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Cook2010web

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Presented at UWM on March 02, 2010.

Presented at UWM on March 02, 2010.

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  • Homology modeling , also known as comparative modeling of protein refers to constructing an atomic-resolution model of the " target " protein from its amino acid sequence and an experimental three-dimensional structure of a related homologous protein (the " template ").
  • Pyridodiindoles- note the loss in binding when R7 is methyl Phenylpyrazoloquinolinones
  • 3 substituted ethers
  • International Union of Pure and Applied Chemistry
  • Here we are studying how betacarbolines fit in the lipopohilic pocket. The excluded receptor volume was generated by using the MVOL option of SYBYL 5.22 by subtracting the union of the volume of the “actives” from the union of volume of the “inactives”.
  • Introduced 2 descriptors in in 1992. Places the hydrogen bond donor 1.84 A away from Nitrogen and A2 1.84 A away from the indole proton Inverse agonist bccm
  • Antagonist- the earliest fit of a benzo in the developing pharmacophore with an imidazo Nitrogen coordinating with ideal bond lengths at 1.84 A and the carbonyl lone pair adjusted to 135 degrees from ( 120)
  • basis set in chemistry is a set of functions used to create the molecular orbitals
  • Monte Carlo Methods and the Metropolis Criterion Other search methods can be much more efficient for sampling conformational space and determining low-energy structures of a protein, such as stochastic searches involving "importance sampling": Start from an initial conformation, and minimize this structure with respect to the energy. Choose an arbitrary number of torsional angles from the initial conformation, vary them by a random amount, and then minimize this conformation. Use a criterion based on the energy to retain or discard this move (see below). If retained, continue; if discarded, go back to step 1. Compare the resulting structure against all structures previously found. If it does not match any of them, then store this structure as a unique one. Return to Step 2 and continue. Stop the search procedure when no new structures are found. Monte Carlo (MC) and Molecular Dynamics (MD) or Stochastic Dynamics (SD) use distinct methods to generate conformational states. In MC changes are assigned to the system by randomly modifying a few degrees of freedom at a time; the atoms are moved truly randomly, not deterministically. This methods allows for significant thermal fluctuations and sampling of higher energy structures, thus producing a wide spectrum of conformational states economicaly.
  • Scf=tight- rigorous algorithm to a achieve identical population of low energy minimizations. Scf-qc is a less rigorour algorithm used in larger proteins that “ fail to converge”
  • PLS IS USEFUL IN MULTIVARIATE SYSTEMS.
  • BCCM (I), pyridodiindole 3, and 3H-2- phenyl-2,5-dihydropyrazolo[4,3-clquinolin-3-on(e4 1). Red and yellow contours surround regions where a more negative electlostatic interaction is predicted to increase dfinity
  • Red – do not want steric interaction. Green/yellow- steric interaction is good
  • Note the CoMFA does not show that negative electrostatic at carbonyl enhances affinity..This is because all ligands used in the comfa were of this scaffold. YOU CANT SEE DIFFERENCES IF YOUR DATASET IS ALL THE SAME.
  • pyrazoloquinoline ligand CGS-8216 , Diazepam 8-carbolines which effect agonist actions (i. ZK-93423, H1 and H2 are hydrogen bond donating groups indicated by the arrows in the left (yellow) and right (orange) hand portions, respectively. L2 and L3 are areas of lipophilic interaction indicated by the arrows on the top right (redorange) and left (cyan), respectively. S1 and S2 are areas of negative interaction shown in purple.
  • Here we are studying how betacarbolines fit in the lipopohilic pocket. The excluded receptor volume was generated by using the MVOL option of SYBYL 5.22 by subtracting the union of the volume of the “actives” from the union of volume of the “inactives”.
  • Using 166 ligands from 9 different families a binding site was proposed and included volumes were build using ligands with had good binding affinities.
  • Figure 6: Overlap between pairs of included volumes derived from receptor subtype selective ligands: a) a1 and a2 , b) a2 and a3 , c) a4 and a6 , d) a1 and a6, e) a1 and a5 . Yellow color indicates overlapping regions and each grid measures 4 Å in width and height. In order to provide the connection between this figure and other figures, f) shows diazepam and the descriptors of the unified pharmacophore model depicted in the included volume requirement of the a1 subtype.
