In silico discovery of inhibitors using

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In silico discovery of inhibitors using
structure-based approaches Jasmita
Gill Structural and Computational Biology Group,
ICGEB, New Delhi Nov 2005
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Computational approach
Target protein 3D structure
Find an inhibitor
Molecular modeling In silico screening
Computational Techniques
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In silico screening
Structure based virtual screening
docking methods to fit putative ligands into 3D
structure of target receptor
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Structure-based inhibitor discovery
Protein Data Bank
Vendors
Public drug-like in silico libraries
3D structure of target protein
Literature, Visual analysis
Binding site (s) identification
FlexX
In silico screening
Post-scoring and analysis of results
Cscore, Visual analysis, Unity
Short listed hits provided for testing in
biological assays
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Tools and Techniques

• Sybyl Molecular modelling suite

Analysis of molecular surfaces of proteins
Preparation of target protein and ligand(s) for
screening Screening utility --
FlexX Post-scoring -- Cscore Data Mining --
Unity

• Public in silico chemical compound libraries used
  

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FlexX an overview
Target protein with pre-defined active site (s)
and Ligands with designated base fragment (s)
Input
Energetically best ranked ligand placements in
target site (s) Each placement has variable
conformations
Output
Thomas Lengauer et. al, J Mol. Bio. 1996
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Considerations in FlexX
Receptor target protein rigid Ligand
Conformational Flexibility

• Multiple conformations determined by torsion
  angles of
• acyclic single bonds in the ligands
• - Low energy conformation of the complex is
  the goal

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Modeling protein-ligand interactions
Interactions types
H-acceptor H-donor
Metal acceptor Metal
Aromatic-ring-atom, Methyl, amide Aromatic-ring-center
Interaction geometries
Main scoring criteria Free energy of binding of
protein-ligand Consensus scoring Cscore
protein
ligand
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Public drug-like in silico libraries

• A database of structures of small molecule
  compounds
• Most libraries are free to download
• Lead-like properties
• Available for purchase

Name No. of Compounds
NCI Diversity set NCI Open Collection 1990 200,000
Maybridge 95,000
Specs 202,000
Peakdale 20,000
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In silico Screening

• Preparation of the target protein structure
• Templates for charged, neutral, non-polar
  residues
• Charges
• Hydrogens

• Preparation of ligand structure
• Charges
• Hydrogens
• Filtering was done based on Lipinskis
  rule of 5
• Mw lt 500 daltons (relaxed,
  lt900)
• H-bond acceptors lt 10
• H-bond donors lt 5
• ClogP (solubility
  indicator) lt 5

• Definition of binding site (s) whole protein
  in case of Pfg27

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Screening results
Final output of screening Ranking based on free
energy of binding of protein-ligand complex
Analysis
Mathematical
Cscore
Visual
Binding sites to which compounds
docked Conformations H-bonding interactions Hydrop
hobic interactions Van Der Waals attractions
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Application to Pfg27
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Binding sites of interest on Pfg27
From literature

• Two RNA binding sites per dimer
• Four SH3 binding sites per dimer
• A dimer interface

Visual/computational analysis

• Revealed a deep cavity on a unique surface

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SH3 binding site
RNA binding site

RNA binding site
SH3 binding site (N)
Deep cavity
Dimer interface
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Colour coding Basic Acidic Non-polar Polar
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Depth
Deepest cavity in Pfg27
Deep cavity
Surface
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Cavities in the dimer interface
Cavities
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Cavities in the SH3 binding site (N)
Cavity
SH3 binding site
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Cavities in the RNA binding site
Multiple cavities of different depths
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NCI-diversity set 1820 compounds
Docking patterns on Pfg27
Visual analysis of top 200
30 in the RNA binding site 30 in the dimer
interface 20 in deep cavity 10 in SH3 binding
site (N) 10 on other sites
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Analysis of top 200 compounds

• Best binding energies observed
• from -44.363 KJ/mol to 24.056 KJ/mol

• Cscore
• 3 to 5 (a good score is 4-5)
• Score of 3 37 compounds
• Score of 4 43 compounds
• Score of 5 48 compounds

• Chemical composition
• Most hits had an electronegative
  character
• N, O-, SO3-, Cl-, F-, Br-
• 
  

• CLogP 3.59 to 1 (-4 to 4 range is acceptable
  for solubility)

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Dockings in the RNA binding site
Most compounds interact with Arg70, Arg74,
Arg78, Arg80 and Val71
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Dockings in the deep cavity
Most compounds interact with Ser107, Lys112 and
Ile122
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Dockings at the dimer interface
Most compounds interact with Asp40, Arg36,
Glu134, Arg131, Phe43, Leu126, Trp127
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Dockings in SH3 binding sites
Most hits interact with Arg34