EMDB-57068: Cryo-EM structure of human DHX9 in complex with ATX-968 and ADP.

Basic information

Entry

Database: EMDB / ID: EMD-57068

• Title: Cryo-EM structure of human DHX9 in complex with ATX-968 and ADP.

Sample

• Complex: Human DHX9 bound to ATX-968 and ADP.
  • Protein or peptide: ATP-dependent RNA helicase A
• Ligand: ADENOSINE-5'-DIPHOSPHATE
• Ligand: MAGNESIUM ION
• Ligand: (4M)-N-[3-chloro-5-(methanesulfonamido)phenyl]-4-(3-methylpyridin-2-yl)thiophene-2-carboxamide
• Ligand: water

• Keywords: RNA helicase / RNA BINDING PROTEIN

Function / homology

Function:

single-stranded 3'-5' DNA helicase activity / 3'-5' DNA/RNA helicase activity / regulatory region RNA binding / CRD-mediated mRNA stability complex / positive regulation of RNA export from nucleus / DNA-templated viral transcription / positive regulation of viral transcription / G-quadruplex unwinding activity / regulation of cytoplasmic translation / negative regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / RISC complex binding / CRD-mediated mRNA stabilization / triplex DNA binding / importin-alpha family protein binding / nucleoside triphosphate diphosphatase activity / protein localization to cytoplasmic stress granule / DEx/H-box helicases activate type I IFN and inflammatory cytokines production / alternative mRNA splicing, via spliceosome / positive regulation of interleukin-18 production / nuclear stress granule / perichromatin fibrils / 3'-5' RNA helicase activity / miRNA-mediated post-transcriptional gene silencing / regulation of defense response to virus by host / regulation of mRNA processing / RISC-loading complex / positive regulation of response to cytokine stimulus / RIP-mediated NFkB activation via ZBP1 / RISC complex assembly / siRNA binding / positive regulation of cytoplasmic translation / sequence-specific mRNA binding / RNA polymerase binding / RISC complex / RNA stem-loop binding / positive regulation of innate immune response / cellular response to exogenous dsRNA / positive regulation of interferon-alpha production / RNA polymerase II complex binding / DNA replication origin binding / pyroptotic inflammatory response / 3'-5' DNA helicase activity / mRNA Splicing - Major Pathway / positive regulation of interferon-beta production / positive regulation of fibroblast proliferation / positive regulation of DNA replication / positive regulation of DNA repair / promoter-specific chromatin binding / chromatin DNA binding / positive regulation of interleukin-6 production / osteoblast differentiation / PKR-mediated signaling / transcription coregulator activity / mRNA Polyadenylation / cytoplasmic ribonucleoprotein granule / positive regulation of inflammatory response / actin cytoskeleton / positive regulation of tumor necrosis factor production / double-stranded RNA binding / single-stranded DNA binding / ribonucleoside triphosphate phosphatase activity / ribosome binding / chromatin organization / protein-containing complex assembly / Dengue Virus-Host Interactions / double-stranded DNA binding / RNA polymerase II-specific DNA-binding transcription factor binding / nuclear body / DNA helicase activity / transcription coactivator activity / RNA helicase activity / single-stranded RNA binding / ribonucleoprotein complex / RNA helicase / RNA polymerase II cis-regulatory region sequence-specific DNA binding / mRNA binding / centrosome / regulation of transcription by RNA polymerase II / nucleolus / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / protein-containing complex / DNA binding / RNA binding / nucleoplasm / ATP binding / membrane / nucleus / cytosol / cytoplasm

Domain/homology:

DHX9, first double-stranded RNA binding domain / DHX9, second double-stranded RNA binding domain / DHX9, DEXH-box helicase domain / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase associated domain (HA2), winged-helix / Helicase-associated domain / Helicase associated domain (HA2) Add an annotation / Double-stranded RNA binding motif / Double-stranded RNA binding motif / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase

Component:

ATP-dependent RNA helicase A

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 2.8 Å
• Authors: Hallett ST / Silva STN / Le Bihan Y / Van Montfort R

Funding support

United Kingdom, 1 items

Organization	Grant number	Country
Cancer Research UK	C309/A8274 and C309/A11566	United Kingdom

