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Development of a cell-free screening assay for the identification of direct PERK activators

Costa, Márcia F D ; Höglinger, Günter U ; Rösler, Thomas W Paudel, Hemant K.

PloS one, 2023-05, Vol.18 (5), p.e0283943-e0283943 [Periódico revisado por pares]

United States: Public Library of Science

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Título:
Development of a cell-free screening assay for the identification of direct PERK activators
Autor: Costa, Márcia F D ; Höglinger, Günter U ; Rösler, Thomas W
Paudel, Hemant K.
Assuntos: Analysis ; Assaying ; Biology and Life Sciences ; Calcineurin ; Cell activation ; Degeneration ; Domains ; Drug development ; eIF-2 Kinase - genetics ; eIF-2 Kinase - metabolism ; Endoplasmic reticulum ; Endoplasmic Reticulum Stress ; Eukaryotic Initiation Factor-2 - metabolism ; Health aspects ; Homeostasis ; Humans ; Kinases ; Light ; Medical examination ; Modulators ; Nervous system ; Neurodegenerative diseases ; Neuromodulation ; Phosphorylation ; Physical Sciences ; Protein folding ; Protein Kinase Inhibitors ; Proteins ; Reagents ; Screening ; Signal Transduction ; Smad3 protein ; Substrates ; Tau protein ; Tau proteins ; Tauopathies ; Unfolded Protein Response
É parte de: PloS one, 2023-05, Vol.18 (5), p.e0283943-e0283943
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
Competing Interests: The authors have declared that no competing interests exist.
Descrição: The activation of the unfolded protein response, particularly via the PERK pathway, has been suggested as a promising therapeutic approach in tauopathies, a group of neurodegenerative disorders characterized by the abnormal phosphorylation and aggregation of tau protein. So far, a shortage of available direct PERK activators has been limiting the progresses in this field. Our study aimed at the development of a cell-free screening assay enabling the detection of novel direct PERK activators. By applying the catalytic domain of recombinant human PERK, we initially determined ideal conditions of the kinase assay reaction, including parameters such as optimal kinase concentration, temperature, and reaction time. Instead of using PERK's natural substrate proteins, eIF2α and NRF2, we applied SMAD3 as phosphorylation-accepting protein and successfully detected cell-free PERK activation and inhibition by selected modulators (e.g., calcineurin-B, GSK2606414). The developed assay revealed to be sufficiently stable and robust to assess an activating EC50-value. Additionally, our results suggested that PERK activation may take place independent of the active site which can be blocked by a kinase inhibitor. Finally, we confirmed the applicability of the assay by measuring PERK activation by MK-28, a recently described PERK activator. Overall, our data show that a cell-free luciferase-based assay with the recombinant human PERK kinase domain and SMAD3 as substrate protein is capable of detecting PERK activation, which enables to screen large compound libraries for direct PERK activators, in a high-throughput-based approach. These activators will be useful for deepening our understanding of the PERK signaling pathway, and may also lead to the identification of new therapeutic drug candidates for neurodegenerative tauopathies.
Editor: United States: Public Library of Science
Idioma: Inglês

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Development of a cell-free screening assay for the identification of direct PERK activators

Costa, Márcia F D ; Höglinger, Günter U ; Rösler, Thomas W Paudel, Hemant K.

United States: Public Library of Science

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