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. |
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| Authors: | Capener JL; Structural Genomics Consortium, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Vasta JD; Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.; Katis VL; ARUK Oxford Drug Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, UK.; Michaud A; Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.; Beck MT; Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.; Daglish SCD; Structural Genomics Consortium, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Cohen-Kedar S; Division of Gastroenterology, Rabin Medical Center, Petah-Tikva, Israel.; Felsenstein Medical Research Center, Faculty of Medical & Health Sciences, Tel-Aviv University, Tel-Aviv, Israel.; Barda ES; Division of Gastroenterology, Rabin Medical Center, Petah-Tikva, Israel.; Felsenstein Medical Research Center, Faculty of Medical & Health Sciences, Tel-Aviv University, Tel-Aviv, Israel.; Howell S; Structural Genomics Consortium, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Dotan I; Division of Gastroenterology, Rabin Medical Center, Petah-Tikva, Israel.; Felsenstein Medical Research Center, Faculty of Medical & Health Sciences, Tel-Aviv University, Tel-Aviv, Israel.; Robers MB; Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.; Axtman AD; Structural Genomics Consortium, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Bashore FM; Structural Genomics Consortium, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. |
| Source: | BioRxiv : the preprint server for biology [bioRxiv] 2024 Jun 13. Date of Electronic Publication: 2024 Jun 13. |
| Publication Type: | Journal Article; Preprint |
| Language: | English |
| Journal Info: | Country of Publication: United States NLM ID: 101680187 Publication Model: Electronic Cited Medium: Internet ISSN: 2692-8205 (Electronic) Linking ISSN: 26928205 NLM ISO Abbreviation: bioRxiv Subsets: PubMed not MEDLINE |
| Abstract: | 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). |
| Competing Interests: | Conflict of Interest Michael T. Beck, Ani Michaud, James D. Vasta, and Matthew B. Robers are employees of Promega, which provided the SYK GoF variant clones and has a commercial interest in kinase NanoBRET assays. |
| Comments: | Update in: Front Chem Biol. 2024;3. doi: 10.3389/fchbi.2024.1447622.. (PMID: 42004161) |
| Grant Information: | U54 AG065187 United States AG NIA NIH HHS |
| Contributed Indexing: | Keywords: NanoBRET; Spleen tyrosine kinase; autoinhibition; gain–of–function; target engagement |
| Entry Date(s): | Date Created: 20240625 Date Completed: 20260422 Latest Revision: 20260422 |
| Update Code: | 20260422 |
| PubMed Central ID: | PMC11195201 |
| DOI: | 10.1101/2024.06.12.598544 |
| PMID: | 38915605 |
| Database: | MEDLINE |
Journal Article; Preprint