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

2005 ◽  
Vol 49 (6) ◽  
pp. 2460-2466 ◽  
Author(s):  
Janet L. Douglas ◽  
Marites L. Panis ◽  
Edmund Ho ◽  
Kuei-Ying Lin ◽  
Steve H. Krawczyk ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Inner Core ◽  
Specific Binding ◽  
Expression System ◽  
Heptad Repeat ◽  
Single Amino Acid ◽  
Cross Resistance ◽  
Resistance Mutations ◽  
F Protein ◽  
Syncytial Virus

ABSTRACT 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.

scholarly journals Inhibition of Respiratory Syncytial Virus Fusion by the Small Molecule VP-14637 via Specific Interactions with F Protein

2003 ◽  
Vol 77 (9) ◽  
pp. 5054-5064 ◽  
Author(s):  
Janet L. Douglas ◽  
Marites L. Panis ◽  
Edmund Ho ◽  
Kuei-Ying Lin ◽  
Steve H. Krawczyk ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Small Molecule ◽  
Respiratory Tract Infections ◽  
Heptad Repeat ◽  
Resistance Mutations ◽  
F Protein ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Tract Infections ◽  
Virus System

ABSTRACT Human respiratory syncytial virus (RSV) is a major cause of respiratory tract infections worldwide. Several novel small-molecule inhibitors of RSV have been identified, but they are still in preclinical or early clinical evaluation. One such inhibitor is a recently discovered triphenol-based molecule, VP-14637 (ViroPharma). Initial experiments suggested that VP-14637 acted early and might be an RSV fusion inhibitor. Here we present studies demonstrating that VP-14637 does not block RSV adsorption but inhibits RSV-induced cell-cell fusion and binds specifically to RSV-infected cells with an affinity corresponding to its inhibitory potency. VP-14637 is capable of specifically interacting with the RSV fusion protein expressed by a T7 vaccinia virus system. RSV variants resistant to VP-14637 were selected; they had mutations localized to two distinct regions of the RSV F protein, heptad repeat 2 (HR2) and the intervening domain between heptad repeat 1 (HR1) and HR2. No mutations arose in HR1, suggesting a mechanism other than direct disruption of the heptad repeat interaction. The F proteins containing the resistance mutations exhibited greatly reduced binding of VP-14637. Despite segregating with the membrane fraction following incubation with intact RSV-infected cells, the compound did not bind to membranes isolated from RSV-infected cells. In addition, binding of VP-14637 was substantially compromised at temperatures of ≤22°C. Therefore, we propose that VP-14637 inhibits RSV through a novel mechanism involving an interaction between the compound and a transient conformation of the RSV F protein.

scholarly journals Neutralization of Human Respiratory Syncytial Virus Infectivity by Antibodies and Low-Molecular-Weight Compounds Targeted against the Fusion Glycoprotein

2010 ◽  
Vol 84 (16) ◽  
pp. 7970-7982 ◽  
Author(s):  
Margarita Magro ◽  
David Andreu ◽  
Paulino Gómez-Puertas ◽  
José A. Melero ◽  
Concepción Palomo
Keyword(s):  
Molecular Weight ◽  
Respiratory Syncytial Virus ◽  
Vaccine Development ◽  
Human Respiratory Syncytial Virus ◽  
Heptad Repeat ◽  
Low Molecular Weight ◽  
Virus Infectivity ◽  
F Protein ◽  
Low Molecular Weight Compounds ◽  
Syncytial Virus

ABSTRACT Human respiratory syncytial virus (HRSV) fusion (F) protein is an essential component of the virus envelope that mediates fusion of the viral and cell membranes, and, therefore, it is an attractive target for drug and vaccine development. Our aim was to analyze the neutralizing mechanism of anti-F antibodies in comparison with other low-molecular-weight compounds targeted against the F molecule. It was found that neutralization by anti-F antibodies is related to epitope specificity. Thus, neutralizing and nonneutralizing antibodies could bind equally well to virions and remained bound after ultracentrifugation of the virus, but only the former inhibited virus infectivity. Neutralization by antibodies correlated with inhibition of cell-cell fusion in a syncytium formation assay, but not with inhibition of virus binding to cells. In contrast, a peptide (residues 478 to 516 of F protein [F478-516]) derived from the F protein heptad repeat B (HRB) or the organic compound BMS-433771 did not interfere with virus infectivity if incubated with virus before ultracentrifugation or during adsorption of virus to cells at 4°C. These inhibitors must be present during virus entry to effect HRSV neutralization. These results are best interpreted by asserting that neutralizing antibodies bind to the F protein in virions interfering with its activation for fusion. Binding of nonneutralizing antibodies is not enough to block this step. In contrast, the peptide F478-516 or BMS-433771 must bind to F protein intermediates generated during virus-cell membrane fusion, blocking further development of this process.

