In silico discovery of inhibitors using

structure-based approaches

Jasmita Gill

Structural and Computational Biology Group,

ICGEB, New Delhi

Nov 2005

Target protein 3D structure

Find an inhibitor

Molecular modeling

In silico screening

Computational Techniques

Computational approach

In silico screening

Structure based virtual screening

docking methods to fit putative ligands into 3D structure of target receptor

Structure-based inhibitor discovery

3D structure of target protein Public drug-like in silico libraries

In silico screening

Short listed hits provided for testing in biological assays

Binding site (s) identification

Post-scoring and analysis of results

Literature, Visual analysis

FlexX

Cscore, Visual analysis, Unity

VendorsProtein Data Bank

Sybyl® – Molecular modelling suite

Tools and Techniques

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

FlexX – an overview

Input

OutputEnergetically best ranked ligand placements in target site (s)

Each placement has variable conformations

Target protein with pre-defined active site (s)

and

Ligands with designated base fragment (s)

Thomas Lengauer et. al, J Mol. Bio. 1996

- Multiple conformations determined by torsion angles of

acyclic single bonds in the ligands

- Low energy conformation of the complex is the goal

Considerations in FlexX

Receptor target protein rigid

Ligand Conformational Flexibility

Modeling protein-ligand interactions

Interaction geometries

protein ligand

Interactions types

H-acceptor H-donor

Metal acceptor Metal

Aromatic-ring-atom,

Methyl, amide

Aromatic-ring-center

Main scoring criteria

Free energy of binding of protein-ligand

Consensus scoring ‘Cscore’

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

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 Lipinski’s rule of 5

Mw < 500 daltons (relaxed, <=900) H-bond acceptors < 10 H-bond donors < 5 ClogP (solubility indicator) < 5

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

Final output of screening:

Ranking based on free energy of binding of protein-ligand complex

Visual

Mathematical

Binding sites to which compounds docked

Conformations

H-bonding interactions

Hydrophobic interactions

Van Der Waals attractions

Cscore

Screening results

Analysis

Application to Pfg27

Binding sites of interest on Pfg27

• Two RNA binding sites per dimer

• Four SH3 binding sites per dimer

• A dimer interface

From literature

• Revealed a deep cavity on a unique surface

Visual/computational analysis

RNA binding site

SH3 binding site (N)

Dimer interface

RNA binding site

Deep cavity

Colour coding

Basic

Acidic

Non-polar

Polar

Deep cavity

Depth

Surface

Deepest cavity in Pfg27

Cavities in the dimer interface

Cavities

SH3 binding site

Cavity

Cavities in the SH3 binding site (N)

Cavities in the RNA binding site

Multiple cavities of different depths

NCI-diversity set: 1820 compounds

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

Docking patterns on Pfg27

Visual analysis of top 200

• Best binding energies observed:

from -44.363 KJ/mol to –24.056 KJ/mol

• 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)

• 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

Analysis of top 200 compounds

Dockings in the RNA binding site

Most compounds interact with Arg70, Arg74, Arg78, Arg80 and Val71

Dockings in the deep cavity

Most compounds interact with Ser107, Lys112 and Ile122

Dockings at the dimer interface

Most compounds interact with Asp40, Arg36, Glu134, Arg131, Phe43, Leu126, Trp127

Dockings in SH3 binding sites

Most hits interact with Arg34