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- EMDB-48528: Locally-Refined Inactive Kappa-Opioid Receptor with Nb6M, NabFab,... -

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Basic information

Entry
Database: EMDB / ID: EMD-48528
TitleLocally-Refined Inactive Kappa-Opioid Receptor with Nb6M, NabFab, and isoquinuclidine compound #020_E1
Map data
Sample
  • Complex: Mouse Kappa-Opioid Receptor
    • Protein or peptide: Kappa-Opioid Receptor
  • Ligand: 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
KeywordsG-protein coupled receptor / Kappa-opioid / isoquinuclidine / antagonist / nanobodies / fabs / MEMBRANE PROTEIN
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsKim JY / Vigneron SF / Billesbolle C / Manglik A / Shoichet BK
Funding support United States, 3 items
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, 2025-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48528.png

Downloads & links

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Map

FileDownload / File: emd_48528.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 360 pix.
= 311.4 Å		0.87 Å/pix.
x 360 pix.
= 311.4 Å		0.87 Å/pix.
x 360 pix.
= 311.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.865 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.616
Minimum - Maximum-3.4046154 - 4.3036766
Average (Standard dev.)-0.0011255649 (±0.08780805)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 311.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_48528_half_map_1.map
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Half map: #2

Fileemd_48528_half_map_2.map
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Sample components

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Entire : Mouse Kappa-Opioid Receptor

EntireName: Mouse Kappa-Opioid Receptor
Components
  • Complex: Mouse Kappa-Opioid Receptor
    • Protein or peptide: Kappa-Opioid Receptor
  • Ligand: 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

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Supramolecule #1: Mouse Kappa-Opioid Receptor

SupramoleculeName: Mouse Kappa-Opioid Receptor / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 46 MDa

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Macromolecule #1: Kappa-Opioid Receptor

MacromoleculeName: Kappa-Opioid Receptor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 43.626902 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: DYKDDDDAME SPIQIFRGDP GPTCSPSACL LPNSSSWFPN WAESDSNGSV GSEDQQLESA HISPAIPVII TAVYSVVFVV GLVGNSLVM FVIIRYTKMK TATNIYIFNL ALADALVTTT MPFQSAVYLM NSWPFGDVLC KIVISIDYYN MFTSIFTLTM M SVDRYIAV ...String:
DYKDDDDAME SPIQIFRGDP GPTCSPSACL LPNSSSWFPN WAESDSNGSV GSEDQQLESA HISPAIPVII TAVYSVVFVV GLVGNSLVM FVIIRYTKMK TATNIYIFNL ALADALVTTT MPFQSAVYLM NSWPFGDVLC KIVISIDYYN MFTSIFTLTM M SVDRYIAV CHPVKALDFR TPLKAKIINI CIWLLASSVG ISAIVLGGTK VREDVDVIEC SLQFPDDEYS WWDLFMKICV FV FAFVIPV LIIIVCYTLM ILRLKSVRLL SGSREKDRNL RRITKLVLVV VAVFIICWTP IHIFILVEAL GSTSHSTAAL SSY YFCIAL GYTNSSLNPV LYAFLDENFK RCFRDFCFPI KMRMERQSTN RVRNTVQDPA SMRDVGGMNK PV

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Macromolecule #2: methyl (1S,3R,4S,6S,8M)-2-[(1-ethyl-1H-pyrazol-4-yl)methyl]-8-(3-...

MacromoleculeName: 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
type: ligand / ID: 2 / Number of copies: 1 / Formula: A1BNM
Molecular weightTheoretical: 395.495 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsPhase plate: OTHER
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 64.1 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 98518
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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