Toward Discovery of Novel Microtubule Targeting Agents: A SNAP-tag–Based High-Content Screening Assay for the Analysis of Microtubule Dynamics and Cell Cycle Progression

Abstract

Microtubule targeting agents (MTAs) are used for the treatment of cancer. Novel MTAs could provide additional and beneficial therapeutic options. To improve the sensitivity and throughput of standard immunofluorescence assays for the characterization of MTAs, we used SNAP-tag technology to produce recombinant tubulin monomers. To visualize microtubule filaments, A549 cells transfected with SNAP-tubulin were stained with a membrane-permeable, SNAP-reactive dye. The treatment of SNAP-tubulin cells with stabilizing MTAs such as paclitaxel resulted in the formation of coarsely structured microtubule filaments, whereas depolymerizing MTAs such as nocodazole resulted in diffuse staining patterns in which the tubulin filaments were no longer distinguishable. By combining these components with automated microscopy and image analysis algorithms, we established a robust high-content screening assay for MTAs with a Z' factor of 0.7. Proof of principle was achieved by testing a panel of 10 substances, allowing us to identify MTAs and to distinguish between stabilizing and destabilizing modes of action. By extending the treatment of the cells from 2 to 20 h, our assay also detected abnormalities in cell cycle progression and in the formation of microtubule spindles, providing additional readouts for the discovery of new MTAs and facilitating their early identification during drug-screening campaigns.

Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

“…Having confirmed the specific induction of apoptosis and cell cycle arrest by the EGFR-specific ADCs, we investigated the effects on the tubulin cytoskeleton by microscopy using EGFR + A549 cells stably transfected with SNAP-TubB3, which facilitates visualization of the tubulin network by covalent labeling with BG fluorophores 29. This cell line was used instead of the former used cell lines, because it was evaluated before and used as model cell line as described by Berges et al29 First, we determined the cytotoxic activity of the ADCs against A549 cells using an XTT-based cell viability assay, and representative results showing cells treated with 1711(scFv)-SNAP-AURIF are presented in Figure 5A.…”

“…This cell line was used instead of the former used cell lines, because it was evaluated before and used as model cell line as described by Berges et al29 First, we determined the cytotoxic activity of the ADCs against A549 cells using an XTT-based cell viability assay, and representative results showing cells treated with 1711(scFv)-SNAP-AURIF are presented in Figure 5A. A concentration-dependent reduction of cell viability was observed when the cells were incubated with 50, 150, and 250 nM of the fusion protein: there was no effect at 50 nM and cell viability declined be ~15% at 250 nM (Figure 5A).…”

“…We inspected the effect of the ADCs by confocal microscopy in EGFR + A549 cells stably expressing SNAP-TubB3. This transfected cell line allows the covalent coupling of BG fluorophores to tubulin so that microtubule dynamics can be analyzed in the living cells 29. The treatment of these cells with the EGFR-specific ADCs resulted in destabilization of the microtubules, which would also fit with the effect of auristatin derivatives such as MMAF (Figure 5B).…”

“…A549 cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose (Thermo Fisher Scientific), also supplemented with fetal calf serum and penicillin and streptomycin as above. A549 cells transfected with pSNAP-tubulin B3 (TubB3) were cultivated in medium supplemented with 800 µg/mL G418 29. All cell lines were cultivated at 37°C in a humidified atmosphere with 5% CO 2 .…”

“…The effects on microtubule dynamics were visualized as previously described,29 using A549 cells stably expressing a recombinant SNAP-TubB3. Briefly, 7×10 3 cells were plated in a flat bottom black 96-well half-area microplate (µclear; Greiner Bio-One) in a total volume of 50 µL cell culture medium without G418.…”