  • 8-substituted
  • 2.74 kcal/mol higher energy in syn conformation of ester for Ro-4513 ( N=N=N at 8 position , ethyl ester , CO at 5) WHAT IS ENERGY DIFFERENCE IN RING A VERSUS B
  • Develop Pharmacophore Models 􀁦 Create GASP Models 􀁦 Create GALAHAD Models 􀁦 Align Compounds to GALAHAD Models Search Databases 􀁦 Modify 3D Queries 􀁦 Conduct Database Searches
  • A2/a3 selective ligands aligned Perform a receptor surface analysis 3D QSAR
  • serotonin–norepinephrine–dopamine reuptake inhibitor Coding of features: blue sphere, positive ionizable; pink spheres, H-bond acceptor; green spheres, hydrophobic; orange sphere, aromatic….maestro created 3 pharmacophore….merged to one. For the triple reuptake inhibitors. Blockade of hERG K + channels is widely regarded as the predominant cause of Cardiac arrhythmia …. data for a diverse set of 194 potent uncharged hERG actives, we propose a pharmacophore for neutral hERG blockers …..used as a screen…. Pharmacophores for Uncharged Human Ether-a-go-go-Related Gene (hERG) Blockers ring…MOE used to build multiple pharmacophores….then the best pharmacophores with fewest (5) green (hydrophobe/aromatic) and red (hydrogen bond acceptor)
  • Homology modeling , also known as comparative modeling of protein refers to constructing an atomic-resolution model of the " target " protein from its amino acid sequence and an experimental three-dimensional structure of a related homologous protein (the " template ").
  • site directed mutagenesis studies have led to the conclusion that the GABA receptor is between an alpha and beta subunit and the Bz receptor is lied between an alpha and gamma subunit. This has made it difficult to crystalize this pentameric protein complex.
  • Brejc in 2001 reports the crystal structure of homopentamer with a ligand in the binding site. This was verified as the residues implicated with agonist binding in nAChR and AChBP were conserved in the receptor. [site directed mutagenesis]
  • Crystal structure of a single alpha subunit of the acetylcholine binding protein.. This was verified as the residues implicated with agonist binding in nAChR and AChBP were conserved in the receptor. [site directed mutagenesis]
  • . The ligand binding domains of human GABA A receptors (a1, a2, a3, a5, b2, and g2) were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB). Originated in 1971 at Brookhaven National Laboratories, this is the home of the Protein Data Bank.
  • Thread amino acid using iterative magic fit.
  • dihedral angles ψ against φ of amino acid residues in protein structure . [1] It shows the possible conformations of ψ and φ angles for a polypeptide . Root mean square deviation
  • Could our compounds be racemic? H NMR or HPLC to elucidate…DONE BY NMR

Cook2010web Cook2010web Presentation Transcript

  • Computational Chemistry Methods SAR, Pharmacophores & Protein Docking Terry S. Clayton 03/10/10
  • Warning!
  • Outline
    • Part 1
      • SAR
      • QSAR
      • Pharmacophore
      • Today’s Methods
    • Part 2
      • Homology Modeling
    • Part 3
      • Protein Ligand Docking
  • Molecular Databases ChemDraw for Excel ChemDBsoft
  • SAR
  • Betacarbolines
  • Pharmacophore
    • Paul Ehrlich in 1909: "a molecular framework that carries the essential features responsible for a drug’s biological activity"
    • IUPAC: "an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a specific biological target and to trigger (or block) its biological response".
    • Typical features are for where a molecule is hydrophobic, aromatic, a hydrogen bond acceptor, a hydrogen bond donor, a cation, or an anion.
    Ehrlich. Dtsch. Chem. Ges. 1909, 42: p.17 Wermuth, C.G.; Ganellin, C.R.; Lindberg, P.; Mitscher, L.A. (1998). "Glossary of terms used in medicinal chemistry". Pure Appl. Chem.
  • 2D pharmacophore Martin et al. J. Med. Chem. 1992,35,4105-4117
  • Hardware for Modeling
    • Current SYBYL Version: SYBYL 8.1
    • OS: Linux RedHat 4
    • 2 Intel(R) Pentium(R) 4 CPU 3.20GHz, 3200.763, 1024 KB
    • Video Card NVIDIA Corporation
    • NX Machine
  • Excluded volume analysis Allen et al. Journal of Medicinal Chemistry, 1990, Vol. 33, No. 9
  • H1 and A2 Descriptor
  • Ro15-1788
  • 3D QSAR
    • X-ray structures
    • Energy minimizations (6-31G*)
    • Biological data ( IC 50 )
    • Align molecules
    • Perform CoMFA (Sybyl)
  • Molecular Databases ChemDraw for Excel ChemDBsoft
  • MacroModel
    • structures energy minimized using MM2 (molecular mechanics program 2) or MMFF (Merck molecular force field) force fields
    • Monte Carlo conformational searches
  • Gaussian
    • Low energy conformations were optimized via molecular orbital calculations at the 3-21G basis, torsional angles fixed.
    • Further calibration with 6-31G* single point calculations at an “SCF=TIGHT” convergence criteria
  • CoMFA
  • Validation
    • Crossvalidation tests a model by omitting compounds (rows), re-deriving the model, and then predicting the omitted observations
    • r2 > 0.6: The model is fairly good.
