Towards the development of next-generation antibiotics

A multidisciplinary team from the University of Cambridge developed a new platform to study membrane proteins in native-like environments and screen the antimicrobial effect of small molecules .

Antimicrobial resistance (AMR) represents a global health threat, yet the number of new antibiotics being developed is extremely limited. As of December 2019, the Pew Charitable Trusts identified that the antibiotic pipeline stood at less than 50 products with the potential to treat serious bacterial infections. New fundamental research is required to sustain new drug discovery and development over the coming decades. One strategy might be to evaluate and validate alternative, non-traditional therapeutic targets.

Membrane transporters with essential roles in cell growth and virulence potentially represent novel targets for antibiotic development. ATP-binding cassette (ABC) transporters couple the hydrolysis of ATP to the uptake or efflux of substrates across cell membranes in prokaryotic and eukaryotic cells. MsbA is homologous to the B-subfamily of mammalian ABC transporters and is essential in the viability and growth of Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Pseudomonas aeruginosa, and Acinetobacter baumannii. It is situated in the cytoplasmic membrane and is involved in the synthesis and maintenance of the outer membrane by mediating the export of Lipid-A, the lipid anchor of lipopolysaccharides (LPS). LPS is a large amphipathic glycoconjugate that contributes to the impermeable nature of the outer membrane. Loss of MsbA in E. coli causes rapid inner membrane disruption and cell death. In most Gram-negative bacteria, even partial inhibition of the LPS biosynthetic pathway results in significant perturbation of bacterial growth, highlighting the vital role LPS plays in outer membrane structure and stability that is required for cell survival. Consequently, the development of inhibitors targeting MsbA could provide an important strategy to advance the treatment of Gram-negative bacterial infections. The first-generation of inhibitory small molecules against MsbA provided a promising start to the development of MsbA-selective antibiotics. However, at present, these inhibitors face severe drawbacks that prevent their application in clinical settings, including low binding affinity to MsbA, interaction with animal serum, and increased membrane permeability.

To this end, a team of scientists at the Department of Chemical Engineering and Biotechnology and the Department of Pharmacology, in the research groups of Profs Róisín Owens and Hendrik van Veen, report the first platform to record electrical responses from MsbA in a supported lipid bilayer (Bali et al.). Karan Bali and Charlotte Guffick, shared-first authors of the paper, said: “The aim of the study was to generate a novel platform in which we could study membrane proteins, like MsbA, in native-like environments to facilitate the screening of inhibitory small molecules. The biosensor we created, offers much promise to accelerating the search for such molecules for MsbA and perhaps other membrane proteins. Interdisciplinary research was key in the success of this project”.

The integration of supported lipid bilayers containing purified and functionally reconstituted MsbA with bioelectronic devices is a particularly exciting new area of research that allows the precise monitoring of membrane protein activity. The active material of an organic bioelectronic device is the conducting polymer, here poly(3,4-ethylenedioxy-thiophene) poly(styrene sulfonate) (PEDOT:PSS) which, owing to its mixed conductivity (both ionic and electronic), can transduce events in this biological system into an electronic output. The combination of electrochemical impedance spectroscopy with optical and biochemical techniques provides a novel method for monitoring ATP-dependent MsbA activity on PEDOT:PSS electrodes as well as identifying and characterising the efficacy of inhibitors that block this activity. 

Substantial biochemical characterisation of MsbA had been carried out.  Furthermore, two MsbA inhibitors were available for research purposes, but are sadly not clinically relevant. One of these (G907) was applied in our study as a tool to provide proof of concept for our biosensor.

The authors report the first platform to record electrical responses associated with MsbA activity in a supported lipid bilayer.  The signals generated in the electrochemical impedance spectroscopy measurements were correlated with biochemical assays of MsbA activity. The microelectrode array system provided a quantitative readout of the blocking effect of the drug G907, thus showing the compatibility of the devices with MsbA and the potential of the system for inhibitor development.

This work holds much exciting potential. Electrical measurements are well-established for ion channels. This study widens the application of this approach to ABC transporters that exhibit much smaller electrical signals in their transport reactions. The application of our biosensor holds much promise for screening future antimicrobial compounds that target MsbA.

The exact contribution of cation and lipid transport by MsbA to signal generation in the device is still unknown, and further work will focus on discerning the detailed characteristics of this ion permeation reaction. Further biochemical study into the transport pathways in MsbA is required to explore our understanding of how this protein is active in this system. These answers will not only aid in the development of antibiotics that can inhibit MsbA, but also increase the understanding of how MsbA operates in the physiological context of the cell.

Karan Bali and Charlotte Guffick were funded by EPSRC-Doctoral Training Partnership (DTP) and BBSRC-DTP-Targeted PhD studentships, respectively.

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Reference:

Bali K., Guffick C., McCoy R., Lu Z., Kaminski C.F., Mela I., Owens R. M. and Hendrik W. van Veen H.W.” Biosensor for multimodal characterization of an essential ABC Transporter for next-generation antibiotic Research”, ACS Appl. Mater. Interfaces 2023, 15, 10, 12766–12776 https://doi.org/10.1021/acsami.2c21556