Restoring E. coli Sensitivity for Antibiotics by blocking TolC-Mediated Efflux
Overexpression of efflux pumps is a major factor for drug resistance in Gram-negative bacteria. In E. coli, the AcrAB-TolC efflux pump complex transports antibiotics from the periplasm or cytoplasm into the external medium. As TolC deletion has been shown to result in increased susceptibilities of E. coli to several antibiotics, it may represent an attractive drug target. Recently, we have identified the first organic small molecule that effectively blocks TolC function using virtual screening in combination with experimental validation of the in silico hits by surface plasmon resonance (SPR) and electrophysiology studies. Building on these results, we propose to further develop this compound and in parallel to identify and develop novel TolC blockers within an interdisciplinary consortium. The already known blocker will be progressed in three rounds of optimisation. Each round comprises compound modifications by medicinal chemistry, assessment of TolC binding and blockage using SPR and electrophysiology, various antimicrobial studies and ADMETox profiling. Novel small molecules blocking TolC will be identified and optimised using the same experimental platform, starting with virtual screening for identification of novel compounds targeting TolC. Experimentally validated TolC blockers will be progressed in two rounds of optimisation. Ultimately, this approach will allow to assess TolC target validity for adjuvants in antimicrobial therapies and result in potent TolC blockers that may be further developed into drugs restoring E. coli susceptibility to antibiotics.
- Björn Windshügel, Fraunhofer Institute for Molecular Biology and Applied Ecology, IME, Germany (Coordinator)
- Mathias Winterhalter, Jacobs University Bremen, Germany (Partner)
- Päivi Tammela, University of Helsinki, Finland (Partner)
- Aigars Jirgensons, Latvian Institute of Organic Synthesis, Latvia (Partner)
- Matteo Ceccarelli, University of Cagliari, Italy (Observer)
Bacteria have developed several resistance mechanisms against antibiotics. One of these mechanisms involvesfastremoval of antibiotics from the bacterium. This results in an impaired effect of antibiotics. For this purpose, bacteria use so-called efflux pumps, which are large protein complexes composed of three different components.In E. coli, this complex consists of a pump located in the inner membrane (AcrB),a periplasmic adapter protein (AcrA)and the outer membrane factor TolC.The aim of our project is to develop molecules that block the outer membrane factor TolCin a way that antibiotics cannot be removed anymore from the bacterium. As a consequence, the efficacy of antibiotics can be improved significantly.In this project, an interdisciplinary team composed of research groups from Germany, Finland, Latvia, and Italy combines different experimental and computational techniques for identification and optimizationof efflux pump blockers which sensitize E. colibacteria for antibiotics. If successful, the approach will be applied also to other human pathogenic bacteria.
- bioRxiv, 2022. Uptake of aminoglycosides through outer membrane porins in Escherichia coli
- Biochimie, 2023. TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology
- Angewandte Chemie International Edition, 2021. Large Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca