Pacific Blue-Taxoids as Fluorescent Molecular Probes of Microtubules.
| Autor: | Andres AE; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA., Rane D; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA., Peterson BR; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA. peterson.1119@osu.edu. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2430, pp. 449-466. |
| DOI: | 10.1007/978-1-0716-1983-4_28 |
| Abstrakt: | Taxoids such as paclitaxel (Taxol) are an important class of anticancer drugs that bind β-tubulin and stabilize cellular microtubules. To provide new chemical tools for studies of microtubules, we synthesized derivatives of paclitaxel modified at the 7-position with the small coumarin-derived fluorophore Pacific Blue (PB). Three of these Pacific Blue-Taxoids termed PB-Gly-Taxol, PB-β-Ala-Taxol, and PB-GABA-Taxol bind purified crosslinked microtubules with affinities of 34-265 nM, where the affinity can be tuned based on the length of an amino acid linker. When added to living cells in the presence of verapamil or probenecid as inhibitors of efflux, these compounds allow visualization of the microtubule network by confocal microscopy. We describe methods for the synthesis of these probes, determination of their affinities for crosslinked tubulin, and imaging of microtubules in living HeLa cells. We further describe their uptake by Caco-2 cells and two transporter-deficient Caco-2 knockout cell lines in the absence and presence of efflux inhibitors by flow cytometry. These studies revealed that p-glycoprotein (MDR1) and multidrug-resistance protein 2 (MRP2) are major mediators of efflux of these molecular probes. These compounds provide useful tools for studies of microtubules and cellular efflux transporters in living cells. (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Databáze: | MEDLINE |
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