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COMMANDEER: Disrupting microbial resistance using rationally designed signalling molecules

Periodic Reporting for period 1 - COMMANDEER (COMMANDEER: Disrupting microbial resistance using rationally designed signalling molecules)

Reporting period: 2015-07-01 to 2017-06-30

Lung disease is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Over time, there has arisen an increasing appreciation for the polymicrobial nature of such infections and the potentially important role for interspecies interactions in influencing both bacterial virulence and response to therapy. Chronic colonisation and infection by P. aeruginosa occurs in 80% of CF patients by 18 years of age, and patients are commonly co-infected by other pathogens such as B. cenocepacia and S. maltophilia. B. cenocepacia has emerged as an important pathogen frequently associated with high mortality in CF patients. This pathogen produces signal molecules of the DSF family, which are simple fatty acids. Molecules that target cell-to-cell signalling (Quorum Sensing/QS) have been proposed as a strategy that could be added to the existing arsenal in treating bacterial infections.

Traditionally, antibiotics have been used to combat bacterial infections. Although these compounds were initially highly effective, they resulted in substantial stress on the target bacterium, which rapidly selected for multi-antibiotic resistant bacteria. Additionally, the number of new effective antibiotics brought to market is steadily falling.

The overall aim of COMMANDEER was to build upon a very promising pilot study demonstrating that DSF analogue compounds interfere with cell-to-cell signalling processes in P. aeruginosa making them significantly more susceptible to antibiotic treatment. We have identified antagonists which can be co-administered with current antibiotics forming the basis of future therapies for CF. We carried out a deeper investigation of the relationship between structure and activity, ability to permeate biofilms and spectrum of activity of these analogues. A collaborative and multidisciplinary approach was used, incorporating chemical and biomolecular techniques, to generate a library of bioisosteric analogues of DSF, evaluate their ability to interfere with DSF cell-to-cell signalling processes in bacteria that colonize the CF lung and select those compounds which improve the efficacy of antibiotic treatment of bacterial infections.
The initial focus of this work was the optimisation of the synthetic route to the BDSF (Burkholderia Diffusible Signal Factor) and DSF (Diffusible Signal Factor) sulfonamide derivatives. The original route used a modified Horner-Wadsworth-Emmons reaction to install the critical cis-alkene double bond. This reaction leads to a mixture of isomers. Accordingly, any new synthetic route should ideally overcome these difficulties. A number of BDSF sulfonamide analogues were prepared using optimised chemistry. These compounds all span a range of pKas and allow for a structure activity analysis based on a comparison of activity and acidity of the bioisosteric sulfonamide. A similar approach was utilised for DSF analogues.

It has previously been found that antibiotics which readily permeate biofilms are the most effective against biofilm-producing bacteria. During the course of this project, a study was undertaken to develop a model system using high performance liquid chromatography (HPLC) retention times as an method of determining lipophilicity. This data is being mapped to biological activity thereby ascertaining the correlation between efficacy and lipophilicity.

Knowing that DSF and BDSF quorum sensing is shared across many different microbial species, we sought to maximise the chances of success of our compound library by seeking out researchers who could use these molecules as potential investigative tools. While in depth biological testing is still on-going, we have already received very promising results. In the case of P. aeruginosa, we have identified that a number of our compounds are indeed active and inhibit biofilm formation at micromolar concentrations. Interestingly, when the same library was tested by our collaborators in Spain, they also found that the same compounds inhibited biofilm formation in S. maltophilia again at micromolar concentrations. This is an especially exciting result as it demonstrates that our compounds have potential application across several microbial species as general agents for disrupting biofilm formation and antibiotic resistance.

This project also included an industry secondment. During this time, we worked to further optimise the synthetic route to these compounds, with a particular emphasis on scale-up of the current route. The co-administration of our active compounds with current antibiotics was investigated. Results from these studies are promising as they indicate that the Minimum Inhibitory Concentration (MIC) of a current antibiotic was reduced 6 fold when one of compounds was co-administered with that same antibiotic. This, again, is an exciting result, as it demonstrates that our compounds do indeed improve the efficacy of existing antibiotics, one of the central aims of this project.
COMMANDEER marks the first concerted effort to identify inhibitors of DSF using a rational approach, preparing and testing a library of bioisosteres. We have already determined that our compounds inhibit biofilm formation in P. aeruginosa at micromolar concentrations. Significantly, we have also determined that these same compounds similarly inhibit biofilm formation in S. maltophilia also at micromolar concentrations. These observations increase both the scientific significance and commercial attractiveness of our compounds, as they constitute potential novel treatments against multiple bacterial infections.

More specifically, in the case of CF patients, it has been observed these patients are invariably co-infected with multiple bacterial species. These infections typically include both P. aeruginosa and S. maltophilia – the two species which have proven sensitive to our compounds thus far. Therefore, our compounds are likely to be especially beneficial to CF patients who have acquired polymicrobial infections. This will lead to improved quality of life for patients with a disease which is especially prevalent in Europe.

A major impetus for this project is growing problem of bacterial resistance and reduced effectiveness of current antibiotics. By co-administering our molecules with existing antibiotics, we could significantly improve their efficacy and give these antibiotics a greatly extended “shelf life”. This would mean that drugs that were otherwise consigned to the dustbin due to bacterial resistance can again be effective therapies. Gratifyingly, over the course of this project, we have proven that co-administration of our compounds with an existing antibiotic results in a 6 fold reduction in the Minimum Inhibitory Concentration (MIC).

This project has boosted University College Cork’s existing research profile in bioactive molecules and has enabled us to expand into the development of novel anti-microbial agents. These compounds can be licensed to the European pharmaceutical sector in future. We have established new collaborations with biologists in Europe and beyond and we will maximise the impact of this fellowship by sharing our compounds with several collaborators. This project has increased European research output with the publication of high impact publications in peer-reviewed journals, presentations at national and international conferences, and attracting external funding to continue this research.
Basis of biofilm formation