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Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1

Tubulin is a protein that plays a critical role in maintaining cellular structure and facilitating cell division. Inhibiting tubulin polymerization has been shown to be an effective strategy for inhibiting the proliferation of cancer cells. In the past, identifying compounds that could inhibit tubul...

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Autores principales: Khachatryan, Harutyun, Olszowy, Bartlomiej, Barrero, Carlos A., Gordon, John, Perez-Leal, Oscar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9953358/
https://www.ncbi.nlm.nih.gov/pubmed/36830618
http://dx.doi.org/10.3390/biom13020249
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author Khachatryan, Harutyun
Olszowy, Bartlomiej
Barrero, Carlos A.
Gordon, John
Perez-Leal, Oscar
author_facet Khachatryan, Harutyun
Olszowy, Bartlomiej
Barrero, Carlos A.
Gordon, John
Perez-Leal, Oscar
author_sort Khachatryan, Harutyun
collection PubMed
description Tubulin is a protein that plays a critical role in maintaining cellular structure and facilitating cell division. Inhibiting tubulin polymerization has been shown to be an effective strategy for inhibiting the proliferation of cancer cells. In the past, identifying compounds that could inhibit tubulin polymerization has required the use of in vitro assays utilizing purified tubulin or immunofluorescence of fixed cells. This study presents a novel approach for identifying tubulin polymerization inhibitors using a CRISPR-edited cell line that expresses fluorescently tagged β-tubulin and a nuclear protein, enabling the visualization of tubulin polymerization dynamics via high-content imaging analysis (HCI). The cells were treated with known tubulin polymerization inhibitors, colchicine, and vincristine, and the resulting phenotypic changes indicative of tubulin polymerization inhibition were confirmed using HCI. Furthermore, a library of 429 kinase inhibitors was screened, resulting in the identification of three compounds (ON-01910, HMN-214, and KX2-391) that inhibit tubulin polymerization. Live cell tracking analysis confirmed that compound treatment leads to rapid tubulin depolymerization. These findings suggest that CRISPR-edited cells with fluorescently tagged endogenous β-tubulin can be utilized to screen large compound libraries containing diverse chemical families for the identification of novel tubulin polymerization inhibitors.
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spelling pubmed-99533582023-02-25 Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1 Khachatryan, Harutyun Olszowy, Bartlomiej Barrero, Carlos A. Gordon, John Perez-Leal, Oscar Biomolecules Article Tubulin is a protein that plays a critical role in maintaining cellular structure and facilitating cell division. Inhibiting tubulin polymerization has been shown to be an effective strategy for inhibiting the proliferation of cancer cells. In the past, identifying compounds that could inhibit tubulin polymerization has required the use of in vitro assays utilizing purified tubulin or immunofluorescence of fixed cells. This study presents a novel approach for identifying tubulin polymerization inhibitors using a CRISPR-edited cell line that expresses fluorescently tagged β-tubulin and a nuclear protein, enabling the visualization of tubulin polymerization dynamics via high-content imaging analysis (HCI). The cells were treated with known tubulin polymerization inhibitors, colchicine, and vincristine, and the resulting phenotypic changes indicative of tubulin polymerization inhibition were confirmed using HCI. Furthermore, a library of 429 kinase inhibitors was screened, resulting in the identification of three compounds (ON-01910, HMN-214, and KX2-391) that inhibit tubulin polymerization. Live cell tracking analysis confirmed that compound treatment leads to rapid tubulin depolymerization. These findings suggest that CRISPR-edited cells with fluorescently tagged endogenous β-tubulin can be utilized to screen large compound libraries containing diverse chemical families for the identification of novel tubulin polymerization inhibitors. MDPI 2023-01-29 /pmc/articles/PMC9953358/ /pubmed/36830618 http://dx.doi.org/10.3390/biom13020249 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Khachatryan, Harutyun
Olszowy, Bartlomiej
Barrero, Carlos A.
Gordon, John
Perez-Leal, Oscar
Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1
title Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1
title_full Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1
title_fullStr Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1
title_full_unstemmed Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1
title_short Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1
title_sort identification of inhibitors of tubulin polymerization using a crispr-edited cell line with endogenous fluorescent tagging of β-tubulin and histone h1
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9953358/
https://www.ncbi.nlm.nih.gov/pubmed/36830618
http://dx.doi.org/10.3390/biom13020249
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