«Aldehyde Dehydrogenase 1 – A Novel Target of Duocarmycins and Activity Based Protein Profiling during Bacterial Invasion Tanja Wirth Vollständiger ...»
Technische Universität München
Lehrstuhl für Organische Chemie II
Aldehyde Dehydrogenase 1 – A Novel Target
Activity Based Protein Profiling during
Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
Vorsitzender: Univ-Prof. Dr. Michael Groll
Prüfer der Dissertation: 1. Univ-Prof. Dr. Stephan A. Sieber
2. TUM Junior Fellow Dr. Sabine Schneider
3. apl. Prof. Dr. Manfred Heuschmann Ludwig-Maximilians-Universität München Die Dissertation wurde am 16.05.2013 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 10.06.2013 angenommen.
Für meine Familie
DANKSAGUNGZuerst bedanke ich mich herzlich bei Prof. Stephan A. Sieber für die Aufnahme in seinen Arbeitskreis, die stete Unterstützung, die große wissenschaftliche Freiheit und sein Vertrauen in meine Forschung.
Den Mitgliedern der Prüfungskommission danke ich für die Bemühungen bei der Bewertung dieser Arbeit.
Des Weiteren bedanke ich mich bei Dr. Sabine Schneider für die Übernahme des Zweitgutachtens, die interessante Rund- und Einführung am ESRF und die tatkräftige und angenehme kristallographische Unterstützung.
Ein ganz besonderer Dank geht an meine Kooperationspartner der Georg-August-Universität Göttingen, Prof. Lutz F. Tietze, Dr. Kianga Schmuck, Dr. Galina Pestel und Dr. Ingrid Schuberth.
Außerdem bedanke ich mich bei meinen Kooperationspartnern des Max-Planck-Instituts für Psychiatrie München, Dr. Theo Rein, Dr. Thomas Kirmeier, Dr. Vanessa Ganal und Anna-Maria Werner für die freundliche Zusammenarbeit und fruchtbaren Diskussionen.
Natürlich dürfen in meiner Danksagung unter gar keinen Umständen unsere nunmehr zwei guten Seelen des Arbeitskreises fehlen, Mona Wolff und Katja Bäuml. Ohne eure Hilfsbereitschaft und unverzichtbare Unterstützung im Laboraltag läge so manches im Argen!
Für das Ertragen sämtlicher Stimmungslagen, jede (un-)sinnige Diskussion, die freundschaftliche Zusammenarbeit und den ein oder anderen Feierabendgin möchte ich mich herzlich bei meinen unmittelbar betroffene Laborkollegen Franziska Mandl, Dr. Oliver Battenberg, Maximilian Koch, Dr. Maximilian Pitscheider, Thomas Menzel und Georg Rudolf bedanken.
Außerdem danke ich ganz besonders Dr. Matthew Nodwell für die Korrektur meiner Dissertation, sein Interesse an meiner Arbeit und die wissenschaftlichen sowie persönlichen Gesprächen.
Meinen letzter und größter Dank gilt Florian Stadler meinen Eltern Erika und Norbert Wirth, meinenGroßeltern Regina und Karl Wismath und Elisabeth Körbl und meiner/nem Lieblingstante/-onkel Andrea Altstetter und Ernst Körbl für den Rückhalt, die Motivation und die Unterstützung während meiner Promotion und drüber hinaus. Und Oma, ich weiß, du hosch’s mir ja glei gset…
INTRODUCTORY REMARKParts of this thesis have been published in international journals
TABLE OF CONTENTS
1 THE HISTORY OF DRUG DISCOVERYThroughout history humans have been threatened by diseases and explored natural resources for abatement and cure. The first attempts can be dated back to prehistoric times, as residuals of medicinal herbs were discovered in 60,000 year old Neanderthal remains. Ancient civilizations such as the Egyptians gathered their knowledge of herbal treatments in written compendia. The Ebers Papyrus (1500 BC) is one important example and contains 800 prescriptions for over 700 cures.[2, 3] But this knowledge was more a result of trial and error or accidental discoveries, rather than systematic research.
Furthermore the tinctures of alcoholic or aqueous plant extracts did not fit an exact chemical composition, since they contained various constituents in differing amounts. The scientific foundation for today’s therapeutic agents as well as for modern drug discovery was laid in the 19th century. Due to the development of organic and analytical chemistry (Figure I-1) the tools for isolation, purification and characterization of active herbal ingredients were provided.
The first generation of “modern” drugs was launched with the isolation of morphine from opium by Sertürner (1815).[2, 5] Through the access of pure alkaloids and synthetic
organic products, the door was opened to a new and independent scientific discipline:
pharmacology. Further disciplines as medicinal chemistry, biochemistry and microbiology followed soon and gave birth to second-generation drugs (e.g. vaccines) and new types of drug therapies such as chemotherapy to treat cancer. An underlying concept of “chemoreceptors” was first introduced by Paul Ehrlich (1872-1874), who postulated that cancer cells, parasites or microorganisms possess particular chemoreceptors, which are absent in host tissues or healthy cells. He further demonstrated these receptors could be exploited for a selective treatment and cure of infectious disease.[2, 5] With the discovery of Penicillin by Alexander Fleming (1929) and Prontosil (the first sulfonamide antibiotic, 1932) a new age of drug discovery had begun. In the following years further sulfonamide derivatives, hormones, vitamins and antibiotics reached the market and revolutionized the pharmaceutical industry. With the emerging knowledge in biological structures and functions in the 1960s, rational drug discovery and design through structural variations grew popular. The resulting fourth generation of drugs furthermore expanded the scope of treatments to central nerve system or cardiovascular diseases.[2, 5] Today we have reached the fifth generation of drugs consisting of small molecule enzyme inhibitors and biologics, such as monoclonal antibodies and recombinant proteins. But apart from these new types of drugs, a huge progress in technology and methodology was achieved, resulting in the new concept of structurebased drug discovery.[2, 5] This strategy is focused on the molecular mechanism of cellular processes and diseases to identify potential drug targets and to optimize drug candidates. Methods and techniques as high-throughput screening, combinatorial chemistry, X-ray crystallography, bioinformatics, genomics and proteomics (to name but a few) are utilized to solve this task.[2, 5] Hence, drug discovery has become a highly interdisciplinary field with increasing complexity. Nonetheless the evolution will go on and further techniques and strategies have to be developed for target identification and drug optimization.
