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.

Synergy by Perturbing the Gram-Negative Outer Membrane: Opening the Door for Gram-Positive Specific Antibiotics

“… 203 − 205 Compared to the dUSCLs, UTBLPs 5 and 6 contain an extra lysine, while an octanoyl group was employed as the lipophilic moiety ( Figure 4 B). 201 , 202 Methylation of the lysine side chain resulted in a reduction of potentiation for rifampicin and novobiocin in both wild-type and resistant Gram-negative strains. 202 A correlation between the number of methyl groups and loss of activity was seen, while the increase in NPN fluorescence of the trimethylated UTBLPs was on par with that of their un- or monomethylated analogues.…”

“…The Schweizer group also recently reported a series of ultrashort tetrabasic lipopeptides (UTBLPs) synergists . These compounds were specifically prepared to assess the effect of lysine N -ζ-methylation on the potentiation of antibiotics, inspired by reports suggesting that N -methylation can lead to reduced hemolysis, increased proteolytic stability, and improved antibacterial activity. − Compared to the dUSCLs, UTBLPs 5 and 6 contain an extra lysine, while an octanoyl group was employed as the lipophilic moiety (Figure B). , Methylation of the lysine side chain resulted in a reduction of potentiation for rifampicin and novobiocin in both wild-type and resistant Gram-negative strains .…”

“…The Schweizer group also recently reported a series of ultrashort tetrabasic lipopeptides (UTBLPs) synergists . These compounds were specifically prepared to assess the effect of lysine N -ζ-methylation on the potentiation of antibiotics, inspired by reports suggesting that N -methylation can lead to reduced hemolysis, increased proteolytic stability, and improved antibacterial activity. − Compared to the dUSCLs, UTBLPs 5 and 6 contain an extra lysine, while an octanoyl group was employed as the lipophilic moiety (Figure B). , Methylation of the lysine side chain resulted in a reduction of potentiation for rifampicin and novobiocin in both wild-type and resistant Gram-negative strains . A correlation between the number of methyl groups and loss of activity was seen, while the increase in NPN fluorescence of the trimethylated UTBLPs was on par with that of their un- or monomethylated analogues …”

“…202 A correlation between the number of methyl groups and loss of activity was seen, while the increase in NPN fluorescence of the trimethylated UTBLPs was on par with that of their un-or monomethylated analogues. 202 1.5. Lipopeptidomimetic Synergists.…”

Antibiotic Adjuvants: A Versatile Approach to Combat Antibiotic Resistance

“…coli. , Dilipid ultrashort cationic lipopeptides (dUSCLs), bearing lysine-rich tetrapeptides and lipopeptides at the N -terminal, improved the activity of various antibiotics against Gram-negative bacteria by permeabilizing the outer membrane and disrupting antibiotic efflux . Ultrashort tetrabasic peptides were also reported by the same group . Moreover, peptidomimetics like dilipid ultrashort tetrabasic peptidomimetics (dUSTBPs) consisting of three l -arginine units and an eight-carbon-long dilipid potentiated novobiocin and rifampicin against multidrug-resistant P. aeruginosa , A. baumannii , and Enterobacteriaceae species .…”