Periodic Reporting for period 1 - AmCaLiStat (Modular Design of Bacterial Lipid Mimics for Next-Generation Antimicrobials)
Période du rapport: 2021-02-22 au 2023-02-21
The action “Modular Design of Bacterial Lipid Mimics for Next-Generation Antimicrobials” aimed to drive the discovery of new compounds active against bacteria, to tackle the growing antimicrobial resistance (AMR) crisis. The lack of new antibacterial compounds appearing in the last 20 years is a major cause for concern for all of society. Many older treatments are now defunct and cannot be used for infections due to the resistance that bacteria have evolved against them. As a result, common infections can once again become fatal. Therefore, it is crucial that new treatments are devised which prevent bacteria from becoming resistant, which this project aims to address. Killing bacteria requires some target component of the cell to be disrupted, to either kill the cells or prevent them multiplying. Targets often include specific parts of the cellular machinery, such as enzymes involved in protein or DNA production. However, through evolution bacteria can adapt several strategies, such as changing the structure of the target or pumping the drug out of the cell.
One route to kill bacteria which is more difficult to develop resistance against is to target the cell membrane: the envelope of lipids that protects it from the surrounding environment. However, the major challenge in targeting a membrane is designing a compound which only affects bacterial cell membranes, while leaving membranes from other types of cells unharmed, i.e. the host animal and plant. One way in which bacterial membranes differ from human cell membranes is in the type of lipid molecules that build them, and also the number of layers of lipids. Therefore, finding molecules that bind and break bacterial lipid membranes selectively requires testing many combinations of compounds. To maximise the chances of locating the “golden bullet” molecule against bacteria, I adopted a “high-throughput” approach to screen a large range of compounds. In this, more than 100 different combinations of molecular building blocks are trialled against bacteria, each of which has a subtly different shape. In this way, hit compounds can more quickly be identified. Previously, it has not been well understood how the shape of a molecule affects its antibacterial activity, nor why shape might be an important factor. This project was designed to study a huge library of molecules with different shapes to gain insight into which molecules can disrupt bacterial membranes and destroy their structure, whilst leaving human or other host cells unharmed.
The main objectives of this project centre around the design of new antibacterial “lipidoids”: molecules that resemble the components of cell membranes but which can also disrupt them. Objectives of the action were to i) synthesise a library of lipidoids and characterise their structure and purity, ii) test the antibacterial properties of the compounds against a range of bacterial species and their toxicity to mammalian cells, and iii) garner information on the mechanism of action of the compounds against biological membranes.
One route to kill bacteria which is more difficult to develop resistance against is to target the cell membrane: the envelope of lipids that protects it from the surrounding environment. However, the major challenge in targeting a membrane is designing a compound which only affects bacterial cell membranes, while leaving membranes from other types of cells unharmed, i.e. the host animal and plant. One way in which bacterial membranes differ from human cell membranes is in the type of lipid molecules that build them, and also the number of layers of lipids. Therefore, finding molecules that bind and break bacterial lipid membranes selectively requires testing many combinations of compounds. To maximise the chances of locating the “golden bullet” molecule against bacteria, I adopted a “high-throughput” approach to screen a large range of compounds. In this, more than 100 different combinations of molecular building blocks are trialled against bacteria, each of which has a subtly different shape. In this way, hit compounds can more quickly be identified. Previously, it has not been well understood how the shape of a molecule affects its antibacterial activity, nor why shape might be an important factor. This project was designed to study a huge library of molecules with different shapes to gain insight into which molecules can disrupt bacterial membranes and destroy their structure, whilst leaving human or other host cells unharmed.
The main objectives of this project centre around the design of new antibacterial “lipidoids”: molecules that resemble the components of cell membranes but which can also disrupt them. Objectives of the action were to i) synthesise a library of lipidoids and characterise their structure and purity, ii) test the antibacterial properties of the compounds against a range of bacterial species and their toxicity to mammalian cells, and iii) garner information on the mechanism of action of the compounds against biological membranes.
Research was conducted in 3 work packages (WPs), each containing a number of work tasks. WP1 comprised the synthetic aspects of the project, involving process optimisation to make the lipidoid compounds in high purity, which was completed in the first 6 months. WP2 concerned the antimicrobial testing, which also required some optimisation to adapt to the screening of a high number of compounds. These experiments were conducted across the whole duration of the MSCA. In addition, WP2 included toxicity testing of lipidoids against mammalian cells. In this WP, the Fellow gained several new research skills and techniques beyond his research background. Finally, WP3 was aimed at understanding how the shape of lipidoids changes depending on molecular structure, and how this impacts on its activity against biological membranes. WP3 also included a trip to the European Synchrotron Radiation Facility (ESRF) for high quality X-ray scattering analysis. The fellow also hired a masters student for 10 months, who contributed significantly to WP2 and 3.
