Weiterentwicklung von Leitstrukturen zur Inhibierung von IpgC in Shigella und Untersuchungen zu Wechselwirkungen zwischen IpgC und seinen natürlichen Interaktionspartnern MxiC und Spa13
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Abstract
Shigellosis is an infection of the large intestine caused by Shigella bacteria. Every year 80-165 million cases of infection and hundreds of thousands of deaths occur. For treatment antibiotics are applied but the development of resistances grows. The class-II chaperone IpgC is a substantial component in Shigella’s Type-III secretion system. It interacts with a variety of proteins and is indispensable for the transport of effector proteins into the target cell. Furthermore, it acts as co-activator for the transcription factor to start the expression of effectors. Due to its essential role in the pathogenicity of Shigella, IpgC was chosen as target protein for drug design.
In previous work, three lead structures (U08, U09, U11) were determined by X-ray crystallography, which served as starting point for further development in the present work. In this work, U08 was validated as a binding molecule in IpgC by a crystal structure (PDB-Code: 8QH6). Furthermore, the three lead structures were validated by assays. Molecules based on the three lead structures that were obtained through substructure searches or in-silico fragment-growing were docked and scored. For ligands U08 and U11 which bind in the homodimer interface of IpgC two strategies were pursued that could lead to inhibition of IpgC. An enlargement of the ligands could either lead to stabilization or to destabilization of the IpgC-homodimer formation. Since the two ligands already occupied the binding pocket well and enlarged molecules mostly did not interact with the protein no promising molecules were identified. In contrast to U08 and U11, ligand U09 binds in the binding site of IpgC’s natural interaction partner IpaB. Interrupting their interaction would lead to the apathogenicity of the Shigella bacteria. For U09 several in-silico molecules were determined which were to be validated experimentally. However, the synthesis of the molecules was not possible so an experimental validation could not be attempted. A docking and pharmacophore search of the DRUID Core Facility – Cheminformatics provided six commercially available molecules based on U09. To confirm these molecules as binders they were used in soaking experiments and assays. No ligand could be validated crystallographically. One of the ligands showed a destabilizing effect on IpgC in the thermal shift assay.
Furthermore, interactions of IpgC and its natural interaction partners MxiC and Spa13 were investigated in this work. Therefore, protocols for the recombinant production and purification of both proteins were established. MxiC was obtained in good yields and used in co-crystallization experiments with IpgC. In addition, a Microscale Thermophoresis Assay was performed and a KD-value of 41.2 ± 3.3 µM determined. Crystals were also obtained but showed no diffraction in the beginning. By the application of various methods – screening of conditions, diverse seeding-methods, the use of additives, deployment of cryo- and dehydration reagents, the use of truncated IpgC1-151 – crystals were obtained that showed diffraction up to 3.7 Å. Based on the calculation of the Matthews-coefficient the crystals could comprise of the protein complex of IpgC and MxiC or only comprise MxiC. A determination of the structure is nevertheless still pending. The reason for this could be the low resolution at which the space group could not be determined unambiguously. Potential solutions in molecular replacement did not result in suitable packing of the molecules. For Spa13 the purification proved to be challenging as the protein stability seemed to depend on contamination of DNA. The protein was purified from other proteins but always contained DNA impurifications. Various methods were tested to obtain pure Spa13. Without cleaving the affinity tag which was necessary for expression of Spa13 the purity of the protein was increased. To increase the crystallization probability Spa131-141+MBP was created and purified. With all Spa13-variants crystallization experiments were conducted, as well as co-crystallization experiments with IpgC. Furthermore, Microscale Thermophoresis was applied to determine the binding site of Spa13 in IpgC. None of the methods used were able to elucidate the binding site.
As support of the experimental results and visualization of the various essential interactions of IpgC AlphaFold predictions were performed and discussed.
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Issued: 2025-12-19
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Has part: https://doi.org/10.1021/acsomega.3c07058
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FB16:Pharmazie
Language
de
Keywords
ShigellaTyp-III-SekretionssystemIpgCWirkstoffdesignMxiCSpa13Kristallisation
DFG-subjects
2.22-08 - Pharmazie
Funding
Project Information:Loewe Zentrum DRUID
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Wallbaum, Johanna: Weiterentwicklung von Leitstrukturen zur Inhibierung von IpgC in Shigella und Untersuchungen zu Wechselwirkungen zwischen IpgC und seinen natürlichen Interaktionspartnern MxiC und Spa13. : 2025-12-19.
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