• Citation: Journal: J Med Chem / Year: 2026; Title: Fragment-Based Discovery of Potent RNA-Competitive Inhibitors of the DEAH-Box RNA Helicase DHX8.; Authors: Benjamin J Read / Caroline Ewens / Federica Gigante / Jemima Thomas / Catarina Felisberto-Rodrigues / Silvia Alvarez Peres / Catherine Tighe / Edgar de Las Heras Ruiz / Kai Schiemann / Andrew G Malcolm / Peter Craig McAndrew / Mark Stubbs / Harshnira Patani / Helena Dos Santos Costa / Katharine Stoodley / Lisa Pickard / Michael Busch / Emma Gunnell / Sara Silva / Alexandra Knopp / Stephen T Hallett / Martin Augustin / Alfred Lammens / Michael Carter / Mirco Meniconi / Marco Ballarotto / Jon Ainsley / Paul Meister / Debarati Sethi / Rosemary Burke / Andrea Scarpino / Yann-Vaï Le Bihan / Ulrich Grädler / Julian Blagg / Paul Workman / Paul A Clarke / Andreas Blum / Christina Esdar / Gurdip Bhalay / Rob L M van Montfort / ; Abstract: Human DHX8 is a spliceosomal DEAH-box RNA helicase involved in releasing mRNA from the spliceosome and crucial in ensuring splicing fidelity. DHX8 was identified as a promising therapeutic oncology target due to its role in regulating stress-adaptive gene expression, including HSF1-dependent transcription, while having broader transcriptional effects in cells under oncogenic stress. We report the discovery of novel RNA-competitive DHX8 inhibitors based on a 2-(phenethylthio)nicotinic acid scaffold, which were optimized using a structure-guided design approach, following a biophysical fragment screen. This yielded compound 53 with nanomolar biochemical potency, good in vitro PK, and activity in a cellular target engagement assay. Optimizing inhibitor binding between Arg647 and the nonconserved His693, coupled with extending into a pocket in the DHX8 Winged-Helix domain, was crucial for potency improvement. By binding in the Winged-Helix domain, these inhibitors restrict the helicase domain's conformational plasticity, stabilizing a closed, inactive conformation while sterically blocking ssRNA translocation.

History

Deposition	Mar 5, 2026	-
Header (metadata) release	Oct 14, 2026	-
Map release	Oct 14, 2026	-
Update	Oct 14, 2026	-
Current status	Oct 14, 2026	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_57068.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 1.017 Å

Density

Contour Level	By AUTHOR: 0.012
Minimum - Maximum	-0.06187148 - 0.094118185
Average (Standard dev.)	0.00000575866 (±0.0015841502)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 260.352 Å α=β=γ: 90.0 °

Supplemental data

Half map: #2

• File: emd_57068_half_map_1.map

Half map: #1

• File: emd_57068_half_map_2.map

Sample components

Entire : Human DHX9 bound to ATX-968 and ADP.

• Entire: Name: Human DHX9 bound to ATX-968 and ADP.

Components

• Complex: Human DHX9 bound to ATX-968 and ADP.
  • Protein or peptide: ATP-dependent RNA helicase A
• Ligand: ADENOSINE-5'-DIPHOSPHATE
• Ligand: MAGNESIUM ION
• Ligand: (4M)-N-[3-chloro-5-(methanesulfonamido)phenyl]-4-(3-methylpyridin-2-yl)thiophene-2-carboxamide
• Ligand: water

Supramolecule #1: Human DHX9 bound to ATX-968 and ADP.

• Supramolecule: Name: Human DHX9 bound to ATX-968 and ADP. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: ATP-dependent RNA helicase A

• Macromolecule: Name: ATP-dependent RNA helicase A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 114.340164 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

String:

