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Antimicrobial Agents and Chemotherapy, June 2005, p. 2460-2466, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2460-2466.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Small Molecules VP-14637 and JNJ-2408068 Inhibit Respiratory Syncytial Virus Fusion by Similar Mechanisms

Janet L. Douglas, Marites L. Panis, Edmund Ho, Kuei-Ying Lin, Steve H. Krawczyk, Deborah M. Grant, Ruby Cai, Swami Swaminathan, Xiaowu Chen, and Tomas Cihlar^*

Gilead, 333 Lakeside Dr., Foster City, CA 94404

Received 29 August 2004/ Returned for modification 28 October 2004/ Accepted 26 December 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here we present data on the mechanism of action of VP-14637 and JNJ-2408068 (formerly R-170591), two small-molecule inhibitors of respiratory syncytial virus (RSV). Both inhibitors exhibited potent antiviral activity with 50% effective concentrations (EC₅₀s) of 1.4 and 2.1 nM, respectively. A similar inhibitory effect was observed in a RSV-mediated cell fusion assay (EC₅₀ = 5.4 and 0.9 nM, respectively). Several drug-resistant RSV variants were selected in vitro in the presence of each compound. All selected viruses exhibited significant cross-resistance to both inhibitors and contained various single amino acid substitutions in two distinct regions of the viral F protein, the heptad repeat 2 (HR2; mutations D486N, E487D, and F488Y), and the intervening domain between HR1 and HR2 (mutation K399I and T400A). Studies using [³H]VP-14637 revealed a specific binding of the compound to RSV-infected cells that was efficiently inhibited by JNJ-2408068 (50% inhibitory concentration = 2.9 nM) but not by the HR2-derived peptide T-118. Further analysis using a transient T7 vaccinia expression system indicated that RSV F protein is sufficient for this interaction. F proteins containing either the VP-14637 or JNJ-2408068 resistance mutations exhibited greatly reduced binding of [³H]VP-14637. Molecular modeling analysis suggests that both molecules may bind into a small hydrophobic cavity in the inner core of F protein, interacting simultaneously with both the HR1 and HR2 domains. Altogether, these data indicate that VP-14637 and JNJ-2408068 interfere with RSV fusion through a mechanism involving a similar interaction with the F protein.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Respiratory syncytial virus (RSV) is a major cause of severe respiratory tract infections in pediatric, elderly, and immunocompromised patients (7, 16, 19). Despite extensive research to develop a RSV vaccine, currently no vaccine has been approved. Prophylactic antibodies have been developed that effectively reduce the incidence and severity of RSV disease in the high-risk pediatric population (8, 9). However, the only antiviral treatment available for patients with RSV disease is ribavirin, a nucleoside analog with a suboptimal clinical efficacy and safety profile (18). Recently, several promising small-molecule inhibitors with in vitro and in vivo anti-RSV activity have been identified. These include the disulfonated stilbenes CL387626 and RFI-641 (15, 22), the benzimidazole derivative JNJ-2408068 (formerly R-170591) (1, 21), the benzotriazole derivative BMS-433771 (4, 23), and the triphenol compound VP-14637 (13) (D. C. Pevear et al., Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1854, 2000). Initial studies indicated that these inhibitors act early in the RSV replication cycle and mutations conferring resistance to these structurally diverse molecules map to various regions of the viral fusion (F) protein (1, 4, 14, 15).

The RSV F protein that mediates the fusion of viral envelope with host cell membrane consists of two disulfide-linked subunits, F1 and F2. The F1 subunit contains a hydrophobic fusion peptide at its N terminus, followed by two heptad repeats (HR1 and HR2) separated by almost 300 amino acids of intervening region (5). It is believed that a conformational change of the F protein homo-trimer leads to the formation of a stable HR1/HR2 six-helix bundle, which triggers the actual fusion of viral and cell membranes (11, 12, 24). Studying inhibitors of this process will not only increase our understanding of the fusion mechanism but may help to design more effective anti-RSV treatments.

