Use and development of chem-bioinformatics tools and methods for drug discovery and target identification INAUGURALDISSERTATION zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Hitesh Patel aus Ahmedabad, India 2014 Vorsitzender des Promotionsausschusses: Prof. Dr. Rolf Schubert Referent: Prof. Dr. Irmgard Merfort Korreferent: Jun.-Prof. Dr. Stefan Günther Drittprüfer: Prof. Dr. Andreas Bechthold Datum der mündlichen Prüfung: 04.11.2014 ii Parts of this work have been published in the following conference presentations and publications: Publications Patel H., Grüning B. A., Merfort I. and Günther S. PyWATER: A PyMOL plugin to find conserved water molecules in protein by clustering. Bioinformatics (Oxford, England), 30(20), 2978–80, 2014 Patel H., Lucas X., Günther S. and Merfort I. Target fishing by cross-docking for explaining polypharmacological effects. (Under preparation) Patel H., Grüning B. A., Merfort I. and Günther S. RECAP Fragmenter-Merger: Exploring the chemical space by knowledge based assembling of fragments. (Under preparation) Grüning B. A., Lucas X., Kuster G., Patel H., Chbeib M., Bleher S., Merfort I. and Günther S. ChemicalToolBoX, a new Galaxy for cheminformatics. (Under revision) Senger C., Grüning B. A., Erxleben A., Döring K., Patel H., Flemming S., Merfort I. and Günther S. Mining and evaluation of molecular relationships in literature. Bioinformatics (Oxford, England), 28(5), 709–14, 2012 iii Conferences - poster presentation Patel H., Grüning B.A., Lucas X., Günther S. and Merfort I. Cross-docking, virtual screening and development of chem-bioinformatics tools for drug discovery. Workshop ‘IT for lifescience @ Bayer’, Leverkusen, Germany, 2014 Patel H., Grüning B.A., Lucas X., Günther S. and Merfort I. Target fishing by cross-docking: A method to explain side effects of drugs. (Ethacrynic acid) 9th Status Seminar Chemical Biology, Frankfurt, Germany, 2014 Patel H., Grüning B.A., Lucas X., Günther S. and Merfort I. Target fishing by cross-docking as a tool to explain side effects of drugs. Faculty day, University of Freiburg, Freiburg, Germany, 2013 Patel H., Grüning B.A., Lucas X., Günther S. and Merfort I. Target fishing by cross-docking: A method to explain side effects of drugs. (Gabapentin, Aminocaproic acid) DPhD-Jahrestagung 2013, Drug discovery inspired by Nature, Freiburg, Germany Grüning B.A., Lucas X., Patel H., Flemming S., Chbeib M., Merfort I. and Günther S. ChemicalToolBoX in Action! DPhD-Jahrestagung 2012, Moleküle, Targets und Tabletten – Translationale Forschung für Arzneimittel der Zukunft, Greifswald, Germany iv Conferences - short talks (*: presented by) Jager C., Patel H., Humar M, Günther S. and Merfort I*. Helenalin derivatives and their impact on transcriptional control of mediators involved in rheumatic diseases. DPhD-Jahrestagung 2012, Moleküle, Targets und Tabletten – Translationale Forschung für Arzneimittel der Zukunft, Greifswald, Germany v Contents 1 Summary ................................................................................................................................. 1 1.1 German version ................................................................................................................ 1 1.2 English version ................................................................................................................. 3 2 STLs mechanism of action and new targets identification ..................................................... 5 2.1 Introduction to sesquiterpene lactones (STLs) ................................................................. 5 2.2 Correlating reversibility of binding reaction with STLs’ biological activity ................... 8 2.2.1 Michael addition reaction, its mechanism and reversible nature .............................. 8 2.2.2 Sesquiterpene lactones (STLs) bind via a Michael addition reaction to proteins ..... 9 2.2.3 Concept of studying the correlation between reversibility of Michael addition reaction and the biological activity of STLs ........................................................................... 9 2.2.4 Possibility of different binding possibilities of STLs via Michael reaction. .......... 10 2.2.5 NF-κB and its subunit p65 ...................................................................................... 11 2.2.6 Aims of this part of the thesis ................................................................................. 12 2.2.7 Results ..................................................................................................................... 12 2.2.8 Discussion ............................................................................................................... 17 2.3 Finding new targets for STLs ......................................................................................... 18 2.3.1 Introduction ............................................................................................................. 18 2.3.2 Results ..................................................................................................................... 21 2.3.3 Discussion ............................................................................................................... 32 3 Chem-bioinformatics tools development .............................................................................. 33 3.1 Introduction .................................................................................................................... 33 3.2 RECAP fragmenter-merger ............................................................................................ 33 3.2.1 Background ............................................................................................................. 33 vi 3.2.2 Availability and usage............................................................................................. 35 3.2.3 Results and discussion ............................................................................................ 35 3.3 PyWATER ..................................................................................................................... 38 3.3.1 Background ............................................................................................................. 38 3.3.2 Availability and usage............................................................................................. 39 3.3.3 Validation ................................................................................................................ 39 4 Virtual screening to target exotoxin A .................................................................................. 41 4.1 Introduction .................................................................................................................... 41 4.2 Results ............................................................................................................................ 44 4.2.1 Validation of grid and constraints by docking of known inhibitors ....................... 44 4.2.2 Molecules obtained from high-throughput virtual screening .................................. 45 4.2.3 Exotoxin A inhibiting activity of selected molecules ............................................. 46 4.3 Conclusion ...................................................................................................................... 49 5 New drug-target pairs identification ..................................................................................... 50 5.1 Introduction .................................................................................................................... 50 5.2 Results and discussion .................................................................................................... 52 5.2.1 Molecules higher ranked than known ligands ........................................................ 52 5.2.2 The docking score analysis ..................................................................................... 53 5.2.3 Similarity of new ligands with known ligands ....................................................... 53 5.2.4 Proposed outcomes from new drug-target interactions .......................................... 54 5.2.5 Interaction of PPAR-γ with ethacrynic acid ........................................................... 57 5.2.6 Interaction of mitochondrial L-arginine:glycine amidinotransferase (AGAT) with gabapentin and aminocaproic acid ........................................................................................ 59 5.2.7 Interaction of exotoxin A with sulfasalazine, 7-azatryptophan and Carazolol ....... 61 vii 5.3 Conclusions .................................................................................................................... 62 6 Materials and methods .......................................................................................................... 63 6.1 Materials and dataset ...................................................................................................... 63 6.1.1 Sesquiterpene lactones dataset ................................................................................ 63 6.1.2 Known exotoxin A inhibitors.................................................................................. 70 6.1.3 Chemical space for virtual screening – Purchasable box ........................................ 72 6.1.4 Protein structures .................................................................................................... 72 6.1.5