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Università degli Studi di Milano Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”. Protein modeling by fragmental approach: connecting global homologies with local peculiarities. Alessandro Pedretti. Molecular docking. Molecular dynamics. Protein modelling. Structure-based studies. - PowerPoint PPT Presentation
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Università degli Studi di MilanoDipartimento di Scienze Farmaceutiche “Pietro Pratesi”
Alessandro Pedretti
Protein modeling by fragmental approach:connecting global homologies
with local peculiarities
Structure-based studies
• In order to perform structure-based studies as:
– ligand optimization;
– virtual screening;
– signal transduction;
– substrate recognition.
the 3D structure of the biological target is required.
• Unluckily, the experimental structure (X-ray diffraction or NMR) is not available for all proteins.
Molecular docking
Molecular dynamics
Protein modelling
What’s the protein modelling ?
• The protein modelling allows to obtain the 3D structure of a protein from its aminoacid sequence (primary structure):
GFGPHQRLEKLDSLLS…
Protein modelling1D structure
3D structure
• It can be classified into two main approaches:
Protein modellingProtein modelling
Comparative modellingComparative modelling
Ab-initio modellingAb-initio modelling
Comparative modelling
• It’s based on the assumption: proteins with high homology of sequence should have similar folding.
Target sequenceTarget sequence
3D template3D template
AlignmentAlignment
Rough 3D modelRough 3D model
3D structure database3D structure database
Homology > 70 %
Structures obtained by experimental approaches (X-ray and NMR).
To refinement workflow
Between target and template
Ab-initio modelling
• It’s based on physical principles and geometric rules obtained by sequence and structure analysis of the 3D experimental models.
Target sequenceTarget sequence
Multiple solutionsMultiple solutions
Global optimizationGlobal optimization
Rough 3D modelRough 3D model
Folding predictionFolding prediction
To refinement workflow
Application of physical and geometric rules
By MM and stochastic approaches
Comparative vs. ab-initio modelling
• The possibility to obtain structural “clones” is very high, submitting whole query sequences of protein families with high homology to a limited number of 3D templates (e.g. transmembrane proteins).
Comparative Ab-initio
3D template Yes No
Success High Low
Computational time Low Very high
Structural “clones”* Yes No
*Models that are structurally similar due to the common template.
Fragmental approach
Target sequenceTarget sequence
Fragmentation in structural domains
Fragmentation in structural domains
Folding prediction of each fragment
Folding prediction of each fragment
Assembling using the global 3D template
Assembling using the global 3D template
Rough modelRough model
Done on the basis of information included indatabases and/or domain finder tools.
Trough multiple comparative modelling procedures.
By geometric superimposition with the 3D structure of the global template, using molecular modelling tools as VEGA ZZ.
To refinement workflow
Model refinement procedure
Missing residuesMissing residues
Side chains addSide chains add
Hydrogens addHydrogens add
Energy minimizationEnergy minimization
Final modelFinal model
Rough modelRough model
VEGA ZZ+
NAMD
Structure checkStructure check
Human 42 nicotinic receptor
• The nicotinic acetylcholine receptors (nAchRs) are composed by five subunits assembled around a central pore permeable to cations.
17 subunit types17 subunit types
1, 1, , 1, 1, , 2-10, 2-42-10, 2-4
MuscleMuscle Nervous systemNervous system
• The therapeutic interest on nicotinic ligands is highlighted by diseases involving the nAchRs as: Alzheimer’s and Parkinson’s disease, autism, epilepsy, schizophrenia, depression, etc.
Human4 subtype
• The complete model didn’t exist.• The design of selective 42 ligands is problematic
due to the low information about the binding mode.
Pedretti A. et Al., Biochemical and Biophysical Research Communications, Vol. 369, 648–53 (2008).
Monomer modeling
Primary structurePrimary structure FragmentationFragmentation
Folding prediction of each fragment
Folding prediction of each fragment
Helices assembly by molecular docking
Helices assembly by molecular docking
Side chainsSide chains
HydrogensHydrogens
MM refinementMM refinement Final monomerFinal monomer
VEGA ZZ
VEGA ZZ + NAMD
ESCHER NG
Fugue
SwissProt
Full assemblyFull assembly
4 transmembrane domains2 cytoplasmic loops1 extracellular loop2 terminal domains
The docking results were filtered the Torpedo Californica nAChR structure.
Complex assembling
+
2x4
3x 2
42
Side view
Top view
Multistep docking:4 + 2 → 422 42 → (4)2(2)2
2 + (4)2(2)2 → (4)2(2)3
ESCHER NG
Model validation
• The soundness of the resulting model was checked docking a set of know nicotinic ligands:
NH
N
Cl
N N
CH3
ON
CH3
N
H
CH3
N
O
NH
N
NH
Nicotine Epibatidine ABT-418 Citisine A-85380
• All these ligands were simulated in their ionized form.
