Developing mass spectrometry to understand molecular mechanisms of antibacterial and antiviral drugs

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

Bacteria and viruses exploit increasingly novel and ingenious pathways to infect their human and animal hosts. Their continued 'inventiveness' has led to two of today's most serious health threats: drug-resistant bacteria and resurgent viral epidemics such as the current COVID-19 pandemic. In both cases, gaps in our understanding of drug mechanisms pose a major barrier to progress. Discovering new antibiotics is painstakingly slow, and designing new ones is difficult because we have a poor grasp of how most existing antibiotics work. We also lack detailed insight into how bacteria achieve resistance. Meanwhile, there is a critical need to better understand existing drugs that are being repurposed to treat COVID-19 infections.

In this research, we aim to fill in the critical gaps in our understanding of both antibiotic and antiviral drugs, including those showing some efficacy against both bacteria and Covid-19. We will study molecular activities taking place where these drugs act - in the membranes that surround bacteria and viruses. A challenge in this area has been the technical difficulty of membrane studies, but we have recently developed specialised techniques, using mass spectrometry, that make such studies possible.

One strand of our research will focus on Darobactin, an antibiotic that was discovered recently. We aim to understand how Darobactin (and modified forms of it) enter bacterial cells - aiding the design of new antibiotics.

As the range of effective antibiotics dwindles, another strategy is to re-engineer the antibiotics we already have. For example, a new version of the last-resort antibiotic vancomycin (with two vancomycin molecules joined together) has proven effective, and we predict its pathway for treating infection is different to that of traditional vancomycin. Uncovering this new mode of action is another focus of our work, and would inform efforts to re-engineer other existing antibiotics.

Another area of our work aims to identify new antibiotic targets. It focuses on enzymes that sit in the membranes of bacteria, building and remodelling the highly protective wall that surrounds bacterial cells. We will monitor the synthesis and assembly of cell wall components, exploring interventions that weaken these bacterial defences and could be developed into new antibiotics.

We will subsequently address one of the ways in which bacteria resist antibiotic treatment - by assembling an efflux pump to flush the antibiotic out of the cell. One pump, AceI, exports chlorhexidine, a low cost antimicrobial used widely in low-income countries - hindering use of what had been a highly effective substance for clinical hygiene. A further strand of our research is devoted to finding inhibitors for this pump. We also aim to discover how other complex drug efflux pumps are assembled, and how they export drugs. If successful, this strategy could enable the continued use of many of our existing antibiotics.

In applying our approaches to COVID-19, we are keen to improve understanding of drugs that are being repurposed for infection treatment - their mechanisms and targets, and the potential to combine them to create 'drug cocktails' (as successfully employed for HIV). We will also investigate how assembly of the virus is coordinated and controlled. As new drug targets continue to emerge for COVID-19 therapies, we will explore their drug binding interactions in combination with other molecules that reside in the membranes of cells.

This research programme will investigate, from multiple perspectives, the interactions between components of bacteria and viruses, and the drugs that can prevent them from infecting us. Together - through identification of new drug targets, design of new drugs and re-engineering and re-purposing of existing drugs - they represent a powerful strategy to help in the search for new antibiotics and to tackle COVID-19.

Technical Summary

The search for new antibiotcs has become more acute as the COVID-19 pandemic has brought a surge in antimicrobial usage that is expected to drive up resistance. Here, we seek to uncover the targets and mechanisms of action of antibiotics and antivirals - aspects that remain, for many drugs, poorly understood or completely unknown.

To investigate the mechanisms of action of antibiotics - both new and re-engineered - we have assembled a series of mass spectrometry-based assays, involving in vitro synthesis of bacterial cell-wall components and a clear indication of bacterial lipid flippase activity. Where targets are unknown, we will eject complexes directly from bacterial membranes, monitoring antibiotic responses over time and thereby identifying targets in situ.

We also aim to identify new antibiotic targets, by establishing a mechanistic understanding of the membrane-embedded enzymes and multiprotein complexes responsible for remodelling and synthesising components of bacterial cell envelopes. Though extremely difficult to study via standard biophysical approaches, these enzymes and complexes are essential for bacterial survival; a better understanding of them will be invaluable for antibiotic targeting.

For our COVID-19 research theme, we will continue our studies with existing antivirals, covering additional drugs that have shown activity in in vitro assays of virus proliferation. Probing interactions among the SARS CoV-2 main proteins, RNA and accessory factors - and, applying our expertise in membrane protein-lipid interactions, we will explore emerging COVID-19 targets, such as the two-pore cation channel in the lysosomal degradation pathway.

Bringing antibacterial and antiviral studies together allows synergistic discoveries and consideration of repurposed drugs active in both scenarios, and enables us to contribute to tackling the biggest global health challenges of our time.

Publications

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