Elsevier

Analytical Biochemistry

Volume 409, Issue 2, 15 February 2011, Pages 273-283
Analytical Biochemistry

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

https://doi.org/10.1016/j.ab.2010.10.033Get rights and content

Abstract

Fluorescence assays employing semisynthetic 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-chloride. 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]1polymyxin B3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxin B3 for LPS was comparable to polymyxin B and colistin, and considerably greater (Kd < 1 μM) than for whole cells (Kd  6–12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxin B3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxin B3–LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxin B3–LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC ⩾16 μg/mL). Dansyl[Lys]1polymyxin B3 may prove useful as a screening tool for drug development.

Section snippets

Materials

Polymyxin B sulfate (Lot No. 453306, ⩾6000 USP units per mg) was purchased from Fluka (Castle Hill, NSW, Australia). Colistin sulfate (Lot No. 036K1374, 15,000 units per mg), dansyl chloride, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 99.5%), chloramphenicol (98%), sodium dodecyl sulfate (SDS; 99%), DNase, and RNase were obtained from Sigma–Aldrich (Sydney, NSW, Australia). Proteinase K was obtained from Promega (Sydney, NSW, Australia). Semisynthetic dansyl-polymyxin B was from

Mass spectrometry (MS) analysis of semisynthetic and commercial DPmB

MS analysis of nonpurified products from the semisynthetic reaction showed that the PmB remained unreacted or in mono-, di-, tri-, or tetra-dansyl-PmB form (Supplementary Figs. 3.1–3.6). Similarly, commercial DPmB was found to be a complex mixture of mono-, di-, tri-, and tetra-dansyl-PmB, although underivatized polymyxin B was not detected.

In positive mode electrospray ionization MS analysis of the commercial DPmB, multiple charged molecular ions between 2 and 4 positive charges were observed

Discussion

The resurgence of polymyxins for the treatment of MDR gram-negative infections [39] has led to the inevitable development of resistance to this last-line agent, prompting urgent efforts to advance our knowledge of the mechanisms of polymyxin action and resistance. To broaden our understanding of the initial binding of polymyxins to LPS, the DPmB fluorescence assay theoretically offers an inexpensive and simple method of quantitatively describing the interaction. Moreover, given the urgent need

Conclusions

In summary, semisynthetic DPmB popularly employed in the literature to investigate the binding affinity of polycationic compounds is poly-substituted with dansyl groups. Despite extensive use, our study has first revealed structural complexities and mechanistic inadequacies associated with the DPmB assay. Our mono-substituted DPmB3 presents an improved probe for this fluorescent assay. From a drug-screening perspective, the DPmB3 assay is thus of considerable utility in medicinal chemistry

Acknowledgments

R.L.N. and J.L. are supported by research grants from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R01A1070896 and R01AI079330). R.L.N., J.L., P.E.T., and T.V. are also supported by the Australian National Health and Medical Research Council (NHMRC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes

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    These authors contributed equally to this work.

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