Work from WP1 and WP3, concerning the synthesis, self-assembly, and molecular shape analysis of the lipidoids was recently accepted for publication in the nanomaterials journal Small. This article outlined the unexpected discovery that a number of different lipidoid structures could assemble into rare cubic liquid crystal phases, and uncovered a new type of molecular stacking within the phase. The second article, which has already been submitted to another journal for publication, from the key results from WP2 and WP3, outlines the wealth of information that was gained by high-throughput screening of the antibacterial properties of lipidoids. The design rules that were discovered could inform other researchers in the field. A third journal relating to the activity of the compounds against different bacteria is also planned to be submitted in the next 2 months. The fellow also co-wrote a book chapter in an upcoming volume on “Membrane Shape and Biological Function”, in which the research findings about lipidoids from the fellowship are summarised. As part of the dissemination, the Fellow attended a total of 6 conferences: 2 international (US and UK), 2 remote, and 2 domestic, in addition to presenting research at departmental seminars.
Work from WP1 and WP3, concerning the synthesis, self-assembly, and molecular shape analysis of the lipidoids was recently accepted for publication in the nanomaterials journal Small. This article outlined the unexpected discovery that a number of different lipidoid structures could assemble into rare cubic liquid crystal phases, and uncovered a new type of molecular stacking within the phase. The second article, which has already been submitted to another journal for publication, from the key results from WP2 and WP3, outlines the wealth of information that was gained by high-throughput screening of the antibacterial properties of lipidoids. The design rules that were discovered could inform other researchers in the field. A third journal relating to the activity of the compounds against different bacteria is also planned to be submitted in the next 2 months. The fellow also co-wrote a book chapter in an upcoming volume on “Membrane Shape and Biological Function”, in which the research findings about lipidoids from the fellowship are summarised. As part of the dissemination, the Fellow attended a total of 6 conferences: 2 international (US and UK), 2 remote, and 2 domestic, in addition to presenting research at departmental seminars.
Results from this project have expanded the state of the art for a number of research fields in several directions. Firstly, the new discoveries about the way that lipidoid molecules assemble together into liquid crystals were largely contradictory to what known about lipid-like materials. In particular, the observation of rare cubic liquid crystal phases in many lipidoids, which consist of complex intertwined networks that persist in 3 dimensions, and are useful in many material applications beyond what was studied here. Therefore, the discovery of a whole new set of molecules from which these liquid crystals can be made could inspire research in diverse scientific fields and will have a significant impact on the direction of the fellow’s future research career.
The investigations into how lipidoids of different shapes disrupt biological membranes also yielded some ground-breaking discoveries. After screening almost 150 different compounds, it was clear that there was no single molecular feature which granted the most effective antibacterial molecules. Instead, particular combinations of features led to the highest activity against bacteria, and this was strongly connected with the specific shape of the molecule. In the future, these shape design rules will inform other researchers working on antibacterials that target membranes. In future, these new antibacterial molecules could be used directly to treat infections, as disinfectants, or as coatings for medical materials that are prone to contamination and the spread of AMR. Alternatively, antibacterial lipidoids could be used in combination with existing antibiotics, as a means to enhance their solubility and delivery into the site of action. New antibacterial platforms such as this are crucial within healthcare environments where AMR is most prevalent. As AMR is responsible for 1 m lives globally per year and is often named “the silent pandemic”, results that increase the breadth of knowledge about methods to overcome pathogenic bacteria will have a broad societal impact.
The investigations into how lipidoids of different shapes disrupt biological membranes also yielded some ground-breaking discoveries. After screening almost 150 different compounds, it was clear that there was no single molecular feature which granted the most effective antibacterial molecules. Instead, particular combinations of features led to the highest activity against bacteria, and this was strongly connected with the specific shape of the molecule. In the future, these shape design rules will inform other researchers working on antibacterials that target membranes. In future, these new antibacterial molecules could be used directly to treat infections, as disinfectants, or as coatings for medical materials that are prone to contamination and the spread of AMR. Alternatively, antibacterial lipidoids could be used in combination with existing antibiotics, as a means to enhance their solubility and delivery into the site of action. New antibacterial platforms such as this are crucial within healthcare environments where AMR is most prevalent. As AMR is responsible for 1 m lives globally per year and is often named “the silent pandemic”, results that increase the breadth of knowledge about methods to overcome pathogenic bacteria will have a broad societal impact.