    • r2 = 0.4 - 0.6: The model is questionable.
    • r2 < 0.4: The model is poor.
  • Troubleshooting
    • Realign molecules
    • Plot the PLS to see troublesome ligands
    • Reevaluate the conformation
    • Reevaluate the chosen alignment
    • Compound may be too unique for the SAR
  • Electrostatic CoMFA Map
  • Steric CoMFA Map
  • DS electrostatic Map
  •  
  • Prediction Accuracy Huang. Dissertation 1998. UWM
  • Pharmacophore Descriptors
  • BzR Pharmacophore L1
  • Cook’s Unified Pharmacophore Figure III . The pyrazolo [3,4-c]quinolin-3-one ligand CGS-9896 (dotted line), diazepam (thick line), and planar diindoles (thin line) fitted to a schematic representation of the inclusive pharmacophore model for the BzR. The descriptors H1 and H2 designate hydrogen bond donor sites on the receptor protein while A2 represents a hydrogen bond acceptor site necessary for potent inverse agonist activity in vivo . L1, L2, L3 and LDi are four lipophilic regions in the binding
  • Excluded volume analysis Allen et al. Journal of Medicinal Chemistry, 1990, Vol. 33, No. 9
  • Classes of Ligands Employed for the Study of Pharmacophore/ Receptor Models of BzR Subtypes
  • Pharmacophore
    • Descriptors
    • Included Volume
  • Overlay of Included Volume of Pharmacophore/Receptor Model for  and  Receptor Subtypes Orthogonal views of the overlap of the included volumes of the pharmacophore/ receptor models for a1b3g2 (green) and a5b3g2 (red) receptor subtypes(overlap in yellow).
  • L 2 L 2 A 1 A 1 H 1 H 2 H 2 H 1 L Di L Di L 3 L 3 L 1 L 1 L 2 L 2 A 1 A 1 H 1 H 2 H 2 H 1 L Di L Di L 3 L 3 L 1 L 1 L 2 L 2 A 1 A 1 H 1 H 2 H 2 H 1 L Di L Di L 3 L 3 L 1 L 1 L 2 L 2 A 1 A 1 H 1 H 2 H 2 H 1 L Di L Di L 3 L 3 L 1 L 1 L 2 L 2 A 1 A 1 H 1 H 2 H 2 H 1 L Di L Di L 3 L 3 L 1 L 1 ) Orthogonal views of the overlap (yellow) volumes of a1b3g2 (red) and a2b3g2 (green) b) Orthogonal views of the overlap (yellow) volumes of a2b3g2 (red) and a3b3g2 (green) c) Orthogonal views of the overlap (yellow) volumes of a4b3g2 (red) and a6b3g2 (green) d) Orthogonal views of the overlap (yellow) volumes of a1b3g2 (red) and a6b3g2 (green) e) Orthogonal views of the overlap (yellow) volumes of a5b3g2 (red) and a1b3g2 (green) f) Diazepam and the unified pharmacophore descriptors depicted in the included volume of the a1b3g2 subtype.
  • The 8-Phenyl Ligands Aligned in the Included Volume of the Pharmacophore/Receptor Model for the  (blue) and  (red) Subtypes
  • Xli-356
  • R and S Conformation S Conformation R Conformation
  • Today’s Pharmacophore
    • Sybyl
    • Ligands are loaded into a MSS (molecular spreadsheet)
    • DISCOtech performs3D alignment of features that define the active conformation
    • GASP to refine the alignment of the molecules
    • Pharmacophore Model Builder creates a query
  • HipHop Module of Catalyst Viijayan et al. J. Mol. Graphics and Modeling, 27 (2008) 286-298.
  • Examples Micheli et al. J. Med. Chem. , Articles ASAP February 19, 2010 Aronov J. Med. Chem. , 2006, 49 (23), pp 6917–6921 Maestro MOE
  • 3D Database Searching
    • UNITY
      • Applies pharmacophore to compound databases
    • National Cancer Institute (NCI) [260,070]
    • PubChem [26,167,050]
    • CambridgeSoft
    Clark, Expert Opinion on Drug Discovery 2008, Vol. 3, No. 8 : Pages 841-851
  • Homology Modeling
    • template selection
    • target-template (sequence) alignment
    • model construction
    • model assessment
  • GABA A Receptor
  • Cross-section of GABA A Receptor Absolute subunit arrangement of the a1b2g2 GABAA receptor when viewed from the synaptic cleft. The GABA binding sites are located at the b+a- subunit interfaces and the modulatory Bz BS (Bz) is located at the a+g- subunit interface.18, [i] [i] Pritchett, D., Sontheimer, H., Shivers, B., Ymer, S., Kettenmann, H., Schofield, P., and Seeburg, P., Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology, Nature , 1989 , 338, 582-585.
