Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants

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

Journal article

Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants

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).

Publication status:: Published

Peer review status:: Peer reviewed

Actions

Email

Email this record

Send the bibliographic details of this record to your email address.

Your Email
Please enter the email address that the record information will be sent to.

-
Your message (optional)
Please add any additional information to be included within the email.
Cite

Cite this record

APA Style

Capener, J. L., Vasta, J. D., Katis, V. L., Michaud, A., Beck, M. T., Daglish, S. C. D., Cohen-Kedar, S., Shaham Barda, E., Howell, S., Dotan, I., Robers, M. B., Axtman, A. D., & Bashore, F. M. (2024). Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants. Frontiers in Chemical Biology, 3.

MLA Style

Capener, J. L., et al. “Development of SYK NanoBRET Cellular Target Engagement Assays for Gain–of–Function Variants.” Frontiers in Chemical Biology, vol. 3, Frontiers Media, 2024.

Chicago Style

Capener, JL, JD Vasta, VL Katis, A Michaud, MT Beck, SCD Daglish, S Cohen-Kedar, et al. 2024. “Development of SYK NanoBRET Cellular Target Engagement Assays for Gain–of–Function Variants.” Frontiers in Chemical Biology 3.
Share
Print