Thesis title: Alternative Therapeutic Strategies in the Fight Against Antibiotic Resistance
Abstract
The excessive and inappropriate use of antibiotics is among the primary drivers of antimicrobial resistance, underscoring the urgent need to develop innovative antimicrobial strategies. Among the approaches currently considered most promising are the use of antibiotic adjuvants, which restore the activity of well-established antibiotics, and the design of structurally modified antimicrobial peptides, which aim to enhance antimicrobial efficacy while overcoming inherent limitations such as instability and cytotoxicity. Being structurally “optimized” by evolution to perform specific biological functions and interact with defined molecular targets, natural compounds represent an invaluable resource for the discovery and development of molecules active against pathogens. The thesis is divided into two major parts, A and B, both of which are built upon naturally derived compounds with well-documented antimicrobial activity and aim to develop molecules that could serve as novel strategies for the treatment of resistant bacteria.
Part A, the main section, focuses on the design and development of diterpene-based colistin adjuvants. Colistin is a last-resort antibiotic used to treat multidrug-resistant Gram-negative bacterial infections. In Pseudomonas aeruginosa, resistance primarily arises through the covalent addition of L-aminoarabinose to the lipid A moiety of lipopolysaccharide. Since the glycosyltransferase ArnT catalyses the final step of this pathway, its inhibition represents a promising strategy to overcome colistin resistance. The natural tetracyclic diterpene ent-beyer-15-en-18-oxalate (FDO) was recently identified and patented for its novel colistin adjuvant activity via ArnT inhibition. However, its very low abundance in the plant, lack of chromophores and poor aqueous solubility have limited its full characterization and pharmaceutical development.
Therefore, to complete the preclinical studies on the hit FDO, we first optimized its isolation from the plant and developed strategies to improve its poor biopharmaceutical properties.
The isolation of the precursor FDA from plant material was optimized using ultrasound-assisted extraction (UAE) combined with automated flash chromatography, yielding FDA with higher purity and improved yield, which enabled the synthesis of FDO and its reduced analogue, FDO-H, in quantitative yield. These compounds will subsequently be evaluated as anti-biofilm agents in combination with colistin against P. aeruginosa strains, as the pathogenicity and antibiotic tolerance of this bacterium are enhanced in biofilm form.
In parallel, to address the low aqueous solubility of these diterpene derivatives, liposome-mediated co-delivery systems for colistin and ent-beyerene-based compounds were developed, using isostevic acid (ISA) as prototype of diterpenoid ArnT inhibitors. Liposomal co-encapsulation of ISA and colistin enhanced activity against Pseudomonas aeruginosa, improved interaction with the bacterial cell surface, and enabled controlled drug release. Collectively, these strategies highlight the potential of ent-beyerane- and abietane-based scaffolds as robust platforms for designing colistin adjuvants with enhanced ArnT-targeted activity.
To optimise the hit compound in terms of potency and synthetic feasibility, we then designed a new series of diterpenoid derivatives based on a simplified abietane scaffold. A function-oriented synthesis (FOS) approach was applied to structurally related aromatic abietanes to overcome the structural complexity and lack of chromophores of ent-beyerene diterpenes. Podocarpic acid (PD-1), an aromatic abietane lacking ring D but sharing key stereochemical and structural features with FDO, was used to generate a small focused library of derivatives incorporating functional groups essential for ArnT inhibition, as established through SAR studies of ent-beyerene derivatives. Through a combination of microbiological evaluation and computational docking in the ArnT binding site of Pseudomonas aeruginosa, a preliminary structure–activity relationship (SAR) for PD-1 derivatives was established. Compounds 2, 13, and 18 exhibited the highest activity, suggesting that C-16 sp³ hybridization, C-12 phenol methylation, and optimal linker length between the lipophilic scaffold and polar groups enhance binding to the ArnT catalytic site. Dehydroabietic acid (DH-1) was also investigated to explore additional chemical modifications and further validate the abietane scaffold. A series of DH-1 derivatives, currently under evaluation, was synthesized with varying substitution patterns on ring C and diverse functional groups at C-18.
Part B investigates the preliminary development of peptide-natural product conjugates with antimicrobial potential, an approach that remains far less explored than conventional antibiotic-peptide conjugates. Temporin L, a short peptide with potent and broad-spectrum antimicrobial activity, was selected as the peptide scaffold and coupled with dehydroabietic acid or naringenin to generate an irreversible amide-linked conjugate and a reversible, pH-responsive hydrazone-linked conjugate, respectively. These hybrid molecules are designed to enhance antimicrobial efficacy by integrating complementary modes of action of both components. Upcoming microbiological assays will evaluate their stability, release kinetics, antibacterial properties, and cytotoxicity, thereby guiding the rational development of peptide-natural product conjugates with improved therapeutic profiles.