Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, Georgia, USA; Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, Georgia, USA.
Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, Georgia, USA; Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, Georgia, USA.
SLAS Discov. 2024 Sep;29(6):100177. doi: 10.1016/j.slasd.2024.100177. Epub 2024 Aug 21.
The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as a TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as an acceptor. We have optimized the assay into a 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.
脾酪氨酸激酶（SYK）和高亲和力免疫球蛋白 E 受体亚基 γ（FCER1G）相互作用在正常的先天和适应性免疫反应中起着重要作用，但这种相互作用的失调与几种人类疾病有关，包括自身免疫性疾病、血液恶性肿瘤和阿尔茨海默病。小分子化学探针的开发可以帮助研究正常和异常情况下的这条途径。在此，我们描述了时间分辨荧光共振能量转移（TR-FRET）测定法的小型化，以测量 SYK 和 FCER1G 之间的相互作用，该测定法采用 SYK 的 His-SH2 结构域，该结构域间接用抗 His-铽标记，用作 TR-FRET 供体，并用 FCER1G 的 FITC 缀合的磷酸化 ITAM 结构域肽作为受体。我们已经将测定法优化为 384 孔 HTS 格式，并进一步将测定法小型化为 1536 孔 uHTS 格式。通过对 2036 种 FDA 批准的和有生物活性的化合物库进行的初步筛选，该测定法具有>0.8 的 Z'因子和>15 的信号与背景（S/B）比值，从而实现了稳健的测定法性能。该 uHTS TR-FRET 测定法已通过对 2036 种 FDA 批准的和有生物活性的化合物库进行的初步筛选得到验证。从初步 uHTS 中已经鉴定出几个主要的命中化合物。一种化合物苏木精被证实可以在正交蛋白质-蛋白质相互作用测定中破坏 SYK/FECR1G 相互作用。因此，我们优化和小型化的 uHTS 测定法可用于未来扩大筛选范围，以鉴定针对 SYK 和 FCER1G 相互作用的小分子抑制剂。
