Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug development
The Raf/MEK/ERK signaling pathway plays a key role in regulating cellular proliferation, differentiation, apoptosis, cytokine production, and immune responses. However, it is also involved in diseases such as cancer, and numerous viruses rely on an active Raf/MEK/ERK pathway for propagation. This pa...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-12-01
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| Series: | Frontiers in Cell and Developmental Biology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcell.2022.1063692/full |
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| author | Lara M. Schüssele Lara M. Schüssele Julia Koch-Heier Julia Koch-Heier Julian Volk Julian Volk Markus W. Löffler Markus W. Löffler Markus W. Löffler Markus W. Löffler Katharina Hoffmann Regina M. Bruyns Oliver Planz |
| author_facet | Lara M. Schüssele Lara M. Schüssele Julia Koch-Heier Julia Koch-Heier Julian Volk Julian Volk Markus W. Löffler Markus W. Löffler Markus W. Löffler Markus W. Löffler Katharina Hoffmann Regina M. Bruyns Oliver Planz |
| author_sort | Lara M. Schüssele |
| collection | DOAJ |
| description | The Raf/MEK/ERK signaling pathway plays a key role in regulating cellular proliferation, differentiation, apoptosis, cytokine production, and immune responses. However, it is also involved in diseases such as cancer, and numerous viruses rely on an active Raf/MEK/ERK pathway for propagation. This pathway, and particularly MEK1/2, are therefore promising therapeutic targets. Assessment of target engagement is crucial to determine pharmacodynamics or the efficacy of a MEK1/2 inhibitor. In the field of infectious diseases, this is usually first determined in clinical trials with healthy volunteers. One method to detect MEK1/2 inhibitor target engagement is to assess the degree of ERK1/2 phosphorylation, as ERK1/2 is the only known substrate of MEK1/2. As healthy subjects, however, only feature a low baseline MEK1/2 activation and therefore low ERK1/2 phosphorylation in most tissues, assessing target engagement is challenging, and robust methods are urgently needed. We hence developed a method using PBMCs isolated from whole blood of healthy blood donors, followed by ex vivo treatment with the MEK1/2 inhibitor zapnometinib and stimulation with PMA to first inhibit and then induce MEK1/2 activation. As PMA cannot activate MEK1/2 upon MEK1/2 inhibition, MEK1/2 inhibition results in impaired MEK1/2 activation. In contrast, PMA stimulation without MEK1/2 inhibition results in high MEK1/2 activation. We demonstrated that, without MEK1/2 inhibitor treatment, MEK1/2 stimulation with PMA induces high MEK1/2 activation, which is clearly distinguishable from baseline MEK1/2 activation in human PBMCs. Furthermore, we showed that treatment with the MEK1/2 inhibitor zapnometinib maintains the MEK1/2 activation at approximately baseline level despite subsequent stimulation with PMA. As our protocol is easy to follow and preserves the cells in an in vivo-like condition throughout the whole handling process, this approach can be a major advance for the easy assessment of MEK1/2 inhibitor target engagement in healthy probands for clinical drug development. |
| first_indexed | 2024-04-12T02:51:50Z |
| format | Article |
| id | doaj.art-570a196fc06649cd97cda4740a096598 |
| institution | Directory Open Access Journal |
| issn | 2296-634X |
| language | English |
| last_indexed | 2024-04-12T02:51:50Z |
| publishDate | 2022-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj.art-570a196fc06649cd97cda4740a0965982022-12-22T03:50:57ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2022-12-011010.3389/fcell.2022.10636921063692Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug developmentLara M. Schüssele0Lara M. Schüssele1Julia Koch-Heier2Julia Koch-Heier3Julian Volk4Julian Volk5Markus W. Löffler6Markus W. Löffler7Markus W. Löffler8Markus W. Löffler9Katharina Hoffmann10Regina M. Bruyns11Oliver Planz12Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, GermanyAtriva Therapeutics GmbH, Tübingen, GermanyDepartment of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, GermanyAtriva Therapeutics GmbH, Tübingen, GermanyDepartment of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, GermanyAtriva Therapeutics GmbH, Tübingen, GermanyDepartment of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, GermanyDepartment of General, Visceral and Transplant Surgery, University Hospital Tübingen, Tübingen, GermanyDepartment of Clinical Pharmacology, University Hospital Tübingen, Tübingen, GermanyCluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tübingen, Tübingen, GermanyNuvisan ICB (Part of Nuvisan), Neu-Ulm, GermanyNuvisan ICB (Part of Nuvisan), Neu-Ulm, GermanyDepartment of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, GermanyThe Raf/MEK/ERK signaling pathway plays a key role in regulating cellular proliferation, differentiation, apoptosis, cytokine production, and immune responses. However, it is also involved in diseases such as cancer, and numerous viruses rely on an active Raf/MEK/ERK pathway for propagation. This pathway, and particularly MEK1/2, are therefore promising therapeutic targets. Assessment of target engagement is crucial to determine pharmacodynamics or the efficacy of a MEK1/2 inhibitor. In the field of infectious diseases, this is usually first determined in clinical trials with healthy volunteers. One method to detect MEK1/2 inhibitor target engagement is to assess the degree of ERK1/2 phosphorylation, as ERK1/2 is the only known substrate of MEK1/2. As healthy subjects, however, only feature a low baseline MEK1/2 activation and therefore low ERK1/2 phosphorylation in most tissues, assessing target engagement is challenging, and robust methods are urgently needed. We hence developed a method using PBMCs isolated from whole blood of healthy blood donors, followed by ex vivo treatment with the MEK1/2 inhibitor zapnometinib and stimulation with PMA to first inhibit and then induce MEK1/2 activation. As PMA cannot activate MEK1/2 upon MEK1/2 inhibition, MEK1/2 inhibition results in impaired MEK1/2 activation. In contrast, PMA stimulation without MEK1/2 inhibition results in high MEK1/2 activation. We demonstrated that, without MEK1/2 inhibitor treatment, MEK1/2 stimulation with PMA induces high MEK1/2 activation, which is clearly distinguishable from baseline MEK1/2 activation in human PBMCs. Furthermore, we showed that treatment with the MEK1/2 inhibitor zapnometinib maintains the MEK1/2 activation at approximately baseline level despite subsequent stimulation with PMA. As our protocol is easy to follow and preserves the cells in an in vivo-like condition throughout the whole handling process, this approach can be a major advance for the easy assessment of MEK1/2 inhibitor target engagement in healthy probands for clinical drug development.https://www.frontiersin.org/articles/10.3389/fcell.2022.1063692/fullpharmacodynamicszapnometinibPBMCsclinical drug developmentMEK1/2 inhibitor |
| spellingShingle | Lara M. Schüssele Lara M. Schüssele Julia Koch-Heier Julia Koch-Heier Julian Volk Julian Volk Markus W. Löffler Markus W. Löffler Markus W. Löffler Markus W. Löffler Katharina Hoffmann Regina M. Bruyns Oliver Planz Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug development Frontiers in Cell and Developmental Biology pharmacodynamics zapnometinib PBMCs clinical drug development MEK1/2 inhibitor |
| title | Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug development |
| title_full | Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug development |
| title_fullStr | Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug development |
| title_full_unstemmed | Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug development |
| title_short | Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug development |
| title_sort | establishment of a novel method to assess mek1 2 inhibition in pbmcs for clinical drug development |
| topic | pharmacodynamics zapnometinib PBMCs clinical drug development MEK1/2 inhibitor |
| url | https://www.frontiersin.org/articles/10.3389/fcell.2022.1063692/full |
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