We previously described the interaction of VP-14637 with the RSV F protein (6). In the present study, we focused on the potential functional similarities between VP-14637 and the structurally unrelated inhibitor JNJ-2408068 (Fig. 1).

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FIG. 1. Structures of VP-14637 and JNJ-2408068.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and viruses. Hep-2 and BHK-21 cell lines were cultured in minimal essential medium plus 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 10% fetal bovine serum. The BHK-21 cells were also supplemented with 10% tryptose phosphate broth. Primary chicken embryonic fibroblasts (CEF) were cultured in Dulbecco's minimal essential medium with 4.5 g/liter glucose, 4 mM glutamine, and 10% fetal bovine serum at 39°C. All cell lines were obtained from the American Type Culture Collection (Manassas, VA). The RSV strain A2 (American Type Culture Collection) and the attenuated vaccinia virus expressing T7 polymerase (MVA-T7), kindly provided by Bernard Moss (National Institutes of Health, Bethesda, MD), were grown and titers were determined as previously described (6).

Plasmids. To generate pCDNA-F construct, the F gene from RSV A2 was obtained by reverse transcription-PCR amplification of RNA isolated from RSV-infected Hep-2 cells and cloned into pCDNA 3.1 expression vector (Invitrogen, Carlsbad, CA). To construct the F protein mutants, site-directed mutagenesis (QuickChange protocol from Stratagene) was performed on a fragment of F gene (nucleotides 916 to 1725), which had been subcloned into pBluescript IIsk⁺ (Stratagene, La Jolla, CA). To verify the mutations, the inserts were sequenced using an ABI Prism BigDye terminator kit on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA). The fragments containing mutations were cloned back into the pCDNA-F plasmid. Plasmids for the T7 expression system were described previously (6).

Compounds. VP-14637 {5,5'-bis[1-(((5-amino-1H-tetrazolyl)imino)methyl)] 2,2',4'' -methylidynetrisphenol} (Fig. 1) was synthesized as described previously (6). [³H]VP-14637 (specific activity, 4 Ci/mmol) was prepared by Moravek Biochemicals (Brea, CA). JNJ-2408068 {2-[[2-[[1-(2-aminoethyl)-4-piperidinyl]amino]-4-methyl-1H-benzimidazol-1-yl]-6-methyl-3-pyridinol]} (Fig. 1) was synthesized in nine steps from the commercially available 2-nitro-3-methyl benzoic acid as described previously (Janssens et al., patent no. WO 01/00611, WO 01/00612, and WO 01/00615). Ribavirin was obtained from Sigma. The HR2-derived peptide T-118 (10) was synthesized by Genemed Synthesis (South San Francisco, CA).

Antiviral activity. Hep-2 cells were seeded into 96-well plates at 1,000 cells per well. After 24 h, the cells were infected with RSV at a multiplicity of infection (MOI) of 0.1 and incubated at 37°C for 2 h. The inoculum was replaced with fresh media containing serial dilutions of the tested inhibitors. Following a 4-day incubation, the media was removed and the cells were stained with crystal violet (0.1% in 20% methanol). The RSV-induced cytopathic effect was determined spectrophotometrically by reading absorbance at 630 nm, and the concentration of inhibitor that reduced the cytopathic effect by 50% relative to untreated control (EC₅₀) was calculated by nonlinear regression using Prizm software (GraphPad, San Diego, CA).

Viral fusion assay. The fusion assay was described previously (6). Briefly, BHK-21 cells were infected with RSV for 24 h at an MOI of 0.5 and then transfected with a T7-driven luciferase plasmid (T7-Luc) for 3 to 5 h. During the infection, another population of BHK-21 cells was transfected with a plasmid expressing T7 polymerase. After transfection, the virus-infected cells were washed and fresh medium containing the tested inhibitor was added to the cells. The second population of cells transfected with the T7 polymerase plasmid was trypsinized and overlaid onto the virus-infected, T7-Luc-transfected cells in a 1:1 ratio. After 6 to 7 h, the mixed population of cells were lysed and analyzed for luciferase activity using the luciferase assay system (Promega, Madison, WI). Luciferase activity was measured as relative light units in a TopCount NXT (Packard Bioscience).

