From Active-Site Mapping to Lead Discovery using Fragment-based Approaches on the Aspartic protease Endothiapepsin Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Pharmazie der PHILIPPS-UNIVERSITÄT MARBURG vorgelegt von Nedyalka Radeva aus Kardzhali Marburg/Lahn 2016 Von Fachbereich Pharmazie der Philipps-Universität Marburg als Dissertation eingereicht am: 01.12.2016 Erstgutachter: Prof. Dr. Gerhard Klebe Zweitgutachter: Prof. Dr. Andreas Heine Tag der mündlichen Prüfung: 25.01.2017 Hochschulkennziffer: 1180 Die Untersuchungen zum vorliegenden Thema wurden auf Anregung von Prof. Dr. Gerhard Klebe am Institut für Pharmazeutische Chemie des Fachbereichs Pharmazie der Philipps-Universität Marburg in der Zeit von August 2012 bis November 2016 durchgeführt. Nicht die Neugier entscheidet über unser Wissen, sondern die Bereitschaft Altes aufzugeben. Meiner Familie This thesis resulted in several scientific contributions: I. Journal Articles: Johannes Schiebel, Nedyalka Radeva, Helene Köster, Alexander Metz, Timo Krotzky, Maren Kuhnert, Wibke E. Diederich, Andreas Heine, Lars Neumann, Cedric Atmanene, Dominique Roecklin, Valérie Vivat-Hannah, Jean-Paul Renaud, Robert Meinecke, Nina Schlinck, Astrid Sitte, Franziska Popp, Markus Zeeb, Gerhard Klebe. One Question, Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists. ChemMedChem 2015, 10, 1511–1521. Nedyalka Radeva, Johannes Schiebel, Xiaojie Wang, Stefan G. Krimmer, Kann Fu, Martin Stieler, Frederik R. Ehrmann, Alexander Metz, Thomas Rickmeyer, Michael Betz, Johan Winquist, Ah Young Park, Franziska U. Huschmann, Manfred S. Weiss, Uwe Mueller, Andreas Heine, Gerhard Klebe. Active-Site Mapping of an Aspartic Protease by Multiple Fragment Crystal Structures: Versatile Warheads to Address a Catalytic Dyad. J.Med.Chem. 2016, 59, 9743–9759. Nedyalka Radeva, Stefan G. Krimmer, Martin Stieler, Kan Fu, Xiaojie Wang, Frederik R. Ehrmann, Alexander Metz, Franziska U. Huschmann, Manfred S. Weiss, Uwe Müller, Johannes Schiebel, Andreas Heine, Gerhard Klebe. Experimental Active-Site Mapping by Fragments – Hot Spots Remote from the Catalytic Center of Endothiapepsin. J.Med.Chem. 2016, 59, 7561–7575. Johannes Schiebel, Nedyalka Radeva, Stefan G. Krimmer, Xiaojie Wang, Martin Stieler, Frederik R. Ehrmann, Kan Fu, Alexander Metz, Franziska U. Huschmann, Manfred S. Weiss, Uwe Müller, Andreas Heine, Gerhard Klebe. Six Biophysical Screening Methods Miss a Large Proportion of Crystallographically Discovered Fragment Hits: A Case Study. ACS Chem.Biol. 2016, 11, 1693–1701. Johannes Schiebel, Stefan G. Krimmer, Karine Röwer, Anna Knörlein, Xiaojie Wang, Ah Young Park, Martin Stieler, Frederik R. Ehrmann, Kan Fu, Nedyalka Radeva, Michael Krug, Franziska U. Huschmann, Steffen Glöckner, Manfred S. Weiss, Uwe Mueller, Gerhard Klebe, Andreas Heine. High-Throughput Crystallography: Reliable and Efficient Identification of Fragment Hits. Structure 2016, 24, 1398–1409. I Milon Mondal, Nedyalka Radeva, Helene Köster, Ah Young Park, Constantinos Potamitis, Maria Zervou, Gerhard Klebe, Anna K. H. Hirsch. Structure-based design of inhibitors of the aspartic protease endothiapepsin by exploiting dynamic combinatorial chemistry. Angew.Chem.Int.Ed. 2014, 53, 3259–3263. Alwin M. Hartman, Milon Mondal. Nedyalka Radeva, Gerhard Klebe, Anna K. H. Hirsch. Structure-based optimization of inhibitors of the aspartic protease endothiapepsin. Int.J.Mol.Sci. 2015, 16, 19184-19194. Milon Mondal, Nedyalka Radeva, Hugo Fanlo-Virgós, Sijbren Otto, Gerhard Klebe, Anna K. H. Hirsch. Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry. Angew.Chem.Int.Ed. 2016, 55, 9422–9426. Jonathan Cramer, Johannes Schiebel, Tobias Wulsdorf, Kristof Grohe, Eszter Eva Najbauer, Frederik R. Ehrmann, Nedyalka Radeva, Nina Zitzer, Dr. Uwe Linne, Prof. Dr. Rasmus Linser, Prof. Dr. Andreas Heine, Prof. Dr. Gerhard Klebe. Watch out for the Red Herring: Surprising Reactivity of a Fragment Results in Biological Activity. Angew.Chem.Int.Ed., DOI: 10.1002/anie.201609824R1 and 10.1002/ange.201609824R1 II. Oral presentations: Design of Novel Aspartic Protease Inhibitors Exploiting Dynamic Combinatorial Chemistry, 22nd Annual Conference of the German Crystallographic Society (DGK), Berlin 2014 Design of Novel Aspartic Protease Inhibitors Exploiting Dynamic Combinatorial Chemistry, International School of Crystallography, 47th Course, Erice 2014 II Table of content 1. Introduction .............................................................................................................................................. 