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Application of Models for Biomolecular Structure and Interactions to Understand and Influence Biological Function

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Application of Models for Biomolecular Structure and Interactions to Understand and Influence Biological Function Application of Models for Biomolecular Structure and Interactions to Understand and Influence Biological Function Zur Erlangung des akademischen Grades eines DOKTORS DER NATURWISSENSCHAFTEN (Dr. rer. nat.) Fakultät für Chemie und Biowissenschaften Karlsruher Institut für Technologie (KIT) - Universitätsbereich genehmigte DISSERTATION von Irene Meliciani aus Rom (Italien) Dekan: Prof. Dr. Martin Bastmeyer Referent: Prof. Dr. Stefan Bräse Korreferent: Apl. Prof. Dr. Wolfgang Wenzel Tag der mündlichen Prüfung: 19.10.2011 a mamma e papà Application of Models for Biomolecular Structure and Interactions to Understand and Influence Biological Function Irene Meliciani Karlsruher Institut für Technologie Institut für Nanotechnologie Intitut für Organische Chemie Zusammenfassung Die Forschung in den Lebenswissenschaften hat in den letzten Jahrzehnten zu einem enormen Wissensgewinns bezüglich der Abläufe zellulärer Prozesse und ihrer Steuerung geführt. Die Steuerung biologischer Prozesse wird vielfach über Kontaktwechselwirkungen der daran beteiligten Biomoleküle bewältigt. Mit dieser Arbeit möchte ich zu einem besseren Verständnis biomolekularer Interaktionen beitragen. Ein Teil dieser Arbeit beschäftigt sich mit der Untersuchung von Proteininteraktionen, wobei als Methoden für die Modellierung von Protein-Protein-Wechselwirkungen (POEM) und für die Protein-Liganden-Wechselwirkung (FLEXSCREEN) eingesetzt wurden. Es wurden die Interaktionen der Chemokinrezeptoren CCR3 und CXCR1 mit für den Entzündungsverlauf wichtigen Steuerungsmolekülen (Chemokinen) untersucht. Dazu wurde ein in silico Alanin-Scanning-Protokoll entwickelt, welches experimentelle Verfahren zur Untersuchung wichtiger Interaktionen in der Protein-Protein-Kontaktfläche ergänzt. Die Untersuchungen zielen auf die Entwicklung neuer Inhibitoren für Chemokinrezeptoren ab, um inflammatorische Krankheiten besser behandeln zu können. In einer weiteren Untersuchung wurde versucht, neue Wirkstoffmoleküle für die Cannabinoidrezeptoren CB1 und CB2 zu entwickeln, die in den Huntington und Alzheimer Krankheiten eine wichtige Rolle spielen. Dazu wurden Strukturmodelle für die Proteinrezeptoren erstellt und das Bindungsverhalten von Cumarinderivaten untersucht. In enger Zusammenarbeit mit dem Experiment wurde eine Reihe neuer Liganden synthetisiert und experimentell validiert, wobei sich eine erhöhte Affinität verglichen mit dem Referenzmolekül und eine gute, quantitative Übereinstimmung zwischen Simulation und Experiment zeigte. In einer letzten Anwendung im Bereich der rechnergestützten Medikamentenentwicklung wurde eine grosse Datenbank einem virtuellen Screening unterzogen, um neune Substanzen zu finden die regulierend in die Blutgerinnung einzugreifen. Ein weiteres wichtiges Anwendungsfeld ist die Identifizierung genetischer Ursachen für Entwicklungsstörungen, die von patientenspezifischen Mutationen herrühren. Darüber hinaus habe ich Methoden der Protein-Strukturvorhersage angewandt, um die Relevanz von Mutationen, die an Patienten, die an Kallmann-Syndrom (KS), sowie normosmic hypogonadotropen hypogonadismus (nHH) leiden für den Krankheitsverlauf aufzuklären. Diese Ergebnisse zeigen, dass rechnergestützte Verfahren zur Modellierung der Struktur von Biomolekülen und deren Wechselwirkungen heute dazu beitragen können, die komplexen zellulären Steuerungsmechanismen zu verstehen und gegebenenfalls auch zu beeinflussen. Damit erwächst aus den rechnergestützten Verfahren ein neues wichtiges Instrument um biologische Prozesse zu verstehen und die pharmazeutische Forschung voranzutreiben. Table of contents 1. Overview ................................................................................................................. 7 2. Introduction ......................................................................................................... 13 2.1. Introduction ........................................................................................................ 13 2.2. Protein Structure ............................................................................................... 14 2.3. Protein Structure Prediction ............................................................................. 19 2.3.1. Comparative Modeling .......................................................................... 20 2.3.2. Model Building ....................................................................................... 21 2.3.3. Loop Modeling ....................................................................................... 23 2.3.4. Side Chain Modeling ............................................................................. 23 2.3.5. Quality Assessment .............................................................................. 24 2.3.6. State of the Art....................................................................................... 24 2.4. Protein Ligand Interactions............................................................................... 26 2.4.1. Docking Methods .................................................................................. 27 2.4.2. Estimates of the Affinity ........................................................................ 29 2.5. Protein Protein Interactions .............................................................................. 30 2.5.1. Protein Protein docking ........................................................................ 31 2.5.2. Alanine scanning mutagenesis ............................................................ 33 3. Application of protein structure prediction to identify genetic causes for human disease .................................................................................................. 37 3.1. Introduction ........................................................................................................ 37 3.2. Methods.............................................................................................................. 39 3.3. Investigation of Mutations in CHD7 ................................................................. 40 3.3.1. Sequence Based Analysis of Mutations ............................................. 41 3.3.2. Structural Models of CHD7 Domains .................................................. 42 3.3.3. Conclusions ........................................................................................... 44 3.4. Investigation of mutations in WDR11 ............................................................. 45 3.4.1. Protein Modeling ................................................................................... 45 3.4.2. Analysis of Mutations ............................................................................ 46 3.4.3. Discussion.............................................................................................. 47 3.5. Investigation of mutations in Nasal Embryonic LHRH Factor (NELF) ......... 51 3.5.1. Protein Modeling ................................................................................... 52 3.5.2. Conclusions ........................................................................................... 53 3.5.3. Discussion ............................................................................................. 54 3.6. Summary ............................................................................................................ 55 4. Protein Protein Interactions ............................................................................. 59 4.1. Introduction ........................................................................................................ 59 4.2. Methods ............................................................................................................. 61 4.2.1. The forcefield PFF02 ............................................................................ 61 4.2.2. Simulation Protocol ............................................................................... 64 4.3. Elucidating specificity for interleukin-8 and its receptor CXCR1 .................. 65 4.4. Elucidating specificity for ERBIN and its receptor ERBB2 ............................ 67 4.5. Elucidating specificity for eotaxin and its receptor CCR3.............................. 69 4.6. Summary ............................................................................................................ 73 5. Protein Ligand Interactions .............................................................................. 77 5.1. Introduction ........................................................................................................ 77 5.2. Methods ............................................................................................................. 78 5.3. Small Molecule Ligands for Binding Chemokines .......................................... 80 5.4. Selective high-affinity inhibitors of cannabinoid receptors CB1/CB2 ........... 83 5.4.1. Model Building and Validation ............................................................. 83 5.4.2. Design of novel ligands ........................................................................ 86 5.4.3. Origins of CB1/CB2 selectivity ............................................................. 89 5.4.4. Conclusions ........................................................................................... 93 5.5. Modulating activation of antithrombin in presence of heparin ...................... 94 5.5.1. In-Silico Alanine Screen ....................................................................... 95 5.5.2. In-Silico Screening ...............................................................................
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