Projects per year
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 I?-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 I?M) than for whole cells (Kd 6a??12 I?M). 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 B3a??LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxin B3a??LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC 16 I?g/mL). Dansyl[Lys]1polymyxin B3 may prove useful as a screening tool for drug development.
Original language | English |
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Pages (from-to) | 273 - 283 |
Number of pages | 11 |
Journal | Analytical Biochemistry |
Volume | 409 |
Issue number | 2 |
DOIs | |
Publication status | Published - 2011 |
Projects
- 2 Finished
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Optimizing dosing of colistin for infections resistant to all other antibiotics.
Nation, R. (Primary Chief Investigator (PCI)) & Li, J. (Chief Investigator (CI))
NIH - National Institutes of Health (United States of America)
15/09/08 → 31/08/12
Project: Research
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Targeting MD hetero-resistant Gram-negatives: PK/PD for rational combinations.
Nation, R. (Primary Chief Investigator (PCI)), Li, J. (Chief Investigator (CI)), Forrest, A. (Partner Investigator (PI)), Paterson, D. L. (Partner Investigator (PI)) & Tsuji, B. T. (Partner Investigator (PI))
NIH - National Institutes of Health (United States of America)
15/07/08 → 30/06/12
Project: Research