Targeting cell wall glycans: an untapped approach for therapeutics and diagnostics to combat antimicrobial resistance?

Tuberculosis (TB) is one of the leading causes of death worldwide from a single infectious agent. In 2013, 1.5 million people died from TB and 9 million people became infected, with an estimated 2 billion latent global infections. TB is caused by the bacterium Mycobacterium tuberculosis (Mtb). Treatment of TB requires a lengthy, complicated drug regimen, which means that many patients fail to comply with the recommended course of treatment. Poor compliance, in turn, has led to the evolution of multi-drug resistant, extensively drug-resistant and totally drug-resistant TB (MDR-TB, XDR-TB and TDR-TB). MDR- and XDR-TB strains are difficult and costly to treat. In some instances XDR and TDR TB, few, if any, therapeutic agents remain. In conjunction with HIV infection, this deadly infection now presents us with a global time bomb that could devastate societies across the globe. The problem of TB is not confined to developing countries and in the last decade cases of TB have doubled in the UK with London now known as the TB capital of Western Europe. Mycobacterium bovis is a growing threat to livestock and can spread to humans causing TB

There have been no new drugs to treat TB for > 40 years and there is thus a clear and pressing need for better, innovative, methods to treat and diagnose TB in the hope that we can find new weapons in the fight against this fearful pathogen.

Mtb is unique from most bacteria that cause infection in that it has a distinctive, unusual cell wall. The cell wall has an unusually high fat and sugar content that provides a barrier to drugs, such as penicillin, and protects Mtb from the immune defence system.

The majority of current antibiotic drugs are reliant on targeting a single biological process inside the bacteria; this approach, however, has lead to the resistance that is now widespread. As these targets are inside the bacteria, the drugs must be able to cross the cell wall to function, which in the case of TB, is hugely challenging.

The aim of this innovative project is to challenge the current beliefs that exist in the field of anti-microbial research. Most drug searches focus on screening libraries of small molecules that fulfill a limited range of criteria (known as Lipinski's rule of 5) and focus on cell-well permeation potential.

In this study the work will be carried out at the University of Warwick. The aim is to identify whether the unique cell wall of mycobacteria can itself prove to be a new target for therapies and/or diagnostics - essentially turning its own cell wall against itself. The objective of this work is to identify specific molecules that are able to bind to the unique cell wall sugars in the cell wall of mycobacteria and then to understand how this affects and disrupts essential cellular processes. This elegantly bypasses the need for drugs to reach an intracellular target - a major obstacle for anti-tubercular drug efficacy - and will exploit the unique nature of Mtb's cell wall to develop weapons against itself. The specific cell-wall binding process will also lead to potential diagnostic agents, increasing the value of this work.

Potential applications and benefits resulting from this work are in the development of a new set of tools that can be used for diagnostics, and in identifying new drug targets for TB treatment. This work will stimulate untapped avenues and lead to paradigm shift in thinking to tackle the huge challenge of AMR.

The cell wall of Mycobacterium tuberculosis has an essential role in its pathogenesis and protection from environmental stresses and common antibiotics. The aim of the project is to undertake an integrated approach to identify probes that specifically cross-link cell wall glycans in mycobacteria and understand how this cross-linking affects the cell's chemical biology to identify new targets for TB drug targeting and diagnostics. Firstly, we will build on studies using metabolic labeling to guide the design of optimal dendrimer-based probes that covalently cross-link the cell wall glycans using bio-orthogonal 'click' chemistry. We will then investigate the effects this cross-linking has on the physiological state/chemical biology of mycobacteria. Growth kinetics will be monitored, effects of nutrient uptake, acquisition and metabolism assessed. Membrane stability will be investigated and susceptibility of cross-linked bacteria to TB drugs determined by standard kill-curves. Shot-gun proteomic studies will profile the changes in protein expression. In addition build-up/depletion of a subset of metabolites (e.g. glucose) will be monitored (GC(/LC)-MS). Guided by these results, a range of molecules that bind specifically to glycans will be screened to find purpose-built probes that will be incorporated onto the optimized dendrimer scaffold. The kinetics and quantification of probe incorporation into the cell wall (Raman, IR, fluorescence, GC/(LC)-MS advanced imaging) investigated. Specificity and sensitivity of these probes for mycobacteria will be explored through development of 'on/off' pro-fluorescent probes.

A multi-disciplinary approach to will be exploited to gain a full fundamental understanding of the effects of cross-linking the mycobacterial cell wall. This will be a major advance in understanding role of cell wall glycans and anticipated that this will be useful for development of new therapies/diagnostics and challenge existing paradigms.

Throughout the project the potential for exploitation and dissemination of the research will be carefully monitored.

This project seeks to provide innovative methodologies to identify new targets in mycobacteria with the long-term goal leading to new therapies and diagnostics for Mycobacterium tuberculosis and the zoonotic disease Mycobacterium bovis. Multi-drug (MDR), extensively-drug (XDR) and totally-drug (TDR) resistant strains of tuberculosis are spreading at an alarming rate. This threat is a 'ticking time bomb' and the threat from antibiotic resistance is ranked alongside the threat of terrorism to the Nation. New weapons are therefore needed to combat the problem of rapidly emerging drug resistant strains.

The potential beneficiaries are many and include:

1. Individuals with TB strains resistant to the current drugs used clinically will benefit through decreased mortality rates. In the case of XDR- and TDR-TB few drug-regimens exist for successful treatment. It is possible that our approach to identifying novel targets in the bacteria will result in shorter treatment time - resulting in improved quality of life, along with improved financial benefits for individuals/family/society affected by the disease.

2. Developing nations with high burdens of TB are trapped in a cycle of poverty since individuals are unable to work due to the debilitating disease. Improving treatment times and enabling people to work will improve the long-term health and economic prosperity of these nations.

3. Global travel threatens health security and economies of nations through the spread of resistant pathogens. New tools in the fight against drug resistant strains that have novel mechanisms of actions may prevent the emergence of drug-resistant strains occurring. This will lead to more effective treatment options and control pathogen spread.

4. Agriculture/farming communities may benefit through the production of a cheap rapid diagnostic test for M. bovis that threatens farming communities, both in the UK and globally. The global economic loss due to M. bovis infection in cows is immense. In addition, M. bovis is a zoonotic disease which can cause TB in humans, predominantly through close contact with cows and through drinking unpasteurised milk. Rapid, low-cost detection, particularly in resource-poor developing countries would be transformative.

5. Academics involved in a wide range of disciplines will be able to use the outputs from this cross-disciplinary research and apply it in own specific fields, generating new lines of research enquiry across the scientific community.

6. Companies interested in drug development/new diagnostic development will potentially benefit from this research in exploiting the tools and methods developed during this project.

7. Early career researchers will benefit from enhanced skill provision: the early career researchers involved in the project will learn to think and communicate in different scientific languages which will only serve to benefit of AMR research community whilst contributing to much needed skills in the workplace.

8. The public: We plan to continue to ensure that the public is aware of the problems of AMR and are educated through our outreach plans. During the time-frame of the award we plan to embark on a outreach program to ensure 'next generation of scientists are inspired and educated. We will also liaise with the press office to ensure research is promoted in a timely manner.

9. Industry engagement will be driven by emerging patentable technology. The research findings will also help drive policy through longer term interactions with e.g NICE, WHO, TB-Alliance.

10. Overall, the work in this project provides a step change in thinking regarding the way we identify targets for anti-microbial therapy and new techniques for diagnostics. This will benefit all academics, industrialists and global scientific community.