Two potent inhibitors based on the crystal structure of influenza virus sialidase have been designed. These compounds are effective inhibitors not only of the enzyme, but also of the virus in cell culture and in animal models. The results provide an example of the power of rational, computer-assisted drug design, as well as indicating significant progress in the development of a new therapeutic or prophylactic treatment for influenza infection.

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Racemic 2'-deoxy-3'-thiacytidine (BCH 189) is a dideoxycytidine analog having a sulfur atom in place of the 3' carbon. The enantiomers of BCH 189 have been resolved and found to be equipotent in antiviral activity against human immunodeficiency virus types 1 and 2. However, the (-)-enantiomer (3TC) is considerably less cytotoxic than the (+)-enantiomer.

The (-)-enantiomer of 2'-deoxy-3'-thiacytidine (3TC) was found to be a potent and selective inhibitor of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) in vitro. We determined its antiviral activity against a number of laboratory strains of HIV-1 and HIV-2 in a range of CD4-bearing lymphocyte cell lines (mean 50% inhibitory concentration [IC50] range, 4 nM to 0.67 microM). 3TC was also active against a range of HIV-1 strains in peripheral blood lymphocytes (mean IC50 range, 2.5 to 90 nM). The IC50 for cytotoxicity in seven lymphocyte cell cultures, including human peripheral blood lymphocytes, ranged from 0.5 to 6 mM. 3TC had no detectable antiviral activity against a range of other viruses or in cells chronically infected with HIV-1 or HIV-2. The effects of time of addition of the compound and varying the multiplicity of infection on the antiviral activity of 3TC were determined. The results showed that 3TC is a potent and selective inhibitor of HIV-1 and HIV-2 replication in vitro.

36:733-739, 1992). The effect of 3TC 5'-triphosphate on both the RNA-dependent and DNA-dependent activities of human immunodeficiency virus type 1 reverse transcriptase and DNA polymerases alpha, beta, and gamma from HeLa cells was investigated. 3TC 5'-triphosphate is a competitive inhibitor (with respect to dCTP) of the RNA-dependent DNA polymerase activity (apparent Ki = 10.6 1.0 to 12.4 5.1 ,uM, depending on the template and primer used); the DNA-dependent DNA polymerase activity is 50%o inhibited by a 3TC 5'-triphosphate concentration of 23.4 + 2.5 ,uM when dCTP is present at a concentration equal to its Km value. Chain elongation studies show that 3TC 5'-triphosphate is incorporated into newly synthesized DNA and that transcription is terminated in a manner identical to that found for ddCTP. The 50%o inhibitory concentrations of 3TC 5'-triphosphate against DNA polymerases alpha, beta, and gamma at concentrations of dCTP equal to the Km were 175 31, 24.8 10.9, and 43.8 16.4 ,uM, respectively. More detailed kinetic studies with 3TC 5'-triphosphate and DNA polymerases beta and gamma are consistent with the fact that inhibition of these enzymes by 3TC 5'-triphosphate is competitive with respect to dCTP. The values of Ki were determined to be 18.7 ,uM for DNA polymerase beta and 15.8 0.8 ,uM for DNA polymerase gamma.

We demonstrate the potent antiviral activity of a novel viral neuraminidase (sialidase) inhibitor, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GG167), administered by the intranasal route in comparison with those of amantadine and ribavirin in experimental respiratory tract infections induced with influenza A and B viruses. In an extended study in which mice were infected (day 0) with influenza A/Singapore/1/57 virus, with treatments given prophylactically plus twice daily over days 0 to 3 and with mice observed to day 10, we show that intranasally administered GG167 at 0.4 and 0.01 mg/kg of body weight per dose reduced mortality, lung consolidation, and virus titers in the lung, with no virus growing back following the cessation of treatment. In other studies with influenza B/Victoria/102/85 virus in which infected mice were culled after the cessation of treatment, the calculated intranasal dose required to reduce virus titers in the lungs of treated animals to 10% of that seen in untreated controls (EDAUC,o [where AUC is area under the virus titer days curve]) was 0.085 mg/kg per dose. GG167 was inactive against influenza viruses A and B when given by the intraperitoneal or oral route (EDAUC,0, >100 mg/kg per dose). GG167 was metabolically stable, with an elimination half-life of 10 min following intravenous administration. While readily bioavailable by systemic routes, it was poorly bioavailable by the oral route. Its potent efficacy by the intranasal route but lack of efficacy by other routes, relative to those of amantadine and ribavirin, was explicable in terms of its in vitro activity, bioavailability, and pharmacokinetic properties and with the extracellular activity of viral sialidase.

The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants. NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture. Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays. Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors. No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found. However, changes in conserved amino acids in the hemagglutinin were detected. These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229. Hence, mutations at these sites could be expected to affect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.

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