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- EMDB-28596: CryoEM Structure of NLRP3 NACHT domain in complex with G2394 -

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Basic information

Entry
Database: EMDB / ID: EMD-28596
TitleCryoEM Structure of NLRP3 NACHT domain in complex with G2394
Map data
Sample
  • Complex: NLRP3 NACHT domain
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: (6S,8R)-N-[(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl]-6-(methylamino)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonamide
Function / homology
Function and homology information


small molecule sensor activity / detection of biotic stimulus / phosphatidylinositol phosphate binding / cysteine-type endopeptidase activator activity / positive regulation of T-helper 2 cell differentiation / interphase microtubule organizing center / NLRP3 inflammasome complex assembly / positive regulation of T-helper 2 cell cytokine production / NLRP3 inflammasome complex / positive regulation of type 2 immune response ...small molecule sensor activity / detection of biotic stimulus / phosphatidylinositol phosphate binding / cysteine-type endopeptidase activator activity / positive regulation of T-helper 2 cell differentiation / interphase microtubule organizing center / NLRP3 inflammasome complex assembly / positive regulation of T-helper 2 cell cytokine production / NLRP3 inflammasome complex / positive regulation of type 2 immune response / osmosensory signaling pathway / peptidoglycan binding / phosphatidylinositol-4-phosphate binding / negative regulation of non-canonical NF-kappaB signal transduction / microtubule organizing center / negative regulation of interleukin-1 beta production / pattern recognition receptor signaling pathway / negative regulation of NF-kappaB transcription factor activity / pyroptosis / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / protein maturation / The NLRP3 inflammasome / negative regulation of acute inflammatory response / positive regulation of interleukin-4 production / signaling adaptor activity / Purinergic signaling in leishmaniasis infection / molecular condensate scaffold activity / positive regulation of interleukin-1 beta production / ADP binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / protein homooligomerization / defense response / Cytoprotection by HMOX1 / Metalloprotease DUBs / negative regulation of inflammatory response / cellular response to virus / positive regulation of inflammatory response / positive regulation of non-canonical NF-kappaB signal transduction / SARS-CoV-1 activates/modulates innate immune responses / positive regulation of NF-kappaB transcription factor activity / DNA-binding transcription factor binding / cellular response to lipopolysaccharide / sequence-specific DNA binding / molecular adaptor activity / inflammatory response / Golgi membrane / innate immune response / apoptotic process / SARS-CoV-2 activates/modulates innate and adaptive immune responses / endoplasmic reticulum / signal transduction / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular region / ATP binding / membrane / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
NACHT-associated domain / Fish-specific NACHT associated domain / Fish-specific NACHT associated domain / NACHT, LRR and PYD domains-containing protein, helical domain HD2 / NLRC4 helical domain HD2 / NOD2, winged helix domain / NOD2 winged helix domain / Leucine rich repeat, ribonuclease inhibitor type / DAPIN domain / DAPIN domain profile. ...NACHT-associated domain / Fish-specific NACHT associated domain / Fish-specific NACHT associated domain / NACHT, LRR and PYD domains-containing protein, helical domain HD2 / NLRC4 helical domain HD2 / NOD2, winged helix domain / NOD2 winged helix domain / Leucine rich repeat, ribonuclease inhibitor type / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / PAAD/DAPIN/Pyrin domain / Leucine Rich repeat / Death-like domain superfamily / Leucine-rich repeat / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
NACHT, LRR and PYD domains-containing protein 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsMurray JM / Johnson MC
Funding support1 items
OrganizationGrant numberCountry
Other private
CitationJournal: J Med Chem / Year: 2022
Title: Overcoming Preclinical Safety Obstacles to Discover ()--((1,2,3,5,6,7-Hexahydro--indacen-4-yl)carbamoyl)-6-(methylamino)-6,7-dihydro-5-pyrazolo[5,1-][1,3]oxazine-3-sulfonamide (GDC-2394): A ...Title: Overcoming Preclinical Safety Obstacles to Discover ()--((1,2,3,5,6,7-Hexahydro--indacen-4-yl)carbamoyl)-6-(methylamino)-6,7-dihydro-5-pyrazolo[5,1-][1,3]oxazine-3-sulfonamide (GDC-2394): A Potent and Selective NLRP3 Inhibitor.
Authors: Christopher McBride / Lynnie Trzoss / Davide Povero / Milos Lazic / Geza Ambrus-Aikelin / Angelina Santini / Rama Pranadinata / Gretchen Bain / Ryan Stansfield / Jeffrey A Stafford / James ...Authors: Christopher McBride / Lynnie Trzoss / Davide Povero / Milos Lazic / Geza Ambrus-Aikelin / Angelina Santini / Rama Pranadinata / Gretchen Bain / Ryan Stansfield / Jeffrey A Stafford / James Veal / Ryan Takahashi / Justin Ly / Shu Chen / Liling Liu / Marika Nespi / Robert Blake / Arna Katewa / Tracy Kleinheinz / Swathi Sujatha-Bhaskar / Nandhini Ramamoorthi / Jessica Sims / Brent McKenzie / Mark Chen / Mark Ultsch / Matthew Johnson / Jeremy Murray / Claudio Ciferri / Steven T Staben / Michael J Townsend / Craig E Stivala /
Abstract: Inappropriate activation of the NLRP3 inflammasome has been implicated in multiple inflammatory and autoimmune diseases. Herein, we aimed to develop novel NLRP3 inhibitors that could minimize the ...Inappropriate activation of the NLRP3 inflammasome has been implicated in multiple inflammatory and autoimmune diseases. Herein, we aimed to develop novel NLRP3 inhibitors that could minimize the risk of drug-induced liver injury. Lipophilic ligand efficiency was used as a guiding metric to identify a series of 6,7-dihydro-5H-pyrazolo[5,1-][1,3]oxazinesulfonylureas. A leading compound from this series was advanced into safety studies in cynomolgus monkeys, and renal toxicity, due to compound precipitation, was observed. To overcome this obstacle, we focused on improving the solubility of our compounds, specifically by introducing basic amine substituents into the scaffold. This led to the identification of GDC-2394, a potent and selective NLRP3 inhibitor, with an in vitro and in vivo safety profile suitable for advancement into human clinical trials.
History
DepositionOct 17, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28596.png

