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Synthesis and characterization of BODIPY‐FL‐cyclosporine A as a substrate for both human and mouse multidrug resistance‐linked P‐glycoprotein

Generation of fluorescent conjugates of small molecules and existing drugs has facilitated their use in addressing cellular functions and pharmacological analysis. These compounds interact with several proteins as substrates or inhibitors, thus allowing the development of unique fluorescence‐based methods to study in vivo localization and the molecular mechanisms. Pglycoprotein (P‐gp) is an ATP‐binding cassette transporter that effluxes most anti‐cancer drugs from cells, contributing to the development of drug resistance in cancer cells. P‐gp, which exports toxic metabolites and xenobiotics, is expressed on the surface of epithelial cells of the intestine, kidney, liver, placenta, adrenal gland and endothelial cells at blood‐brain barrier. With the aim of developing an effective probe to study drug‐transport by P‐gp in various model systems including cultured cells, mice and zebra fish, we synthesized a BODIPY‐FL conjugate of cyclosporine A (BD‐CsA). After synthesis and characterization of its chemical purity, BD‐CsA was compared with the currently available 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl (NBD)‐CsA probe in terms of its usage as a substrate of human and mouse P‐gp. Using a BacMam baculovirus HeLa cell expression system and a flow cytometry‐based transport assay, we found that BD‐CsA is recognized as a substrate by both human and mouse P‐gp. The rate of efflux of BD‐CsA by human P‐gp is similar to that of NBD‐CsA (T1/2 of 5.0 min and 3.0 min for BD‐CsA and NBD‐CsA, respectively). In flow cytometry assays, the fluorescence intensity of BD‐CsA was almost 10‐times higher than that of NBD‐CsA due to the high fluorescence quantum yield of the BODIPY fluorophore, allowing us to use significantly lower concentrations of BD‐CsA to achieve the same fluorescence levels. The transport of BD‐CsA was inhibited by the P‐gp inhibitor tariquidar, with similar IC50 values as for NBD‐CsA transport (21.6 nM for BD‐CsA and 25.8 nM for NBD‐CsA). We also observed that both BD‐CsA and NBD‐CsA partially inhibited the ATPase activity of P‐gp. In silico docking of BD‐CsA and NBD‐CsA to the homology model of human P‐gp indicates that both probes bind in the drug‐binding pocket of Pgp with similar docking scores. Thus, we demonstrate that BD‐CsA is a sensitive and efficient fluorescent substrate of P‐gp that can be used to study the transporter's function in both in vitro and in vivo settings.