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- PDB-9axy: Crystal structure of BRAF/MEK complex with NST-628 and inactive RAF -

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Basic information

Entry
Database: PDB / ID: 9axy
TitleCrystal structure of BRAF/MEK complex with NST-628 and inactive RAF
Components
  • Dual specificity mitogen-activated protein kinase kinase 1
  • Serine/threonine-protein kinase B-raf
KeywordsTRANSFERASE / Inhibitor / complex / SIGNALING PROTEIN
Function / homology
Function and homology information


epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / JUN kinase kinase activity / CD4-positive, alpha-beta T cell differentiation / regulation of axon regeneration / mitogen-activated protein kinase kinase / placenta blood vessel development / MAP-kinase scaffold activity / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / labyrinthine layer development ...epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / JUN kinase kinase activity / CD4-positive, alpha-beta T cell differentiation / regulation of axon regeneration / mitogen-activated protein kinase kinase / placenta blood vessel development / MAP-kinase scaffold activity / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / labyrinthine layer development / negative regulation of synaptic vesicle exocytosis / cerebellar cortex formation / type B pancreatic cell proliferation / Signalling to p38 via RIT and RIN / myeloid progenitor cell differentiation / head morphogenesis / ARMS-mediated activation / endothelial cell apoptotic process / Signaling by MAP2K mutants / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / negative regulation of fibroblast migration / positive regulation of D-glucose transmembrane transport / establishment of protein localization to membrane / positive regulation of axonogenesis / regulation of Golgi inheritance / regulation of T cell differentiation / trachea formation / mitogen-activated protein kinase kinase binding / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / regulation of stress-activated MAPK cascade / MAPK3 (ERK1) activation / Frs2-mediated activation / ERBB2-ERBB3 signaling pathway / positive regulation of axon regeneration / stress fiber assembly / endodermal cell differentiation / face development / MAP kinase kinase activity / Bergmann glial cell differentiation / synaptic vesicle exocytosis / Uptake and function of anthrax toxins / thyroid gland development / somatic stem cell population maintenance / protein kinase activator activity / MAP kinase kinase kinase activity / positive regulation of protein serine/threonine kinase activity / postsynaptic modulation of chemical synaptic transmission / negative regulation of endothelial cell apoptotic process / Schwann cell development / keratinocyte differentiation / response to cAMP / positive regulation of peptidyl-serine phosphorylation / positive regulation of stress fiber assembly / positive regulation of substrate adhesion-dependent cell spreading / myelination / ERK1 and ERK2 cascade / protein serine/threonine/tyrosine kinase activity / substrate adhesion-dependent cell spreading / insulin-like growth factor receptor signaling pathway / cellular response to calcium ion / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / thymus development / animal organ morphogenesis / Signal transduction by L1 / cell motility / RAF activation / cellular response to nerve growth factor stimulus / Spry regulation of FGF signaling / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / visual learning / response to peptide hormone / centriolar satellite / long-term synaptic potentiation / epidermal growth factor receptor signaling pathway / neuron differentiation / chemotaxis / Signaling by RAF1 mutants / Negative regulation of MAPK pathway / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / cellular senescence / Signaling by BRAF and RAF1 fusions / MAPK cascade / late endosome / cellular response to xenobiotic stimulus / presynapse / T cell receptor signaling pathway / regulation of cell population proliferation / T cell differentiation in thymus / heart development / cell body / protein tyrosine kinase activity / scaffold protein binding / negative regulation of neuron apoptotic process / eukaryotic translation initiation factor 2alpha kinase activity
Similarity search - Function
: / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / : / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. ...: / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / : / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / C1-like domain superfamily / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Ubiquitin-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
: / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Serine/threonine-protein kinase B-raf / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.6 Å
AuthorsQuade, B. / Huang, X.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cancer Discov / Year: 2024
Title: The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers.
Authors: Meagan B Ryan / Bradley Quade / Natasha Schenk / Zhong Fang / Marshall Zingg / Steven E Cohen / Brooke M Swalm / Chun Li / Ayşegül Özen / Chaoyang Ye / Maria Stella Ritorto / Xin Huang / ...Authors: Meagan B Ryan / Bradley Quade / Natasha Schenk / Zhong Fang / Marshall Zingg / Steven E Cohen / Brooke M Swalm / Chun Li / Ayşegül Özen / Chaoyang Ye / Maria Stella Ritorto / Xin Huang / Arvin C Dar / Yongxin Han / Klaus P Hoeflich / Michael Hale / Margit Hagel /
Abstract: Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the ...Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.
History
DepositionMar 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Serine/threonine-protein kinase B-raf
B: Dual specificity mitogen-activated protein kinase kinase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)76,8957
Polymers75,3462
Non-polymers1,5495
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5750 Å2
ΔGint-39 kcal/mol
Surface area25310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)117.097, 117.097, 129.424
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Space group name HallP312"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+1/3
#3: -x+y,-x,z+2/3
#4: x-y,-y,-z+2/3
#5: -x,-x+y,-z+1/3
#6: y,x,-z

