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Drug Repurposing for Influenza Virus Polymerase Acidic (PA) Endonuclease Inhibitor.

ABSTRACT: Drug repurposing can quickly and effectively identify novel drug repurposing opportunities. The PA endonuclease catalytic site has recently become regarded as an attractive target for the screening of anti-influenza drugs. PA N-terminal (PA_N) inhibitor can inhibit the entire PA endonuclease activity. In this study, we screened the effectivity of PA_N inhibitors from the FDA database through in silico methods and in vitro experiments. PA_N and mutant PA_N-I38T were chosen as virtual screening targets for overcoming drug resistance. Gel-based PA endonuclease analysis determined that the drug lifitegrast can effectively inhibit PA_N and PA_N-I38T, when the IC₅₀ is 32.82 ± 1.34 μM and 26.81 ± 1.2 μM, respectively. Molecular docking calculation showed that lifitegrast interacted with the residues around PA or PA-I38 T's active site, occupying the catalytic site pocket. Both PA_N/PA_N-I38T and lifitegrast can acquire good equilibrium in 100 ns molecular dynamic simulation. Because of these properties, lifitegrast, which can effectively inhibit PA endonuclease activity, was screened through in silico and in vitro research. This new research will be of significance in developing more effective and selective drugs for anti-influenza therapy.

SUBMITTER: Meng X

PROVIDER: S-EPMC8659148 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Endonuclease substrate selectivity characterized with full-length PA of influenza A virus polymerase.

Project description:The influenza A polymerase is a heterotrimer which transcribes viral mRNAs and replicates the viral genome. To initiate synthesis of mRNA, the polymerase binds a host pre-mRNA and cleaves a short primer downstream of the 5' end cap structure. The N-terminal domain of PA has been demonstrated to have endonuclease activity in vitro. Here we sought to better understand the biochemical nature of the PA endonuclease by developing an improved assay using full-length PA protein. This full-length protein is active against both RNA and DNA in a cap-independent manner and can use several different divalent cations as cofactors, which affects the secondary structure of the full-length PA. Our in vitro assay was also able to demonstrate the minimal substrate size and sequence selectivity of the PA protein, which is crucial information for inhibitor design. Finally, we confirmed the observed endonuclease activity of the full-length PA with a FRET-based assay.

| S-EPMC3647620 | biostudies-literature

A novel small-molecule inhibitor of influenza A virus acts by suppressing PA endonuclease activity of the viral polymerase.

Project description:The RNA-dependent RNA polymerase of influenza A virus comprises conserved and independently-folded subdomains with defined functionalities. The N-terminal domain of the PA subunit (PA(N)) harbors the endonuclease function so that it can serve as a desired target for drug discovery. To identify a class of anti-influenza inhibitors that impedes PA(N) endonuclease activity, a screening approach that integrated the fluorescence resonance energy transfer based endonuclease inhibitory assay with the DNA gel-based endonuclease inhibitory assay was conducted, followed by the evaluation of antiviral efficacies and potential cytotoxicity of the primary hits in vitro and in vivo. A small-molecule compound ANA-0 was identified as a potent inhibitor against the replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2, in cell cultures. Combinational treatment of zanamivir and ANA-0 exerted synergistic anti-influenza effect in vitro. Intranasal administration of ANA-0 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. In summary, ANA-0 shows potential to be developed to novel anti-influenza agents.

| S-EPMC4783701 | biostudies-literature

Mutational and metal binding analysis of the endonuclease domain of the influenza virus polymerase PA subunit.

Project description:Influenza virus polymerase initiates the biosynthesis of its own mRNAs with capped 10- to 13-nucleotide fragments cleaved from cellular (pre-)mRNAs. Two activities are required for this cap-snatching activity: specific binding of the cap structure and an endonuclease activity. Recent work has shown that the cap-binding site is situated in the central part of the PB2 subunit and that the endonuclease activity is situated in the N-terminal domain of the PA subunit (PA-Nter). The influenza endonuclease is a member of the PD-(D/E)XK family of nucleases that use divalent metal ions for nucleic acid cleavage. Here we analyze the metal binding and endonuclease activities of eight PA-Nter single-point mutants. We show by calorimetry that the wild-type active site binds two Mn(2+) ions and has a 500-fold higher affinity for manganese than for magnesium ions. The endonuclease activity of the isolated mutant domains are compared with the cap-dependent transcription activities of identical mutations in trimeric recombinant polymerases previously described by other groups. Mutations that inactivate the endonuclease activity in the isolated PA-Nter knock out the transcription but not replication activity in the recombinant polymerase. We confirm the importance of a number of active-site residues and identify some residues that may be involved in the positioning of the RNA substrate in the active site. Our results validate the use of the isolated endonuclease domain in a drug-design process for new anti-influenza virus compounds.

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PA-E18G substitution in influenza A virus confers resistance to ZX-7101, a cap-dependent endonuclease inhibitor.

Project description:Cap-dependent endonuclease (CEN) in the polymerase acidic protein (PA) of influenza A virus (IAV) represents a promising drug target due to its critical role in viral gene transcription. The CEN inhibitor, baloxavir marboxil (BXM), was approved in Japan and the US in 2018 and several other countries subsequently. Along with the clinical use of BXM, the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern. Herein, we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A, an analogue of BXM. The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes, including pH1N1, H3N2, H7N9 and H9N2, in MDCK cells, and the 50% effective concentration (EC50) was calculated to nanomole level and comparable to that of baloxavir acid (BXA), the active form of BXM. Furthermore, in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice, with reduced viral RNA loads and alleviated pulmonary damage. Importantly, serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage. Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA. Taken together, our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance, which provides critical clues for future drug development and drug resistance surveillance.

| S-EPMC10436051 | biostudies-literature

Virtual Screening and Biological Validation of Novel Influenza Virus PA Endonuclease Inhibitors.