2 FROM GENOME TO PROTEOMEWith finishing the sequence of the human genome (Human Genome Project, 1990 –
2003) the foundation for functional analysis at the molecular level of cellular processes was laid. However, the genome – the total set of genes of an organism - is a static system, independent from environmental conditions or cell type. Compared to the genome of nematodes (∼12,000 – 24,000 genes, depending on the species) the human genome (∼20,000 genes) itself cannot explain the obviously increased biological complexity. Therefore, an explanation can only be found at the level of the dynamic and highly diverse transcriptome and proteome (Figure I-2).
Figure I-2: From genome to proteome. The proteome complexity is increased through protein complexes and post-translational modifications. The examples shown here are only a few of the over 400 types of known protein modifications (Ubi = ubiquitination, P = Phosphorylation, G = Glycosylation).The figure was partially prepared by the Biological and Environmental Research Information System, Oak Ridge National Laboratory.
Due to changes in the environmental conditions, such as stress, drug exposure or disease, their composition can alter dramatically, as a result of transcriptional regulation, RNA processing, protein synthesis and modification. The transcriptome – the total set of mRNA in a given cell or cellular state – is generated from the genome, whereby one gene may encode different transcripts by alternative splicing or alternative promoters.
The transcriptome in turn is translated to produce the proteome, the total set of proteins in a given cell or cellular state. The expressed proteins can act as single molecules or as
subunits of homo- (same subunits) and heteromeric (different subunits) protein complexes. Moreover, the proteome complexity is increased through post-translational modifications (PTMs), which can emerge at any stage of a proteins life cycle. Taking PTMs into account, there are over one million possible protein variations according to estimations. These chemical modifications exert influence over protein activity, localization, degradation, expression and the interaction with other macromolecules.
Today’s major goal for drug discovery and basic research is focused on the detailed understanding of biological systems, to create pathways and networks. One approach to resolve the functions of genes and their products is based on the transcriptome (transcriptomics). But DNA and mRNA are “solely” information carriers, which cannot predict the structure, interaction, abundance or activity of a protein. However, the activity and function of a protein depends greatly on the cellular localization and eventual post-translational modifications. In contrast to DNA and mRNA, proteins provide a direct link to the various biological functions in the cell, as they control biochemical reactions, intracellular transport, cell shape, signal transduction and many more. Hence the analysis of the proteome (proteomics) presents a powerful tool to determine altered protein expression, interaction or activity.
3 ENZYME INHIBITORSEnzyme catalysis and the control of biochemical reactions are essential for all life.
Furthermore altered regulation of activity, substrate specificity and expression can either be caused by diseases or result in a pathologic state, making enzymes attractive drug targets. Beside the attractiveness and pathophysiologic relevance, enzymes are highly druggable for several reasons. First the active site and ligand binding pockets are well attuned to interact with small molecule drugs. That is because enzyme pockets are small, shielded from bulk solvent and show large surfaces relative to volume with a distinct topography, which contains specific groups for hydrogen bonding and electrostatic interactions with ligands.[13, 14] Second minor changes in the surface topography, induced through ligand or drug binding, are enough to alter the enzymatic activity drastically.
Third enzymes are highly dynamic molecules, rearranging shape and structure to
INTRODUCTIONfacilitate the catalysis of chemical reactions. Thus one enzyme offers various conformational forms and transition states, whereas each form represents a unique point of application that can be addressed by small molecule drugs.[13, 14] As mentioned before, proteins are responsible for diverse biological functions and can therefore be further divided into subclasses, such as cytoskeletal proteins, coagulation factors, ion channels, receptors, chaperones, enzymes and many more. But concerning the druggability and the actual application in modern medicine, enzymes and G-proteincoupled receptors represent the predominant drug targets (Figure I-3A/B).[15, 16] Figure I-3: A. Target classes of approved drugs. Similar target molecules of different drugs were counted once. B. Molecular mechanism of action (MMOA) of the 50 first-in-class small-molecule drugs (with new MMOA) approved by the FDA between 1999 and 2008. C. Number of successful drug targets sorted by drug classes. The Data was derived from the Therapeutic Target Database (TTD 2012) and considers also targets of multiple drug classes. Therefore the successful target entries are shown in absolute numbers.
A more detailed view on the molecular mode of action is shown in Figure I-3C. Successful and clinically used targets are divided by the classes of attacking drugs, revealing enzyme inhibitors as major class. As a conclusion the pathophysiologic relevance and druggability of enzymes makes inhibitors attractive for drug discovery and applicable in any human disease, such as cancer, infectious, metabolic, inflammatory or cardiovascular diseases.