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Strategies to Evade Resistance: Combining Biophysical and Biochemical Approaches to Discover Compounds Addressing New Bacterial

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Strategies to Evade Resistance: Combining Biophysical and Biochemical Approaches to Discover Compounds Addressing New Bacterial Strategies to Evade Resistance: Combining Biophysical and Biochemical Approaches to Discover Compounds Addressing New Bacterial Target Systems DISSERTATION zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät III Chemie, Pharmazie, Bio- und Werkstoffwissenschaften der Universität des Saarlandes Von Christian Brengel Saarbrücken 2016 Tag des Kolloquiums: 22.09.2016 Dekan: Prof. Dr.-Ing. Dirk Bähre Vorsitz: Prof. Dr. Andriy Luzhetskyy Berichterstatter: Prof. Dr. Rolf W. Hartmann Prof. Dr. Claus-Michael Lehr Akad. Mitarbeiter: Dr. Stefan Boettcher I Die vorliegende Arbeit wurde von Januar 2012 bis Januar 2016 unter Anleitung von Herrn Univ.- Prof. Dr. Rolf W. Hartmann in der Fachrichtung 8.2 Pharmazeutische und Medizinische Chemie der Naturwissenschaftlich-Technischen Fakultät III der Universität des Saarlandes sowie am Helmholtz-Institut für Pharmazeutische Forschung Saarland (HIPS) angefertigt. II III „Man sollte alles so einfach wie möglich sehen - aber auch nicht einfacher“ Albert Einstein IV SUMMARY The rising number of resistant bacteria combined with the innovation gap in the antibiotic development has become a huge health problem. Such infections are insufficient to be treated with the approved antibiotic agents on the market resulting in an increasing number of deaths and a financial burden on the healthcare system. Thus, there is a need for antiinfectives with new modes of action. P. aeruginosa, a Gram-negative pathogen often associated with antibiotic resistance, controls the production of major virulence factors and biofilm formation via a quorum sensing system. PqsD and PqsR are key player proteins in this system. The interruption of their function by small molecules should lower virulence of the bacterium without exerting a selection pressure on it. A biophysical method-guided binding mode characterization supports the optimization of these small molecules. Influence of the compounds on biofilm formation validates their pharmacological efficiency on this lifestyle characteristic for chronic infections. M. tuberculosis that still kills millions of people worldwide is hard to be eradicated because of a dormant state and a huge number of resistances. The high content of CYP enzymes encoded in the genome provides a promising base for new drug targets. CYP121 is essential for growth in vitro; CYP125 plays an important role for survival in vivo. Biophysical and biochemical test systems are used for identification and characterization of the first hit molecules. V ZUSAMMENFASSUNG Das zunehmende Auftreten von resistenten Bakterien verbunden mit einer Innovationslücke in der Antibiotikaentwicklung führen zu einer ansteigenden Zahl an Todesopfern und einer finanziellen Belastung des Gesundheitssystems. Folglich besteht ein dringender Bedarf an Antiinfektiva mit neuen Wirkmechanismen. P. aeruginosa, ein Gram-negativer Krankheitserreger, der oft im Zusammenhang mit Antibiotikaresistenzen steht, kontrolliert die Produktion von Virulenzfaktoren sowie die Biofilmbildung mittels eines „Quorum Sensing-Systems“. PqsD und PqsR sind Schlüsselproteine in diesem System. Das Stören ihrer Wirkungsweise mittels kleiner Moleküle sollte die Virulenz des Bakteriums herabsetzen, ohne einen Selektionsdruck auszuüben. Eine biophysikalische Methode zur Aufklärung des Bindungsmodus dieser kleinen Moleküle unterstützt hier auch deren Optimierung. Der Einfluss der Verbindungen auf die Biofilmbildung, die für chronische Infektionen essentiell ist, validiert deren pharmakologische Wirksamkeit. M. tuberculosis tötet immer noch Million Menschen auf der ganzen Welt und ist wegen seiner Dormanz und einer großen Anzahl an Resistenzen schwer zu behandeln. Eine hohe Anzahl an CYP Enzymen im Genom dieser Bakterien bietet eine vielversprechende Grundlage für neue Wirkstofftargets. CYP121 ist essentiell für das in vitro Wachstum; CYP125 spielt eine wichtige Rolle für das in vivo Überleben. Biophysikalische und biochemische Methoden werden verwendet, um Hit- Verbindungen zu identifizieren und biologisch zu charakterisieren. VI PAPERS INCLUDED IN THIS THESIS This thesis is divided into four publications and two manuscripts submitted for publication, which are referred to in the text by their letters. A Biophysical Screening of a Focused Library for the Discovery of Novel Antimycobacterials targeting CYP121 Christian Brengel,* Andreas Thomann,* Alexander Schifrin, Giuseppe Allegretta, Ahmed Kamal, Jörg Haupenthal, Sang Hyun Cho, Scott G. Franzblau, Jens Eberhard, and Rolf W. Hartmann Manuscript in preparation *These authors contributed equally B Discovery and biophysical