Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants
Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based...
| Autores principales: | , , , , , , , , , , , , |
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| Formato: | Journal article |
| Lenguaje: | English |
| Publicado: |
Frontiers Media
2024
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| _version_ | 1826313844567310336 |
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| author | Capener, JL Vasta, JD Katis, VL Michaud, A Beck, MT Daglish, SCD Cohen-Kedar, S Shaham Barda, E Howell, S Dotan, I Robers, MB Axtman, AD Bashore, FM |
| author_facet | Capener, JL Vasta, JD Katis, VL Michaud, A Beck, MT Daglish, SCD Cohen-Kedar, S Shaham Barda, E Howell, S Dotan, I Robers, MB Axtman, AD Bashore, FM |
| author_sort | Capener, JL |
| collection | OXFORD |
| description | Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y). |
| first_indexed | 2024-09-25T04:22:55Z |
| format | Journal article |
| id | oxford-uuid:f0f726f4-bb42-450d-8f8f-1a7e11716b3b |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:22:55Z |
| publishDate | 2024 |
| publisher | Frontiers Media |
| record_format | dspace |
| spelling | oxford-uuid:f0f726f4-bb42-450d-8f8f-1a7e11716b3b2024-08-12T19:35:19ZDevelopment of SYK NanoBRET cellular target engagement assays for gain–of–function variantsJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:f0f726f4-bb42-450d-8f8f-1a7e11716b3bEnglishJisc Publications RouterFrontiers Media2024Capener, JLVasta, JDKatis, VLMichaud, ABeck, MTDaglish, SCDCohen-Kedar, SShaham Barda, EHowell, SDotan, IRobers, MBAxtman, ADBashore, FMSpleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y). |
| spellingShingle | Capener, JL Vasta, JD Katis, VL Michaud, A Beck, MT Daglish, SCD Cohen-Kedar, S Shaham Barda, E Howell, S Dotan, I Robers, MB Axtman, AD Bashore, FM Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants |
| title | Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants |
| title_full | Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants |
| title_fullStr | Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants |
| title_full_unstemmed | Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants |
| title_short | Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants |
| title_sort | development of syk nanobret cellular target engagement assays for gain of function variants |
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