Abstract
Cationic amphiphilic drugs (CADs) comprise a wide variety of different substance classes such as antidepressants, antipsychotics, and antiarrhythmics. It is well recognized that CADs accumulate in certain intracellular compartments leading to specific morphological changes of cells. So far, no adequate technique exists allowing for ultrastructural analysis of CAD in intact cells. Azidobupramine, a recently described multifunctional antidepressant analogue, allows for the first time to perform high-resolution studies of CADs on distribution pattern and morphological changes in intact cells. We showed here that the intracellular distribution pattern of azidobupramine strongly depends on drug concentration and exposure time. The mitochondrial compartment (mDsRed) and the late endo-lysosomal compartment (CD63-GFP) were the preferred localization sites at low to intermediate concentrations (i.e. 1 μM, 5 μM). In contrast, the autophagosomal compartment (LC3-GFP) can only be reached at high concentrations (10 μM) and long exposure times (72 hrs). At the morphological level, LC3-clustering became only prominent at high concentrations (10 μM), while changes in CD63 pattern already occurred at intermediate concentrations (5 μM). To our knowledge, this is the first study that establishes a link between intracellular CAD distribution pattern and morphological changes. Therewith, our results allow for gaining deeper understanding of intracellular effects of CADs.
Highlights
IntroductionThe aim of this study was to re-evaluate the intracellular distribution pattern of CADs using the recently synthetized and characterized CAD azidobupramine, a chemically modified antidepressant harnessing the power of modern medicinal chemistry to overcome above mentioned limitations[28]: azidobupramine carries an azide-group amenable for photoaffinity labelling (PAL) and an alkyne-group enabling chemical bonding of fluorescent tags by click-chemistry (CuAAC)
The fluorescent signal was recorded after azidobupramine was immobilized on its target site by UV-crosslinking and the azidobupramine-target complexes were labelled with fluorescent dyes by click-chemistry (S-Fig. 1)
Using azidobupramine as model substance, we demonstrate here for the first time that the vesicular accumulation of CADs is accompanied by an expansion of CD63-positive vesicles[33,34]
Summary
The aim of this study was to re-evaluate the intracellular distribution pattern of CADs using the recently synthetized and characterized CAD azidobupramine, a chemically modified antidepressant harnessing the power of modern medicinal chemistry to overcome above mentioned limitations[28]: azidobupramine carries an azide-group amenable for photoaffinity labelling (PAL) and an alkyne-group enabling chemical bonding of fluorescent tags by click-chemistry (CuAAC). These modifications allow for UV-induced immobilization of this CAD in living cells and subsequent for labeling with fluorophores without hampering subcellular structures. We employed cells stably expressing markers targeting mitochondria, late endosomes and autophagosomes
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