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.
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.
1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Division Molecular Biology Aachen, Germany.
Nina
N
Berges
1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Division Molecular Biology Aachen, Germany.|Paul-Ehrlich-Institute, Federal Institute for Vaccines and Biomedicines, Langen, Germany.
Katharina
K
Arens
1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Division Molecular Biology Aachen, Germany.
Verena
V
Kreusch
1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Division Molecular Biology Aachen, Germany.|2 Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany.
Rainer
R
Fischer
1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Division Molecular Biology Aachen, Germany.
Stefano
S
Di Fiore
D000072283:A549 Cells; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D015739:Nocodazole / Q000494:pharmacology; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry; D017239:Paclitaxel / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000494:pharmacology; D014404:Tubulin / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*
D000970:Antineoplastic Agents; D054852:Small Molecule Libraries; C498585:TUBB3 protein, human; D014404:Tubulin; D050257:Tubulin Modulators; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D017239:Paclitaxel; D015739:Nocodazole
SNAP-tag; cell-based assays; high-content screening; microtubule targeting agents; tubulin cytoskeleton