Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry.

Drugs such as paclitaxel (Taxol) that bind microtubules are widely used for the treatment of cancer. Measurements of the affinity and selectivity of these compounds for their targets are largely based on studies of purified proteins, and only a few quantitative methods for the analysis of interactions of small molecules with microtubules in living cells have been reported. We describe here a novel method for rapidly quantifying the affinities of compounds that bind polymerized tubulin in living HeLa cells. This method uses the fluorescent molecular probe Pacific Blue-GABA-Taxol in conjunction with verapamil to block cellular efflux. Under physiologically relevant conditions of 37 °C, this combination allowed quantification of equilibrium saturation binding of this probe to cellular microtubules (K d = 1.7 µM) using flow cytometry. Competitive binding of the microtubule stabilizers paclitaxel (cellular K i = 22 nM), docetaxel (cellular K i = 16 nM), cabazitaxel (cellular K i = 6 nM), and ixabepilone (cellular K i = 10 nM) revealed intracellular affinities for microtubules that closely matched previously reported biochemical affinities. By including a cooperativity factor (α) for curve fitting of allosteric modulators, this probe also allowed quantification of binding (K b) of the microtubule destabilizers colchicine (K b = 80 nM, α = 0.08), vinblastine (K b = 7 nM, α = 0.18), and maytansine (K b = 3 nM, α = 0.21). Screening of this assay against 1008 NCI diversity compounds identified NSC 93427 as a novel microtubule destabilizer (K b = 485 nM, α = 0.02), illustrating the potential of this approach for drug discovery.

Synthesis of a fluorinated pyronin that enables blue light to rapidly depolarize mitochondria.

Fluorinated analogues of the fluorophore pyronin B were synthesized as a new class of amine-reactive drug-like small molecules. In water, 2,7-difluoropyronin B was found to reversibly react with primary amines to form covalent adducts. When this fluorinated analogue is added to proteins, these adducts undergo additional oxidation to yield fluorescent 9-aminopyronins. Irradiation with visible blue light enhances this oxidation step, providing a photochemical method to modify the biological properties of reactive amines. In living HeLa cells, 2,7-difluoropyronin B becomes localized in mitochondria, where it is partially transformed into fluorescent aminopyronins, as detected by spectral profiling confocal microscopy. Further excitation of these cells with the blue laser of a confocal microscope can depolarize mitochondria within seconds. This biological activity was only observed with 2,7-difluoropyronin B and was not detected with analogues such as pyronin B or 9-methyl-2,7-difluoropyronin B. This irradiation with blue light enhances the cellular production of reactive oxygen species (ROS), suggesting that increased ROS in mitochondria promotes the formation of aminopyronins that inactivate biomolecules critical for maintenance of mitochondrial membrane potential. The unique reactivity of 2,7-difluoropyronin B offers a novel tool for photochemical control of mitochondrial biology.

Pacific Blue-Taxoids as Fluorescent Molecular Probes of Microtubules.

Taxoids such as paclitaxel (Taxol) are an important class of anticancer drugs that bind ß-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-ß-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.