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- EMDB-47889: Cryo-EM structure of USP1-UAF1-Ubiquitin in complex with TNG348 -

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Basic information

Entry
Database: EMDB / ID: EMD-47889
TitleCryo-EM structure of USP1-UAF1-Ubiquitin in complex with TNG348
Map datafull map
Sample
  • Complex: USP1-UAF1-Ub complex with TNG348
    • Protein or peptide: Ubiquitin
    • Protein or peptide: WD repeat-containing protein 48
    • Protein or peptide: Ubiquitin carboxyl-terminal hydrolase 1
  • Ligand: 3-(methanesulfonyl)propan-1-amine
  • Ligand: ZINC ION
  • Ligand: 2-(4-cyclopropyl-6-methoxypyrimidin-5-yl)-9-({4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenyl}methyl)-7-(2,2,2-trifluoroethyl)-7,9-dihydro-8H-purin-8-imine
Keywordsdeubiquitination / PCNA / allosteric inhibitor / HYDROLASE
Function / homology
Function and homology information


regulation of protein monoubiquitination / positive regulation of error-prone translesion synthesis / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / monoubiquitinated protein deubiquitination / seminiferous tubule development / deubiquitinase activator activity / symbiont entry into host cell via disruption of host cell glycocalyx / skeletal system morphogenesis / symbiont entry into host cell via disruption of host cell envelope / skin development ...regulation of protein monoubiquitination / positive regulation of error-prone translesion synthesis / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / monoubiquitinated protein deubiquitination / seminiferous tubule development / deubiquitinase activator activity / symbiont entry into host cell via disruption of host cell glycocalyx / skeletal system morphogenesis / symbiont entry into host cell via disruption of host cell envelope / skin development / virus tail / homeostasis of number of cells / protein deubiquitination / embryonic organ development / single fertilization / positive regulation of double-strand break repair via homologous recombination / response to UV / regulation of DNA repair / Fanconi Anemia Pathway / ubiquitin binding / Recognition of DNA damage by PCNA-containing replication complex / positive regulation of epithelial cell proliferation / skeletal system development / double-strand break repair via homologous recombination / positive regulation of receptor signaling pathway via JAK-STAT / regulation of protein stability / multicellular organism growth / late endosome / peptidase activity / single-stranded DNA binding / double-stranded DNA binding / spermatogenesis / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / lysosome / Ub-specific processing proteases / cysteine-type endopeptidase activity / DNA repair / intracellular membrane-bounded organelle / DNA damage response / proteolysis / DNA binding / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / Domain of unknown function (DUF3337) / : / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase ...WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / Domain of unknown function (DUF3337) / : / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase / Ubiquitin specific protease domain / Ubiquitin specific protease (USP) domain profile. / Pectate lyase superfamily protein / Rhamnogalacturonase A/epimerase, pectate lyase-like / Pectin lyase fold / Pectin lyase fold/virulence factor / Papain-like cysteine peptidase superfamily / WD domain, G-beta repeat / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Ubiquitin carboxyl-terminal hydrolase 1 / Tail fiber / WD repeat-containing protein 48
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWhittington DA
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Mol Cancer Ther / Year: 2025
Title: Characterization of TNG348: A Selective, Allosteric USP1 Inhibitor That Synergizes with PARP Inhibitors in Tumors with Homologous Recombination Deficiency.
Authors: Antoine Simoneau / Charlotte B Pratt / Hsin-Jung Wu / Shreya S Rajeswaran / Charlotte Grace Comer / Sirimas Sudsakorn / Wenhai Zhang / Shangtao Liu / Samuel R Meier / Ashley H Choi / Tenzing ...Authors: Antoine Simoneau / Charlotte B Pratt / Hsin-Jung Wu / Shreya S Rajeswaran / Charlotte Grace Comer / Sirimas Sudsakorn / Wenhai Zhang / Shangtao Liu / Samuel R Meier / Ashley H Choi / Tenzing Khendu / Hannah Stowe / Binzhang Shen / Douglas A Whittington / Yingnan Chen / Yi Yu / William D Mallender / Tianshu Feng / Jannik N Andersen / John P Maxwell / Scott Throner /
Abstract: Inhibition of the deubiquitinating enzyme USP1 can induce synthetic lethality in tumors characterized by homologous recombination deficiency (HRD) and represents a novel therapeutic strategy for the ...Inhibition of the deubiquitinating enzyme USP1 can induce synthetic lethality in tumors characterized by homologous recombination deficiency (HRD) and represents a novel therapeutic strategy for the treatment of BRCA1/2-mutant cancers, potentially including patients whose tumors have primary or acquired resistance to PARP inhibitors (PARPi). In this study, we present a comprehensive characterization of TNG348, an allosteric, selective, and reversible inhibitor of USP1. TNG348 induces dose-dependent accumulation of ubiquitinated protein substrates both in vitro and in vivo. CRISPR screens show that TNG348 exerts its antitumor effect by disrupting the translesion synthesis pathway of DNA damage tolerance through RAD18-dependent ubiquitinated PCNA. Although TNG348 and PARPi share the ability to selectively kill HRD tumor cells, CRISPR screens reveal that TNG348 and PARPi do so through discrete mechanisms. Particularly, knocking out PARP1 causes resistance to PARPi but sensitizes cells to TNG348 treatment. Consistent with these findings, combination of TNG348 with PARPi leads to synergistic antitumor effects in HRD tumors, resulting in tumor growth inhibition and regression in multiple mouse xenograft tumor models. Importantly, our data on human cancer models further show that the addition of TNG348 to PARPi treatment can overcome acquired PARPi resistance in vivo. Although the clinical development of TNG348 has been discontinued because of unexpected liver toxicity in patients (NCT06065059), the present data provide preclinical and mechanistic support for the continued exploration of USP1 as a drug target for the treatment of patients with BRCA1/2-mutant or HRD cancers.
History
DepositionNov 13, 2024-
Header (metadata) releaseJan 29, 2025-
Map releaseJan 29, 2025-
UpdateSep 17, 2025-
Current statusSep 17, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47889.png

