Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions.
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 (K(d)<1 μM) than for whole cells (K(d)∼6-12μM). 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≥16μg/mL). Dansyl[Lys]¹polymyxin B₃ may prove useful as a screening tool for drug development.
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
Rachel L
RL
Soon
Tony
T
Velkov
Francis
F
Chiu
Philip E
PE
Thompson
Rashmi
R
Kancharla
Kade
K
Roberts
Ian
I
Larson
Roger L
RL
Nation
Jian
J
Li
D001665:Binding Sites; D002151:Calorimetry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D010713:Phosphatidylcholines / Q000737:chemistry; D011112:Polymyxin B / Q000737:chemistry* / Q000378:metabolism; D011113:Polymyxins / Q000138:chemical synthesis* / Q000737:chemistry
C496906:1-myristoyl-2-(12-((5-dimethylamino-1-naphthalenesulfonyl)amino)dodecanoyl)-sn-glycero-3-phosphocholine; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D010713:Phosphatidylcholines; D011113:Polymyxins; C558797:polymyxin B3, dansyllysyl(1)-; D011112:Polymyxin B