Blocking Respiratory Syncytial Virus Entry: A Story with Twists

Weisshaar, Marco; Cox, Robert; Plemper, Richard K.

doi:10.1089/dna.2015.2896

Cited by 7 publications

(9 citation statements)

References 37 publications

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“…However, recent mapping of resistance mutations (61) and cocrystallization of structurally different inhibitor classes with the RSV fusion (F) protein (62) spotlighted a defined microdomain located at the base of the prefusion RSV F protein head domain as the common docking site. Analogous to our prediction for the different HA inhibitor classes, all RSV compounds tested engage the same F microdomain, but different avenues exist to productively populate the target (60,62,63). Viral resistance against current therapeutics constitutes a major driving force for the development of novel influenza virus inhibitors; clinical use of the adamantanes is no longer advised, and preexisting resistance increasingly compromises the neuraminidase inhibitors (11,22).…”

Section: Discussionmentioning

confidence: 60%

“…Second, a bias toward interaction of small-molecule screening hits with viral glycoproteins and the presence of a single predominant target site is not entirely unprecedented for small-molecule drug screens against myxovirus family members. A number of anti-RSV HTS campaigns were conducted in recent years that likewise yielded entry inhibitors at a high frequency (60). The molecular target site of these chemically diverse compound classes was originally discussed controversially.…”

Section: Discussionmentioning

confidence: 99%

“…We propose that cooperative resistance is mediated by a secondary escape mechanism, based on several observations, as follows: sterically, a small molecule cannot engage both mutation sites simultaneously; the compound binding affinity for an HA protein with a cooperative double mutation was virtually identical to that for standard HA; and the cooperative double mutations raised the HA trigger pH, indicating a reduced conformational stability of the mutant prefusion HA trimer. Interestingly, different escape mechanisms also seem to be responsible for RSV pan-resistance to all entry inhibitor classes that were profiled in regard to resistance (60)(61)(62). In the case of RSV, however, secondary resistance does not depend on cooperative mutations; rather, the actual nature of amino acid substitutions at the same position controls the mechanism of escape.…”

Section: Discussionmentioning

confidence: 99%

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Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Weisshaar

Cox

Morehouse

et al. 2016

J Virol

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Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCEThis study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale highthroughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for smallmolecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism ...

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Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Weisshaar

Cox

Morehouse

et al. 2016

J Virol

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“…Firstly, several hot spots have been identified that mediate universal escape from inhibition by all advanced RSV entry inhibitors tested, despite the structural diversity of the different chemotypes [68]. The existence of these pan-resistance sites suggests that it may be challenging to proactively counteract viral escape through synthetic scaffold optimization [69]. Supporting this notion, a large-scale high-throughput drug screen using a recombinant RSV strain carrying a signature pan-resistance mutation did not return any hits blocking F protein activity, although entry inhibitors typically emerge readily and are often pharmacodominant in anti-RSV drug screens [70,71].…”

Section: Synthetic F Protein Blockersmentioning

confidence: 99%

Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition

Aggarwal

Plemper

2020

Viruses

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Paramyxoviruses and pneumoviruses infect cells through fusion (F) protein-mediated merger of the viral envelope with target membranes. Members of these families include a range of major human and animal pathogens, such as respiratory syncytial virus (RSV), measles virus (MeV), human parainfluenza viruses (HPIVs), and highly pathogenic Nipah virus (NiV). High-resolution F protein structures in both the metastable pre-and the postfusion conformation have been solved for several members of the families and a number of F-targeting entry inhibitors have progressed to advanced development or clinical testing. However, small-molecule RSV entry inhibitors have overall disappointed in clinical trials and viral resistance developed rapidly in experimental settings and patients, raising the question of whether the available structural information may provide a path to counteract viral escape through proactive inhibitor engineering. This article will summarize current mechanistic insight into F-mediated membrane fusion and examine the contribution of structural information to the development of small-molecule F inhibitors. Implications are outlined for future drug target selection and rational drug engineering strategies.Viruses 2020, 12, 342 2 of 16 from its natural bat host to domestic animals, resulting in case/fatality rates reaching from 40% to over 90% [9]. Continued spillover into the human population must be expected, and human-to-human transmission through respiratory secretions, urine, and saliva has been documented [10].Of the pneumoviruses, RSV infects almost all children before two years of age and is responsible for over 100,000 hospitalizations yearly in the United States alone. The monoclonal antibody palivizumab has been approved for immunoprophylaxis against RSV infection [11], however, use is restricted to high-risk patients due to the high-cost and need for prophylactic administration.Given the health, medical and economic burden associated with paramyxo-and pneumovirus infections, a major and currently unmet clinical need exists to expedite the development of novel safe and effective therapeutics for improved disease management and outbreak control. Current direct-acting therapeutics predominantly focus on preventing viral entry through neutralizing antibodies (nAbs) and on small-molecules targeting the envelope glycoproteins or inhibiting the viral RNA-dependent RNA polymerase (RdRP) complex. Reflecting major efforts to identify a cost-effective alternative to high-price passive immunization with anti-RSV nAbs, a number of compounds have entered advanced preclinical development and clinical testing in recent years (Table 1). Breakthroughs in the structural and functional characterization of the viral entry machinery and polymerase complexes in the past decade [12][13][14][15][16][17][18][19][20][21] have furthermore created a novel opportunity for structure-informed mechanistic characterization and ligand optimization.

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“…4). The F protein then folds back in on itself to bring the viral and target membranes together causing membrane fusion [136][137][138][139][140]. Initial work on designing antibodies and small molecules against the F protein were hampered by the multiple confirmations the protein forms; indeed, targeting the post-fusion F protein form had little clinical benefit.…”

Section: F Proteinmentioning

confidence: 99%

Revisiting respiratory syncytial virus’s interaction with host immunity, towards novel therapeutics

Efstathiou

Abidi

Harker

et al. 2020

Cell. Mol. Life Sci.

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Every year there are > 33 million cases of Respiratory Syncytial Virus (RSV)-related respiratory infection in children under the age of five, making RSV the leading cause of lower respiratory tract infection (LRTI) in infants. RSV is a global infection, but 99% of related mortality is in low/middle-income countries. Unbelievably, 62 years after its identification, there remains no effective treatment nor vaccine for this deadly virus, leaving infants, elderly and immunocompromised patients at high risk. The success of all pathogens depends on their ability to evade and modulate the host immune response. RSV has a complex and intricate relationship with our immune systems, but a clearer understanding of these interactions is essential in the development of effective medicines. Therefore, in a bid to update and focus our research community's understanding of RSV's interaction with immune defences, this review aims to discuss how our current knowledgebase could be used to combat this global viral threat.

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Blocking Respiratory Syncytial Virus Entry: A Story with Twists

Cited by 7 publications

References 37 publications

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition

Revisiting respiratory syncytial virus’s interaction with host immunity, towards novel therapeutics

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