Downloads & links

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Map

FileDownload / File: emd_47889.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map
Projections & slices

Image control

Size
Brightness
Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 300 pix.
= 253.8 Å		0.85 Å/pix.
x 300 pix.
= 253.8 Å		0.85 Å/pix.
x 300 pix.
= 253.8 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.846 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-2.1330411 - 3.3903952
Average (Standard dev.)-0.0010856558 (±0.0727039)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 253.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47889_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Half map: half-map A

Fileemd_47889_half_map_1.map
Annotationhalf-map A
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map B

Fileemd_47889_half_map_2.map
Annotationhalf-map B
Projections & Slices
AxesZYX

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Sample components

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Entire : USP1-UAF1-Ub complex with TNG348

EntireName: USP1-UAF1-Ub complex with TNG348
Components
  • Complex: USP1-UAF1-Ub complex with TNG348
    • Protein or peptide: Ubiquitin
    • Protein or peptide: WD repeat-containing protein 48
    • Protein or peptide: Ubiquitin carboxyl-terminal hydrolase 1
  • Ligand: 3-(methanesulfonyl)propan-1-amine
  • Ligand: ZINC ION
  • Ligand: 2-(4-cyclopropyl-6-methoxypyrimidin-5-yl)-9-({4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenyl}methyl)-7-(2,2,2-trifluoroethyl)-7,9-dihydro-8H-purin-8-imine

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Supramolecule #1: USP1-UAF1-Ub complex with TNG348

SupramoleculeName: USP1-UAF1-Ub complex with TNG348 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: USP1-UAF1 co-expressed and purified; Ub-VS then used to covalently modify USP1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 174 KDa

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Macromolecule #1: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 1 / Details: vinyl sulfone modification at C-terminus / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.519778 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRG

UniProtKB: Tail fiber

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Macromolecule #2: WD repeat-containing protein 48

MacromoleculeName: WD repeat-containing protein 48 / type: protein_or_peptide / ID: 2 / Details: C-terminal Flag tag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 77.309359 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAAHHRQNTA GRRKVQVSYV IRDEVEKYNR NGVNALQLDP ALNRLFTAGR DSIIRIWSVN QHKQDPYIAS MEHHTDWVND IVLCCNGKT LISASSDTTV KVWNAHKGFC MSTLRTHKDY VKALAYAKDK ELVASAGLDR QIFLWDVNTL TALTASNNTV T TSSLSGNK ...String:
MAAHHRQNTA GRRKVQVSYV IRDEVEKYNR NGVNALQLDP ALNRLFTAGR DSIIRIWSVN QHKQDPYIAS MEHHTDWVND IVLCCNGKT LISASSDTTV KVWNAHKGFC MSTLRTHKDY VKALAYAKDK ELVASAGLDR QIFLWDVNTL TALTASNNTV T TSSLSGNK DSIYSLAMNQ LGTIIVSGST EKVLRVWDPR TCAKLMKLKG HTDNVKALLL NRDGTQCLSG SSDGTIRLWS LG QQRCIAT YRVHDEGVWA LQVNDAFTHV YSGGRDRKIY CTDLRNPDIR VLICEEKAPV LKMELDRSAD PPPAIWVATT KST VNKWTL KGIHNFRASG DYDNDCTNPI TPLCTQPDQV IKGGASIIQC HILNDKRHIL TKDTNNNVAY WDVLKACKVE DLGK VDFED EIKKRFKMVY VPNWFSVDLK TGMLTITLDE SDCFAAWVSA KDAGFSSPDG SDPKLNLGGL LLQALLEYWP RTHVN PMDE EENEVNHVNG EQENRVQKGN GYFQVPPHTP VIFGEAGGRT LFRLLCRDSG GETESMLLNE TVPQWVIDIT VDKNMP KFN KIPFYLQPHA SSGAKTLKKD RLSASDMLQV RKVMEHVYEK IINLDNESQT TSSSNNEKPG EQEKEEDIAV LAEEKIE LL CQDQVLDPNM DLRTVKHFIW KSGGDLTLHY RQKSTDYKDD DDK

