COmbined Docking And Similarity Searching

1. CODASS: Using virtual screening to target protein-peptide interactions Douglas R. Houstona, Gillian McCormackb, Gillian Stepekb, Antony Pageband Malcolm D. Walkinshawa aInstituteof Structural and Molecular Biology, University of EdinburghbInstitute of Infection, Immunity and Inflammation, University of Glasgow INTRODUCTION METHODS - CODASS process • COmbined Docking And Similarity Searching • Utilises both major classes of in silico ligand discovery: • Structure-based methods: • Docking • Ligand-based methods: • Topology • Pharmacophoric • Descriptor RECEPTOR • Trichostrongylus gut parasites cause widespread debilitating infections in small ruminants and cattle • Estimated cost of the disease is £84m in the UK sheep farming industry alone • Infections occur via ingestion of infective larvae from contaminated feed or water Parallelised rigid-body docking; screens millions of compounds in hours2 25m Generate conformers LIDAEUS 0.5m Parallelised fast flexible docking 5k Vina 100 Parallelised rigorous flexible docking models electrostatics 100k 5m Autodock Pharmaco-phore search 100k Binding modes are compared - matches are 82% likely to be correct PoseMatch EDULISS • All nematodes are encased in a proteinaceous exoskeleton known as the cuticle • The cuticle must be softened prior to shedding • A battery of proteases work to modify the cuticle collagen • Unsuccessful moulting causes death X-Score + DSX Docked poses are graded using best scoring algorithms currently available Pharmacophore search • CODASS integrates all of these approaches into one workflow • The flexible and modular nature of CODASS means diverse types of target data can be incorporated and utilised in any virtual screen Searchable database of 5m commercially available 3D compounds Visual analysis LIGAND CONFORMATIONS Purchase & test • Metalloproteases have been shown to be essential for cuticle collagen processing and moulting • Of these, C. elegans possesses 40 different astacin metalloproteases belonging to the M12A family • Gene knockout studies have demonstrated lethal cuticle moulting defect phenotypes1 METHODS - CODASS input RESULTS • NAS35 had never been crystallised, therefore a homology model using the structure of crayfish astacin was constructed; 76% similarity between them meant a good quality model was possible • 69 compounds selected from the list of predicted hits were tested • The effects of the compounds on C. elegans were monitored in both liquid and plate growth assays • CODASS can take advantage of all information available, including: • Receptor structure • Ligand structures • Ligand binding conformation • Compound database Knocking out the dpy-31 gene causes the dumpy phenotype; the nematodes cannot remodel their cuticle properly and are stunted • All known astacin inhibitors have one thing in common - zinc coordination • Zinc-coordinating functional groups were specified for the ligand-based methods 500µM actinonin positive control 500µM 6525968 500µM T5513853 • The particular template structure chosen contained a ligand in the active site • The protein was therefore in a correct conformation for inhibitor binding Actinonin is a broad spectrum astacin inhibitor Some compounds elicited cuticle shedding defects, suggesting inhibition of more than one astacin Some compounds weakened the cuticle so much the nematodes ruptured • 38 compounds elicited the “dumpy” phenotype in the more sensitive liquid culture assay • 13 compounds elicited the “dumpy” phenotype in the less sensitive plate assay • Several compounds were at least as potent as actinonin • EDULISS is our in-house chemical database3 • It contains 10m commercially available compounds from multiple suppliers • 5m of these are “lead-like” - they meet the Oprea rules for cLogP, cLogS, rigidity, MW and H-bond donors/acceptors CONCLUSIONS RESULTS Hydroxamic acid • Hits from 3 different classes were found • Ligand-based methods hits included phosphinic and hydroxamate compounds • Structure-based methods produced a more diverse range of chemical families • Both the ligand- and structure-based components of CODASS contributed to successful hit discovery. • CODASS can offer a cost-effective alternative to High-Throughput Screening for inhibitor discovery. • CODASS is applicable to any target protein for which the structure is known. • Further characterisation of the hit compounds is ongoing, including confirmation of activity using in vitro biophysical assays. • Elucidation of structure-activity relationships will enable design of more potent compounds. Phosphinic acid Docking hit example • Summary of virtual screening success rates against a range of targets using our in-house tools: • Average hit rate: 49% • Compare to 1% and 5% “industry standards” for HTS and vHTS, respectively REFERENCES & ACKNOWLEDGMENTS • StepekG, McCormack G, Page AP. Collagen processing and cuticle formation is catalysed by the astacin metalloprotease DPY-31 in free-living and parasitic nematodes. Int J Parasitol. 2010 Apr;40(5):533-42. • Taylor P, Blackburn E, Sheng YG, Harding S, Hsin KY, Kan D, Shave S, Walkinshaw MD. Ligand discovery and virtual screening using the program LIDAEUS. Br J Pharmacol. 2008 Mar;153 Suppl 1:S55-67. Epub 2007 Nov 26. • HsinKY, Morgan HP, Shave SR, Hinton AC, Taylor P, Walkinshaw MD. EDULISS: a small-molecule database with data-mining and pharmacophore searching capabilities. Nucleic Acids Res. 2011 Jan;39(Database issue):D1042-8. • The authors would like to thank Dr. Paul Taylor and Dr. Steven Shave for their assistance.