Retrieving hits through in silico screening and expert assessment M. N. Drwal a,b and R. Griffith a

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1 Retrieving hits through in silico screening and expert assessment M.. Drwal a,b and R. Griffith a a: School of Medical Sciences/Pharmacology, USW, Sydney, Australia b: Charité Berlin, Germany Abstract: A strategy will be presented involving combination of ligand- and structure-based drug design methods to retrieve structurally novel compounds through virtual screening of large chemical databases. In particular, various pharmacophore methods (ligand-based, complex-based and structure-based) form an integral part of this strategy and will be discussed. It will be shown that the combination of pharmacophores, docking, and expert assessment can be successfully applied in database screening to retrieve structurally novel compounds as topoisomerase I and II inhibitors. [1-3] References: [1] M.. Drwal, et al.: Development of purely structure-based pharmacophores for the topoisomerase I-DA- ligand binding pocket. J Comput-Aided Mol Des, 27, , [2] M.. Drwal, R. Griffith: Combination of Ligand- and Structure-Based Methods in Virtual Screening. Drug Discov Today: Tech, 10, , [3] M.. Drwal, et al.: Exploring DA Topoisomerase I Ligand Space in Search of ovel Anticancer Agents. PLoS E, 6(9), e25150, 2011.

2 Background oncovalent complex Rotation Covalent complex Topoisomerases are validated anticancer drug targets: overexpressed in tumour cells Topo I covalent complex is vulnerable to trapping by agents intercalating between DA base pairs at cleavage site + forming interactions with the protein (poisons) Ternary complex: protein-da-drug Main group of topo I poisons: camptothecin (CPT) derivatives used clinically Topo II enzymes induce DA double-strand breaks Poisons trap DA/topo II complexes Topo II poisons used in the clinic, all exhibit severe side effects: etoposides, anthracyclines (doxorubicin), anthraquinones (mitoxantrone), acridines (amsacrine)

3 Methodology What is a hit and why do we want to retrieve hits? Hits in a drug discovery program: molecules with activity against drug target µm affinity for a receptor or µm inhibitory activity against an enzyme usually retrieved through high throughput screening (HTS) in silico or in vitro The ideal hit: structurally novel, accessible to chemical synthesis and modification drug-like properties promise of selectivity: has been counter-screened against an off-target How do we retrieve such hits? General methodology: stepwise in silico screening of large databases with pharmacophores expert assessment of resulting lists docking of selected molecules selection of a small number of molecules for biological testing

4 Pharmacophores for Discovery and Design of Bioactive Small Molecules Classical or LIGAD-BASED: 3D pattern of features explaining biological activity of a series of small molecules = TRAIIG SET features: hydrogen bond donors/acceptors, hydrophobic groups assumption: same binding mode, same binding site Receptor- or CMPLEX-BASED describe interactions observed in a 3D structure of a complex between a small molecule and its target STRUCTURE-BASED describe potential interactions in the binding pocket Pharmacophores extremely useful: design of new or modified bioactive molecules fast in silico database mining

5 Ligand-Based Pharmacophores for Topo I Inhibitors R R Selection of training set (27 compounds) Definition of CyPi feature Generation of pharmacophore models (Catalyst) R R Camptothecin derivatives IC 50 values from DA topo I cleavage assay, performed by the same lab, spanning 4 orders of magnitude Selection based on structural or functional diversity (if possible) Most topo I inhibitors intercalate into the cleaved DA π-interactions important Software used ( Catalyst, now in Discovery Studio, Accelrys) has no π-interaction feature Extension of aromatic feature definition of CyPi feature Model 1 Model 2 Correlation: 0.96 Stat. significance: 99 % H Correlation: 0.94 Stat. significance: 99 % Features used: HBA, HBD, hydrophobic, CyPi, excluded volumes (max. 5) H

6 Complex-Based Pharmacophores Two X-ray structures of topo I DA camptothecin derivative complexes available and used for identification of ligand interactions camptothecin, PDB: 1t8i topotecan, PDB: 1k4t Generation of pharmacophore: interactions present in both crystal structures used averaged features placement of excluded volumes to mimic the shape of binding pocket placement of additional excluded volume for discrimination between active/inactive stereoisomers of CPT Lys Thr H Arg364 Asp533 Lys532 ligand-protein interactions ligand-da interactions

7 Screening of a Virtual Database, Selection of Hits CI database: compounds Screening with ligand-based pharmacophores: 7175 hits Screening with complex-based pharm. (no excl. vol.): 3474 hits Druglikeness filter: 1763 hits Excl. volumes added to complex-based pharmacophore (mimic shape of pocket): 746 hits Generation 1 compounds 21.0% with confirmed anticancer activity Positive control: 2.7 % camptothecin derivatives Generation 2 compounds 14.6 % with confirmed anticancer activity Positive control: 6.2 % camptothecin derivatives Top 20 of generation 1 and generation 2 hit lists inspected individually, selected according to criteria: not a camptothecin derivative not tested for topo I inhibition dissimilar to compounds already selected 22 compounds selected and docked (round 1) Assessment based on a combination of criteria: docking score comparable to control small number of clusters intercalation between DA bases at cleavage site hydrogen bonds to protein formed 6 compounds eliminated in round 1: failed more than one criterion 9 compounds survived round 2: passed all criteria Requested for biological testing, 7 available

8 Biological Assays - Conclusions DA cleavage assay to determine topo I inhibition (K. Agama a ; Y. Pommier a ) 3 -radiolabelled DA substrates-da-topo I-potential inhibitor incubated denaturation, polyacrylamide gel electrophoresis o direct correlation between activity and docking score or rank in hitlist 5 out of 7 tested compounds active = 71 % hit rate Hit rate difficult to assess: top scoring compounds not available compounds with known activity or similar to camptothecins not tested Structurally novel topoisomerase I inhibitors found, some are also toxic to cancer cell lines ewsflash: unpublished results topo II inhibitors identified using very similar strategies (J. Marinello b ; J. Capranico b ) a: Laboratory of Molecular Pharmacology, Center for Cancer Research, ational Cancer Institute, Bethesda, USA b: Department of Pharmacy and Biotechnology Bologna University, Bologna, Italy