Selection and characterization of drug-resistant viruses. Viruses resistant to VP-14637 or JNJ-2408068 were selected by passaging RSV in Hep-2 cells in the presence of increasing concentrations of either inhibitor. The starting drug concentration for VP-14637 was 5 nM. For JNJ-2408068, two concentration ranges were used for selection. The low range was 10 to 100 nM and the high range was 25 to 2,000 nM. Control viruses were grown in parallel to the same passage without drug. Susceptibility of the resultant viruses to VP-14637 and JNJ-2408068 was determined in the cytopathic assay, and the F genes were sequenced.

Compound binding assays. Hep-2 cells were seeded into 12-well plates at 1 x 10⁵ cells/well and infected with RSV at an MOI of 0.05 for 48 h followed by a 2-h incubation with 10 nM [³H]VP-14637 alone or in the presence of various concentrations of the competing inhibitor (JNJ-2408068 or T-118) at 37°C. After incubation, the media was removed and the cells were washed three times in phosphate-buffered saline, lysed in 1% Igepal, added to 5 ml of EcoLite scintillation fluid (ICN), and read in an LS6500 scintillation counter (Beckman Coulter, Fullerton, CA). Expression using MVA-T7 vaccinia virus in CEF cells (20) was employed to assess the direct binding of VP-14637 to the wild-type and mutant RSV F proteins. Cells were plated at 4 x 10⁵ cells/well in 12-well plates and incubated for 24 h at 39°C. After the incubation, the cells were infected with MVA-T7 at an MOI of 1 for 1.5 to 2 h, washed in serum-free media, and then transfected with pCDNA-F or an empty control plasmid pCDNA-3.1 using Lipofectamine Plus according to the manufacturer's directions (Invitrogen). After an additional 24 h, the cells were incubated with [³H]VP-14637 for 6 h. Compound binding was quantified as described above. No significant differences were found in the cell surface expression of the wild-type and various mutant forms of F protein when compared by fluorescence-activated cell sorting (FACS) analysis performed according to previously described protocol (6).

Molecular modeling. Docking of small-molecule inhibitors to the HR1 hydrophobic pocket was carried out using Tripos' Sybyl modeling suite of programs (Tripos, St. Louis, MO) on SGI's Octane workstation (SGI, Mountain View, CA). Optimal binding modes for VP-14637 and JNJ-2408068 were identified by a manual docking process, since automatic docking did not yield satisfactory results. All the images were generated with Sybyl.

Top Abstract Introduction Materials and Methods Results Discussion References

 
To determine whether VP-14637 and JNJ-2408068 inhibit fusion in a similar manner, we utilized a RSV-induced cell fusion assay. RSV-infected BHK-21 cells were transfected with a plasmid containing a T7-driven luciferase gene and overlaid with uninfected BHK-21 cells transfected with a plasmid containing the T7 RNA polymerase gene. Luciferase expression was quantified 6 h after mixing the cells as a measure of RSV-mediated cell-cell fusion. In Table 1, the results from the fusion assay are compared to a standard antiviral assay in Hep-2 cells. Ribavirin and HR2-derived peptide T-118 (10) were included as controls. Both VP-14637 and JNJ-2408068 were approximately 1,000- and 10,000-fold more potent than T-118 and ribavirin, respectively, at inhibiting RSV replication in the antiviral assay. Similarly for the fusion assay, both inhibitors had EC₅₀ values in the low nanomolar range compared to 3.5 µM for T-118. These data confirm that the antiviral effects of both VP-14637 and JNJ-2408068 are mediated by their ability to inhibit the RSV fusion process.

