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 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 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 DEVELOPMENT AND PROPERTIES OF NEUTRALIZING MONOCLONAL ANTIBODIES FOR FUSION PROTEIN OF RESPIRATORY SYNCYTIAL VIRUS

2019 ◽  
Vol 64 (2) ◽  
pp. 90-96
Author(s):  
A. A. Kushch ◽  
R. R. Klimova ◽  
N. E. Fedorova ◽  
O. V. Masalova ◽  
A. A. Niconova ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
Culture Method ◽  
The Elderly ◽  
F Protein ◽  
Wide Range ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Tract Infections

Introduction. Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants and the elderly. The absence of a wide range of therapeutic drugs and vaccines indicates to the high relevance of the development of new effective drugs for the prevention and treatment of RSV infections. Purpose: to obtain highly active and specific monoclonal antibodies (MAbs) capable of detecting RSV in infected cells and neutralizing the infectious activity of the virus in vitro. Material and methods. RSV reference strains of group A 2 subgroups (A2 and Long) were propagated in HEp-2 and MA-104 cell lines, respectively. Mice were immunized with purified RSV A2 virus. MAbs were obtained using hybridoma technology. Results. A panel of 6 MAbs reacting with RSV strains А2 and Long has been obtained. Four MAbs were IgG (IgG2a or IgG2b subtype), two MAbs were IgM. All MAbs reacted with RSV F-protein in immunochemical tests. The MAbs actively reacted with RSV in ELISA, in immufluorescence and peroxidase staining of infected cells, and in immunodot test. Five out of 6 MAbs neutralized of RSV in cell culture. Different properties of MAbs suggest that they target different antigenic sites of F-protein. Discussion. Comparative analysis suggests that the obtained MAbs can be used for the development of diagnostic preparations, for RSV detection in clinical materials and confirmation of infection etiology by rapid culture method. Conclusion. High activity and specificity of MAbs indicate that they can serve as a basis for development vaccines and preventive medicines.

scholarly journals Extracellular amoebal-vesicles: potential transmission vehicles for respiratory viruses

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Rafik Dey ◽  
Melanie A. Folkins ◽  
Nicholas J. Ashbolt
Keyword(s):  
Respiratory Syncytial Virus ◽  
Respiratory Tract ◽  
Respiratory Tract Infections ◽  
Respiratory Viruses ◽  
Environmental Persistence ◽  
Viral Dissemination ◽  
Acute Respiratory Tract Infections ◽  
High Concentrations ◽  
Syncytial Virus ◽  
Tract Infections

AbstractHuman respiratory syncytial virus (RSV) is a major cause of acute respiratory tract infections in children and immunocompromised adults worldwide. Here we report that amoebae-release respirable-sized vesicles containing high concentrations of infectious RSV that persisted for the duration of the experiment. Given the ubiquity of amoebae in moist environments, our results suggest that extracellular amoebal-vesicles could contribute to the environmental persistence of respiratory viruses, including potential resistance to disinfection processes and thereby offering novel pathways for viral dissemination and transmission.

Reappraisal of respiratory syncytial virus as an aetiology of severe acute lower respiratory tract infections in children younger than 5 years in Nigeria

2019 ◽  
Vol 113 (8) ◽  
pp. 446-452
Author(s):  
Damilola M Oladele ◽  
Dimeji P Oladele ◽  
Rasheedat M Ibraheem ◽  
Mohammed B Abdulkadir ◽  
Rasaki Adewole Raheem ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Respiratory Tract ◽  
Lower Respiratory Tract ◽  
Respiratory Tract Infections ◽  
Preventive Strategy ◽  
Lower Respiratory Tract Infections ◽  
Cross Sectional ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Tract Infections

Abstract Background Acute lower respiratory tract infections (ALRIs) especially severe ALRIs, constitute a global high burden of morbidity and mortality in children <5 y of age and respiratory syncytial virus (RSV) has been documented to a play a major aetiological role. However, Nigerian reports on severe childhood RSV ALRIs are rare and most reports are old. With recent advances in RSV preventive strategy, arises the need for a recent appraisal of RSV infection in children with severe ALRI. The current study thus set out to determine the prevalence of RSV infection among hospitalized children <5 y of age and describe the related social determinants. Methods We performed a descriptive cross-sectional study conducted over 1 y of 120 children, ages 2–59 months, diagnosed with ALRI. Relevant data were obtained and an antigen detection assay was used for viral studies. Results The prevalence of RSV infection was 34.2% and its peak was in the rainy months. The proportion of infants in the RSV-positive group was significantly higher than that in the RSV-negative group (82.9% vs 54.4%; p=0.002). These findings were largely consistent with those of earlier reports. Conclusions RSV has remained a common cause of severe ALRI in infants, especially during the rainy months in Nigeria. It is thus suggested that more effort be focused towards implementing the current global recommendations for the prevention of RSV-associated LRI, particularly in infants.

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