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

Abstract

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.

Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells

“…As shown in Figure , in nontransfected living cells (the P1 gate shown in Figure ), probes 1 and 2 exhibited low background levels of linear dose-dependent blue fluorescence. This is consistent with our previous observations − of cellular efflux of PB-linked probes. This nonspecific binding was expected to result from interactions of the probe with membranes, endogenous C1 domain-containing proteins, and other cellular biomolecules.…”

“…After treatment with these probes for 2 h, dilution with the medium resulted in rapid loss of blue fluorescence for probes 1 and 2 (efflux t 1/2 ( 1 ) = 3 min and ( 2 ) = 8 min) but much slower efflux of probe 3 (efflux t 1/2 ( 3 ) >90 min). This rapid loss of signal for probe 1 is consistent with active efflux previously observed , with other cell-permeable probes linked to Pacific Blue. This rapid efflux of 1 and 2 indicates that these probes reversibly interact with the expressed target protein, another key criterion for studies of equilibrium binding.…”

“…PB was chosen because of its potential for cellular permeability and facile detection in cells by flow cytometry. This fluorophore has been shown to promote cellular efflux when linked to the small molecule paclitaxel, − and we hypothesized that this property might reduce nonspecific binding of other linked compounds to cells and facilitate quantification of higher affinity specific binding to overexpressed protein targets. To detect binding of small molecules to specific target proteins, we coexpressed proteins in cells with the yellow-fluorescent protein mVenus. , This spectrally orthogonal fluorescent protein was investigated both fused to full-length target proteins and coexpressed as a separate marker with more physiologically relevant full-length untagged protein targets using an internal ribosomal entry site (IRES) vector.…”

Design, synthesis and biological evaluation of a novel colchicine-magnolol hybrid for inhibiting the growth of Lewis lung carcinoma in Vitro and in Vivo

“…Microtubules, which maintain the shape of the cell through the dynamic assembly of tubulin heterodimers, are generally accepted as an attractive target for the development of anti-cancer drugs ( Andres et al, 2022 ). Specifically, colchicine binding site agents bind to colchicine binding domain and prevent the polymerization of tubulin proteins, thereby destabilize microtubules and provide antitumor potential ( Wang et al, 2016 ; Sueth-Santiago et al, 2017 ).…”

Synthesis of Monofluorinated 7-Hydroxycoumarin-3-Carboxamides as Cell-Permeable Fluorescent Molecular Probes

“…This new method uses flow cytometry for quantitative studies of the binding of cell-permeable fluorescent probes and competitors to specific native (untagged) proteins that are overexpressed in living cells. Although FPCBA was previously validated using allosteric activators of Protein Kinase C isozymes, our previous studies − of interactions of fluorescent derivatives of the anticancer drug paclitaxel (Taxol) with microtubules in living cells were instrumental in developing this method. Key to this approach is the use of cell-permeable fluorophores that can be efficiently detected by flow cytometry when linked to ligands of protein targets.…”