scholarly journals The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

2017 ◽  
pp. JVI.01323-17 ◽  
Author(s):  
Imogen M. Bermingham ◽  
Keith J. Chappell ◽  
Daniel Watterson ◽  
Paul R. Young
Keyword(s):  
Amino Acids ◽  
Respiratory Syncytial Virus ◽  
Membrane Fusion ◽  
Functional Characterization ◽  
Proteolytic Processing ◽  
Heptad Repeat ◽  
Alanine Scanning Mutagenesis ◽  
Viral Pathogen ◽  
F Protein ◽  
Syncytial Virus

Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. RSV F comprises a trimer of disulfide bonded F1and F2subunits that is present on the virion surface in a ‘metastable' pre-fusion state. This pre-fusion form is readily triggered to undergo refolding to bring two heptad repeats (HRA and HRB) into close proximity to form a six-helix bundle that stabilizes the post-fusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75-97), an amphipathic α-helix that lies at the interface of the pre-fusion F trimer and is a major structural feature of the F2subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Herein we propose a model in which HRC plays a stabilizing role within the globular head for the pre-fusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the post-fusion state.IMPORTANCERSV is recognized as the most important viral pathogen amongst pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of pre-fusion stabilized F protein based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here we present a comprehensive mutagenesis based study of a region of the RSV F2subunit (amino acids 75 - 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the pre-fusion form.

scholarly journals GS-5806 Inhibits a Broad Range of Respiratory Syncytial Virus Clinical Isolates by Blocking the Virus-Cell Fusion Process

2015 ◽  
Vol 60 (3) ◽  
pp. 1264-1273 ◽  
Author(s):  
Michel Perron ◽  
Kirsten Stray ◽  
April Kinkade ◽  
Dorothy Theodore ◽  
Gary Lee ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cell Fusion ◽  
Clinical Investigation ◽  
Temperature Shift ◽  
Airway Epithelial Cells ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
F Protein ◽  
Syncytial Virus ◽  
Tract Infections

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and young children. In addition, RSV causes significant morbidity and mortality in hospitalized elderly and immunocompromised patients. Currently, only palivizumab, a monoclonal antibody against the RSV fusion (F) protein, and inhaled ribavirin are approved for the prophylactic and therapeutic treatment of RSV, respectively. Therefore, there is a clinical need for safe and effective therapeutic agents for RSV infections. GS-5806, discovered via chemical optimization of a hit from a high-throughput antiviral-screening campaign, selectively inhibits a diverse set of 75 RSV subtype A and B clinical isolates (mean 50% effective concentration [EC50] = 0.43 nM). The compound maintained potency in primary human airway epithelial cells and exhibited low cytotoxicity in human cell lines and primary cell cultures (selectivity > 23,000-fold). Time-of-addition and temperature shift studies demonstrated that GS-5806 does not block RSV attachment to cells but interferes with virus entry. Follow-up experiments showed potent inhibition of RSV F-mediated cell-to-cell fusion. RSV A and B variants resistant to GS-5806, due to mutations in F protein (RSV A, L138F or F140L/N517I, and RSV B, F488L or F488S), were isolated and showed cross-resistance to other RSV fusion inhibitors, such as VP-14637, but remained fully sensitive to palivizumab and ribavirin. In summary, GS-5806 is a potent and selective RSV fusion inhibitor with antiviral activity against a diverse set of RSV clinical isolates. The compound is currently under clinical investigation for the treatment of RSV infection in pediatric, immunocompromised, and elderly patients.

scholarly journals Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit Are Critical for Its Fusion Activity