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Components

#1: Protein Serine/threonine-protein kinase B-raf / Proto-oncogene B-Raf / p94 / v-Raf murine sarcoma viral oncogene homolog B1


Mass: 31826.807 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRAF, BRAF1, RAFB1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P15056, non-specific serine/threonine protein kinase
#2: Protein Dual specificity mitogen-activated protein kinase kinase 1 / MAP kinase kinase 1 / MAPKK 1 / MKK1 / ERK activator kinase 1 / MAPK/ERK kinase 1 / MEK 1


Mass: 43518.988 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP2K1, MEK1, PRKMK1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q02750, mitogen-activated protein kinase kinase
#3: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-A1AHE / N-[3-fluoro-4-({7-[(3-fluoropyridin-2-yl)oxy]-4-methyl-2-oxo-2H-1-benzopyran-3-yl}methyl)pyridin-2-yl]-N'-methylsulfuric diamide


Mass: 488.464 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H18F2N4O5S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.4 Å3/Da / Density % sol: 63.82 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 0.2 M Lithium chloride, 0.1 M TRIS pH 8, 20% w/v PEG 8000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL02U1 / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER2 S 9M / Detector: PIXEL / Date: Dec 16, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 3.6→53.34 Å / Num. obs: 12215 / % possible obs: 99.5 % / Redundancy: 12.6 % / Biso Wilson estimate: 85.16 Å2 / Rmerge(I) obs: 0.257 / Net I/σ(I): 8.5
Reflection shellResolution: 3.6→3.94 Å / Rmerge(I) obs: 0.918 / Num. unique obs: 2810

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
REFMAC5refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.6→47.21 Å / SU ML: 0.3801 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 22.2411
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2375 592 4.85 %
Rwork0.1879 11614 -
obs0.1903 12206 99.41 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 84.54 Å2
Refinement stepCycle: LAST / Resolution: 3.6→47.21 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4655 0 98 0 4753
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00314855
X-RAY DIFFRACTIONf_angle_d0.65016565
X-RAY DIFFRACTIONf_chiral_restr0.044718
X-RAY DIFFRACTIONf_plane_restr0.0045827
X-RAY DIFFRACTIONf_dihedral_angle_d9.967658
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.6-3.960.31211370.2512810X-RAY DIFFRACTION98.04
3.96-4.530.24221620.18882867X-RAY DIFFRACTION100
4.54-5.710.23071400.17762917X-RAY DIFFRACTION100
5.72-47.210.21011530.1673020X-RAY DIFFRACTION99.59
Refinement TLS params.Method: refined / Origin x: -52.3876393731 Å / Origin y: 22.7845663519 Å / Origin z: 4.09307028667 Å
111213212223313233
T0.500983411737 Å2-0.0978084271347 Å2-0.0914469790958 Å2-0.407496621825 Å20.0412817516241 Å2--0.44750486462 Å2
L0.671954758028 °20.00933789090019 °2-0.524735175279 °2-0.902006410429 °2-0.0757051212351 °2--1.25136892392 °2
S0.110072846096 Å °-0.0978260456651 Å °-0.0742572634063 Å °0.106508482254 Å °-0.127980508531 Å °-0.152034818033 Å °-0.0076702681622 Å °-0.013269034622 Å °0.00228494568146 Å °
Refinement TLS groupSelection details: all

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