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ORIGINAL RESEARCH article

Front. Chem. Biol.
Sec. Molecular Sciences
Volume 3 - 2024 | doi: 10.3389/fchbi.2024.1447622
This article is part of the Research Topic Investigating Small Molecule Selectivity in Chemical Biology: Protein Kinases and GPCRs View all articles

Development of SYK NanoBRET Cellular Target Engagement Assays for Gain-of-Function Variants

Provisionally accepted
Jacob L. Capener Jacob L. Capener 1James D. Vasta James D. Vasta 2Vittorio L. Katis Vittorio L. Katis 3Ani Michaud Ani Michaud 2Michael T. Beck Michael T. Beck 2Sabrina C. Daglish Sabrina C. Daglish 1Sarit Cohen-Kedar Sarit Cohen-Kedar 4Efrat Shaham Barda Efrat Shaham Barda 4Stefanie Howell Stefanie Howell 1Iris Dotan Iris Dotan 4Matthew B. Robers Matthew B. Robers 2Alison D. Axtman Alison D. Axtman 1Frances M. Bashore Frances M. Bashore 1*
  • 1 Structural Genomics Consortium, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, United States
  • 2 Promega Corporation (United States), Madison, Wisconsin, United States
  • 3 ARUK Oxford Drug Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, England, United Kingdom
  • 4 Felsenstein Medical Research Center, Faculty of Medical & Health Sciences, Tel Aviv University, Tel Aviv, Tel Aviv, Israel

The final, formatted version of the article will be published soon.

    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 tyrosinebased 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).

    Keywords: Spleen tyrosine kinase, NanoBRET, gain-of-function, autoinhibition, Target engagement

    Received: 11 Jun 2024; Accepted: 04 Jul 2024.

    Copyright: © 2024 Capener, Vasta, Katis, Michaud, Beck, Daglish, Cohen-Kedar, Shaham Barda, Howell, Dotan, Robers, Axtman and Bashore. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Frances M. Bashore, Structural Genomics Consortium, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, United States

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