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

• Published in: Antimicrobial agents and chemotherapy 2015-12, Vol.59 (12), p.7489-7496
• Main Authors: Yun, Bo, Azad, Mohammad A K, Nowell, Cameron J, Nation, Roger L, Thompson, Philip E, Roberts, Kade D, Velkov, Tony, Li, Jian
• Format: Article
• Language: English
• Subjects: Acinetobacter baumannii - drug effects; Acinetobacter baumannii - growth & development; Animals; Anti-Bacterial Agents; Anti-Bacterial Agents - chemical synthesis; Anti-Bacterial Agents - metabolism; Anti-Bacterial Agents - pharmacology; Apoptosis - drug effects; Biological Transport; Biosynthesis; Cell Line; Chemistry; Chemistry; Biosynthesis; Dansyl Compounds; Dansyl Compounds - chemical synthesis; Dansyl Compounds - metabolism; Dansyl Compounds - pharmacology; Drug Design; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Epithelial Cells; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Epithelial Cells - ultrastructure; Fluorescent Dyes; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - metabolism; Fluorescent Dyes - pharmacology; Hydrophobic and Hydrophilic Interactions; Kidney Tubules - drug effects; Kidney Tubules - metabolism; Kidney Tubules - ultrastructure; Klebsiella pneumoniae - drug effects; Klebsiella pneumoniae - growth & development; Microbial Sensitivity Tests; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondria - ultrastructure; Molecular Imaging - methods; Polymyxin B; Polymyxin B - analogs & derivatives; Polymyxin B - chemical synthesis; Polymyxin B - metabolism; Polymyxin B - pharmacology; Pseudomonas aeruginosa - drug effects; Pseudomonas aeruginosa - growth & development; Rats
• ISSN: 0066-4804; 1098-6596
• DOI: 10.1128/AAC.01216-15

Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity.