PhD Thesis Molecular Determinants of Serine and Cysteine Protease Substrate Recognition and Implications for Rational Drug Design by Birgit Waldner submitted to the Faculty of Chemistry and Pharmacy of the Leopold Franzens University of Innsbruck in partial fulfillment of the requirements for the degree of Doctor rerum naturalium (Dr. rer. nat.) LEOPOLD-FRANZENS-UNIVERSITY INNSBRUCK FACULTY OF CHEMISTRY AND PHARMACY INSTITUTE OF GENERAL, INORGANIC AND THEORETICAL CHEMISTRY Innsbruck, October 2017 Acknowledgements Many people accompanied me on the way to finishing my PhD thesis and helped me become the person and researcher I am today. First, I would like to thank Univ. -Prof. Dr. Dr. Klaus Liedl for giving me the opportunity to be a PhD student in his group and for all of the discussions during the course of my PhD thesis, without whom this work would not be what it is today. His supervision allowed me to grow as a researcher and could not prepare me better for all the challenges that are to come. Secondly, I would like to thank Prof. Gabriele Cruciani of the University of Perugia and Prof. Rafaela Ferreira of the UFMG in Belo Horizonte, who both allowed me to spend research stays in their laboratories and shared their knowledge with me to help me improve my work. I also would like to thank Prof. Hans Brandstetter, for giving me the possibility to travel to his lab and produce factor Xa. I am grateful to the Austrian Academy of Sciences (ÖAW) for funding my PhD through the DOC-grant. Thank you to all the members and former members of the Liedl group, for not only scientific discussions, but also sledging fun and cooking evenings. Alex, Anna, Christian, Dennis, Florian, Hannes, Julian, Maren, Meli, Michael, Moni, Niko, Radu, Roland, Stefania, Sonja, Suse, Ursula and Wang Yin. Thank you to Sarah and Martina for taking me to Aqua-Boxing, Magician Shows and Italian Aperitifo and to Giovanni for going climbing with me in Perugia. Thank you also to Daniela, Fabrizio, Laura, Massimo, Paulo, Simon and Susi of the Cruciani group for making my exchange stay in Perugia such a good experience. I am also glad to have met Silvia during my stay in Perugia, who is still a dear friend to me. I don’t know what I would have done without Elany of the Ferreira group, who showed me around Brazil and together with Viviane, Lorrena, Rafael and Glaecia made me feel welcome i from the first moment on I arrived. Thank you also to Lucianna for connecting me with her sister Marianna-Luiza, without whom travelling to Rio would not have been as much fun. Life as a PhD student would have been half as good, if it were not for the “Bombsquad” filling every lunch break with action. Christoph, David, Gabs, Johann, Johannes, Kathi, Richie, Theresa, Sti, Wolle and all of the guest players, thank you for all the laughs we had. Thank you also to the members of the Thursday lunch group: Christiane, Dani, Danny, Lisa, Maren, Theresa, Sebastian, Willi. I would like to thank my parents and my little brother for their endless support and patience. I know how lucky I am to have them. Last, but not least I would like to thank Tobi, whom I love more than words could describe. He lived with me through more than one PhD crisis and never got tired of putting the smile back into my face. For his constant love and support I am grateful every day. ii The work presented in this thesis led to the following three articles as first author in peer- reviewed academic journals: "Electrostatic Recognition as First Step of Substrate Binding to Serine Proteases", Waldner, B. J., Kramml, J., Kahler, U., Spinn, A., Schauperl, M., Podewitz, M., Fuchs, J. E., Cruciani, G., Liedl, K. R., Bioinformatics (2017), submitted June 2017. "Protease Inhibitors in View of Peptide Substrate Databases", Waldner, B. J., Fuchs, J. E., Schauperl, M., Kramer, C., & Liedl, K. R.. Journal of chemical information and modeling (2016). DOI: 10.1021/acs.jcim.6b00064 "Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases", Waldner, B. J., Fuchs, J. E., Huber, R. G., von Grafenstein, S., Schauperl, M., Kramer, C., & Liedl, K. R. The Journal of Physical Chemistry B (2015). DOI: 10.1021/acs.jpcb.5b10637 Furthermore, the work presented in this thesis led to the following contributions as co- author in articles in peer-reviewed journals: "Benzimidazoles as potent cysteine protease inhibitors: characterization against rhodesain and molecular basis for structure-activity relationships and selectivity between trypanosomal enzymes", Santos, L. ; Pereira, G. ; Villela, F.; Dessoy, M.; Dias, L.; Andricopulo, A.; Costa, M.; Nagem, R.; Caffrey, C.; Waldner, B.J.; Fuchs, J.; Liedl, K.R.; Caffarena, E.; Ferreira, R.. Journal of Medicinal Chemistry (2017), submitted September 2017. "A Binding Pose Flip Explained via Enthalpic and Entropic Contributions", Schauperl, M., Czodrowski, P., Fuchs, J. E., Huber, R. G., Waldner, B. J., Podewitz, M., Kramer, C., Liedl, K.R.. Journal of Chemical Information and Modeling (2017). DOI: 10.1021/acs.jcim.6b00483 iii "Enthalpic and Entropic Contributions to