evaluation of first low nanomolar hits targeting CYP125 of M. tuberculosis Christian Brengel,* Andreas Thomann,* Jens Eberhard, and Rolf W. Hartmann Manuscript in preparation *These authors contributed equally C Biochemical and Biophysical Analysis of a Chiral PqsD Inhibitor Revealing Tight-Binding Behavior and Enantiomers with Contrary Thermodynamic Signatures Michael P. Storz,* Christian Brengel,* Elisabeth Weidel, Michael Hoffmann, Klaus Hollemeyer, Anke Steinbach, Rolf Müller, Martin Empting, and Rolf W. Hartmann ACS Chem. Biol. 2013, 8, 2794-2801. *These authors contributed equally D Combining in Silico and Biophysical Methods for the Development of Pseudomonas aeruginosa Quorum Sensing Inhibitors: An Alternative Approach for Structure-Based Drug Design J. Henning Sahner,* Christian Brengel,* Michael P. Storz, Matthias Groh, Alberto Plaza, Rolf Müller, and Rolf W. Hartmann J. Med. Chem. 2013, 56, 8656-8664. *These authors contributed equally E Structure Optimization of 2-Benzamidobenzoic Acids as PqsD Inhibitors for Pseudomonas aeruginosa Infections and Elucidation of Binding Mode by SPR, STD NMR, and Molecular Docking VII Elisabeth Weidel, Johannes C. de Jong, Christian Brengel, Michael P. Storz, Matthias Negri, Alberto Plaza, Anke Steinbach, Rolf Müller, and Rolf W. Hartmann J. Med Chem. 2013, 56, 6146-6155. F Application of dual inhibition concept within looped autoregulatory system toward antivirulence agents against Pseudomonas aeruginosa infections Andreas Thomann, Antonio G. Gomes de Mello Martins, Christian Brengel, Martin Empting and Rolf W. Hartmann ACS Chem. Biol. 2016, 11, 1279–1286 VIII CONTRIBUTION REPORT The author wishes to clarify his contributions to the Publications/Manuscript A−F in the thesis. A The author heterologously expressed CYP121 and designed the LC-MS/MS-based functional enzyme assay. He was involved in the synthesis of Mycocyclosin. Furthermore, he planned, executed, measured, and analysed the SPR and heme assay screening. Additionally, he developed the antimycobacterial growth assay and determined the MIC values on M. bovis. He planned the project, conceived and wrote the manuscript. B The author heterologously expressed CYP121, CP125, and CYP142. Furthermore, he planned, executed, measured, and analysed the SPR and heme assay screening. He planned the project, conceived and wrote the manuscript. C The author designed and generated the site-directed mutagenesis constructs. Furthermore, he heterologously expressed PqsD and the mutants of the enzyme. He planned, executed, measured, and analysed the ITC experiments. He was significantly involved in the interpretation of the results and contributed to writing of the manuscript. D The author designed and generated the site-directed mutagenesis constructs. Furthermore, he heterologously expressed PqsD and the mutants of the enzyme. He planned, executed, measured, and analysed the SPR and ITC experiments. He was significantly involved in the interpretation of the results and contributed to writing of the manuscript. E The author designed and generated the site-directed mutagenesis constructs. Furthermore, he heterologously expressed PqsD and the mutants of the enzyme. He planned, executed, measured, and analysed the ITC experiments. Furthermore, he contributed to the interpretation of the results. F The author designed and performed the biofilm assay and the antibiotic susceptibility test. Furthermore, he contributed to the interpretation of the results. IX FURTHER PAPERS OF THE AUTHOR THAT ARE NOT PART OF THIS DISSERTATION A Mechanistic details for anthraniloyl transfer in PqsD: the initial step in HHQ biosynthesis Michael C. Hutter, Christian Brengel, Matthias Negri, Claudia Henn, Christina Zimmer, Rolf W. Hartmann, Anke Steinbach, Rolf Müller, and Martin Empting J. Mol. Model. 2014, 2:2255. B Molecular basis of HHQ biosynthesis: molecular dynamic simulation, enzyme kinetic and surface plasmon resonance studies Anke Steinbach Christine K. Maurer, Elisabeth Weidel, Christian Brengel, Rolf W. Hartmann, and Matthias Negri BMC Biophys. 2013, 6:10. C Validation of PqsD as an Anti-biofilm Target in Pseudomonas aeruginosa by Development of Small-Molecule Inhibitors Michael P. Storz, Christine K. Maurer, Christina Zimmer, Nathalie Wagner, Christian Brengel, Johannes C. de Jong, Simon Lucas, Mathias Müsken, Susanne Häussler, Anke Steinbach, and Rolf W. Hartmann J. Am. Chem. Soc. 2012, 134, 16143-16146 D QSAR-guided Optimization of 2-sulfonylpyrimidines to tackle biofilm formation and eDNA release of Pseudomonas aeruginosa Andreas Thomann, Christian Brengel, Carsten Börger, Dagmar Kail, Anke Steinbach, Martin Empting, and Rolf W. Hartmann Manuscript in preparation X Abbreviations 3D Three dimensional AQ 2-alkyl-4-quinolones ACoA Anthraniloyl coenzyme A ATP Adenosine triphosphate BCG Mycobacterium bovis BCG CLSM Confocal laser scanning microscopy CoMFA Comparative
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