Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin–lipopolysaccharide binding interactions

Abstract

Fluorescence assays employing semi-synthetic or commercial dansyl-polymyxin B, have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric-acid residues of polymyxin B are potentially derivatized with dansyl-cholride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra- dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]1polymyxinB3. The affinity of polymyxin for purified Gram-negative LPS, and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxinB3 for LPS was comparable to polymyxin B and colistin, and considerably greater (kd < 1 μM) than for whole cells (kd ~6 to 12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxinB3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxinB3-LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxinB3-LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC ≥ 16 μg/mL). Dansyl[Lys]1polymyxinB3 may prove useful as a screening tool for drug development.

Measuring Polymyxin Uptake by Renal Tubular Cells: Is BODIPY-Polymyxin B an Appropriate Probe?

“…Binding of polymyxins to LPS involves an initial electrostatic interaction of the positively charged diaminobutyric acid (Dab) residues with the negatively charged phosphate groups of lipid A, displacing divalent cations (Ca 2ϩ and Mg 2ϩ ) that bridge adjacent LPS molecules (8)(9)(10). We have previously highlighted the deficiencies of directly amine coupling dansyl groups onto the Dab side chains in semisynthetic preparations of dansyl-PMB (11). Analysis of these semisynthetic dansyl-PMB preparations revealed a mixture of mono-, di-, and tri-dansyl-substituted species (Fig.…”

“…2). Considering the loss of native antibacterial activity universally seen across polymyxin analogs modified at the Dab residues (10), results of cell uptake studies with BODIPY-PMB (1) or dansyl-PMB as imaging probes for polymyxins may be very misleading (11) and must be interpreted with caution. The binding of such probes with mammalian cells may differ substantially from that of PMB; confirmation of the intracellular localization of BODIPY-PMB is essential using techniques such as confocal microscopy, rather than ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) quantitation of whole-cell lysate (1).…”

“…1). Furthermore, as PMB is comprised of two major components, the potential for each component to be replaced at any of the five Dab residues with multiple dansyl molecules results in an extremely variable mixture of dansylated derivatives (11).…”

Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes

“…Such studies require suitable fluorescent probes representative of the polymyxins to enable imaging of their uptake and localization within cells. The fluorescent probes pre-viously utilized (e.g., semisynthetic preparations of dansyl-and BODIPY-polymyxin B) for studying the mechanism of action, uptake, and localization of polymyxin (17)(18)(19)(20)(21) are not representative of the parent polymyxin (22)(23)(24). Our group recently reported the design and synthesis of a regioselectively monodansylated polymyxin B probe (MIPS-9541) for imaging uptake into bacterial cells (22).…”

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

“…13) The mechanism by which these synthetic peptides exclude the fluorescent probe bound to LPS could be explained as follows. 25) N-terminal PMB analogs with Ser and/or Dap ((1)-(4)) had low E. coli LPS binding activity compared with PMB (Fig. 2).…”