UniProtKB: WD repeat-containing protein 48

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Macromolecule #3: Ubiquitin carboxyl-terminal hydrolase 1

MacromoleculeName: Ubiquitin carboxyl-terminal hydrolase 1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: ubiquitinyl hydrolase 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 88.406344 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GMPGVIPSES NGLSRGSPSK KNRLSLKFFQ KKETKRALDF TDSQENEEKA SEYRASEIDQ VVPAAQSSPI NCEKRENLLP FVGLNNLGN TCYLNSILQV LYFCPGFKSG VKHLFNIISR KKEALKDEAN QKDKGNCKED SLASYELICS LQSLIISVEQ L QASFLLNP ...String:
GMPGVIPSES NGLSRGSPSK KNRLSLKFFQ KKETKRALDF TDSQENEEKA SEYRASEIDQ VVPAAQSSPI NCEKRENLLP FVGLNNLGN TCYLNSILQV LYFCPGFKSG VKHLFNIISR KKEALKDEAN QKDKGNCKED SLASYELICS LQSLIISVEQ L QASFLLNP EKYTDELATQ PRRLLNTLRE LNPMYEGYLQ HDAQEVLQCI LGNIQETCQL LKKEEVKNVA ELPTKVEEIP HP KEEMNGI NSIEMDSMRH SEDFKEKLPK GNGKRKSDTE FGNMKKKVKL SKEHQSLEEN QRQTRSKRKA TSDTLESPPK IIP KYISEN ESPRPSQKKS RVKINWLKSA TKQPSILSKF CSLGKITTNQ GVKGQSKENE CDPEEDLGKC ESDNTTNGCG LESP GNTVT PVNVNEVKPI NKGEEQIGFE LVEKLFQGQL VLRTRCLECE SLTERREDFQ DISVPVQEDE LSKVEESSEI SPEPK TEMK TLRWAISQFA SVERIVGEDK YFCENCHHYT EAERSLLFDK MPEVITIHLK CFAASGLEFD CYGGGLSKIN TPLLTP LKL SLEEWSTKPT NDSYGLFAVV MHSGITISSG HYTASVKVTD LNSLELDKGN FVVDQMCEIG KPEPLNEEEA RGVVENY ND EEVSIRVGGN TQPSKVLNKK NVEAIGLLAA QKSKADYELY NKASNPDKVA STAFAENRNS ETSDTTGTHE SDRNKESS D QTGINISGFE NKISYVVQSL KEYEGKWLLF DDSEVKVTEE KDFLNSLSPS TSPTSTPYLL FYKKL

UniProtKB: Ubiquitin carboxyl-terminal hydrolase 1

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Macromolecule #4: 3-(methanesulfonyl)propan-1-amine

MacromoleculeName: 3-(methanesulfonyl)propan-1-amine / type: ligand / ID: 4 / Number of copies: 1 / Formula: A1A4Y
Molecular weightTheoretical: 137.201 Da

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: 2-(4-cyclopropyl-6-methoxypyrimidin-5-yl)-9-({4-[1-methyl-4-(trif...

MacromoleculeName: 2-(4-cyclopropyl-6-methoxypyrimidin-5-yl)-9-({4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenyl}methyl)-7-(2,2,2-trifluoroethyl)-7,9-dihydro-8H-purin-8-imine
type: ligand / ID: 6 / Number of copies: 1 / Formula: A1BHF
Molecular weightTheoretical: 603.522 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

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
50.0 mMHEPES
150.0 mMsodium chloride
5.0 %glycerol
GridMaterial: GOLD
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 6175 / Average exposure time: 2.5 sec. / Average electron dose: 51.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3700674
CTF correctionSoftware - Name: cryoSPARC (ver. v3.3) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5cvo

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3) / Number images used: 151407
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.3)
Final 3D classificationSoftware - Name: cryoSPARC (ver. v3.3)

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Atomic model buiding 1

Initial model
PDB IDChain

5cvo

chain_id: A, source_name: PDB, initial_model_type: experimental model

7ay2

chain_id: B, source_name: PDB, initial_model_type: experimental model

7ay2

chain_id: C, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9ebs:
Cryo-EM structure of USP1-UAF1-Ubiquitin in complex with TNG348

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