LigandLigand
42 receptor42 receptor
+ DockingDockingBinding site selectionTrp182, Cys225, Cys226 in 4
Binding site selectionTrp182, Cys225, Cys226 in 4 MinimizationMinimization
Final complexFinal complex
VEGA ZZ FRED 2 NAMD
Docking results
• After the final MM minimization, the docking scores were recalculated by Fred 2 (ChemGauss2 scoring function):
CompoundKi
(nM)Score
(Kcal/mol)
Epibatidine 0.009 -48.7
A-85380 0.05 -45.1
Citisine 0.16 -42.6
Nicotine 1.0 -38.9
ABT-418 4.6 -35.9
Cys225 4
Cys226 4
Trp182 4Phe144 2
Asn134 2
Trp82 2
42 – nicotine complex
Human glutamate transporter EAAT1
Pedretti A. et Al., ChemMedChem, Vol. 3, 79-90 (2008).
• L-glutamate is the main excitatory neurotransmitter in the CNS.
Glutamate
Synaptic cleft
Excitatory effects
Axon Dendrite
Metabotropic receptor
Ionotropic receptor
EAAT1-5
• It can also overactivate the ionotropic receptors, inducing a series of destructive processes involved in acute and chronic neurological diseases (e.g. amyotrophic lateral sclerosis, Alzheimer’s disease, epilepsy, CNS ischemia, etc).
EAAT ligand classification
• They can be classified in:
• Natural substrates.
• Substrate inhibitors.
• Non transported uptake blockers.
• The last two classes are interesting because in pathological conditions, when the electrochemical gradient is damaged, EAATs can operate in reverse mode, overactivating the post-synaptic receptors.
Research aims:
• Human EAAT-1 3D structure by homology modeling.
• Pharmacophore models for all ligand classes.
Monomer modeling
Primary structurePrimary structure FragmentationFragmentation
VEGA ZZ
MM refinementMM refinement Final monomerFinal monomerVEGA ZZ + NAMD
Folding prediction of each fragment
Folding prediction of each fragmentFugue
SwissProt
HydrogensHydrogens
Side chainsSide chains
Full assemblyFull assembly
The domains were found aligning the sequences of EAAT1 and glutamate transporter from Pyrococcus horikoshii.
The assembly was carried out using the crystal structure of glutamate transporter homologue from Pyrococcus horikoshii.
Complex assembling
ESCHER NG VEGA ZZ + NAMD
DEEP surface
Monomer
Homotrimer
Complex refinement protocol:• 1 ns of simulation time;• restrained transmembrane segments;• final conjugate gradients minimization.
Docking studies
• Two ligand subsets were docked:
• natural substrates and competitive substrates inhibitors (16);• non-transported blockers (16).
• The following procedure was applied to all ligands:
LigandLigand MinimizationMinimization DockingDocking
EAAT1 monomerEAAT1 monomer
ComplexComplex
Mopac 7 FlexX
• The docking analyses were focused on residues enclosed in a sphere centered on Arg479 (TM4). Mutagenesis studies showed this residue plays a pivotal role in the substrate interaction.
Docking results: substrate inhibitors
pKm = 4.88 (±0.04) – 1.52 (±0.12) Vover
N = 15, r2 = 0.93, s = 0.11, F = 174.11
Where Vover is maximum overlapping volume between the ligand and EAAT1 computed by FlexX.
Gln445
Thr450
Val449Met451
Arg479
Gln204
EAAT1 – (2S, 4R)-methylglutamate complex
Docking results: non-transported blockers
pIC50 = 0.4446(±0.07) – 0.141(±0.02)ScoreFlexX
N = 16, r2 = 0.77, s = 0.55, F = 43.46
Ile468
Trp473
Arg479
Gln204Gln445
Thr450
Val449
Leu448
Ile465
EAAT1 – L-TBOA complex
• Mapping the docking results onto the pharmacophores, it’s possible to highlight the two approaches are successfully overlapped.
Pharmacophore mapping
Natural and substrate inhibitors Non-transported blockersL-glutamate TFB-TBOA
• The two pharmacophore models were obtained by Catalyst 4 software.• Both models highlight the key features required for the interaction.
En = excluded volumeAn = H-bond acceptors
P = ionisable group (positively charged)Y = hydrophobic region
Conclusions
• We obtained the full model of two transmembrane protein through the fragmental approach.
• Performing molecular docking studies, we highlighted the main interaction between ligands and the proteins that were confirmed by experimental data, obtained by mutagenesis studies.
• Although the number of considered ligands isn’t statistically relevant, we obtained good relationships between the docking scores and the experimental data, confirming the soundness of both models.
• All these results show the power and the goodness of the fragmental approach that is able to overcame the problems introduced by global homologies and the possibility to obtain structural clones.
Acknowledgments
www.ddl.unimi.itwww.vegazz.net
• Giulio Vistoli
• Cristina Marconi
• Cristina Sciarrillo
• Laura De Luca