after HIV-1 infection. A slight increase in RT activity was also observed in the supernatant of H9 cell cultures exposed to TNF. However, TNF did not increase either RT activity in MT-4 cell supernatants or viral antigen expression in HUT-78 cells. Thus, TNF is able to stimulate the replication of HIV-1 in de novo infected T4 cells although not all T4 cells seem to be sensitive to this stimulatory effect.

2',3'-Dideoxycytidinene (ddeCyd), the 2',3'-unsaturated derivative of 2',3'-dideoxycytidine (ddCyd) is, like ddCyd itself, a potent and selective inhibitor of HTLV-III/LAV in vitro. This conclusion is based on the relatively high ratio of effective antiviral dose (0.3 microM) versus cell growth inhibitory concentration (20-35 microM) and the lack of any appreciable inhibitory activity against a series of non-oncogenic RNA and DNA viruses. Both compounds were considerably more inhibitory to human lymphoid cell lines than human nonlymphoid or murine cell lines. They were highly dependent on prior activation by deoxycytidine kinase to exert their anti-HTLV-III/LAV and cytostatic effects. In contrast with ddCyd, ddeCyd lost part of its anti-retrovirus effect upon prolonged incubation (10 days) with the virus-infected cells in culture.