Polymyxins with Potent Antibacterial Activity against Colistin-Resistant Pathogens: Fine-Tuning Hydrophobicity with Unnatural Amino Acids

Abstract

In view of the increased prevalence of antimicrobial resistance among human pathogens, antibiotics against multidrug-resistant (MDR) bacteria are in urgent demand. In particular, the rapidly emerging resistance to last-resort antibiotic colistin, used for severe Gram-negative MDR infections, is critical. Here, a series of polymyxins containing unnatural amino acids were explored, and some analogues exhibited excellent antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Hydrophobicity of the compounds within this series (as measured by retention in reversed-phase analytical HPLC) exhibited a discernible correlation with their antimicrobial activity. This trend was particularly pronounced for colistin-resistant pathogens. The most active compounds demonstrated competitive activity against a panel of Gram-negative pathogens, while exhibiting low in vitro cytotoxicity. Importantly, most of these hits also retained (or even had increased) potency against colistin-susceptible strains. These findings infer that fine-tuning hydrophobicity may enable the design of polymyxin analogues with favorable activity profiles.

Figure 1

Structures of polymyxin B₃ and lipid A (from E. coli) as well as common lipid A modifications with 4-l-aminoarabinose and/or PEtN (sites for modification are indicated by arrows).

Scheme 1. Synthesis Strategy for Polymyxin Analogues

Fmoc-Dab-OAll, DIPEA, CH₂Cl₂, 2 h. (b) Assembly by Fmoc-SPPS; (i) 20% piperidine-DMF 2 × 5 min; and (ii) Fmoc-AA-OH/octanoic acid, HBTU, DIPEA, DMF, 1 h. (c) (i) 4% NH₂NH₂-H₂O, DMF, 3 × 5 min; and (ii) Fmoc-Thr(tBu)-OH, HBTU, DIPEA, DMF, 1 h. (d) (i) 4% NH₂NH₂-H₂O, DMF, 3 × 5 min; (ii) Fmoc-Thr(tBu)-OH, HBTU, DIPEA, DMF, 1 h; (iii) 20% piperidine-DMF 2 × 5 min; and (iv) Fmoc-AA-OH, HBTU, DIPEA, DMF, 1 h. (e) (i) 20% piperidine-DMF 2 × 5 min; (ii) Pd(PPh₃)₄, PhSiH₃, CH₂Cl₂, 2 × 15 min; (iii) PyAOP, HOAt, DIPEA, DMF, 2 h; and (iv) TFA-TIPS-H₂O (95:2.5:2.5), 1 h.

Scheme 2. Synthetic Route to Fmoc-Dab-OAll

Figure 2

A semilogarithmic graph showing the correlation between hydrophobicity of polymyxin analogues and log 2 of their antimicrobial activity against E. coli ATCC 25922, E. coli mcr-1 CstR, K. pneumoniae ATCC 13883, P. aeruginosa PA01, P. aeruginosa 7197/19 CstR, A. baumannii ATCC 19606, and A. baumannii 3658/17 CstR. Hydrophobicity is measured as % MeCN (at peak elution in analytical RP-18 HPLC gradient elution with 10–60% eluent B over 10 min). Antimicrobial activity is stated as MICs in mg/L; CstR denotes colistin-resistance.

Figure 3

SYTOX green uptake of the three hit compounds 2h, 5d, and 5e, as well as of colistin at MIC and 2× MIC. Data are shown as changes in fluorescence intensity over time. The assay was performed on the strain E. coli MG1655, which is colistin-susceptible. All compounds had a MIC of 0.25 μg/mL toward E. coli MG1655.

Figure 4

Hemolytic properties of polymyxin analogues. Compounds were tested for their hemolysis in whole blood at 800 and 400 μg/mL. Data are shown as % hemolysis with SEM.

Figure 5

Effect on the viability of HepG2 cells by selected compounds. Compounds were tested in the range of 10–1280 μg/mL to determine or estimate IC₅₀. Results are shown in % survival as a function of the log concentration of the compound in μg/mL.