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