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Stephanie N. Hicks ◽  
Supranee Chaiwatpongsakorn ◽  
Heather M. Costello ◽  
Jason S. McLellan ◽  
William Ray ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Target Cell ◽  
Antiviral Drug ◽  
High Energy ◽  
Heptad Repeat ◽  
Alanine Scanning Mutagenesis ◽  
F Protein ◽  
Syncytial Virus ◽  
Apical Loop ◽  
Tract Infections

ABSTRACT The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both virus-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F2 by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F protein. IMPORTANCE RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545–1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577–587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749–1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110–128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development.

scholarly journals Pharmacological Characterization of TP0591816, a Novel Macrocyclic Respiratory Syncytial Virus Fusion Inhibitor with Antiviral Activity against F Protein Mutants

2020 ◽  
Vol 65 (1) ◽  
pp. e01407-20
Author(s):  
Ippei Yoshida ◽  
Kaho Arikawa ◽  
Yusuke Honma ◽  
Shoko Inatani ◽  
Mitsukane Yoshinaga ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antiviral Activity ◽  
Heptad Repeat ◽  
Fusion Inhibitor ◽  
Rich Region ◽  
F Protein ◽  
Protein Mutants ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Tract Infections

ABSTRACTHuman respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in early childhood. However, no vaccines have yet been approved for prevention of RSV infection, and the treatment options are limited. Therefore, development of effective and safe anti-RSV drugs is needed. In this study, we evaluated the antiviral activity and mechanism of action of a novel macrocyclic anti-RSV compound, TP0591816. TP0591816 showed significant antiviral activities against both subgroup A and subgroup B RSV, while exerting no cytotoxicity. Notably, the antiviral activity of TP0591816 was maintained against a known fusion inhibitor-resistant RSV strain with a mutation in the cysteine-rich region or in heptad repeat B. Results of a time-of-addition assay and a temperature shift assay indicated that TP0591816 inhibited fusion of RSV with the cell membrane during viral entry. In addition, TP0591816 added after cell infection also inhibited cell-cell fusion. A TP0591816-resistant virus strain selected by serial passage had an L141F mutation, but no mutation in the cysteine-rich region or in heptad repeat B in the fusion (F) protein. Treatment with TP0591816 reduced lung virus titers in a dose-dependent manner in a mouse model of RSV infection. Furthermore, the estimated effective dose of TP0591816 for use against F protein mutants was thought to be clinically realistic and potentially tolerable. Taken together, these findings suggest that TP0591816 is a promising novel candidate for the treatment of resistant RSV infection.

scholarly journals Recombinant Soluble Respiratory Syncytial Virus F Protein That Lacks Heptad Repeat B, Contains a GCN4 Trimerization Motif and Is Not Cleaved Displays Prefusion-Like Characteristics

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0130829 ◽  
Author(s):  
Ivy Widjaja ◽  
Alan Rigter ◽  
Shamir Jacobino ◽  
Frank J. M. van Kuppeveld ◽  
Kees Leenhouts ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Heptad Repeat ◽  
F Protein ◽  
Syncytial Virus

scholarly journals Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein

2021 ◽  
Author(s):  
Wei Tang ◽  
Yueyue Li ◽  
Qiaoyun Song ◽  
Ziqin Wang ◽  
Manmei Li ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Membrane Fusion ◽  
Small Molecule ◽  
Cross Resistance ◽  
Fusion Activity ◽  
F Protein ◽  
Fusion Inhibitors ◽  
Molecule Inhibitor ◽  
Fusion Glycoprotein ◽  
Syncytial Virus