MSRKEEQEVQ ATLESEEVDL NAGLHGNWTL ENAKARLNQY FQKEKIQGEY KYTQVGPDHN RSFIAEMTIY IKQLGRRIFA REHGSNKKL AAQSCALSLV RQLYHLGVVE AYSGLTKKKE GETVEPYKVN LSQDLEHQLQ NIIQELNLEI LPPPEDPSVP V ALNIGKLA QFEPSQRQNQ VGVVPWSPPQ SNWNPWTSSN IDEGPLAFAT PEQISMDLKN ELMYQLEQDH DLQAILQERE LL PVKKFES EILEAISQNS VVIIRGATGC GKTTQVPQFI LDDFIQNDRA AECNIVVTQP RRISAVSVAE RVAFERGEEP GKS CGYSVR FESILPRPHA SIMFCTVGVL LRKLEAGIRG ISHVIVDEIH ERDINTDFLL VVLRDVVQAY PEVRIVLMSA TIDT SMFCE YFFNCPIIEV YGRTYPVQEY FLEDCIQMTH FVPPPKDKKK KDKDDDGGED DDANCNLICG DEYGPETRLS MSQLN EKET PFELIEALLK YIETLNVPGA VLVFLPGWNL IYTMQKHLEM NPHFGSHRYQ ILPLHSQIPR EEQRKVFDPV PVGVTK VIL STNIAETSIT INDVVYVIDS CKQKVKLFTA HNNMTNYATV WASKTNLEQR KGRAGRVRPG FCFHLCSRAR FERLETH MT PEMFRTPLHE IALSIKLLRL GGIGQFLAKA IEPPPLDAVI EAEHTLRELD ALDANDELTP LGRILAKLPI EPRFGKMM I MGCIFYVGDA ICTIAAATCF PEPFINEGKR LGYIHRNFAG NRFSDHVALL SVFQAWDDAR MGGEEAEIRF CEHKRLNMA TLRMTWEAKV QLKEILINSG FPEDCLLTQV FTNTGPDNNL DVVISLLAFG VYPNVCYHKE KRKILTTEGR NALIHKSSVN CPFSSQDMK YPSPFFVFGE KIRTRAISAK GMTLVTPLQL LLFASKKVQS DGQIVLVDDW IKLQISHEAA ACITGLRAAM E ALVVEVTK QPAIISQLDP VNERMLNMIR QISRPSAAGI NLMIDYKDDD DK

UniProtKB: ATP-dependent RNA helicase A

Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: ADP
• Molecular weight: Theoretical: 427.201 Da

Chemical component information

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

Macromolecule #3: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Macromolecule #4: (4M)-N-[3-chloro-5-(methanesulfonamido)phenyl]-4-(3-methylpyridin...

• Macromolecule: Name: (4M)-N-[3-chloro-5-(methanesulfonamido)phenyl]-4-(3-methylpyridin-2-yl)thiophene-2-carboxamide; type: ligand / ID: 4 / Number of copies: 1 / Formula: A1BKX
• Molecular weight: Theoretical: 421.921 Da

Macromolecule #5: water

• Macromolecule: Name: water / type: ligand / ID: 5 / Number of copies: 22 / Formula: HOH
• Molecular weight: Theoretical: 18.015 Da

Chemical component information

ChemComp-HOH:
WATER

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.5 mg/mL
• Buffer: pH: 7.5 / Details: 20 mM HEPES pH 7.5, 300 mM NaCl, 2 mM TCEP
• Grid: Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 50 sec. / Pretreatment - Atmosphere: AIR / Details: 15 W
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV; Details: Sample was centrifuged at 21,000 x g for 10 minutes at 6 degrees Celsius and diluted to 0.5 mg/mL (4.34 micromolar) just before grid preparation. 3 microlitres of the sample was applied to the grid, blotted for 3 seconds with a blot force of 3, and immediately flash frozen..
• Details: Sample was fairly monodisperse.

Electron microscopy

• Microscope: TFS KRIOS
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV; Details: Due to potential preferred particle orientation additional movies were collected from the same grid at a tilt of 30 degrees.
• Details: Preliminary grid screening was performed in a Glacios.
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 35037 / Average exposure time: 1.55 sec. / Average electron dose: 60.0 e/Ų; Details: Images collected in movie mode (60 frames per movie).
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 165000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• CTF correction: Software - Name: CTFFIND (ver. 4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
• Startup model: Type of model: OTHER; Details: Used internally produced model, obtained by X-ray crystallography, as starting model.
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0) / Number images used: 985972
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0)
• Final 3D classification: Software - Name: RELION (ver. 5.0); Details: Particles from CryoSPARC were re-extracted in RELION using a box size of 256 x 256 pixels with no downscaling. 3D auto-refinement was performed followed by per particle CTF refinement, another round of 3D auto-refinement and then Bayesian polishing. During polishing particles were extracted in a box size of 512 x 512 pixels downscaled to 256 x 256 pixels to maintain a pixel size of 1.017 Angstrom.

Atomic model buiding 1

• Initial model: Chain - Source name: Other / Chain - Initial model type: experimental model / Details: Model produced in-house by X-ray crystallography.
• Details: Initial fit was done in Chimera, followed by chain adjustments in Coot and refinement in Phenix.
• Refinement: Space: REAL / Protocol: OTHER / Overall B value: 64.98

Output model

PDB-29by:
Cryo-EM structure of human DHX9 in complex with ATX-968 and ADP.