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TABLE 1. Effect of RSV inhibitors on virus replication, virus-induced cell fusion, and [3H]VP-14637 binding to virus-infected cells

To further characterize the interactions of these compounds, a VP-14637 binding assay with RSV-infected cells was performed. Cells were incubated with [³H]VP-14637 in the presence of various concentrations of unlabeled VP-14637, JNJ-2408068, or T-118. Both VP-14637 and JNJ-2408068 interfered with the binding of [³H]VP-14637 to infected cells at concentrations similar to those inhibiting RSV fusion and replication, suggesting a similar binding mode for the two small molecules (Table 1). In contrast, 20 µM T-118 did not affect [³H]VP-14637 binding to infected cells. T-118 is thought to inhibit RSV fusion by binding to the HR1 domain, thus preventing the heptad repeats from interacting and forming a stable fusion-competent six-helix complex (10).

Drug-resistant RSV variants were selected by culturing the virus for six to eight passages in the presence of increasing concentrations of each inhibitor to further assess their mode of action. Drug susceptibilities of the resultant viruses were determined by the cytopathic antiviral assay, and viral F genes were amplified and sequenced. Two viruses that were independently selected in the presence of VP-14637 showed >1,000-fold reduced susceptibility to both VP-14637 and JNJ-2408068 (Table 2). Similarly, both low and high concentrations of JNJ-2408068 selected for viruses with diminished susceptibility to VP-14637 and JNJ-2408068. Hence, all selected viruses were cross resistant to both inhibitors. Genotypic analysis of the resistant viruses revealed various mutations located either in HR2 or the intervening domain of the F protein (Table 2 and Fig. 2). Although VP-14637 and JNJ-2408068 did not select for the exact same resistance mutations, it is interesting that the mutations in each region were within one to two amino acids of each other. There were no apparent defects in F protein expression or transport to the cell surface for any of the resistant viruses as verified by Western blot and FACS analysis (data not shown).

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TABLE 2. Characterization of RSV variants selected in the presence of VP-14637 or JNJ-2408068

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FIG. 2. Location of the resistance mutations selected in the presence of VP-14637 and JNJ-2408068. Schematic of the RSV F polyprotein indicating salient features: F1 and F2 subunits; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain. The numbers represent amino acid designations, the asterisks above the line indicate the resistance mutations selected by VP-14637, and the arrows below the line indicate the resistance mutations selected by JNJ-2408068.

To determine whether a decreased inhibitor binding to mutant F protein could account for the resistance, the VP-14637 binding assay was performed in CEF cells expressing drug-resistant mutant F proteins by using the MVA-T7 system (6, 20). The binding of [³H]VP-14637 to all F proteins containing the drug resistance mutations, including those selected by JNJ-2408068, was considerably reduced (Fig. 3A). Only the wild-type F protein exhibited efficient binding of VP-14637. FACS analysis confirmed that all of the F proteins were expressed at equivalent levels on the cell surface (data not shown). In addition to the mutations selected in our studies, S398L, a previously reported resistance mutation for JNJ-2408068 (1), also showed reduced binding of [³H]VP-14637 (Fig. 3B). Thus, the reduced activity of both inhibitors against the selected RSV mutants is likely due to their inability to interact with the mutant forms of the F protein. To further investigate the binding mode of VP-14637, several negatively charged amino acids in the HR2 region were substituted by neutral residues. D479N had no apparent effect on VP-14637 binding (Fig. 3B). Interestingly, despite a profound effect of the E487D mutation on the activity and binding of [³H]VP-14637, the E487Q mutant did not show diminished compound binding, suggesting that the length rather than the charge of the amino acid residue affected the compound interaction at this site. Lastly, the binding of [³H]VP-14637 to the D489N mutant was greatly diminished, indicating the potential importance of an acidic residue for this interaction. Although no resistance mutations were selected at D489 in our study, D489Y was previously selected by JNJ-2408068 (14).

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FIG. 3. VP-14637 does not bind to F proteins containing resistance mutations selected by either VP-14637 or JNJ-2408068. Genes encoding wild-type F (WT), (A) one of the five selected resistance mutations (K399I, D486N, E487D, T400A, and F488Y), or (B) one of the following point mutations (S398L, D479N, E487Q, and D489N) were expressed in CEF cells using the MVA-T7 system. Cells were incubated with 10 nM [3H]VP-14637 for 90 min, and the cell-associated radioactivity was determined. The control was generated by the transfection of an empty expression plasmid. The data presented are the average of four replicates from a representative experiment. The error bars represent the standard deviations of the replicates.

