Quantitation of Polymyxin-Lipopolysaccharide Interactions Using an Image-Based Fluorescent Probe

Area of Science:

• Microbiology
• Drug Discovery
• Biochemistry

Background:

• Increasing polymyxin resistance in Gram-negative bacteria is a significant clinical threat.
• Lipopolysaccharide (LPS) modification is a key mechanism of resistance, impacting polymyxin binding.
• Understanding polymyxin-LPS interactions is crucial for developing new antibiotics.

Purpose of the Study:

• To evaluate MIPS-9451, a fluorescent polymyxin analogue, as a reporter molecule for quantifying polymyxin-LPS binding affinities.
• To assess MIPS-9451's utility across diverse Gram-negative bacterial LPS types.
• To validate MIPS-9451's performance in displacement assays for ranking polymyxin B affinities.

Main Methods:

• Titration of MIPS-9451 against LPS from 17 Gram-negative species/strains.
• Determination of dissociation constants (Kd) for MIPS-9451-LPS interactions.
• Displacement assays using MIPS-9451 and polymyxin B to determine inhibition constants (Ki).

Main Results:

• MIPS-9451 exhibited varying binding affinities across LPS types, with Kd values ranging from 0.14 ± 0.01 μM (E. coli) to 0.90 ± 0.42 μM (P. gingivalis).
• Displacement assays showed inhibition constants for polymyxin B against K. pneumoniae, P. aeruginosa, and S. enterica (6.2 μM ± 33%, 7.2 μM ± 30%, 0.95 μM ± 13%, respectively).
• Results align with existing polymyxin probe data, confirming MIPS-9451's quantitative potential.

Conclusions:

• MIPS-9451 is a valuable fluorescent reporter molecule for assessing polymyxin-LPS binding.
• This probe facilitates the quantitative analysis of polymyxin affinities, supporting novel antibiotic discovery programs.
• MIPS-9451 aids in understanding resistance mechanisms and developing strategies against Gram-negative pathogens.