The Contribution of the N-Terminal Structure of Polymyxin B Peptides to Antimicrobial and Lipopolysaccharide Binding Activity

Abstract

To elucidate the N-terminal structure–activity relationships of polymyxin B peptides, seven polymyxin B component peptides, the structures of which having been elucidated, and seven N-terminal fatty acid and/or amino acid deletion analogs were synthesized, and their antimicrobial activities determined. The lipopolysaccharide (LPS) binding activities of synthetic peptides were evaluated using [Dab(Dansyl-Gly)1]-polymyxin B3 (Dab; l-α,γ-diaminobutyric acid) as a fluorescent probe. The results indicated that the fatty acyl moiety was not indispensable for LPS binding, but the C9 fatty acyl groups of polymyxin B peptides contributed to the binding affinity to a slightly greater extent than C8 or C7. The fatty acyl moieties of polymyxin B contributed greatly to the antimicrobial activity, while the distinct N-terminal structures of polymyxin B1–B6, bearing normal-, iso-, or anteiso-fatty acids, or 3-hydroxy-fatty acid with chain lengths between C7 and C9, did not affect bactericidal potency.

Novel Des-Fatty Acyl-Polymyxin B Derivatives with Pseudomonas aeruginosa-Specific Antimicrobial Activity

“…1). 13,17) The product was extensively purified by HPLC and gel-filtration prior to examination of its biological activity. The purity of the synthetic peptides was Ͼ98%.…”

“…]-PMB(1-10) (5) retained antimicrobial activity against E. coli, S. Typhimurium, and P. aeruginosa (MIC: 4, 2, and 1 nmol/ml, respectively), and its activity was higher than that of des-FA-PMB decapeptide (MIC: 8, 16, and 4 nmol/ml against the same three bacteria, previously reported). 13) Guanyl-[Thr 2 -Dab 3 ]-PMB(2-10) (6) and Guanyl-[Dab 2 -Dab 3 ]-PMB(2-10) (7) have much higher activity than PMBN (MIC values of Ͼ256, Ͼ256, and 128 nmol/ml, respectively) against the three bacteria. Guanyl- [Dab 3 ] -PMB(3-10) (8) and N a,g -diguanyl-[Dap 3 ]-PMB(3-10) (10) also have higher activity (MIC: 16, 16, and 2 nmol/ml and 16, 32, and 0.5 nmol/ml, respectively) than Des-FA-PMB octapeptide (MIC: 128, 256, and 64 nmol/ml, respectively, against the same three bacteria).…”

“…In a previous study we and Urakawa et al found that PMB heptapeptide and its derivatives have no significant antimicrobial activity against Gram-negative bacteria, but the binding activity to LPS is retained. 13,22) Therefore, PMB heptapeptide analogs with better permeability into the outer membranes were designed. Only Guanyl-PMB(4-10) (10), which is the lowest molecular weight analog studied, had antimicrobial activity against P. aeruginosa.…”

“…13) [Dap 3 ]-PMB(3-10) (3) and [Ser 3 ]-PMB(3-10) (4) showed high antimicrobial activities, with MIC values of 4 and 2 nmol/ml, respectively, compared with PMB octapeptide against P. aeruginosa. These results show that small hydrophilic amino acids (Dap, Ser) at the N-terminal are essential for antimicrobial specificity towards P. aeruginosa.…”

“…Assay of LPS Binding of the Synthetic Peptides As described previously, 13) a solution of [Dab(N g -dansyl-Gly) 1 ]-PMB 3 in H 2 O (1 mmol/ml) (4 ml, 4 nmol) was added to a quartz cuvette containing N-(2-hydroxyethyl)piperazine-NЈ-(2-ethanesulfonic acid) buffer (HEPES; 5 mmol/l, pH 7.2) (1 ml), followed by a solution of E. coli LPS (serotype 055:B5) (Sigma) in H 2 O (3 mg/ml) (10 ml, 30 mg). The solution was kept at 30°C for 60 min, by which time the fluorescence intensity of [Dab(N g -dansyl-Gly) 1 ]-PMB 3 reached a plateau.…”