Effects of Lysine N-ζ-Methylation in Ultrashort Tetrabasic Lipopeptides (UTBLPs) on the Potentiation of Rifampicin, Novobiocin, and Niclosamide in Gram-Negative Bacteria

Abstract

Outer membrane (OM) drug impermeability typically associated with a molecular weight above 600 Da and high hydrophobicity prevents accumulation of many antibiotics in Gram-negative bacteria (GNB). Previous studies have shown that ultrashort tetrabasic lipopeptides (UTBLPs) containing multiple lysine residues potentiate Gram-positive bacteria (GPB)-selective antibiotics in GNB by enhancing OM permeability. However, there is no available information on how N-substitution at the ζ-position of lysine in UTBLPs affects antibiotic potentiation in GNB. To study these effects, we prepared a series of branched and linear UTBLPs that differ in the degree of N-ζ-methylation and studied their potentiating effects with GPB-selective antibiotics including rifampicin, novobiocin, niclosamide, and chloramphenicol against wild-type and multidrug-resistant GNB isolates. Our results show that increasing N-ζ-methylation reduces or abolishes the potentiating effects of UTBLPs with rifampicin, novobiocin, and niclosamide against GNB. No trend was observed with chloramphenicol that is largely affected by efflux. We were unable to observe a correlation between the strength of the antibiotic potentiating effect to the increase in fluorescence in the 1-N-phenylnaphthylamine (NPN) OM permeability assay suggesting that other factors besides OM permeability of NPN play a role in antibiotic potentiation. In conclusion, our study has elucidated crucial structure-activity relationships for the optimization of polybasic antibiotic potentiators in GNB.

Keywords: Acinetobacter baumannii; Escherichia coli; Pseudomonas aeruginosa; antibiotic adjuvant; antibiotic potentiator; niclosamide; novobiocin; outer membrane permeabilizer; rifampicin.

Figure 1

Structures of (a) branched and (b) linear UTBLPs.

Figure 2

Potentiation of (a) RIF, (b) NOV, (c) NIC, and (d) CHL by 6 μM UTBLP against wild-type GNB.

Figure 2

Potentiation of (a) RIF, (b) NOV, (c) NIC, and (d) CHL by 6 μM UTBLP against wild-type GNB.

Figure 3

Potentiation of (a) RIF and (b) NOV by 6 μM UTBLP against a panel of MDR and carbapenem-resistant GNB isolates.

Figure 4

Dose-dependent increase in fluorescence of NPN in the presence of (a) UTBLP 1, (b) UTBLP 2, (c) UTBLP 3, (d) UTBLP 4, and PMBN (control) in A. baumannii ATCC 17978.