Top Abstract Introduction Materials and Methods Results Discussion References

 
By using a RSV-induced cell fusion assay we have confirmed that JNJ-2408068, like VP-14637, inhibits fusion at similar drug concentrations that prevent RSV replication in a standard antiviral assay. In addition, we have shown that these same concentrations of JNJ-2408068 can block the binding of VP-14637 to RSV-infected cells, suggesting an overlapping mode of interaction for both compounds. The observations that mutant viruses selected in the presence of either drug were cross resistant to both compounds and that F proteins containing resistance mutations selected by either VP-14637 or JNJ-2408068 could no longer bind VP-14637 further support the conclusion that the two inhibitors act via a similar mechanism.

Structural analysis of the F protein core formed by HR1 and HR2 peptides has revealed a hydrophobic cavity formed by six amino acids from two adjacent HR1 repeats. This pocket can presumably accommodate two hydrophobic residues from HR2 (F483 and F488) during the process of six-helix formation (Fig. 4A) (24). VP-14637 selected for a resistance mutation at F488, and the other HR2 mutations selected by both VP-14637 and JNJ-2408068 (E487 and D486) were in close proximity. Therefore, this hydrophobic pocket might potentially be a site for VP-14637 and JNJ-2408068 binding, and the various substitutions in and around F488 may change these interactions without impairing the F protein function. Photoaffinity labeling experiments with a structural analog of another recently identified RSV fusion inhibitor BMS-433771 showed that this compound reacted specifically with Y198 of HR1 repeat, one of the amino acids that form this hydrophobic cavity (4a), providing direct evidence that the pocket is indeed capable of accommodating a potent RSV-specific inhibitor.

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FIG. 4. (A) Hydrophobic cavity in RSV F protein formed by the HR1 inner core trimer. Surface representation of the cavity is shown in cyan. HR1 heptad repeats are represented by purple ribbons, and the HR2 helix is shown as a yellow ribbon. Three HR2 residues that bind the pocket (Phe483, Phe488, and Ile492) and other key HR2 residues mutated in resistant viruses (Glu 486, Glu 487, Asp 489) are shown in green sticks. (B) A potential binding mode of JNJ-2408068 to the HR1 hydrophobic cavity. (C) A potential binding mode of VP-14637 to the HR1 hydrophobic cavity.

We applied molecular modeling techniques to assess the feasibility of VP-14637 and JNJ-2408068 binding to the F hydrophobic pocket. Docking studies identified several possible binding modes for these two compounds. Figure 4B shows a representative binding mode for JNJ-2408068. Similar binding modes were obtained for VP-14637 (Fig. 4C). These results are consistent with the observed inhibition of VP-14637 binding by JNJ-2408068.

Even though the binding of a small-molecule inhibitor to the hydrophobic pocket may prevent the formation of a fusion-competent HR1 and HR2 helical bundle, it would probably not eliminate all interactions between HR1 and HR2 peptides. Structural analysis indicates that significant interactions between HR1 and HR2 are possible even in the presence of inhibitor bound to the hydrophobic pocket. A region encompassing approximately 37 HR2 residues (L481-N517) is involved in the interaction with the HR1 inner core trimer. Interactions between the HR1 trimer and approximately 12 of those 37 HR2 residues (L481-I492) would be affected by the binding of an inhibitor to the hydrophobic pocket, leaving 25 amino acid residues from HR2 (S493-N517) still available for the interaction with the HR1 trimer. The interaction between HR1 and HR2 can be envisioned as a zipper that can close all the way in the absence of an inhibitor. However, with the inhibitor bound, the zipper can only go approximately two thirds of the way. The disruption of the last one third of the HR1/HR2 interaction is likely sufficient to prevent the formation of a fusion-competent conformation of F protein, resulting in a potent inhibition of virus replication.

