Andreas Rohwer

A comparative chemogenomics strategy to predict potential drug targets in the metazoan pathogen, Schistosoma mansoni.

Schistosomiasis is a prevalent and chronic helmintic disease in tropical regions. Treatment and control relies on chemotherapy with just one drug, praziquantel and this reliance is of concern should clinically relevant drug resistance emerge and spread. Therefore, to identify potential target proteins for new avenues of drug discovery we have taken a comparative chemogenomics approach utilizing the putative proteome of Schistosoma mansoni compared to the proteomes of two model organisms, the nematode, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster. Using the genome comparison software Genlight, two separate in silico workflows were implemented to derive a set of parasite proteins for which gene disruption of the orthologs in both the model organisms yielded deleterious phenotypes (e.g., lethal, impairment of motility), i.e., are essential genes/proteins. Of the 67 and 68 sequences generated for each workflow, 63 were identical in both sets, leading to a final set of 72 parasite proteins. All but one of these were expressed in the relevant developmental stages of the parasite infecting humans. Subsequent in depth manual curation of the combined workflow output revealed 57 candidate proteins. Scrutiny of these for ‘druggable’ protein homologs in the literature identified 35 S. mansoni sequences, 18 of which were homologous to proteins with 3D structures including co-crystallized ligands that will allow further structure-based drug design studies. The comparative chemogenomics strategy presented generates a tractable set of S. mansoni proteins for experimental validation as drug targets against this insidious human pathogen.

Molecular Visualization in the Rational Drug Design Process

The visualization of molecular scenarios on an atomic level can help to interpret experimental and theoretical findings. This is demonstrated in this review article with the specific field of drug design. State-of-the-art visualization techniques are described and applied to the different stages of the rational design process. Numerous examples from the literature, in which visualization was used as a major tool in the data analysis and interpretation, are provided to show that images are not only useful for drawing the attention of the reader to a specific paper in a scientific journal.

Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: Gene identification, cloning, expression, assay development, identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse

Glutamate decarboxylase (l-glutamate 1-carboxylyase, E.C. 4.1.1.15, GAD) is the rate-limiting enzyme for the production of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrates and invertebrates. We report the identification, isolation and characterization of cDNAs encoding GAD from the parasitic arthropods Ctenocephalides felis (cat flea) and Rhipicephalus microplus (cattle tick). Expression of the parasite GAD genes and the corresponding Drosophila melanogaster (fruit fly) GAD1 as well as the mouse GAD(65) and GAD(67) genes in Escherichia coli as maltose binding protein fusions resulted in functional enzymes in quantities compatible with the needs of high throughput inhibitor screening (HTS). A novel continuous coupled spectrophotometric assay for GAD activity based on the detection cascade GABA transaminase/succinic semialdehyde dehydrogenase was developed, adapted to HTS, and a corresponding screen was performed with cat flea, cattle tick and fruit fly GAD. Counter-screening of the selected 38 hit substances on mouse GAD(65) and GAD(67) resulted in the identification of non-specific compounds as well as inhibitors with preferences for arthropod GAD, insect GAD, tick GAD and the two mouse GAD forms. Half of the identified hits most likely belong to known classes of GAD inhibitors, but several substances have not been described previously as GAD inhibitors and may represent lead optimization entry points for the design of arthropod-specific parasiticidal compounds.

Die biologischen Grundlagen der Bioinformatik

Nukleinsauren und Proteine sind die beiden Makromolekulklassen, die in der belebten Natur eine besondere Rolle spielen. Die Desoxyribonukleinsaure (DNS oder DNA) ist der Trager der Erbinformation, wahrend die Ribonukleinsauren (RNS oder RNA) an der Biosynthese der Proteine beteiligt sind. Die Proteine mit ihren vielfaltigen Funktionen steuern die zellularen Prozesse des Lebens. Die monomeren Grundbausteine der Nukleinsauren sind die Nukleotide, die Bausteine der Proteine sind die Aminosauren.

Computer, Betriebssysteme und Internet

In der Bioinformatik ist der Computer das wichtigste Werkzeug des Wissenschaftlers. Neben den bekannten Betriebssystemen Windows und MacOS sind fur die Bioinformatik besonders Unix-Betriebssysteme von Bedeutung.

Die funktionelle Analyse von Genomen

Im Rahmen des humanen Genomprojektes wurde 2001 das Genom des Menschen veroffentlicht. Nach ersten Schatzungen besitzt der Mensch etwa 30000–35 000 Gene. Jede menschliche Zelle auser Spermien und Eizellen besitzt einen kompletten Satz dieser Gene. Jedoch unterscheidet sich beispielsweise eine Blutzelle in ihrer Morphologie und Physiologie sehr stark von einer Leberzelle. Wie sind diese Unterschiede zu erklaren, wenn alle Zellen das gleiche genetische Material besitzen? Die Antwort ist vergleichsweise einfach. Nicht jedes Gen wird in jeder Zelle transkribiert und exprimiert. Daraus folgt, dass in einer Zelle in der Regel nur die Proteine vorliegen, die zu einem bestimmten Zeitpunkt im Leben dieser Zelle benotigt werden. Das Proteom einer Zelle oder eines Gewebes ist also vom Zelltyp und seinem momentanen Zustand abhangig. Ebenfalls ist daraus zu ersehen, dass die alleinige Kenntnis einer gesamten genomischen Sequenz inklusive aller Gene nicht ausreicht, um die Funktionsweise eines Gens, einer Zelle bzw. eines Organismus zu erklaren. Um das komplexe biologische System zu verstehen, sind zusatzliche Informationen uber die Regulation und Expression der Gene, uber die Funktion von Proteinen und die Funktion von Zellen und Geweben notwendig.