Colchicine-BODIPY Probes: Evidence for the Involvement of Intracellular Membranes in the Targeting of Colchicine to Tubulin

The natural product colchicine (<b>Col</b>) is a medication used to treat severe inflammatory conditions. Although its mechanism of action at the level of the cytoskeleton is known, its subcellular distribution has not yet been properly studied. In this work, we present the first rational approach to assess the intracellular localization and biological activity of this alkaloid. We conjugated <b>Col</b> to green-emitting BODIPY dyes (<b>CBs</b>) with alternative linkers of different lengths (<b>CB1</b>-<b>CB12</b>) via different types of linkages. Connections of <b>Col</b> with BODIPY generally reduced its cytotoxicity to different levels depending on the type of linker. From the analysis of <b>CB</b> effects on cytotoxicity, cell cycle, and tubulin polymerization, we selected the most potent substances for fluorescence microscopy. Treatment of cells with 10 μM conjugates for 15 h showed different effects on microtubule organization. Live-cell imaging revealed that <b>CBs</b> rapidly associated with cellular membranes. Double label experiments unveiled that the <b>CB4</b>, which was the most effective in inhibiting tubulin polymerization, binds to the endoplasmic reticulum (ER) and mitochondria. <i>In silico</i> modeling and SPR analyses confirmed the high potency of <b>CB4</b> to bind to the colchicine site on tubulin.