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- PDB-9mql: Locally-Refined Inactive Kappa-Opioid Receptor with Nb6M, NabFab,... -

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Basic information

Entry
Database: PDB / ID: 9mql
TitleLocally-Refined Inactive Kappa-Opioid Receptor with Nb6M, NabFab, and isoquinuclidine compound #020_E1
ComponentsKappa-Opioid Receptor
KeywordsMEMBRANE PROTEIN / G-protein coupled receptor / Kappa-opioid / isoquinuclidine / antagonist / nanobodies / fabs
Function / homology:
Function and homology information
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsKim, J.Y. / Vigneron, S.F. / Billesbolle, C. / Manglik, A. / Shoichet, B.K.
Funding support United States, 3items
OrganizationGrant numberCountry
Defense Advanced Research Projects Agency (DARPA)HR0011-19-2-0020 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122481 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122473 United States
CitationJournal: bioRxiv / Year: 2025
Title: Docking 14 million virtual isoquinuclidines against the mu and kappa opioid receptors reveals dual antagonists-inverse agonists with reduced withdrawal effects.
Authors: Seth F Vigneron / Shohei Ohno / Joao Braz / Joseph Y Kim / Oh Sang Kweon / Chase Webb / Christian Billesbølle / Karnika Bhardwaj / John Irwin / Aashish Manglik / Allan I Basbaum / Jonathan ...Authors: Seth F Vigneron / Shohei Ohno / Joao Braz / Joseph Y Kim / Oh Sang Kweon / Chase Webb / Christian Billesbølle / Karnika Bhardwaj / John Irwin / Aashish Manglik / Allan I Basbaum / Jonathan A Ellman / Brian K Shoichet /
Abstract: Large library docking of tangible molecules has revealed potent ligands across many targets. While make-on-demand libraries now exceed 75 billion enumerated molecules, their synthetic routes are ...Large library docking of tangible molecules has revealed potent ligands across many targets. While make-on-demand libraries now exceed 75 billion enumerated molecules, their synthetic routes are dominated by a few reaction types, reducing diversity and inevitably leaving many interesting bioactive-like chemotypes unexplored. Here, we investigate the large-scale enumeration and targeted docking of isoquinuclidines. These "natural-product-like" molecules are rare in the current libraries and are functionally congested, making them interesting as receptor probes. Using a modular, four-component reaction scheme, we built and docked a virtual library of over 14.6 million isoquinuclidines against both the μ- and -opioid receptors (MOR and KOR, respectively). Synthesis and experimental testing of 18 prioritized compounds found nine ligands with low μM affinities. Structure-based optimization revealed low- and sub-nM antagonists and inverse agonists targeting both receptors. Cryo-electron microscopy (cryoEM) structures illuminate the origins of activity on each target. In mouse behavioral studies, a potent member of the series with joint MOR-antagonist and KOR-inverse-agonist activity reversed morphine-induced analgesia, phenocopying the MOR-selective anti-overdose agent naloxone. Encouragingly, the new molecule induced less severe opioid-induced withdrawal symptoms compared to naloxone during withdrawal precipitation, and did not induce conditioned-place aversion, likely reflecting a reduction of dysphoria due to the compound's KOR-inverse agonism. The strengths and weaknesses of bespoke library docking, and of docking for opioid receptor polypharmacology, will be considered.
History
DepositionJan 3, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 12, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 12, 2025Group: Data collection / Source and taxonomy / Structure summary
Category: em_admin / em_entity_assembly ...em_admin / em_entity_assembly / em_entity_assembly_naturalsource / em_entity_assembly_recombinant / entity_src_gen
Item: _em_admin.last_update / _em_entity_assembly.name ..._em_admin.last_update / _em_entity_assembly.name / _em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism / _em_entity_assembly_recombinant.ncbi_tax_id / _em_entity_assembly_recombinant.organism / _entity_src_gen.gene_src_common_name / _entity_src_gen.host_org_common_name / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name
Revision 1.1Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Experimental summary / Source and taxonomy
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Category: em_admin / em_entity_assembly ...em_admin / em_entity_assembly / em_entity_assembly_naturalsource / em_entity_assembly_recombinant / entity_src_gen
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Item: _em_admin.last_update / _em_entity_assembly.name ..._em_admin.last_update / _em_entity_assembly.name / _em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism / _em_entity_assembly_recombinant.ncbi_tax_id / _em_entity_assembly_recombinant.organism / _entity_src_gen.gene_src_common_name / _entity_src_gen.host_org_common_name / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Kappa-Opioid Receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)44,0222
Polymers43,6271
Non-polymers3951
Water00
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 Kappa-Opioid Receptor


Mass: 43626.902 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#2: Chemical ChemComp-A1BNM / methyl (1S,3R,4S,6S,8M)-2-[(1-ethyl-1H-pyrazol-4-yl)methyl]-8-(3-hydroxyphenyl)-3,4-dimethyl-2-azabicyclo[2.2.2]oct-7-ene-6-carboxylate


Mass: 395.495 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H29N3O3 / Feature type: SUBJECT OF INVESTIGATION
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: Mouse Kappa-Opioid Receptor / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 46 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Mus musculus (house mouse)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2100 nm / Nominal defocus min: 800 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64.1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsPhase plate: OTHER

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286: / Category: model refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98518 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0022360
ELECTRON MICROSCOPYf_angle_d0.4733219
ELECTRON MICROSCOPYf_dihedral_angle_d7.376322
ELECTRON MICROSCOPYf_chiral_restr0.037394
ELECTRON MICROSCOPYf_plane_restr0.003380

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