Because of the relatively small size and shallowness of the hydrophobic pocket, it is unlikely that the binding of an inhibitor to this pocket alone can result in such potent antiviral activity. A possible scenario is that HR2 is bound to the HR1 trimer through anchoring interactions of 25 residues (S493-N517, approximately 70% of the "zipper" length) with the remaining 12 HR2 residues (L481-I492) acting as a cover over the hydrophobic pocket and the bound inhibitor, effectively enhancing the interaction between the bound inhibitor and the HR1/HR2 complex (Fig. 5A). This could explain why the resistance mutations found within the six-helix bundle (positions 486 to 488) were all located in the HR2 peptide and not in the HR1 hydrophobic pocket. Amino acids 486 to 488 are among the residues that form the putative inhibitor cover and therefore could make direct contacts with the bound inhibitor (Fig. 5B). Mutations at these positions would then directly affect interactions with the inhibitor, resulting in altered virus susceptibility. This model would also be consistent with the inability of the HR2-derived peptide T-118 to inhibit VP-14637 binding.

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FIG. 5. (A) Binding of JNJ-2408068 to the hydrophobic cavity may disrupt the HR1-HR2 interaction. The green ribbon represents the part of HR2 that is affected by the presence of inhibitor. (B) Surface representation suggests improved burial of JNJ-2408068 in the presence of the HR2 cover. The HR1 and HR2 surfaces are shown in cyan and green, respectively. Drug-selected resistance mutations in HR2 (Glu486, Glu487, Phe488, and Asp489) are highlighted in red.

The role of F protein residues 398 to 400 in the interaction with inhibitors is not clear since no specific function has been determined yet for the intervening domain between HR1 and HR2. A homology model of the RSV F protein derived from the crystal structure of the NDV fusion protein suggests that these residues would be located in a predicted immunoglobulin-like ß-sandwich domain on the surface of the F protein (3, 17). Since the intervening domain has to undergo a dramatic conformational change during the process of viral fusion, mutations located in this region may affect the kinetics of this conformational change. It may result in a shortening of the time window during which the inhibition-sensitive conformation of F protein is available for the inhibitor to bind. Alternatively, the intervening sequences might be involved in an early transient conformation of F protein present prior to the heptad repeat interaction. The small molecules might be acting at this earlier metastable state instead of directly preventing the formation of the six-helix fusion core. However, experimental evidence to support this type of interaction has yet to be generated.

In addition to RSV, multiple other fusogenic viruses rely on the formation of HR1/HR2 helical bundle within the structure of their fusion protein as the mechanism that allows for their efficient entry into a host cell. Among these, gp41 of human immunodeficiency virus type 1 (HIV-1) is both functionally and structurally equivalent to RSV F protein, i.e., it also contains an inner coiled coil formed by three N-terminal HR1 helices, which interact with three C-terminal HR2 helices to form a stable six-helix bundle that drives the HIV-1 fusion process. Similar to RSV HR1 helical trimer, there is a pocket present in the HR1 coiled coil of gp41 (2). Even though both pockets are hydrophobic in nature, they accommodate different HR2 amino acid residues—two Trp residues in the case of gp41 and two Phe residues in RSV F protein. A comparative structural analysis suggested that the RSV pocket is deeper and better defined whereas the pocket present in HIV-1 gp41 is slightly larger but shallower and more open. This might be one of the reasons why, despite extensive screening efforts, no promising small-molecule gp41 inhibitors have been identified thus far. When we tested VP-14637 and JNJ-2408068 against HIV-1, no antiviral activity was observed at concentrations as high as 10 µM, a result consistent with the observed differences in the size and shape of the pocket present in RSV F protein and that in HIV-1 gp41.

In summary, VP-14637 and JNJ-2408068 are two small molecules from the growing collection of RSV inhibitors that inhibit viral fusion by interacting with the F protein in a similar manner despite their apparent structural dissimilarity. Further detailed understanding of the mechanisms of interaction between these inhibitors and the F protein should facilitate the design of a new generation of RSV fusion inhibitors.

 
We acknowledge Bernard Moss and Robert Lamb for providing necessary reagents. In addition, we thank William Lee and Mick Hitchcock of Gilead Sciences for their critical comments on the manuscript.

