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- PDB-9hli: Influenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor -

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Basic information

Entry
Database: PDB / ID: 9hli
TitleInfluenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor
ComponentsNeuraminidase
KeywordsVIRAL PROTEIN / Influenza / Neuraminidase
Function / homology
Function and homology information


exo-alpha-sialidase / exo-alpha-sialidase activity / viral budding from plasma membrane / carbohydrate metabolic process / host cell plasma membrane / virion membrane / membrane / metal ion binding
Similarity search - Function
Sialidase, Influenza viruses A/B / Glycoside hydrolase, family 34 / Neuraminidase / Sialidase superfamily
Similarity search - Domain/homology
Biological speciesInfluenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2 Å
AuthorsMoran, E. / Davies, G.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: Proc Natl Acad Sci U S A / Year: 2026
Title: Oseltamivir aziridines are potent influenza neuraminidase inhibitors and imaging agents.
Authors: Merijn B L Vriends / Elisha Moran / Martín Calvelo / Thomas Hansen / Isabelle B Pickles / Xincheng Xin / Marieke Biezeno / Zachary W B Armstrong / Maria J Ferraz / Lei Li / Alice Lilley / ...Authors: Merijn B L Vriends / Elisha Moran / Martín Calvelo / Thomas Hansen / Isabelle B Pickles / Xincheng Xin / Marieke Biezeno / Zachary W B Armstrong / Maria J Ferraz / Lei Li / Alice Lilley / Ruth Harvey / Dmitri V Filippov / Qinghua Liao / Sybrin P Schröder / Gijsbert A van der Marel / Marta Artola / Johannes M F G Aerts / James N Blaza / Jeroen D C Codée / Carme Rovira / Herman S Overkleeft / Gideon J Davies /
Abstract: Influenza neuraminidase (NA) is a critical target for seasonal and pandemic antivirals, including the strains of current concern. Current treatments, such as Zanamivir and Oseltamivir, are limited by ...Influenza neuraminidase (NA) is a critical target for seasonal and pandemic antivirals, including the strains of current concern. Current treatments, such as Zanamivir and Oseltamivir, are limited by noncovalent binding and emerging resistance. We hypothesized that Oseltamivir aziridines would unite transition-state mimicry for tight binding, with aziridine-enabled covalent capture of the catalytic tyrosine, thereby supporting both therapy and activity-based quantification. Here, we present oseltamivir-based aziridines, inspired by cyclophellitol chemistry, that act as covalent inhibitors and activity-based probes via an -acylaziridine warhead. Free-energy calculations, and NMR observations, indicate a H half-chair preference consistent with the NA transition state, and selected analogues inhibit multiple NA subtypes with low nanomolar binding constants. Diverse evidence establishes covalency: time-dependent inactivation, inhibitor washout, intact-mass shifts, MS/MS identification of a tyrosine adduct, and QM/MM reaction profiles, while cryoEM of N1 aligns with the proposed binding mode, revealing an elimination product. The inhibitors demonstrate formidable activity against diverse viral neuraminidases, including H5N1, and further enable imaging and quantification of active NA. With their dual therapeutic and diagnostic potential, these first-in-class inhibitors indeed benefit from transition state mimicry and covalency, and thus offer a powerful platform for antiviral development and neuraminidase imaging, addressing urgent global health needs in influenza treatment and prevention.
History
DepositionDec 4, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 17, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Neuraminidase
B: Neuraminidase
C: Neuraminidase
D: Neuraminidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,52116
Polymers169,9984
Non-polymers2,52312
Water6,882382
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Neuraminidase


Mass: 42499.523 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Gene: NA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: C7FH46, exo-alpha-sialidase
#2: Chemical
ChemComp-A1IVX / (3~{S},4~{R},5~{R},6~{R})-4-acetamido-3-azanyl-6-(butanoylamino)-5-pentan-3-yloxy-cyclohexene-1-carboxylic acid


Mass: 369.456 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C18H31N3O5 / Feature type: SUBJECT OF INVESTIGATION
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 382 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Influenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Influenza A virus
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7 / Details: 25 mM Tris pH 7.0 150 mM NaCl, 100 mM CaCl2
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisTris1
2150 mMSodium chlorideNaCl1
3100 mMCalcium chlorideCaCl21
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot force -10 and blot time 3 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1400 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 43.99 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
4CTFFINDCTF correction
7Coot0.9.8.92model fitting
9PHENIX1.21.1_5286:model refinement
10RELIONinitial Euler assignment
13RELION53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 216385 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311792
ELECTRON MICROSCOPYf_angle_d0.64616032
ELECTRON MICROSCOPYf_dihedral_angle_d6.7811724
ELECTRON MICROSCOPYf_chiral_restr0.0521704
ELECTRON MICROSCOPYf_plane_restr0.0052080

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