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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.
- Source :
-
SLAS discovery : advancing life sciences R & D [SLAS Discov] 2017 Apr; Vol. 22 (4), pp. 387-398. Date of Electronic Publication: 2017 Jan 10. - Publication Year :
- 2017
-
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.
- Subjects :
- A549 Cells
Cell Cycle genetics
Dose-Response Relationship, Drug
Drug Discovery
Guanine analogs & derivatives
Guanine chemistry
Humans
Microtubules metabolism
Microtubules ultrastructure
Nocodazole pharmacology
O(6)-Methylguanine-DNA Methyltransferase chemistry
Paclitaxel pharmacology
Small Molecule Libraries pharmacology
Tubulin genetics
Tubulin metabolism
Antineoplastic Agents pharmacology
Cell Cycle drug effects
High-Throughput Screening Assays
Microtubules drug effects
Tubulin agonists
Tubulin Modulators pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 2472-5560
- Volume :
- 22
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- SLAS discovery : advancing life sciences R & D
- Publication Type :
- Academic Journal
- Accession number :
- 28328318
- Full Text :
- https://doi.org/10.1177/2472555216685518