 
* Corresponding author. Mailing address: Gilead, 333 Lakeside Dr., Foster City, CA 94404. Phone: (650) 522-5637. Fax: (650) 522-5890. E-mail: tcihlar{at}gilead.com.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Andries, K., M. Moeremans, T. Gevers, R. Willebrords, C. Sommen, J. Lacrampe, F. Janssens, and P. R. Wyde. 2003. Substituted benzimidazoles with nanomolar activity against respiratory syncytial virus. Antiviral Res. 60:209-219.[CrossRef][Medline]
2
2. Chan, D. C., C. T. Chutkowski, and P. S. Kim. 1998. Evidence that a prominent cavity in the coiled coil of HIV type 1 gp41 is an attractive drug target. Proc. Natl. Acad. Sci. USA 95:15613-15617.[Abstract/Free Full Text]
3
3. Chen, L., J. J. Gorman, J. McKimm-Breschkin, L. J. Lawrence, P. A. Tulloch, B. J. Smith, P. M. Colman, and M. C. Lawrence. 2001. The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion. Structure 9:255-266.[Medline]
4
4. Cianci, C., K. L. Yu, K. Combrink, N. Sin, B. Pearce, A. Wang, R. Civiello, S. Voss, G. Luo, K. Kadow, E. V. Genovesi, B. Venables, H. Gulgeze, A. Trehan, J. James, L. Lamb, I. Medina, J. Roach, Z. Yang, L. Zadjura, R. Colonno, J. Clark, N. Meanwell, and M. Krystal. 2004. Orally active fusion inhibitor of respiratory syncytial virus. Antimicrob. Agents Chemother. 48:413-422.[Abstract/Free Full Text]
4
5. Cianci, C., D. R. Langley, D. D. Dischino, Y. Sun, K. L. Yu, A. Stanley, J. Roach, Z. Li, R. Dalterio, R. Colonno, N. A. Meanwell, and M. Krystal. 2004. Targeting a binding pocket within the trimer-of-hairpins: small-molecule inhibition of viral fusion. Proc. Natl. Acad. Sci. USA 101:15046-15051.[Abstract/Free Full Text]
5
6. Collins, P. L., R. M. Chanock, and B. R. Murphy. 2001. Respiratory syncytial virus, p. 1443-1485. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed., vol. 1. Lippincott Williams & Wilkins, Philadelphia, Pa.
6
7. Douglas, J. L., M. L. Panis, E. Ho, K. Y. Lin, S. H. Krawczyk, D. M. Grant, R. Cai, S. Swaminathan, and T. Cihlar. 2003. Inhibition of respiratory syncytial virus fusion by the small molecule VP-14637 via specific interactions with F protein. J. Virol. 77:5054-5064.[Abstract/Free Full Text]
7
8. Falsey, A. R. 1998. Respiratory syncytial virus infection in older persons. Vaccine 16:1775-1778.[CrossRef][Medline]
8
9. Groothuis, J. R., E. A. Simoes, M. J. Levin, C. B. Hall, C. E. Long, W. J. Rodriguez, J. Arrobio, H. C. Meissner, D. R. Fulton, R. C. Welliver, D. A. Tristram, G. R. Siber, G. A. Prince, M. Van Raden, and V. G. Hemming. 1993. Prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. N. Engl. J. Med. 329:1524-1530.[Abstract/Free Full Text]
9
10. Johnson, S., C. Oliver, G. A. Prince, V. G. Hemming, D. S. Pfarr, S. C. Wang, M. Dormitzer, J. O'Grady, G. Koenig, J. K. Tamura, R. Woods, G. Bansal, D. Couchenour, E. Tsao, C. Hall, and J. F. Young. 1997. Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J. Infect. Dis. 176:1215-1224.[Medline]
10
11. Lambert, D. M., S. Barney, A. L. Lambert, K. Guthrie, R. Medinas, D. E. Davis, T. Bucy, J. Erickson, G. Merutka, and S. R. Petteway, Jr. 1996. Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion. Proc. Natl. Acad. Sci. USA 93:2186-2191.[Abstract/Free Full Text]
11
