Cargando…
Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors
Tyrosine kinases are crucial signaling components of diverse biological processes and are major therapeutic targets in various malignancies and immune-mediated disorders. A critical step of development of novel tyrosine kinase inhibitors is the transition from the confirmation of the in vitro effect...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10117656/ https://www.ncbi.nlm.nih.gov/pubmed/37089957 http://dx.doi.org/10.3389/fphar.2023.1056154 |
_version_ | 1785028638277632000 |
---|---|
author | Futosi, Krisztina Bajza, Boglárka Deli, Dorottya Erdélyi, András Tusnády, Simon Mócsai, Attila |
author_facet | Futosi, Krisztina Bajza, Boglárka Deli, Dorottya Erdélyi, András Tusnády, Simon Mócsai, Attila |
author_sort | Futosi, Krisztina |
collection | PubMed |
description | Tyrosine kinases are crucial signaling components of diverse biological processes and are major therapeutic targets in various malignancies and immune-mediated disorders. A critical step of development of novel tyrosine kinase inhibitors is the transition from the confirmation of the in vitro effects of drug candidates to the analysis of their in vivo efficacy. To facilitate this transition, we have developed a rapid in vivo assay for the analysis of the effect of oral tyrosine kinase inhibitors on basal tyrosine phosphorylation of circulating mouse neutrophils. The assay uses a single drop of peripheral blood without sacrificing the mice. Flow cytometry using intracellular staining by fluorescently labeled anti-phosphotyrosine antibodies revealed robust basal tyrosine phosphorylation in resting circulating neutrophils. This signal was abrogated by the use of isotype control antibodies or by pre-saturation of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids or tyrosine-phosphorylated peptides. Basal tyrosine phosphorylation was dramatically reduced in neutrophils of triple knockout mice lacking the Src-family tyrosine kinases Hck, Fgr, and Lyn. Neutrophil tyrosine phosphorylation was also abrogated by oral administration of the Abl/Src-family inhibitor dasatinib, a clinically used anti-leukemic agent. Detailed dose-response and kinetic studies revealed half-maximal reduction of neutrophil tyrosine phosphorylation by 2.9 mg/kg dasatinib, with maximal reduction observed 2 h after inhibitor administration. Taken together, our assay allows highly efficient analysis of the in vivo effect of orally administered tyrosine kinase inhibitors, and may be used as a suitable alternative to other existing approaches. |
format | Online Article Text |
id | pubmed-10117656 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-101176562023-04-21 Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors Futosi, Krisztina Bajza, Boglárka Deli, Dorottya Erdélyi, András Tusnády, Simon Mócsai, Attila Front Pharmacol Pharmacology Tyrosine kinases are crucial signaling components of diverse biological processes and are major therapeutic targets in various malignancies and immune-mediated disorders. A critical step of development of novel tyrosine kinase inhibitors is the transition from the confirmation of the in vitro effects of drug candidates to the analysis of their in vivo efficacy. To facilitate this transition, we have developed a rapid in vivo assay for the analysis of the effect of oral tyrosine kinase inhibitors on basal tyrosine phosphorylation of circulating mouse neutrophils. The assay uses a single drop of peripheral blood without sacrificing the mice. Flow cytometry using intracellular staining by fluorescently labeled anti-phosphotyrosine antibodies revealed robust basal tyrosine phosphorylation in resting circulating neutrophils. This signal was abrogated by the use of isotype control antibodies or by pre-saturation of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids or tyrosine-phosphorylated peptides. Basal tyrosine phosphorylation was dramatically reduced in neutrophils of triple knockout mice lacking the Src-family tyrosine kinases Hck, Fgr, and Lyn. Neutrophil tyrosine phosphorylation was also abrogated by oral administration of the Abl/Src-family inhibitor dasatinib, a clinically used anti-leukemic agent. Detailed dose-response and kinetic studies revealed half-maximal reduction of neutrophil tyrosine phosphorylation by 2.9 mg/kg dasatinib, with maximal reduction observed 2 h after inhibitor administration. Taken together, our assay allows highly efficient analysis of the in vivo effect of orally administered tyrosine kinase inhibitors, and may be used as a suitable alternative to other existing approaches. Frontiers Media S.A. 2023-04-06 /pmc/articles/PMC10117656/ /pubmed/37089957 http://dx.doi.org/10.3389/fphar.2023.1056154 Text en Copyright © 2023 Futosi, Bajza, Deli, Erdélyi, Tusnády and Mócsai. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Pharmacology Futosi, Krisztina Bajza, Boglárka Deli, Dorottya Erdélyi, András Tusnády, Simon Mócsai, Attila Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors |
title | Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors |
title_full | Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors |
title_fullStr | Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors |
title_full_unstemmed | Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors |
title_short | Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors |
title_sort | analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors |
topic | Pharmacology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10117656/ https://www.ncbi.nlm.nih.gov/pubmed/37089957 http://dx.doi.org/10.3389/fphar.2023.1056154 |
work_keys_str_mv | AT futosikrisztina analysisofintracellulartyrosinephosphorylationincirculatingneutrophilsasarapidassayfortheinvivoeffectoforaltyrosinekinaseinhibitors AT bajzaboglarka analysisofintracellulartyrosinephosphorylationincirculatingneutrophilsasarapidassayfortheinvivoeffectoforaltyrosinekinaseinhibitors AT delidorottya analysisofintracellulartyrosinephosphorylationincirculatingneutrophilsasarapidassayfortheinvivoeffectoforaltyrosinekinaseinhibitors AT erdelyiandras analysisofintracellulartyrosinephosphorylationincirculatingneutrophilsasarapidassayfortheinvivoeffectoforaltyrosinekinaseinhibitors AT tusnadysimon analysisofintracellulartyrosinephosphorylationincirculatingneutrophilsasarapidassayfortheinvivoeffectoforaltyrosinekinaseinhibitors AT mocsaiattila analysisofintracellulartyrosinephosphorylationincirculatingneutrophilsasarapidassayfortheinvivoeffectoforaltyrosinekinaseinhibitors |