Hydrophobicity", Schauperl, M., Podewitz, M., Waldner, B. J., & Liedl, K. R. Journal of Chemical Theory and Computation (2016). DOI: 10.1021/acs.jctc.6b00422 "Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin", Fuchs, J. E., Huber, R. G., Waldner, B. J., Kahler, U., von Grafenstein, S., Kramer, C., & Liedl, K. R., PloS one (2015), 10(10), e0140713. DOI: https://doi.org/10.1371/journal.pone.0140713 "Characterizing Protease Specificity: How Many Substrates Do We Need?" Schauperl, M., Fuchs, J. E., Waldner, B. J., Huber, R. G., Kramer, C., & Liedl, K. R., PloS one (2015), 10(11), e0142658. DOI: https://doi.org/10.1371/journal.pone.0142658 "Independent Metrics for Protein Backbone and Side-Chain Flexibility: Time Scales and Effects of Ligand Binding", Fuchs, J. E., Waldner, B. J., Huber, R. G., von Grafenstein, S., Kramer, C., & Liedl, K. R., Journal of Chemical Theory and Computation (2015), 11(3), 851-860. DOI: 10.1021/ct500633u iv Abstract Proteases are enzymes that catalyze the cleavage of peptide bonds and play a crucial role in a plethora of biological pathways. They recognize their substrates in eight subpockets termed S4-S4’ according to the corresponding substrate binding site (P4-P4’) with the peptide’s scissile bond lying between P1 and P1’. Despite having the same fold, proteases often show distinct specificities, depending on the biological function(s) they bear. Starting with a test set of nine serine proteases with chymotrypsin fold, this thesis is concerned with elucidating the key molecular drivers of substrate recognition in proteases and applying the findings in rational drug design. The methods devised for the serine protease test set are subsequently applied to parasitic and human cysteine proteases, both in selective drug design efforts and for the investigation of molecular mechanisms in the (de)activation of enzymatic function upon ligand binding. Substrate binding in biomolecular recognition is governed by the interplay of entropic and enthalpic factors. Therefore, within this thesis, an ensemble of computational methods is employed to characterize entropic and enthalpic contributions to substrate recognition. Quantification of protease substrate specificity is achieved through the so-called cleavage- entropy metric, which is a value between 0 and 1, with 0 indicating a completely specific subpocket, accepting only one single type of amino acid, and 1 indicating a completely unspecific subpocket, accepting each of the 20 amino acids at the corresponding substrate position. As entropic factors, enzyme dynamics are investigated at different time scales by means of molecular dynamics (MD) simulations. The investigation of enthalpic factors is carried out with the program GRID, which allows for the calculation of molecular interaction fields (MIFs) of selected probe molecules by moving probe molecules along an equi-distant grid placed over the binding site and calculating the interaction potentials of the probe molecules with the binding interface of serine proteases at each grid point. In addition, water thermodynamics, both in terms of water enthalpy and entropy are investigated, using Grid Inhomogeneous Solvation Theory (GIST). v While for some examples of the serine protease test set, enzyme dynamics, quantified as backbone and sidechain flexibility of subpockets, seem to be the determining factor in substrate recognition, in other cases water thermodynamics and binding enthalpy were found to also play a crucial role in defining substrate specificity. Looking solely at binding enthalpy for X- ray structures of three selected serine protease examples, resulted in low correlation with substrate specificity quantified as cleavage entropy. Considering conformational variability through calculation of a conformational binding enthalpy incorporating the binding enthalpy of several representative conformations extracted from MD simulations led to an improvement in the correlation, but was still not able to explain substrate specificity satisfactorily. In collaboration with Prof. Gabriele Cruciani from the University of Perugia, Italy, exclusively concentrating on electrostatic interactions of the binding site with a positive probe and a negative probe bearing charges of +1 and -1 respectively, resulted in high correlation between electrostatic molecular interaction field (eMIF) similarity and electrostatic substrate readout similarity, a metric based on the binning of substrate amino acids into three bins according to their charge. Based on the knowledge gained on serine proteases, a peptide substrate-based shape-based virtual screening approach using vROCS, solely requiring information on the substrate sequences of cleaved peptide substrates, was developed and validated for four test cases. In collaboration with Prof. Rafaela Ferreira from the UFMG in Belo Horizonte, Brazil, the approach developed was employed to screen for new potential small molecule inhibitors of the parasitic cysteine protease cruzain, an important drug target in fighting the neglected tropical Chagas disease.