Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5 and 3 ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5 cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

The in vitro resistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335.T he hepatitis C virus (HCV)-encoded NS3 protease is essential for viral replication and has long been considered an attractive target in drug design efforts (3, 5). NS3 protease inhibitors (PIs) can induce substantial reductions in HCV RNA plasma levels, and several candidates have progressed through clinical development to offer improved treatment options (for a review, see reference 27). Two PIs, boceprevir and telaprevir, were recently approved for use in combination with pegylated interferon (Peg-IFN) and ribavirin (1,6,7,19). The selection of drug-resistant variants is commonly observed in patients experiencing virologic rebound during treatment with PIs (16,[20][21][22]24).BI 201335 is a potent HCV NS3/4A PI (15, 28) currently in phase 3 clinical trials in combination with Peg-IFN and ribavirin as well as phase 2 assessment with other HCV direct acting antivirals in IFN-sparing regimens. BI 201335 exhibited a profound reduction in viral load when administered for 14 days as monotherapy in treatment-naïve patients or for 28 days in combination with Peg-IFN and ribavirin in treatment-experienced patients (16). In these studies, viral breakthrough was observed in most patients on monotherapy, whereas breakthrough was less frequent in patients undergoing combination treatment. Distinct resistant NS3 variants R155K and D168V predominated for genotype 1a and 1b (GT 1a and GT 1b), respectively (8,16).This study was designed to evaluate the genotypic and phenotypic profiles of the resistant variants that emerged during in vitro selection in the presence of BI 201335 in the replicon system and to relate these results to clinical observations. Replicons resistant to BI 201335 were selected in GT 1a H77 and GT 1b CON-1 replicon cell lines in the presence of 2 concentrations (100ϫ and 1,000ϫ drug concentration required to reduce HCV RNA or the luciferase reporter levels by 50% [EC 50 ]) of drug for 3 weeks and G-418 as previously described (9). With the lower concentration of BI 201335, resistant variants encoding NS3 changes at residues 155, 156, and 168 were selected with the GT 1b replicon, with D168G as the predominant variant (55%). R155K was the predominant variant (68%) selected with the GT 1a replicon (Table 1) and is consistent with the predominant variant selected in GT 1a HCV-infected patients (16). At the higher concentration of BI 201335, essentially only D168 variants were selected with D168 A and V as the predominant variants in both genotypes.In order to confirm that the mutations ob...

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Previous studies have shown that herpes virus ribonucleotide reductase can be inhibited by a synthetic nonapeptide whose sequence is identical to the C-terminal of the small subunit of the enzyme. This peptide is able to interfere with normal subunit association that takes place through the C-terminal of the small subunit. In this report, we illustrate that inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of subunit R2 is also observed for the enzyme isolated from Escherichia coli, hamster, and human cells. The nonapeptide corresponding to the bacterial C-terminal sequence was found to inhibit E. coli enzyme with an IC50 of 400 microM, while this peptide had no effect on mammalian ribonucleotide reductase. A corresponding synthetic peptide derived from the C-terminal of the small subunit of the human enzyme inhibited both human and hamster ribonucleotide reductases with IC50 values of 160 and 120 microM, respectively. However, this peptide had no inhibitory activity against the bacterial enzyme. Equivalent peptides derived from herpes virus ribonucleotide reductase had no effect on either the bacterial or mammalian enzymes. Thus, subunit association at the C-terminal of the small subunit appears to be a common feature of ribonucleotide reductases. In addition, the inhibitory phenomenon observed with peptides corresponding to the C-terminal appears not only to be universal, but also specific to the primary sequence of the enzyme.

Nucleoside analogs such as acyclovir (ACV) and penciclovir and prodrugs thereof have been approved as drugs of choice for the treatment of herpes simplex virus (HSV) infections (1, 11). While nucleoside-based therapeutics are quite effective for the treatment of primary and recurrent mucocutaneous infections, present medications are not effective for the treatment of nucleoside-resistant herpesvirus infections in immunocompromised individuals (4,5,29). Moreover, nucleoside-based antiviral therapeutics have limited effects on the establishment of latent HSV infections (6,17,31). Therefore, significant improvements in therapy may be achievable with distinct inhibitors with improved efficacy and pharmacokinetic (PK) properties and a new mechanism of action. We have reported previously on the discovery of specific non-nucleoside-based inhibitors of the HSV type 1 (HSV-1) helicase-primase (10). This enzyme is composed of the virus-encoded UL5, UL8, and UL52 gene products, which are all essential for HSV DNA replication and growth (3,7,9,19,23). The most optimized aminothiazolyl-phenyl compounds exhibited potent antiviral activity against a series of HSV strains analyzed in vitro, including HSV-1 and HSV-2 strains that are ACV r . One of the inhibitors, BILS 179 BS, showed antiviral activity in murine models of wild-type HSV disease after oral administration (10), but its activity against ACV r HSV disease had not been evaluated. In the present report, we provide a detailed examination of the PK and pharmacodynamic properties of a helicase-primase inhibitor, BILS 45 BS, in a mouse model of ACV r HSV infection. PK studies were done with hairless mice, where BILS 45 BS demonstrated in vivo efficacy against wild-type HSV-1 infection with a 50% effective dose (ED 50 ) of 56 mg/kg. The athymic nude mouse model was chosen for the present study because ACV r HSV-1 fails to induce significant disease in normal mice (2, 14-16). In addition, since ACV r HSV infections cause significant disease mainly in the immunocompromised patient population, information obtained with these immunodeficient animals may have clinical relevance (14-16). Our results show that BILS 45 BS, an analog structurally related to BILS 179 BS, exhibited excellent oral efficacy against ACV r HSV-1 infections in nude mice, highlighting the potential of this novel class of antiherpetic agents for the treatment of ACV r HSV disease in humans. MATERIALS AND METHODSCell culture and viruses. All of the cell culture reagents and media used in this study were obtained from Gibco BRL (Burlington, Ontario, Canada). Cells were from the American Type Culture Collection (Manassas, Va.). Vero (African green monkey kidney) cells were grown in Dulbecco's modified Eagle's medium supplemented with 8% fetal bovine serum, 100 U of penicillin per ml, 100 g of streptomycin sulfate per ml, and 100 g of kanamycin sulfate per ml. Baby hamster kidney (BHK) 21/C13 (ATCC CCL10) cells were grown in ␣-MEM instead of Dulbecco's modified Eagle's medium. All cells were grown at 37°C in an ...