脾酪氨酸激酶(SYK)是一种非受体酪氨酸激酶,由FcR下游的磷酸化事件激活,B细胞和T细胞受体,整合素,和C型凝集素受体。当SYK的串联Src同源2(SH2)结构域与这些免疫受体中包含的磷酸化免疫受体酪氨酸活化基序(pITAMs)结合时,或者当SYK在域间区A和B中磷酸化时,SYK已激活。先前在6名具有较高水平的磷酸化SYK和磷酸化下游蛋白JNK和ERK的患者中鉴定了SYK功能获得(GoF)变体。此外,SYK激活增加导致免疫失调的临床表现,器官炎症,和淋巴瘤的易感性。利用SYKGoF变体具有增强活性的知识来开发具有SYKGoF变体的构建体的完整活细胞中的SYKNanoBRET细胞靶标接合测定。在这里,我们使用SYK捐赠的化学探针开发了一种有效的SYK靶向NanoBRET示踪剂,MRL-SYKi,这使得SYKGoF变体的NanoBRET细胞靶标接合测定成为可能,SYK(S550Y),SYK(S550F),和SYK(P342T)。我们确定ATP竞争性SYK抑制剂与完整活细胞中的这些SYK变体有效结合。此外,我们证明MRL-SYKi可以有效降低SYK变体的催化活性,和稳定表达SYK(S550Y)的上皮细胞系(SW480)中SYK(S550Y)的磷酸化水平。
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).