  • ACBP XRAY Brejc et al. Nature 2001 , 411 , 269-276.
  • ACBP  Subunit Belongs to the same LGIC family as GABA A Template for GABA A !!!!
  • AcBP Sequence
    • Data Table 1. Amino Acid Sequence of the alpha subunit of the acetylcholine receptor in the Lymnaea Stagnalis(Great Pond Snail) > sp|P58154|ACHP_LYMST Acetylcholine-binding protein OS=Lymnaea stagnalis PE=1 SV=1 MRRNIFCLACLWIVQACLSLDRADILYNIRQTSRPDVIPTQRDRPVAVSVSLKFINILEV
    • NEITNEVDVVFWQQTTWSDRTLAWNSSHSPDQVSVPISSLWVPDLAAYNAISKPEVLTPQ
    • LARVVSDGEVLYMPSIRQRFSCDVSGVDTESGATCRIKIGSWTHHSREISVDPTTENSDD
    • SEYFSQYSRFEILDVTQKKNSVTYSCCPEAYEDVEVSLNFRKKGRSEIL
    Data Table 3. Amino Acid Sequence of the alpha 1 subunit of the rat GABA A receptor   >sp|P62813|GBRA1_RAT Gamma-aminobutyric acid receptor subunit alpha-1 OS=Rattus norvegicus GN=Gabra1 PE=1 SV=1 MKKSRGLSDYLWAWTLILSTLSGRSYGQPSQDELKDNTTVFTRILDRLLDGYDNRLRPGL GERVTEVKTDIFVTSFGPVSDHDMEYTIDVFFRQSWKDERLKFKGPMTVLRLNNLMASKI WTPDTFFHNGKKSVAHNMTMPNKLLRITEDGTLLYTMRLTVRAECPMHLEDFPMDAHACP LKFGSYAYTRAEVVYEWTREPARSVVVAEDGSRLNQYDLLGQTVDSGIVQSSTGEYVVMT THFHLKRKIGYFVIQTYLPCIMTVILSQVSFWLNRESVPARTVFGVTTVLTMTTLSISAR NSLPKVAYATAMDWFIAVCYAFVFSALIEFATVNYFTKRGYAWDGKSVVPEKPKKVKDPL IKKNNTYAPTATSYTPNLARGDPGLATIAKSATIEPKEVKPETKPPEPKKTFNSVSKIDR LSRIAFPLLFGIFNLVYWATYLNREPQLKAPTPHQ
  • Sequence Alignment
    • ClustalW or Clustal X
      • [http://www.ch.embnet.org/software/ClustalW.html]
    • MAFFT
    • Muscle – built into Swiss
    • Multalin
    • BlastAlign
  • Swiss Model
  • Refinements
    • Loop regions aligned with Loop databases
    • PHD or PREDICT PROTEIN
      • Loop sequences are sent to Columbia University
      • Potential alignments are sent back.. www.rostlab.org
  • Further refinements
    • -hydrophobicity
    • -solvent accessibility of sites- after pentamer assembly.
    • -acidic and basic, and non polar maps
    • -sybyl minimizations
    • -rotamer search to fix steric clashes
    • Ramachandran plot
    • RMSD of alpha carbons
    • Check location of disulfide bond
  • Send to Swiss Server
    • Automated Server
    • 79% of submitted projects are under 3 angstrom off the crystal structure.
    • Force field, conformation, electrostatics
  • GABA Ion Channel
  • GABA ligand docking
  • Protein Binding Site  
  • AutoDock 4.2
    • Prepare PDB files of Protein and Ligand
    • 30 steps
    • Gasteiger (partial )charges are added
    • non-polar hydrogens were merged
    • aromatic carbons were identified
    • rotatable bonds detected, and TORSDOF set
  • Protein
    • Identify Key residues
    • Remove water molecules
    • Add gasteiger charges
    • Create a flexible residue file
    • Choose an algorithm (unique)
  • Define binding site
  • Molecular Modeling
  • Recommendation
    • MOE [www.chemcomp.com] Sybyl [Tripos]
      • workhorse
    • Glide [Schrodinger], Gold or [www.ccdc.com.ac.uk]
      • docking
    • pKa calculator [www.acdlabs.com]
    • Open Eye Package [www.eyesopen.com]
      • High throughput virtual screening
    • GAMESS [www.msg.ameslab.gov/GAMESS]
      • -Quantum Chemistry tool
  • Thank you
  • Benzodiazepine Conformation   Scheme 1. Dynamic Chirality of 1a - c and Stereochemical Cooperativity in 2b (   G    Values Were Determined by 1 H NMR Spectroscopy (Coalescense)) Copyright © 2006 American Chemical Society