An ELISA DYRK1A non-radioactive assay suitable for the characterization of inhibitors [version 1; referees: 2 approved]
The DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) gene encodes a proline-directed Ser/Thr kinase. Elevated expression and/or altered distribution of the kinase have been implicated in the neurological impairments associated with Down syndrome (DS) and Alzheimer’s disease (AD...
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| Format: | Article |
| Language: | English |
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F1000 Research Ltd
2017-01-01
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| Online Access: | https://f1000research.com/articles/6-42/v1 |
| _version_ | 1818282967664951296 |
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| author | Yong Liu Tatyana Adayev Yu-Wen Hwang |
| author_facet | Yong Liu Tatyana Adayev Yu-Wen Hwang |
| author_sort | Yong Liu |
| collection | DOAJ |
| description | The DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) gene encodes a proline-directed Ser/Thr kinase. Elevated expression and/or altered distribution of the kinase have been implicated in the neurological impairments associated with Down syndrome (DS) and Alzheimer’s disease (AD). Consequently, DYRK1A inhibition has been of significant interest as a potential strategy for therapeutic intervention of DS and AD. Many classes of novel inhibitors have been described in the past decade. Although non-radioactive methods for analyzing DYRK1A inhibition have been developed, methods employing radioactive tracers are still commonly used for quantitative characterization of DYRK1A inhibitors. Here, we present a non-radioactive ELISA assay based on the detection of DYRK1A-phosphorylated dynamin 1a fragment using a phosphorylation site-specific antibody. The assay was verified by the use of two well-characterized DYRK1A inhibitors, epigallocatechin gallate (EGCG) and harmine. The IC50s for EGCG and harmine determined by the ELISA method were found to be comparable to those previously measured by radioactive tracing methods. Furthermore, we determined the mode of inhibition for EGCG and harmine by a modification of the ELISA assay. This assay confirms the mode of inhibition of EGCG (non-ATP-competitive) and harmine (ATP-competitive), as previously determined. We conclude that the ELISA platform demonstrated here is a viable alternative to the traditional radioactive tracer assays for analyzing DYRK1A inhibitors. |
| first_indexed | 2024-12-13T00:29:26Z |
| format | Article |
| id | doaj.art-376cbc963abc493a8005b1505e8c458d |
| institution | Directory Open Access Journal |
| issn | 2046-1402 |
| language | English |
| last_indexed | 2024-12-13T00:29:26Z |
| publishDate | 2017-01-01 |
| publisher | F1000 Research Ltd |
| record_format | Article |
| series | F1000Research |
| spelling | doaj.art-376cbc963abc493a8005b1505e8c458d2022-12-22T00:05:22ZengF1000 Research LtdF1000Research2046-14022017-01-01610.12688/f1000research.10582.111404An ELISA DYRK1A non-radioactive assay suitable for the characterization of inhibitors [version 1; referees: 2 approved]Yong Liu0Tatyana Adayev1Yu-Wen Hwang2Molecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USAMolecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USAMolecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USAThe DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) gene encodes a proline-directed Ser/Thr kinase. Elevated expression and/or altered distribution of the kinase have been implicated in the neurological impairments associated with Down syndrome (DS) and Alzheimer’s disease (AD). Consequently, DYRK1A inhibition has been of significant interest as a potential strategy for therapeutic intervention of DS and AD. Many classes of novel inhibitors have been described in the past decade. Although non-radioactive methods for analyzing DYRK1A inhibition have been developed, methods employing radioactive tracers are still commonly used for quantitative characterization of DYRK1A inhibitors. Here, we present a non-radioactive ELISA assay based on the detection of DYRK1A-phosphorylated dynamin 1a fragment using a phosphorylation site-specific antibody. The assay was verified by the use of two well-characterized DYRK1A inhibitors, epigallocatechin gallate (EGCG) and harmine. The IC50s for EGCG and harmine determined by the ELISA method were found to be comparable to those previously measured by radioactive tracing methods. Furthermore, we determined the mode of inhibition for EGCG and harmine by a modification of the ELISA assay. This assay confirms the mode of inhibition of EGCG (non-ATP-competitive) and harmine (ATP-competitive), as previously determined. We conclude that the ELISA platform demonstrated here is a viable alternative to the traditional radioactive tracer assays for analyzing DYRK1A inhibitors.https://f1000research.com/articles/6-42/v1Cognitive Neurology & DementiaMedical GeneticsMethods for Diagnostic & Therapeutic Studies |
| spellingShingle | Yong Liu Tatyana Adayev Yu-Wen Hwang An ELISA DYRK1A non-radioactive assay suitable for the characterization of inhibitors [version 1; referees: 2 approved] F1000Research Cognitive Neurology & Dementia Medical Genetics Methods for Diagnostic & Therapeutic Studies |
| title | An ELISA DYRK1A non-radioactive assay suitable for the characterization of inhibitors [version 1; referees: 2 approved] |
| title_full | An ELISA DYRK1A non-radioactive assay suitable for the characterization of inhibitors [version 1; referees: 2 approved] |
| title_fullStr | An ELISA DYRK1A non-radioactive assay suitable for the characterization of inhibitors [version 1; referees: 2 approved] |
| title_full_unstemmed | An ELISA DYRK1A non-radioactive assay suitable for the characterization of inhibitors [version 1; referees: 2 approved] |
| title_short | An ELISA DYRK1A non-radioactive assay suitable for the characterization of inhibitors [version 1; referees: 2 approved] |
| title_sort | elisa dyrk1a non radioactive assay suitable for the characterization of inhibitors version 1 referees 2 approved |
| topic | Cognitive Neurology & Dementia Medical Genetics Methods for Diagnostic & Therapeutic Studies |
| url | https://f1000research.com/articles/6-42/v1 |
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