New polymyxin analogs to combat Gram-negative biofilm-associated infections

• Autor(es): Graínha, Tânia Raquel Rodrigues ; Martins, Elsa ; Costa, Susana P. ; Jorge, Paula ; Lopes, Susana P.
• Data: 2024
• Identificador Persistente: https://hdl.handle.net/1822/97695
• Origem: RepositóriUM - Universidade do Minho
• Tipo de Documento: Outro
• Idioma: Inglês
• Contribuidor(es): Universidade do Minho

Descrição

Introduction: Antimicrobial resistance is a major global health threat, with which the antibiotic discovery pipeline is not keeping up. Polymyxins (PM), notably B (PMB) and E (PME) have resurrected as last-resort drugs (given their toxicity) against difficult-to-treat Gram-negative bacterial infections (often biofilm-related), but PM resistance has already been reported. In recent years, a fast-growing no. of PM analogs have been reported, but with varied success in improving PM activity and ameliorating toxicity. ,-Dialkylglycines (DAG) are unnatural amino acids with unique characteristics (bioactivity, metabolic resistance, hydrophobicity, stability), making them innovative building blocks in PM analog design. Objectives: By exploiting PMB/PME valuable lipopeptide scaffolds, we set out to design new PM analogs aiming at higher antimicrobial activity and/or lower toxicity. Here, we report the synthesis of 6 new PM analogs and their activity against planktonic and biofilm cultures, including PM analog combination with common antibiotics. Methods: The Ugi multicomponent reaction was applied to synthesise 2 DAGs, diisobutyl glycine (Dibg) and dibenzyl glycine (Dbng), which were assembled in situ at positions 6 and/or 7 of native PMB/E through a microwave assisted solid-phase peptide synthesis. Antibacterial and antibiofilm activities were assessed against Pseudomonas aeruginosa ATCC 27853 by broth microdilution and culturable cell quantification after treatment of 24-h old biofilms, respectively. Synergy with ciprofloxacin (CIP) and tobramycin (TOB) was assessed through checkerboard assays and determination of antibiofilm activity of the combinatorial treatment. Results: The 2 analogs with Dibg at position 7 (PMBa/PMEa) were the most promising, with PMBa having MIC/MBC of 4 mg/L and no cytotoxicity towards A549 lung epithelial cells. Preliminary HPLC analysis indicates ~66% purity, meaning that pure analog concentrations are probably ~2 mg/L, close to that of PMB (1 mg/L). Furthermore, 512 mg/L (~340 mg/L pure analog) of PMBa decreased biofilm cell numbers by ~3.7 log(CFU/mL), which is promising considering that PMB achieved a similar reduction, ~3.9 log(CFU/mL), for a higher effective concentration (512 mg/L). In turn, PMBb/PMEb (Dbng at position 6) and PMBc (Dbng at position 6 + Dibg at position 7) analogs were the most promising in combination scenarios, achieving synergic/additive outcomes (FICI ranging from 0.3125 to 0.75) when combined with TOB and CIP against planktonic cells. Regarding biofilms, preliminary results show potential synergy of PMEb+TOB with decreased biofilm cell numbers by ~3.4 log(CFU/mL). Conclusions: We successfully designed 6 new PM analogs, with 5 showing promising antimicrobial and anti-biofilm activity against P. aeruginosa, including in combination with antibiotics, revealing the suitability of DAGs in PM analog design. Further testing will include analog purification and nephrotoxicity evaluation.