Auffinden von Inhibitoren der tRNA-Guanin Transglykosylase (TGT) und der Insulin-like Growth Factor 1 Receptor Tyrosine Kinase (IGF-1-RTK) aus Pflanzenextrakten durch Ligandenfischen sowie deren Isolierung und Identifizierung
Die rasante Entwicklung von resistenten und multiresitenten Keimen ist ein weltweites Problem, das im Falle der Shigellen besorgniserregende Ausmaße angenommen hat. Die tRNA-Guanin-Transglykosylase (TGT) stellt ein neues Target für die Entwicklung antibiotisch wirkender Substanzen gegen Shigellen da...
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Format: | Doctoral Thesis |
Language: | German |
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Philipps-Universität Marburg
2005
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Online Access: | PDF Full Text |
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The rapid development of resistances and multiresistances against the nowadays used antibiotics has become a worldwide problem. This development has attained alarming magnitudes particularly in the case of Shigella sp.. A new target for the development of antibiotic active substances against Shigella sp. is represented by the tRNA-guanine-transglycosylase (TGT). It was the purpose of this study to identify and to isolate new leading structures or potential inhibitors of the TGT as well as of the insulin-like growth factor 1 receptor tyrosine kinase (IGF-1-RTK) from plant extracts. IGF-1-RTK is a part of the intracellular subunit of the IGF-1-receptor. This receptor takes a key position in the progression of cancer. Its function could be inhibited by blockage of the catalytic kinase domain. Thus an excessive cell proliferation would be prevented while a controlled crossing into the apoptotic pathway would be allowed. For the screening of the plant extracts the innovative method “Fishing of Ligands” was used. Compared with the High-Throughput-Screening this method is the less time-consuming and mainly cheaper procedure for the discovery of active ingredients in extensive substance libraries. With the method “Fishing of Ligands” 15 extracts of traditional medicinal plants were screened for the existence of TGT-binding substances and six more substances were analysed for a TGT-binding. In the extracts of Passiflora incarnata L., Solanum dulcamara L. and Centella asiatica (L). URBAN ingredients were found which bind to TGT. The ingredient of the methanolic extract of the herb of Passiflora incarnata L. was isolated with preparative HPLC and could be identified with NMR and MS as isovitexin-2”-O-ß-glycoside. By usage of high-field NMR spectrometers it was possible to give a complete interpretation of the 1H-NMR spectrum of this substance the first time. Furthermore 4 other flavonoids could be isolated and identified from 4 different fractions of the extract. All of the 5 flavonoids have the basic structure of apigenine. However, only isovitexin-2”-O-ß-glycoside could be selected from the extract by the TGT. For this reason an investigation of the influence of the substitutional pattern of the apigenine basic structure on the enzyme-binding was necessary. Therefore experiments to structure-response-relationships were accomplished by the method “Fishing of Ligands” as well as by determination of Ki values and crystallographic methods in collaboration with the work group of Prof. Dr. G. Klebe (Philipps-University of Marburg). The results of the research with the “Fishing of Ligands” gave indices on a preferred binding of C 6 substituted apigenine derivatives. For apigenine itself a binding to TGT was not observed by this assay. By determination of the Ki values of apigenine as well as of the 5 apigenine derivatives it could be shown that the pattern of substitution correlates with the inhibitory effect. Isovitexin-2”-O-ß-glycoside was tested exhibiting a Ki value of 20 µm. Thus the scope of application for “Fishing of Ligands” could be extended by factor 48. Unfortunately, crystallisation of this substance in complex with TGT was not successful. Therefore the determination of a binding modus was not possible. Isovitexin-2”-O-ß-glycoside and isovitexin are representatives of a new inhibitor class of the TGT. For further investigations, e.g. with the bacteria itself, the substances should be modified for increasing the inhibitory effect. For the second target, IGF-1-RTK, binding substances from medical plants could be obtained with the method “Fishing of Ligands” too. 12 different plant extracts were screened. A binding of one or more ingredients on IGF-1-RTK could be observed while examining the 6 extracts obtained from the roots of Glycyrrhiza glabra L., the herbs of Passiflora incarnata L., Lichen islandicus and from the cortex of Aspidosperma quebracho-blanco SCHLECHT.. The extracts of this cortex were examined particularly. Two substances of the dichloromethane extract which bind to IGF-1-RTK could be isolated. The isolation was performed by an “Extrography”, a normal-phase chromatography based method, followed by further purification steps. One of the isolated substances could be identified as aspidospermine. However, for both substances only a binding on IGF-1-RTK could be observed in consequence of the test procedure of the “Fishing of Ligands”. The inhibitory effect of these substances should be confirmed with additional tests, ideally with a cell test. In this study it was possible to isolate ingredients with new structures for inhibitors for the two targets TGT and the IGF-1-RTK. By the results of this study it could be shown that a successfully quest for new leading structures or active substances is possible in a reasonable way with the method “Fishing of Ligands” and that investigations in known and unknown plant extracts are still worthwhile.