Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging

Abstract

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12.Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively.Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division.Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells.

Live applications of norbormide-based fluorescent probes in Drosophila melanogaster

“…These data show that both compounds, but particularly NRB MC009 , can be used to visualize and distinguish most organs/tissues of the dissected living larvae, and allow good definition of their intracellular structures. In addition, the co-localization studies revealed that both NRB MC009 and NRB ZLW0047 labelled subcellular organelles, that they preferentially targeted the ER and mitochondria, and that they were totally absent from the nuclei, plasma membranes and lysosomes, which is in agreement with data reported in mammalian cell studies [6]. Moreover, the efficiency of the probes was tested with two different approaches: 1) in combination with commercially available dyes for live imaging, and 2) together with green fluorescent proteins that label specific cellular structures.…”

“…In fact, besides the well-known actin and myosin filaments and their associated proteins, muscles also contain a cytoskeleton, intracellular organelles of the endo-lysosomal pathway, and well-defined endoplasmic reticulum and mitochondrial networks [25]. We focused on these particular structures because we recently showed that they contained a significant density of binding sites for NRB MC009 [6]. To study the intracellular localization of NRB MC009 we co-loaded it into larval muscle with organelle-specific red fluorescent dyes.…”

“…NRB MC009 , a BODIPY FL derivative of norbormide, was synthesized as previously reported [6]. Stock solutions 1 mM in DMSO were prepared and maintained at -20°C and diluted to the desired concentration before each experiment.…”

“…In an attempt to identify the cellular targets of NRB, we previously developed fluorescent derivatives of the parent compound by linking it to either nitrobenzoxadiazole (NBD) or boron-dipyrromethene (BODIPY FL) fluorophores, and found that both were able to clearly label intracellular structures such as endoplasmic reticulum (ER), Golgi apparatus, mitochondria, and lipid droplets (LDs) in various cell lines, in the absence of cytotoxic effects. Based on these results, we proposed NRB as a scaffold for the development of new, high performing, non-toxic fluorescent probes for live cell imaging [5,6].…”

Live applications of norbormide-based fluorescent probes in Drosophila melanogaster

“…These data show that both compounds, but particularly NRB MC009 , can be used to visualize and distinguish most organs/tissues of the dissected living larvae, and allow good definition of their intracellular structures. In addition, the co-localization studies revealed that both NRB MC009 and NRB ZLW0047 labelled subcellular organelles, that they preferentially targeted the ER and mitochondria, and that they were totally absent from the nuclei, plasma membranes and lysosomes, which is in agreement with data reported in mammalian cell studies (6). Moreover, the efficiency of the probes was tested with two different approaches: 1) in combination with commercially available dyes for live imaging, and 2) together with GFP tagged protein markers that bound to specific cellular structures.…”