Novel boronic acids for combating anti-microbial resistance in P.aeruginosa

This article is based on a poster originally authored by Arshnous Marandi, Vishal Gulati, Stéphane Mesnage, Ruhksana Quyoum and Asad Fallah. 

The silent pandemic of antibiotic resistance is growing right before our eyes. 1.27 million people die from Antimicrobial Resistance (AMR) every year. 7.7 million people die from bacterial infections every year.¹ Furthermore, bacteria see no borders; we all are connected (humans-animals-environment) - so the challenge goes beyond just human health.

Pseudomonas is a type of Gram-negative bacterium that is commonly found in the environment such as soil and water. Pseudomonas aeruginosa is the variation that can cause infections in humans, resulting in infections in blood, lungs (pneumonia) and other parts of the body after surgery.²

Vulnerable patients are most at risk from infection, such as cancer patients, newborns, and people with severe burns, chronic lung disease or cystic fibrosis. Unfortunately, it is estimated to be one of the major causes of hospital-acquired infection, with 10,000 cases reported each year in the UK alone.³ For some multidrug-resistant types of Pseudomonas aeruginosa, treatment options might be limited.

β-lactam antibiotic effect is hampered by β-lactamases. Boron-containing inhibitors have been shown to be potent serine-β-lactamase inhibitors at the Penicillin Binding Protein (PBP). Therefore, developing new classes of boron-based antibiotics can be a potential route for combating P. aeruginosa.⁴

Scheme of Inhibitors Interfering with Bacterial Cell Wall Synthesis. The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boroncontaining inhibitors are potent serine-β-lactamase inhibitors.⁵ The PBP is a monofunctional peptidoglycan transpeptidase, which is associated with cell division, where it cross-links stem peptides of polymerized molecules, essential for bacterial survival. By interfering with this mechanism the cell wall loses its stability and ultimately kills the bacteria.^6,7

Hypothesis

Our aims were to use rational drug design, pharmacophore analysis and structure-based virtual screening methods to identify novel covalent small molecule inhibitors which will target the PBP of P. aeruginosa. The novel molecules were identified, some were synthesized accordingly, and their antimicrobial activities were and (are being) evaluated in vitro using various gram-positive and negative strands of bacteria such as P. aeruginosa and E. coli.

Method

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).⁸ 

Result

a) Boron containing groups have the ability to change hybridization states from sp2 to sp3. The sp3 form of B mimic tetrahedral transition states and thus bind tightly to the PBP active site. b) Demonstrating the PBP binding site shared with three bacterial organisms P. aeruginosa, Staphylococcus aureus, Neisseria gonorrhoea. These data from Protein Data Bank (PDB) have been applied for structural studies and ligand design to allow elucidation of the molecular details of PBP inhibition and designing more a broad-spectrum antibiotic for all these bacteria.

Example of boronic acid compound (AF 11-4121) and its antimicrobial activity. a) Crystal Structure of PBP from P. aeruginosa (PDB code: 3PBN) shown in blue and the boronic acid compound AF-11-4121 docked in the binding site highlighted in yellow which deactivates the enzyme; (b) Inhibition curves against P. aeruginosa and E. Coli. An increase in the concentration of AF-11-4121 results in acceleration of bacterial death.

Conclusion

• P. aeruginosa is an opportunistic pathogen that is a leading cause of morbidity and mortality in immune-compromised patients.
• Eradication of P. aeruginosa has become increasingly difficult due to its extraordinary capacity to resist antibiotics.
• Boron-containing compounds represent an exciting new class of antibiotics.
• Preliminary in vitro studies have shown that AF 11-4121 has potent antibacterial properties.
• Further analogs are currently under evaluation and being synthesized to be tested.

Future studies

• Further studies investigating the efficacy of these compounds at different concentrations as well as in combinations are tested.
• Bacterial resistance to the lead compounds is also evaluated.
• Further kinetic assays are under development and are looking into PBP assays to conduct structure active relationship (SAR) studies.
• Efficacy assays are also in progress, looking into different strains of bacterium.
• Optimization of the leads is also introduced to examine if the compounds can have dual activity against different classes of bacteria.

References

1. Holloway B. 15 things that need to happen in 2023 - for a robust response on antibiotic resistance! – 2022. ReAct. 2022. Available at www.ReAct.org
2. Aguilar-Rodea P, Estrada-Javier EL. New Variants of Pseudomonas aeruginosa High-Risk CloneST233 Associated with an Outbreak in a Paediatric Hospital. Microorganisms. 2022 Aug 1;10(8):1533.
3. Pseudomonas aeruginosa: guidance, data and analysis. GOV.UK. 2018. Available from: https://www.gov.uk/government/collections/pseudomonasaeruginosa- guidance-data-and-analysis
4. CDC. Pseudomonas aeruginosa in Healthcare Settings. Centres for Disease Control and Prevention. CDC; 2019.
5. Newman H. et al. High-Throughput Crystallography Reveals Boron-Containing Inhibitors of a Penicillin-Binding Protein with Di- and Tricovalent Binding Modes. JMedChem. 2021;64:11379−11394.
6. Sauvage, E.; Kerff, F.; Terrak, M.; Ayala, J. A.; Charlier, P. The Penicillin- Binding Proteins: Structure and Role in Peptidoglycan Biosynthesis. FEMS Microbiol. Rev. 2008, 32, 234−258.
7. Adam, M et al. The Bimodular G57-V577 Polypeptide Chain of the Class B Penicillin-Binding Protein 3 of Escherichia Coli Catalyzes Peptide Bond Formation from Thiolesters and Does Not Catalyze Glycan Chain Polymerization from the Lipid II Intermediate. J. Bacteriol. 1997, 179, 6005−6009
8. Minimum Inhibitory Concentration (MIC). Emery Pharma. 2023. Available from: https://emerypharma.com/solutions/cell-microbiology-services/minimuminhibitory-concentration/