Outer Membrane Interaction Kinetics of New Polymyxin B Analogs in Gram-Negative Bacilli

Abstract

Infections caused by drug-resistant Gram-negative bacilli are a severe global health threat, limiting effective drug choices for treatment. In this study, polymyxin analogs designed to have reduced nephrotoxicity, direct activity, and potentiating activity were assessed for inhibition and outer membrane interaction kinetics against wild-type (WT) and polymyxin or multidrug-resistant (MDR) Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae In MIC assays, two polymyxin B (PMB) analogs (SPR1205 and SPR206) and a polymyxin E analog (SPR946), with shortened peptide side chains and branched aminobutyryl N termini, exhibited promising activity compared with PMB and previously tested control polymyxin analogs SPR741 and polymyxin B nonapeptide (PMBN). Using dansyl-polymyxin (DPX) binding to assess the affinity of interaction with lipopolysaccharide (LPS), purified or in the context of intact cells, SPR206 exhibited similar affinities to PMB but higher affinities than the other SPR analogs. Outer membrane permeabilization measured by the 1-N-phenyl-napthylamine (NPN) assay did not differ significantly between the polymyxin analogs. Moreover, Hill numbers were greater than 1 for most of the compounds tested on E. coli and P. aeruginosa strains which indicates that the disruption of the outer membrane by one molecule of compound cooperatively enhances the subsequent interactions of other molecules against WT and MDR strains. The high activity demonstrated by SPR206 as well as its ability to displace LPS and permeabilize the outer membrane of multiple strains of Gram-negative bacilli while showing cooperative potential with other membrane disrupting compounds supports further research with this polymyxin analog.

Keywords: MDR Gram-negative bacilli; cell membrane interactions; polymyxin B derivatives.

FIG 1

Polymyxin analogs tested in this study. (A) Each analog shared the same polymyxin cyclic core. The substituents R¹ and R² shown on the core structure in (A) were substituted as follows, where R¹ is shown in the left hand box and R² in the right hand box: (B) polymyxin B (PMB). (C) Deacylated polymyxin nonapeptide (PMBN). (D) SPR741 has the same cyclic core as PMB, a shorter N terminus and a d-serine residue at the position adjacent to the cyclic core. (E) SPR1205 and (F) SPR206 have a diaminopropionate (l-Dap) residue adjacent to the cyclic core in place of diaminobutyrate (l-Dab) in PMB, and β-branched aminobutyryl N termini. (G) SPR946 is based on the polymyxin E scaffold with l-Dap adjacent to the cyclic core and with a shorter N-terminal side chain.

FIG 2

Whole-cell DPX displacement by PMB (solid black squares) and SPR compounds. SPR206 (solid gray squares), SPR1205 (solid black triangles), SPR946 (solid gray triangles), PMBN (open triangles), and SPR741 (open squares) were used to displace DPX from various intact bacterial cells leading to a reduction in fluorescence. Maximal displacement was observed at ∼35 μg/ml of each compound except for PMBN and SPR741 that required up to 200 μg/ml. (A) E. coli SC9251 and (B) E. coli NCTC 13846 whole cells. (C) P. aeruginosa PAO1 and (D) P. aeruginosa 9BR whole cells. (E) Acinetobacter baumannii ATCC 17978 and (F) A. baumannii C4 whole cells. (G) K. pneumoniae VA360 and (H) Klebsiella pneumoniae 13883-PXR whole cells. (Left) show WT or polymyxin susceptible cells; (right) show polymyxin or MDR derivatives.

FIG 3

Influence of SPR206 on uptake of NPN across the outer membrane. This was performed with WT (black symbols) and MDR (gray symbols) strains and the corresponding Hill plots for (A) E. coli SC9251 and E. coli NCTC 13846, (B) P. aeruginosa PAO1 and P. aeruginosa 9BR, (C) A. baumannii ATCC 17978 and A. baumannii C4, and (D) K. pneumoniae VA360 (black symbols) and K. pneumoniae 13883-PXR (gray symbols). Percent fluorescence intensity is normalized to maximum fluorescence of PMB for each strain.