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- PDB-9ay7: Crystal structure of CRAF/MEK1 complex with NST-628 and inactive RAF -

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Basic information

Entry
Database: PDB / ID: 9ay7
TitleCrystal structure of CRAF/MEK1 complex with NST-628 and inactive RAF
Components
  • Dual specificity mitogen-activated protein kinase kinase 1
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsTRANSFERASE / Inhibitor / complex / SIGNALING PROTEIN
Function / homology
Function and homology information


death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / JUN kinase kinase activity / regulation of axon regeneration / mitogen-activated protein kinase kinase / placenta blood vessel development / MAP-kinase scaffold activity / labyrinthine layer development ...death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / JUN kinase kinase activity / regulation of axon regeneration / mitogen-activated protein kinase kinase / placenta blood vessel development / MAP-kinase scaffold activity / labyrinthine layer development / cerebellar cortex formation / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / Signaling by MAP2K mutants / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / positive regulation of axonogenesis / insulin secretion involved in cellular response to glucose stimulus / regulation of Golgi inheritance / trachea formation / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / regulation of stress-activated MAPK cascade / IFNG signaling activates MAPKs / MAPK3 (ERK1) activation / GP1b-IX-V activation signalling / Frs2-mediated activation / ERBB2-ERBB3 signaling pathway / endodermal cell differentiation / face development / MAP kinase kinase activity / pseudopodium / neurotrophin TRK receptor signaling pathway / Bergmann glial cell differentiation / regulation of cell differentiation / Uptake and function of anthrax toxins / thyroid gland development / somatic stem cell population maintenance / protein kinase activator activity / extrinsic apoptotic signaling pathway via death domain receptors / MAP kinase kinase kinase activity / type II interferon-mediated signaling pathway / positive regulation of protein serine/threonine kinase activity / negative regulation of protein-containing complex assembly / Schwann cell development / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / keratinocyte differentiation / activation of adenylate cyclase activity / positive regulation of peptidyl-serine phosphorylation / response to muscle stretch / myelination / ERK1 and ERK2 cascade / CD209 (DC-SIGN) signaling / protein serine/threonine/tyrosine kinase activity / insulin-like growth factor receptor signaling pathway / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / thymus development / Signal transduction by L1 / cell motility / RAF activation / wound healing / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / Stimuli-sensing channels / 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 / insulin receptor signaling pathway / heart development / protein tyrosine kinase activity / scaffold protein binding / regulation of apoptotic process / mitochondrial outer membrane / eukaryotic translation initiation factor 2alpha kinase activity / early endosome / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / Rho-dependent protein serine/threonine kinase activity / histone H2BS36 kinase activity / histone H3S57 kinase activity / histone H2AT120 kinase activity / AMP-activated protein 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 / 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
: / ACETATE ION / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / NICKEL (II) ION / RAF proto-oncogene serine/threonine-protein kinase / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.41 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 7, 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

Downloads & links

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Assembly

Deposited unit
A: RAF proto-oncogene serine/threonine-protein kinase
B: Dual specificity mitogen-activated protein kinase kinase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)77,46014
Polymers75,4952
Non-polymers1,96512
Water3,135174
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6990 Å2
ΔGint-49 kcal/mol
Surface area23960 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.030, 107.950, 116.240
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein RAF proto-oncogene serine/threonine-protein kinase / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 31975.775 Da / Num. of mol.: 1 / Mutation: Y340D Y341D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Production host: Homo sapiens (human)
References: UniProt: P04049, 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 / Mutation: S218A S222A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP2K1, MEK1, PRKMK1 / Production host: Homo sapiens (human)
References: UniProt: Q02750, mitogen-activated protein kinase kinase

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Non-polymers , 7 types, 186 molecules

#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-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ni
#6: Chemical
ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C2H3O2
#7: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#8: 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
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 174 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.49 Å3/Da / Density % sol: 50.69 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 8 / Details: 0.2M sodium acetate pH 8.0, 20% w/v PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL45XU / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: May 25, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.41→48.95 Å / Num. obs: 29864 / % possible obs: 100 % / Redundancy: 13.3 % / Biso Wilson estimate: 49.48 Å2 / Rmerge(I) obs: 0.112 / Net I/σ(I): 15.8
Reflection shellResolution: 2.41→2.5 Å / Rmerge(I) obs: 1.148 / Num. unique obs: 2786

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
REFMAC5refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.41→48.95 Å / SU ML: 0.2948 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 24.6551
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2359 1458 4.88 %
Rwork0.2012 28404 -
obs0.2029 29862 99.9 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 58.5 Å2
Refinement stepCycle: LAST / Resolution: 2.41→48.95 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4449 0 123 174 4746
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00174666
X-RAY DIFFRACTIONf_angle_d0.43596326
X-RAY DIFFRACTIONf_chiral_restr0.0406708
X-RAY DIFFRACTIONf_plane_restr0.0038803
X-RAY DIFFRACTIONf_dihedral_angle_d14.34661681
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.41-2.50.26481450.25312786X-RAY DIFFRACTION99.69
2.5-2.60.34391300.26432772X-RAY DIFFRACTION99.83
2.6-2.710.33241510.24722806X-RAY DIFFRACTION99.83
2.71-2.860.34771240.23482841X-RAY DIFFRACTION99.93
2.86-3.040.25091470.23052800X-RAY DIFFRACTION99.93
3.04-3.270.28831300.24472831X-RAY DIFFRACTION99.9
3.27-3.60.25841630.20622824X-RAY DIFFRACTION100
3.6-4.120.19391610.18412836X-RAY DIFFRACTION99.93
4.12-5.190.18361630.15822862X-RAY DIFFRACTION100
5.19-48.950.23211440.1923046X-RAY DIFFRACTION99.91
Refinement TLS params.Method: refined / Origin x: -17.5524925115 Å / Origin y: 1.37446586767 Å / Origin z: -22.9292303617 Å
111213212223313233
T0.333718760208 Å20.00312468701454 Å2-0.0591761575368 Å2-0.384538651809 Å2-0.0454542533311 Å2--0.361897271103 Å2
L0.517926656466 °20.488215751231 °2-0.170984302136 °2-1.67116128224 °2-0.470424321678 °2--0.644519300977 °2
S0.0604136394723 Å °-0.1120695595 Å °0.0362191058286 Å °0.108995845345 Å °-0.163627815825 Å °0.00678417936498 Å °-0.0676164874781 Å °0.0737388832891 Å °0.0772170099863 Å °
Refinement TLS groupSelection details: all

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