1 1.1 Structure-Based Drug Design ......................................................................................................... 1 1.2 Fragment-Based Drug Discovery ................................................................................................... 2 1.3 Experimental Methods used for Identifying Fragment Binding ........................................................... 7 1.3.1 X-ray Crystallography ................................................................................................................... 7 1.3.1.1 Bragg’s Law ........................................................................................................................... 7 1.3.1.2 Crystals and the Unit Cell ...................................................................................................... 9 1.3.1.3 Friedel Pairs .......................................................................................................................... 10 1.3.1.4 Methods for Solving the Phase Problem .............................................................................. 11 1.3.2 Microscale Thermophoresis (MST) ............................................................................................ 12 1.4 Dynamic Combinatorial Chemistry ................................................................................................... 14 1.5 Aspartic Proteases .............................................................................................................................. 15 1.5.1 The Family of Aspartic Proteases ............................................................................................... 15 1.5.2 Functional Mechanism of Aspartic Proteases ............................................................................. 16 1.5.3 BACE1 ........................................................................................................................................ 17 1.5.4 Renin ........................................................................................................................................... 18 1.5.5 Secreted Aspartic Proteases ........................................................................................................ 18 1.5.6 Plasmepsins ................................................................................................................................. 18 1.5.7 HIV1 Protease ............................................................................................................................. 19 1.5.8 Endothiapepsin ............................................................................................................................ 19 1.6 Preliminary Work and Aims of the Thesis ......................................................................................... 22 1.7 References .......................................................................................................................................... 23 2. One Question, Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists .................................................................................................................... 29 2.1 Introductory Remarks ......................................................................................................................... 29 2.2 Abstract .............................................................................................................................................. 30 2.3 Introduction ........................................................................................................................................ 30 2.4 Results and Discussion ....................................................................................................................... 33 2.5 Conclusions ........................................................................................................................................ 44 2.6 Experimental Section ......................................................................................................................... 45 2.6.1 Protein material ........................................................................................................................... 45 2.6.2 Fragment library .......................................................................................................................... 45 2.6.3 Biochemical assay ....................................................................................................................... 45 III 2.6.4 Thermal Shift assay ...................................................................................................................