Downloads & links

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Map

FileDownload / File: emd_28596.map.gz / Format: CCP4 / Size: 2.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1093 Å
Density
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-2.8268428 - 4.4969797
Average (Standard dev.)-1.035472e-11 (±0.31531385)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin126113100
Dimensions849378
Spacing788493
CellA: 86.5254 Å / B: 93.1812 Å / C: 103.1649 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_28596_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28596_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NLRP3 NACHT domain

EntireName: NLRP3 NACHT domain
Components
  • Complex: NLRP3 NACHT domain
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: (6S,8R)-N-[(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl]-6-(methylamino)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonamide

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Supramolecule #1: NLRP3 NACHT domain

SupramoleculeName: NLRP3 NACHT domain / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: NACHT domain only
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: NACHT, LRR and PYD domains-containing protein 3

MacromoleculeName: NACHT, LRR and PYD domains-containing protein 3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 63.38034 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: KDYRKKYRKY VRSRFQCIED RNARLGESVS LNKRYTRLRL IKEHRSQQER EQELLAIGKT KTCESPVSPI KMELLFDPDD EHSEPVHTV VFQGAAGIGK TILARKMMLD WASGTLYQDR FDYLFYIHCR EVSLVTQRSL GDLIMSCCPD PNPPIHKIVR K PSRILFLM ...String:
KDYRKKYRKY VRSRFQCIED RNARLGESVS LNKRYTRLRL IKEHRSQQER EQELLAIGKT KTCESPVSPI KMELLFDPDD EHSEPVHTV VFQGAAGIGK TILARKMMLD WASGTLYQDR FDYLFYIHCR EVSLVTQRSL GDLIMSCCPD PNPPIHKIVR K PSRILFLM DGFDELQGAF DEHIGPLCTD WQKAERGDIL LSSLIRKKLL PEASLLITTR PVALEKLQHL LDHPRHVEIL GF SEAKRKE YFFKYFSDEA QARAAFSLIQ ENEVLFTMCF IPLVCWIVCT GLKQQMESGK SLAQTSKTTT AVYVFFLSSL LQP RGGSQE HGLCAHLWGL CSLAADGIWN QKILFEESDL RNHGLQKADV SAFLRMNLFQ KEVDCEKFYS FIHMTFQEFF AAMY YLLEE EKEGRTNVPG SRLKLPSRDV TVLLENYGKF EKGYLIFVVR FLFGLVNQER TSYLEKKLSC KISQQIRLEL LKWIE VKAK AKKLQIQPSQ LELFYCLYEM QEEDFVQRAM DYFPKIEINL STRMDHMVSS FCIENCHRV

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: (6S,8R)-N-[(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl]-6-(me...

MacromoleculeName: (6S,8R)-N-[(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl]-6-(methylamino)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonamide
type: ligand / ID: 4 / Number of copies: 1 / Formula: WTN
Molecular weightTheoretical: 431.509 Da
Chemical component information

ChemComp-WTN:
(6S,8R)-N-[(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl]-6-(methylamino)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonamide

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
0.15 MNaClSodium chloridesodium chloride
20.0 mMTris-HClTrisTris buffer
10.0 %glycerolgycerol
1.0 mMTCEP(tris(2-carboxyethyl)phosphine)

Details: 0.15M NaCl, 20mM Tris pH 7.5, 10% glycerol, 1mM TCEP, 2.5mM ATP, 2mM MgCl2.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 25.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Pressure: 0.007 kPa
Details: Holey gold grids (UltrAuFoil 25 nm R 1.2/1.3) were glow discharged for 20 seconds using a Solarus plasma cleaner (Gatan, Pleasanton, CA, USA), and grids were blotted and plunge frozen in ...Details: Holey gold grids (UltrAuFoil 25 nm R 1.2/1.3) were glow discharged for 20 seconds using a Solarus plasma cleaner (Gatan, Pleasanton, CA, USA), and grids were blotted and plunge frozen in liquid ethane using a Vitrobot (ThermoFisher Scientific, Waltham, MA), operating at 4C, 100 relative humidity, blot force 7, and with a 4 second blot time.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
Detailsmono disperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
SoftwareName: SerialEM
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 3.0 sec. / Average electron dose: 64.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1054244
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: OTHER / Details: cisTEM ab initio
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cisTEM (ver. 2.0)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 3.1) / Details: 94,527 monomers, 68,368 dimers
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 231263
FSC plot (resolution estimation)

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Atomic model buiding 1

SoftwareName: Coot
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8etr:
CryoEM Structure of NLRP3 NACHT domain in complex with G2394

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