Departments of 1 Virology and 2 Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA
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Abstract |
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Keywords: respiratory syncytial virus , trimer-of-hairpins , heptad repeats , BMS-433771
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Introduction |
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Evolution of BMS-433771: a potential clinical candidate |
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Mechanism of action of the BMS-433771 chemotype |
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Resistant viruses were generated using several structurally related inhibitors in this series in order to elucidate the molecular target. Gene sequencing of the resistant viruses revealed that amino acid changes were detected only in the F1 subunit. A single K394R mutation in the F1 protein inserted into an RSV A2 infectious clone conferred resistance to BMS-433771, further confirming the importance of the F1 polypeptide in the mode of action of these inhibitors.5 The F1 protein contains the hydrophobic fusion peptide at the N-terminus and two hydrophobic heptad repeat domains, one adjacent to the fusion peptide (HR-N) and one adjacent to the transmembrane-spanning domain (HR-C). A conformational change in the F1 protein exposes the fusion peptide, which then inserts into the opposing host cell membrane. Following a second rearrangement of F1, the two heptad repeat domains associate into a six-helix bundle structure, stabilizing a trimer-of-hairpins configuration in the F1 that catalyses the fusion of viral and host cell membranes. The peptide inhibitor enfuvirtide blocks this interaction by binding directly to the HR-N region of the HIV gp41 protein. Similar peptides have been identified for RSV, as well as other class I fusion proteins.7 BMS-433771 is a small molecule that presumably blocks the functional interaction of the HR-N to the HR-C peptide by binding tightly in a pocket formed in the HR-N region.
In order to demonstrate directly binding of the BMS-433771 chemotype to the F protein, a radiolabelled photoaffinity probe, [125I]-BMS-356188, was synthesized (Figure 1).8 Ultraviolet ray activation of this probe yields a highly chemically reactive carbene species that can covalently insert into proximal amino acids within the inhibitor binding site. The photoaffinity probe labelled the RSV F1 polypeptide exclusively in a reaction that could be prevented by the addition of a molar excess of BMS-433771. Peptide mapping indicated that the probe was binding within the N-terminal heptad repeat domain. Further photoaffinity labelling studies conducted with isolated peptides showed that the compound specifically reacted with the tyrosine residue at position 198 of the F protein.9 The structure of a key portion of the RSV fusion protein six-helix bundle has been solved using the N- and C-terminal heptad repeat peptides that associate into the fusogenic trimer-of-hairpins.10 This crystal structure revealed a hydrophobic pocket in the HR-N trimer, where two key phenylalanines (F-483 and F-488) from the HR-C bind. Tyrosine 198 (Y-198) is present in this pocket and BMS-356188 can be readily modelled into this cavity in an orientation where the photo-reactive diazirine is directed towards this tyrosine. Based upon these results, we propose that these inhibitors interfere with the interaction of the key amino acids residues F-483 and F-488 from the HR-C with the hydrophobic cavity in the HR-N trimer, thereby destabilizing the trimer-of-hairpins structure required for fusion (Figure 2).
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BMS-433771 in vitro and in vivo anti-RSV activity |
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BMS-433771 exhibited oral efficacy in two rodent models of RSV infection when administered 1 h prior to RSV inoculation.11 Significant reductions in viral titres were reproducibly achieved with a 5.0 mg/kg dose in RSV-infected mice. For infected cotton rats, a five-fold higher dose of 25 mg/kg of BMS-433771 was required to obtain an equivalent reduction in viral titre. No evidence for the selection of virus resistant to BMS-433771 was observed in either model. Although the exact basis for the disparity in efficacy levels between the two rodent models is not known, potential explanations may be related to the higher permissiveness of the cotton rat to RSV infection compared with the mouse, a pharmacodynamic divergence between the two rodent models, or differences in the pathogenesis and compartments of RSV infection in the respiratory tract of these rodents.
In order to establish that the mechanism of action of BMS-433771 in vivo is due to inhibition of virus fusion, a BMS-433771-resistant virus was used to infect BALB/c mice. Consistent with a fusion inhibitory mode of action in vivo, mice infected with resistant virus were insensitive to compound treatment at a dosage that effectively suppressed wild-type virus infection.11 BMS-433771 was also found to be effective against RSV in immunosuppressed mice, indicating that the host immune response does not contribute to the antiviral efficacy of the compound. In the mouse model, BMS-433771 was only marginally active when dosed in a therapeutic mode in which the drug is administered 1 h post-virus infection. This was consistent with the findings that a single oral prophylactic dose of BMS-433771 was as effective in reducing RSV lung titres as a multiple 4 day twice-daily dosing regimen in which only one dose was given before virus inoculation. The lack of therapeutic efficacy in mice may be related to the pathogenesis of the virus in this animal model or, alternatively, the mechanism of action of BMS-433771 as a fusion inhibitor may be a contributing factor. To that end, similar findings have been obtained for the topically active RSV fusion inhibitor, RFI-641 (CL387626) in cotton rats.12 RFI-641 was found to be highly active when dosed topically in cotton rats 45 days prior to infection, but was inactive when dosed via therapeutic regimens. However, RFI-641 has demonstrated therapeutic efficacy in an African Green Monkey infection model, where it reduced viral titres in both the nasal and throat compartments.13 RSV infection in African Green Monkeys results in a disease state similar to that seen in humans, whereas RSV infection in either mice or cotton rats does not cause overt symptoms. Interestingly, JNJ 2408068 (R170591), another RSV fusion inhibitor, has been reported to exhibit both prophylactic and therapeutic activity in a cotton rat model of infection after delivery via small particle aerosol.14 The reason for the different inhibitory profiles of these topical inhibitors and the implications for clinical application is not clear, but could be a reflection of unknown dynamics associated with host speciesvirus interactions. Moreover, since the small animal models of RSV infection poorly recapitulate the clinical syndrome, the efficacy of RSV inhibitors under prophylactic and therapeutic conditions will need to be determined in a Phase 2a study, where the timing of infection is carefully controlled. A third RSV fusion inhibitor has also been recently disclosed,15 although BMS-433771 is unique among the four, as it is the sole member of this class of fusion inhibitor, with significant oral bioavailability and the ability to inhibit virus infection in rodent models after oral administration. Another class of RSV inhibitors with good oral bioavailability, but an unknown mechanism of action, is also currently in clinical development, although no animal efficacy data have yet been presented.16
Clinical experience with the influenza neuraminidase inhibitors may provide a blueprint for the development and clinical use of RSV inhibitors.17 Studies clearly show that this class of antiviral against influenza virus can have both prophylactic and therapeutic activity. However, maximum therapeutic activity against influenza-related disease is observed if treatment initiates soon after infection, making early diagnosis an important treatment factor. RSV usually begins with a 35 day upper respiratory tract infection before progressing to more serious lower respiratory tract illness.18 This upper respiratory tract incubation period may therefore provide a window, whereby therapeutic intervention with a small molecule inhibitor may be most effective. A key element of this is the need to diagnose and treat the infection before it can spread to the lower respiratory tract. Although diagnostic tests are currently available for RSV, additional studies are needed to determine whether these test kits would be sensitive enough to detect RSV infection in its early stages. In addition, for maximum effect, these kits should be available as a point-of-care tool for the practising physician.
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Conclusions |
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References |
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