The fusion glycoprotein (F) is essential for respiratory syncytial virus (RSV) entry and has become an attractive target for anti-RSV drug development. Despite the promising prospect of RSV F inhibitors, issues of drug resistance remain challenging. In this study, we established a dual-luciferase protocol for RSV fusion inhibitor discovery. A small-molecule inhibitor, salvianolic acid R (LF-6), was identified to inhibit virus-cell and cell-cell fusion mediated by RSV F protein. Sequence analysis of the resultant resistant viruses identified a K394R mutation in the viral F protein. The K394R mutant virus also conferred cross-resistance to multiple RSV fusion inhibitors, including several inhibitors undergoing clinical trials. Our study further showed that K394R mutation not only increased the triggering rate of F protein in prefusion conformation but also enhanced fusion activity of F protein, both of which were positively correlated with the resistance to fusion inhibitors. Moreover, the K394R mutation also showed cooperative effects with other escape mutations to increase the fusion activity of F protein. By substitution of K394 into different amino acids, we found that K394R or K394H substitution resulted in hyperfusiogenic F proteins, whereas F variants with other substitutions exhibited lower fusion activity. Both K394R and K394H in F protein exhibited cross-resistance to RSV fusion inhibitors. Collectively, these findings reveal a positive correlation existed between membrane fusion activity of F protein and the resistance of corresponding inhibitors. All the results demonstrate that K394R in F protein confers cross-resistance to fusion inhibitors through destabilizing F protein and increasing its membrane fusion activity. IMPORTANCE Respiratory syncytial virus (RSV) causes serious respiratory tract disease in children and the elderly. Therapeutics against RSV infection are urgently needed. This study reports the discovery of a small-molecule inhibitor of RSV fusion glycoprotein by using a dual-luciferase protocol. The escape mutation (K394R) of this compound also confers cross-resistance to multiple RSV fusion inhibitors that have been reported previously, including two candidates currently in clinical development. The combination of K394R with other escape mutations can increase the resistance of F protein to these inhibitors through destabilizing F protein and enhancing the membrane fusion activity of F protein. By amino acid deletion or substitution, we found that positively charged residue at the 394 th site is crucial for the fusion ability of F protein as well as for the cross-resistance against RSV fusion inhibitors. These results reveal the mechanism of cross-resistance conferred by K394R mutation and the possible cross-resistance risk of RSV fusion inhibitors.

scholarly journals A single amino acid in the F2 subunit of respiratory syncytial virus fusion protein alters growth and fusogenicity

2013 ◽  
Vol 94 (12) ◽  
pp. 2627-2635 ◽  
Author(s):  
Heather A. Lawlor ◽  
Jeanne H. Schickli ◽  
Roderick S. Tang
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Syncytium Formation ◽  
Comparative Sequence Analysis ◽  
Single Amino Acid ◽  
Live Attenuated Vaccine ◽  
F Protein ◽  
Vero Cell Line ◽  
Single Deletion ◽  
Syncytial Virus

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in children, especially in infants less than 1 year of age. There are currently no licensed vaccines against RSV. rA2ΔM2-2 is a promising live-attenuated vaccine candidate that is currently being evaluated in the clinic. Attenuation of rA2ΔM2-2 is achieved by a single deletion of the M2-2 gene, which disrupts the balance between viral transcription and replication. Whilst performing a manufacturing feasibility study in a serum-free adapted Vero cell line, differences in growth kinetics and cytopathic effect (CPE) were identified between two rA2ΔM2-2 vaccine candidates. Comparative sequence analysis identified four amino acid differences between the two vaccine viruses. Recombinant rA2ΔM2-2 viruses carrying each of the four amino acid differences identified a K66E mutation in the F2 fragment of the fusion (F) protein as the cause of the growth and CPE differences. Syncytium-formation experiments with RSV F protein carrying mutations at aa 66 suggested that a change in charge at this residue within the F2 fragment can have a significant impact on fusion.

scholarly journals Shifting immunodominance pattern of two cytotoxic T-lymphocyte epitopes in the F glycoprotein of the Long strain of respiratory syncytial virus

2004 ◽  
Vol 85 (11) ◽  
pp. 3229-3238 ◽  
Author(s):  
Carolina Johnstone ◽  
Patricia de León ◽  
Francisco Medina ◽  
José A. Melero ◽  
Blanca García-Barreno ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Vaccinia Virus ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Recombinant Vaccinia Virus ◽  
Recombinant Vaccinia ◽  
F Protein ◽  
Syncytial Virus ◽  
Lymphocyte Responses

Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2Kd-restricted CTL epitopes (F85–93 and F92–106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85–93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249–258, which is presented by Kd as a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous Kd ligand. The CD8+ T-lymphocyte responses to epitopes F85–93 and F249–258 present in the F protein of RSV Long were found to be strongly skewed to F85–93 in in vitro multispecific CTL lines and in vivo during a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8+ T-lymphocyte responses to F85–93 and F249–258 epitopes was observed in vivo during a primary response.

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