Epidermal growth factor receptor (EGFR) is among the first discovered transmembrane receptor tyrosine kinases. Ligand binding to the extracellular domain of EGFR results in the formation of homo-dimeric or hetero-dimeric complexes, activation, and auto-phosphorylation in the intracellular tyrosine kinase domain, then regulates the cell's proliferation, differentiation, migration, and invasion. It has been reported that EGFR is overexpressed, amplified, or mutated in several epithelial tumors. EGFR tyrosine kinase inhibitors (EGFR-TKIs) specifically bind to the tyrosine kinase domain of EGFR. Homogeneous time-resolved fluorescence (HTRF) assay is successfully used in kinase assays and is a crucial tool in the drug discovery of various kinases. Moreover, the HTRF assay has been determined to exhibit many advantages, such as being easy to miniaturize, simple to read, and excellent to repeat. In these guidelines, the theory, materials, and reagent preparation of HTRF kinase assay are described and emphasized. Furthermore, the protocol and optimization of the assay are determined to establish an HTRF screening platform, then an optimized HTRF kinase assay is performed to test the compounds for screening potential EGFR-TKIs. In addition, these guidelines can contribute to verifying the high-throughput screening results of HTRF and help to reveal the underlying mechanism of the EGFR kinase and tested compounds.
Yan Li,Yajing Xing,Dongxia Huang,Jiuqing Xie,Yangrui Peng,Zhengfang Yi,Yihua Chen
Aim To develop potent B cell lymphoma 6 (BCL6) inhibitors, novel N-phenyl-4-pyrimidine-diamine analogs were designed and synthesized via structure-based and computer-aided drug design.Methods Starting from the hit compound ZB979, we synthesized three series of pyrimidinediamine BCL6 inhibitors (13a-13e, 14a-14c and 15a-15g) and evaluated their inhibitory activities on BCL6-SMRT interaction using homogeneous time-resolved fluorescence (HTRF) assays. The most promising candidate, compound 15d, was further assessed for its anti-proliferative activity and modulation of BCL6 downstream target genes, suppression of germinal center (GC) formation.Results Compound 15d demonstrated significant BCL6-SMRT inhibition with favorable physicochemical properties (Calculated LogP (ClogP) and topological polar surface area (tPSA)).Conclusion These findings highlight the potential of pyrimidinediamine-based scaffolds as novel BCL6 inhibitors, warranting further structural optimization to improve their efficacy.
危险品化学品经营许可证（带存储）