Project description:The influenza virus RNA-dependent RNA polymerase complex (RdRp), a heterotrimeric protein complex responsible for viral RNA transcription and replication, represents a primary target for antiviral drug development. One particularly attractive approach is interference with the endonucleolytic "cap-snatching" reaction by the RdRp subunit PA, more precisely by inhibiting its metal-dependent catalytic activity which resides in the N-terminal part of PA (PA-Nter). Almost all PA inhibitors (PAIs) thus far discovered bear pharmacophoric fragments with chelating motifs able to bind the bivalent metal ions in the catalytic core of PA-Nter. More recently, the availability of crystallographic structures of PA-Nter has enabled rational design of original PAIs with improved binding properties and antiviral potency. We here present a coupled pharmacophore/docking virtual screening approach that allowed us to identify PAIs with interesting inhibitory activity in a PA-Nter enzymatic assay. Moreover, antiviral activity in the low micromolar range was observed in cell-based influenza virus assays.

| S-EPMC4538433 | biostudies-literature

Mutational analysis of the binding pockets of the diketo acid inhibitor L-742,001 in the influenza virus PA endonuclease.

Project description:The influenza virus PA endonuclease, which cleaves capped host pre-mRNAs to initiate synthesis of viral mRNA, is a prime target for antiviral therapy. The diketo acid compound L-742,001 was previously identified as a potent inhibitor of the influenza virus endonuclease reaction, but information on its precise binding mode to PA or potential resistance profile is limited. Computer-assisted docking of L-742,001 into the crystal structure of inhibitor-free N-terminal PA (PA-Nter) indicated a binding orientation distinct from that seen in a recent crystallographic study with L-742,001-bound PA-Nter (R. M. DuBois et al., PLoS Pathog. 8:e1002830, 2012). A comprehensive mutational analysis was performed to determine which amino acid changes within the catalytic center of PA or its surrounding hydrophobic pockets alter the antiviral sensitivity to L-742,001 in cell culture. Marked (up to 20-fold) resistance to L-742,001 was observed for the H41A, I120T, and G81F/V/T mutant forms of PA. Two- to 3-fold resistance was seen for the T20A, L42T, and V122T mutants, and the R124Q and Y130A mutants were 3-fold more sensitive to L-742,001. Several mutations situated at noncatalytic sites in PA had no or only marginal impact on the enzymatic functionality of viral ribonucleoprotein complexes reconstituted in cell culture, consistent with the less conserved nature of these PA residues. Our data provide relevant insights into the binding mode of L-742,001 in the PA endonuclease active site. In addition, we predict some potential resistance sites that should be taken into account during optimization of PA endonuclease inhibitors toward tight binding in any of the hydrophobic pockets surrounding the catalytic center of the enzyme.

| S-EPMC3807387 | biostudies-literature

N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes.

Project description:Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics. This is an enzyme with divalent metal ion(s) (Mg(2+) or Mn(2+)) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity. Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity. Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones. Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein's active site.

| S-EPMC4980666 | biostudies-literature

An anti-influenza A virus microbial metabolite acts by degrading viral endonuclease PA.

Project description:The emergence of new highly pathogenic and drug-resistant influenza strains urges the development of novel therapeutics for influenza A virus (IAV). Here, we report the discovery of an anti-IAV microbial metabolite called APL-16-5 that was originally isolated from the plant endophytic fungus Aspergillus sp. CPCC 400735. APL-16-5 binds to both the E3 ligase TRIM25 and IAV polymerase subunit PA, leading to TRIM25 ubiquitination of PA and subsequent degradation of PA in the proteasome. This mode of action conforms to that of a proteolysis targeting chimera which employs the cellular ubiquitin-proteasome machinery to chemically induce the degradation of target proteins. Importantly, APL-16-5 potently inhibits IAV and protects mice from lethal IAV infection. Therefore, we have identified a natural microbial metabolite with potent in vivo anti-IAV activity and the potential of becoming a new IAV therapeutic. The antiviral mechanism of APL-16-5 opens the possibility of improving its anti-IAV potency and specificity by adjusting its affinity for TRIM25 and viral PA protein through medicinal chemistry.

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Structural and biochemical basis for development of influenza virus inhibitors targeting the PA endonuclease.

Project description:Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or "snatched" from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.

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Exploration of the 2,3-dihydroisoindole pharmacophore for inhibition of the influenza virus PA endonuclease.

Project description:Seasonal influenza A and B viruses represent a global concern. Antiviral drugs are crucial to treat severe influenza in high-risk patients and prevent virus spread in case of a pandemic. The emergence of viruses showing drug resistance, in particular for the recently licensed polymerase inhibitor baloxavir marboxil, drives the need for developing alternative antivirals. The endonuclease activity residing in the N-terminal domain of the polymerase acidic protein (PAN) is crucial for viral RNA synthesis and a validated target for drug design. Its function can be impaired by molecules bearing a metal-binding pharmacophore (MBP) able to coordinate the two divalent metal ions in the active site. In the present work, the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold is explored for the inhibition of influenza virus PA endonuclease. The structure-activity relationship was analysed by modifying the substituents on the lipophilic moiety linked to the MBP. The new compounds exhibited nanomolar inhibitory activity in a FRET-based enzymatic assay, and a few compounds (15-17, 21) offered inhibition in the micromolar range, in a cell-based influenza virus polymerase assay. When investigated against a panel of PA-mutant forms, compound 17 was shown to retain full activity against the baloxavir-resistant I38T mutant. This was corroborated by docking studies providing insight into the binding mode of this novel class of PA inhibitors.

| S-EPMC8838982 | biostudies-literature

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