Cargando…
Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry
[Image: see text] 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 anal...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2022
|
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585582/ https://www.ncbi.nlm.nih.gov/pubmed/36281300 http://dx.doi.org/10.1021/acsbiomedchemau.2c00031 |
_version_ | 1784813524025868288 |
---|---|
author | Andres, Angelo E. Mariano, Andres Rane, Digamber Peterson, Blake R. |
author_facet | Andres, Angelo E. Mariano, Andres Rane, Digamber Peterson, Blake R. |
author_sort | Andres, Angelo E. |
collection | PubMed |
description | [Image: see text] 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. |
format | Online Article Text |
id | pubmed-9585582 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-95855822022-10-22 Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry Andres, Angelo E. Mariano, Andres Rane, Digamber Peterson, Blake R. ACS Bio Med Chem Au [Image: see text] 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. American Chemical Society 2022-08-09 /pmc/articles/PMC9585582/ /pubmed/36281300 http://dx.doi.org/10.1021/acsbiomedchemau.2c00031 Text en © 2022 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Andres, Angelo E. Mariano, Andres Rane, Digamber Peterson, Blake R. Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry |
title | Quantification
of Engagement of Microtubules by Small
Molecules in Living Cells by Flow Cytometry |
title_full | Quantification
of Engagement of Microtubules by Small
Molecules in Living Cells by Flow Cytometry |
title_fullStr | Quantification
of Engagement of Microtubules by Small
Molecules in Living Cells by Flow Cytometry |
title_full_unstemmed | Quantification
of Engagement of Microtubules by Small
Molecules in Living Cells by Flow Cytometry |
title_short | Quantification
of Engagement of Microtubules by Small
Molecules in Living Cells by Flow Cytometry |
title_sort | quantification
of engagement of microtubules by small
molecules in living cells by flow cytometry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585582/ https://www.ncbi.nlm.nih.gov/pubmed/36281300 http://dx.doi.org/10.1021/acsbiomedchemau.2c00031 |
work_keys_str_mv | AT andresangeloe quantificationofengagementofmicrotubulesbysmallmoleculesinlivingcellsbyflowcytometry AT marianoandres quantificationofengagementofmicrotubulesbysmallmoleculesinlivingcellsbyflowcytometry AT ranedigamber quantificationofengagementofmicrotubulesbysmallmoleculesinlivingcellsbyflowcytometry AT petersonblaker quantificationofengagementofmicrotubulesbysmallmoleculesinlivingcellsbyflowcytometry |