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

Noble, Erin; Cox, Andrew D.; Deval, Jérôme; Kim, Baek

doi:10.1016/j.virol.2012.07.008

Cited by 40 publications

(68 citation statements)

References 27 publications

(44 reference statements)

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“…We here obtained the same IC 50 value (i.e., 0.5 mM; Table 1) against isolated PA-Nter. This supports the view that the endonuclease catalytic site has the same structure in isolated PA-Nter as in native polymerase, and implicates that enzymatic assays with PA-Nter are relevant for developing catalytic site inhibitors Noble et al, 2012). Regarding the direct analogs of L-742,001 that we tested (Table 1), the IC 50 data demonstrate that the activity is unchanged when the phenyl moiety of L-742,001 is replaced by Fold increase at 60 minutes using 100 nM MB.…”

Section: Resultssupporting

confidence: 78%

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An Integrated Biological Approach to Guide the Development of Metal-Chelating Inhibitors of Influenza Virus PA Endonuclease

et al. 2014

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The influenza virus PA endonuclease, which cleaves capped cellular pre-mRNAs to prime viral mRNA synthesis, is a promising target for novel anti-influenza virus therapeutics. The catalytic center of this enzyme resides in the N-terminal part of PA (PA-Nter) and contains two (or possibly one or three) Mg 21 or Mn 21 ions, which are critical for its catalytic function. There is great interest in PA inhibitors that are optimally designed to occupy the active site and chelate the metal ions. We focused here on a series of b-diketo acid (DKA) and DKA-bioisosteric compounds containing different scaffolds, and determined their structure-activity relationship in an enzymatic assay with PA-Nter, in order to build a three-dimensional pharmacophore model. In addition, we developed a molecular beacon (MB)-based PA-Nter assay that enabled us to compare the inhibition of Mn 21 versus Mg 21, the latter probably being the biologically relevant cofactor. This real-time MB assay allowed us to measure the enzyme kinetics of PA-Nter or perform highthroughput screening. Several DKA derivatives were found to cause strong inhibition of PA-Nter, with IC 50 values comparable to that of the prototype L-742,001 (i.e., below 2 mM). Among the different compounds tested, L-742,001 appeared unique in having equal activity against either Mg 21 or Mn 21 . Three compounds (10, with a pyrrole scaffold, and 40 and 41, with an indole scaffold) exhibited moderate antiviral activity in cell culture (EC 99 values 64-95 mM) and were proven to affect viral RNA synthesis. Our approach of integrating complementary enzymatic, cellular, and mechanistic assays should guide ongoing development of improved influenza virus PA inhibitors.

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Section: Resultssupporting

confidence: 78%

“…The IC 50 value for DPBA in our plasmid assay was 2.7 mM, which is comparable to the data reported by others (Dias et al, 2009;Kowalinski et al, 2012;Noble et al, 2012). This value was unchanged upon addition of a methoxy in ortho (14) or para position (16).…”

Section: +supporting

confidence: 91%

An Integrated Biological Approach to Guide the Development of Metal-Chelating Inhibitors of Influenza Virus PA Endonuclease

et al. 2014

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“…Using a previously developed FRET-based assay (20,32), we first confirmed that PA N ΔLoop has a robust nuclease activity. Fig.…”

Section: δLoopmentioning

confidence: 72%

“…We followed previously developed procedures for both assays. For the FRET-based activity assay, the substrate is 5′-Cy5-GAATACTCAAGCTATGCATC-3IAbRQSp (20,32). Details of our fluorescent binding probe and the fluorescent polarization assay have been described (25).…”

Section: Viral Growth Kinetics In Vitromentioning

confidence: 99%

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

Song

Kumar

Shadrick

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitorresistant influenza strains.

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“…PB1 binds to the C-terminal domain of PA (14,15), while the N-terminal domain of PA (PAn) can function as an endonuclease to generate small RNA primers that are essential for initiation of the viral gene transcription (16,17). Recently, it has been shown that both PAn (18) and full-length PA (19) cleave the host-derived RNA in a sequence-specific manner.…”

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confidence: 99%

The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity

Tefsen

Zhu

et al. 2014

J Virol

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e Influenza imposes a great burden on society, not only in its seasonal appearance that affects both humans and domesticated animals but also through the constant threat of potential pandemics. Migratory birds are considered to be the reservoir hosts for influenza viruses, but other animals must also be considered. The recently identified influenza-like virus genome, from H17N10 in bats, was shown to be markedly different from genomes of other known influenza viruses, as both its surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) do not have canonical functions. However, no studies on other individual proteins from this particular virus have been reported until now. Here, we describe the structure of the N-terminal domain of PA from H17N10 influenza-like virus at 2.7-Å resolution and show that it has a fold similar to those of homologous PA domains present in more familiar influenza A virus strains. Moreover, we demonstrate that it possesses endonuclease activity and that the histidine residue in the active site is essential for this activity. Although this particular influenza virus subtype is probably not infectious for humans (even its virus state has not been confirmed in bats, as only the genome has been sequenced), reassortment of canonical influenza viruses with certain segments from H17N10 cannot be ruled out at this stage. Therefore, further studies are urgently needed for the sake of influenza prevention and control.

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

Cited by 40 publications

References 27 publications

An Integrated Biological Approach to Guide the Development of Metal-Chelating Inhibitors of Influenza Virus PA Endonuclease

An Integrated Biological Approach to Guide the Development of Metal-Chelating Inhibitors of Influenza Virus PA Endonuclease

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity

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