Periodic Reporting for period 1 - G4-AntiBac (Targeting G-quadruplex DNA Structures in Bacteria to Combat Antimicrobial Resistance)
Periodo di rendicontazione: 2021-04-06 al 2023-04-05
Infectious diseases remain a leading cause of death worldwide and a developing resistance to antimicrobial drugs is a significant threat to humans. The World Health Organization has identified antimicrobial resistance as one of the most important problems that affects human health. Pseudomonas aeruginosa, is a dreadful Gram-negative bacterium pathogen associated with severe acute and chronic human diseases. The emergence of a number of Gram-negative pathogens, including a number of strains of P. aeruginosa, that are resistant to the front-line antibiotic therapies is a global concern – accounting 10 – 15 % of nosocomial (hospital-acquired) infections world-wide, and the pipeline of antibiotics is essentially empty. P. aeruginosa has been ranked by WHO in the top three of organisms that are critical and needing immediate attention. Therefore, it is urgent to identify and validate alterative biomolecular targets to develop new antibacterial agents capable of either killing these multi-drug resistant (MDR) bacteria or make them susceptible again against current antibiotics. Recently, bioinformatic studies have shown that G-quadruplex DNA (G4 DNA) sequences are prevalent in bacteria, particularly in gene promoter regions of pathogenic bacteria. Furthermore, a number of recent studies have demonstrated that quadruplexes do indeed play a role in virulence of Gram-negative bacteria such as E. coli. Therefore, G4 DNA structures are potentially interesting new targets for the development of antibacterial drugs. The proposed project was aimed at developing a new approach to tackle antimicrobial resistance through targeting G4 DNA structures of relevance to bacteria. To achieve this, following scientific objectives have been proposed.
1. Develop small molecules to target G4 DNA structures of relevance to P. aeruginosa.
2. Fully characterise the interaction of a library of compounds against the three G4 structures (i.e. from murE, ftsB and MexC) and study their selectivity over other DNA topologies
3. Establish the activity and uptake profile of new G4 DNA binders against P.aeruginosa strains.
4. Determine the ability of new molecules to modulate the expression of murE, ftsB and MexC genes.
1. Develop small molecules to target G4 DNA structures of relevance to P. aeruginosa.
2. Fully characterise the interaction of a library of compounds against the three G4 structures (i.e. from murE, ftsB and MexC) and study their selectivity over other DNA topologies
3. Establish the activity and uptake profile of new G4 DNA binders against P.aeruginosa strains.
4. Determine the ability of new molecules to modulate the expression of murE, ftsB and MexC genes.
This report presents synthetic, biophysical and biological studies carried as part of the proposed project to address the proposed scientific objectives – aimed at developing novel anti-bacterial agents. As part of this project, new metal complexes that could potentially exhibit selective bacterial uptake and toxicity and show affinity towards G4 DNA have been designed. The basis for designing these new complexes was based on the proven ability of metal-salphen complexes to bind G4 DNA sequences. In addition to synthesis, the biophysical studies on the bacterial G4 DNA sequences have been performed. Finally, in vitro assays to assess the bacterial toxicity of current and previously developed metal-salphen complexes, with G4 DNA binding ability, have been performed against a wide range of Gram-positive and Gram-negative bacterial strains. The studies performed in this project suggest that only two out of seventeen previously synthesised complexes have shown modest activity against the Gram-positive bacterial strains. The three new metal salphen complexes synthesised in this project have also not shown activity against the bacterial strains. The CD spectroscopic studies on the modified DNA sequences from bacterial genes show that two out of three DNA sequences from these genes form a G4 structure. The FRET melting studies, using three previously synthesised Nickel salphen complexes, on the modified bacterial ftsB DNA sequence showed that two complexes (TK48 and Ni-NMe3+) showed stabilisation of G4 structure of ftsB (at 5:1 ratio of compound to DNA) with higher affinity compared to that observed for the third complex (Ni-Pip). Further studies are required to exploit and disseminate the data obtained in this project.
The scientific impacts of the project were aimed at identification and validation of alternative biological targets for developing new antibacterial agents capable of killing the multi-drug resistant (MDR) pathogenic bacteria. Although further studies are required, the outcomes of this project show the potential of metal-salphen complexes to target G4 DNA structures of relevance to anti-microbial resistance. This data provides basis for future development of new metal-salphen complexes with improved bacterial uptake and ability to target G4 DNA sequences in the promoter regions of bacterial strains and potentially down regulate the genes responsible for anti-microbial resistance.
The other impacts of the projects involve improving the future career prospects of the researcher after the fellowship and effective dissemination of the results obtained in this project. Securing this fellowship has broaden my scientific expertise, strengthen my skills and knowledge in chemical biology related disciplines, and helped me to become a high-level researcher in both chemistry and biology. The training I have received from postdoctoral fellow development centre (PFDC) at Imperial College, contributed significantly for my professional and career development and I was able to secure a full-time permanent job within the Government sector in the UK, where I am currently utilising the technical and professional skills gained from this fellowship project at Imperial. Furthermore, working in a highly interdisciplinary environment at Imperial provided me an excellent opportunity to establish new professional contacts and collaborative network.
The other impacts of the projects involve improving the future career prospects of the researcher after the fellowship and effective dissemination of the results obtained in this project. Securing this fellowship has broaden my scientific expertise, strengthen my skills and knowledge in chemical biology related disciplines, and helped me to become a high-level researcher in both chemistry and biology. The training I have received from postdoctoral fellow development centre (PFDC) at Imperial College, contributed significantly for my professional and career development and I was able to secure a full-time permanent job within the Government sector in the UK, where I am currently utilising the technical and professional skills gained from this fellowship project at Imperial. Furthermore, working in a highly interdisciplinary environment at Imperial provided me an excellent opportunity to establish new professional contacts and collaborative network.