12. Lawless-Delmedico, M. K., P. Sista, R. Sen, N. C. Moore, J. B. Antczak, J. M. White, R. J. Greene, K. C. Leanza, T. J. Matthews, and D. M. Lambert. 2000. Heptad-repeat regions of respiratory syncytial virus F1 protein form a six-membered coiled-coil complex. Biochemistry 39:11684-11695.[CrossRef][Medline]
12
13. Matthews, J. M., T. F. Young, S. P. Tucker, and J. P. Mackay. 2000. The core of the respiratory syncytial virus fusion protein is a trimeric coiled coil. J. Virol. 74:5911-5920.[Abstract/Free Full Text]
13
14. McKimm-Breschkin, J. 2000. VP-14637 ViroPharma. Curr. Opin. Investig. Drugs 1:425-427.[Medline]
14
15. Morton, C. J., R. Cameron, L. J. Lawrence, B. Lin, M. Lowe, A. Luttick, A. Mason, J. McKimm-Breschkin, M. W. Parker, J. Ryan, M. Smout, J. Sullivan, S. P. Tucker, and P. R. Young. 2003. Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay. Virology 311:275-288.[CrossRef][Medline]
15
16. Razinkov, V., A. Gazumyan, A. Nikitenko, G. Ellestad, and G. Krishnamurthy. 2001. RFI-641 inhibits entry of respiratory syncytial virus via interactions with fusion protein. Chem. Biol. 8:645-659.[CrossRef][Medline]
16
17. Simoes, E. A. 2003. Environmental and demographic risk factors for respiratory syncytial virus lower respiratory tract disease. J. Pediatr. 143:S118-S126.[CrossRef][Medline]
17
18. Smith, B. J., M. C. Lawrence, and P. M. Colman. 2002. Modelling the structure of the fusion protein from human respiratory syncytial virus. Protein Eng. 15:365-371.[Abstract/Free Full Text]
18
19. Vujovic, O., and J. Mills. 2001. Preventive and therapeutic strategies for respiratory syncytial virus infection. Curr. Opin. Pharmacol. 1:497-503.[CrossRef][Medline]
19
20. Welliver, R. C. 2003. Review of epidemiology and clinical risk factors for severe respiratory syncytial virus (RSV) infection. J. Pediatr. 143:S112-S117.[CrossRef][Medline]
20
21. Wyatt, L. S., B. Moss, and S. Rozenblatt. 1995. Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells. Virology 210:202-205.[CrossRef][Medline]
21
22. Wyde, P. R., S. N. Chetty, P. Timmerman, B. E. Gilbert, and K. Andries. 2003. Short duration aerosols of JNJ 2408068 (R170591) administered prophylactically or therapeutically protect cotton rats from experimental respiratory syncytial virus infection. Antiviral Res. 60:221-231.[CrossRef][Medline]
22
23. Wyde, P. R., D. K. Moore-Poveda, B. O'Hara, W. D. Ding, B. Mitsner, and B. E. Gilbert. 1998. CL387626 exhibits marked and unusual antiviral activity against respiratory virus in tissue culture and in cotton rats. Antiviral Res. 38:31-42.[CrossRef][Medline]
23
24. Yu, K. L., Y. Zhang, R. L. Civiello, K. F. Kadow, C. Cianci, M. Krystal, and N. A. Meanwell. 2003. Fundamental structure-activity relationships associated with a new structural class of respiratory syncytial virus inhibitor. Bioorg. Med. Chem. Lett. 13:2141-2144.[CrossRef][Medline]
24
25. Zhao, X., M. Singh, V. N. Malashkevich, and P. S. Kim. 2000. Structural characterization of the human respiratory syncytial virus fusion protein core. Proc. Natl. Acad. Sci. USA 97:14172-14177.[Abstract/Free Full Text]

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Antimicrobial Agents and Chemotherapy, June 2005, p. 2460-2466, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2460-2466.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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