Structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-CTD conformation

Abdella, R.; Aggarwal, Megha; Okura, Takashi; Lamb, Robert A.; He, Yuan

doi:10.1073/pnas.1919837117

Cited by 83 publications

(149 citation statements)

References 80 publications

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“…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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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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confidence: 99%

Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition

Aggarwal

Plemper

2020

Viruses

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“…Several cryo-electron microscopy-based reconstructions of mononegavirus polymerase proteins have been reported, including structural models of PIV-5 and RSV L polymerases that are closely related to MeV L (31,32). We have previously identified signature resistance hot-spots for ERDRP-0519 in MeV and CDV L through viral adaptation (10,12).…”

Section: Resultsmentioning

confidence: 99%

“…We favor the mononegavirus RdRP complex as a premier druggable target, based on its diverse enzymatic activities, its unique catalytic activity that lacks a cellular equivalent, and its critical importance for both viral replication and the expression of non-structural immunomodulatory viral proteins that counteract the host innate antiviral response (45). Groundbreaking progress in the structural understanding of the organization of mononegavirus polymerase complexes (29)(30)(31)(32) has established a foundation to define distinct druggable sites in the L polymerase.…”

Section: Discussionmentioning

confidence: 99%

“…For mechanism-of-action characterization, we applied purified recombinant MeV P-L complexes to in vitro RdRP assays in the presence and absence of ERDRP-0519, using 32 A catalytically defective L mutant harboring an N774A substitution in the polymerase active site confirmed that RNA synthesis was MeV P-L specific and not due to co-purified cellular contaminants.…”

Section: Erdrp-0519 Resistance Mutations Restore Initiation Of Rna Symentioning

confidence: 99%

“…Backpriming refers to the spontaneous formation of a circular hairpin structure of the non-encapsidated synthetic templates (35) that allows extension of the resulting paired 3'-ends beyond the actual length of the template (figure 4A). To assess whether the same limitation applies to ERDRP-0519, we applied the MeV polymerase complex to a previously described 25-mer RNA template derived from an authentic RSV promoter sequence that is capable of efficient backpriming (35) We therefore concentrated further analysis on peptides 1 and 2, which are near the intersection between the polymerase capping, connector and MTase domains, and in close proximity to highly conserved polymerase motifs such as the proposed PRNTase domain (HR moiety of motif D and G1156 of motif A) as well as both the postulated paramyxovirus L priming and "intrusion" loops (32) ( figure 5E).…”

Section: Erdrp-0519 Blocks All Phosphodiester Bond Formationmentioning

confidence: 99%

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Therapeutic Targeting of Measles Virus Polymerase with ERDRP-0519 Suppresses All RNA Synthesis Activity

Cox

Sourimant

Govindarajan

et al. 2020

Preprint

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Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA.ImportanceThe mononegavirus order contains major established and recently emerged human pathogens. Despite the threat to human health, antiviral therapeutics directed against this order remain understudied. The mononegavirus polymerase complex represents a promising drug target due to its central importance for both virus replication and viral mitigation of the innate host antiviral response. In this study, we have mechanistically characterized a clinical candidate small-molecule MeV polymerase inhibitor. The compound blocked all phosphodiester bond formation activity, a unique mechanism of action unlike all other known mononegavirus polymerase inhibitors. Photocrosslinking-based target site mapping demonstrated that this class-defining prototype inhibitor stabilizes a pre-initiation conformation of the viral polymerase complex that sterically cannot accommodate template RNA. Function-equivalent druggable sites exist in all mononegavirus polymerases. In addition to its direct anti-MeV impact, the insight gained in this study can therefore serve as a blueprint for indication spectrum expansion through structure-informed scaffold engineering or targeted drug discovery.

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