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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 Computational approach Target protein 3D structure Find an inhibitor Computational Techniques Molecular modeling In silico screening In silico screening Structure based virtual screening docking methods to fit putative ligands into 3D structure of target receptor Structure-based inhibitor discovery Protein Data Bank 3D structure of target protein Vendors Public drug-like in silico libraries Binding site (s) identification In silico screening Literature, Visual analysis FlexX Post-scoring and analysis of results Short listed hits provided for testing in biological assays Cscore, Visual analysis, Unity 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 FlexX – an overview Target protein with pre-defined active site (s) Input and Ligands with designated base fragment (s) Output Energetically best ranked ligand placements in target site (s) Each placement has variable conformations Thomas Lengauer et. al, J Mol. Bio. 1996 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 Modeling protein-ligand interactions Interactions types Interaction geometries H-acceptor H-donor Metal acceptor Metal Aromatic-ringatom, Aromatic-ringcenter Methyl, amide Main scoring criteria Free energy of binding of protein-ligand Consensus scoring ‘Cscore’ protein ligand 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 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 Hydrophobic interactions Van Der Waals attractions Application to Pfg27 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 RNA binding site RNA binding site SH3 binding site (N) Deep cavity Dimer interface Colour coding Basic Acidic Non-polar Polar Depth Deepest cavity in Pfg27 Deep cavity Surface Cavities in the dimer interface Cavities Cavities in the SH3 binding site (N) Cavity SH3 binding site Cavities in the RNA binding site Multiple cavities of different depths 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 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) 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