Towards time-resolved molecular interaction assays in living bacteria

Abstract: Rare and neglected diseases such as multidrug resistant (MDR) tuberculosis, malaria and trypanosomiasis are re-emerging in Europe. New strategies are needed to accelerate drug discovery to fight these pathogens. AEGIS is a Pan-European project that combines different technologies to accelerate the discovery of molecules suitable for drug development in selected neglected diseases. This thesis is part of the AEGIS research area that considers time in a multidisciplinary approach, combining biology, physics and mathematics to provide tools to characterize biological events for improving drug development and information about the target diseases and lead compounds.Real-time cell binding assays (RT-CBA) of receptor-ligand interactions are fundamental in basic research and drug discovery. However, this kind of assays are still rare on living cells, especially in the microbiology field. In this project, we apply the same high-precision assay type on bacterial systems and explored the interior of the cell with a time resolved assay.The effect of temperature was evaluated in the RT-CBA using LigandTracer to ensure that it was possible to use the technology in a range of temperatures suitable for bacteria. A method for attaching Gram positive and negative bacteria on the surface of a normal Petri dish, showing a high reproducibly and a high cellular viability after 16 h. With these two key steps, an RT-CBA fit for microbiology is available.Next, to answer biological questions, intracellular interactions were explored by expression and validation of intracellular proteins with fluorescent tags suitable for RT-CBAs. First, we used the subunit B from the Shiga toxin (STxB) as a model to understand different aspects about the internalization processes. RT-CBAs allowed to discovery new features of STxB binding and mechanism to deliver small molecules or small proteins into cancer cells. Then, for exploring intracellular interactions, insect cells were bioengineered for evaluating the ability of small molecules to internalize and bind to its target. Using Carbonic anhydrase II – sulfonamides as a model system, the molecular interaction in the cytoplasm could be measured using a quencher label approach. The development of this kind of novel RT-CBA tools provide new information about drug candidates for targets that are not properly expressed in bacterial cells.The assays in this project can make drug design more efficient. Furthermore, the evaluation of binding activity of the new compounds developed by AEGIS, focusing on rare/neglected diseases, in a biological environment has the potential to accelerate drug discovery for the targeted emerging diseases.