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目的:建立表皮生长因子受体(epidermal growth factor receptor,EGFR)酪氨酸激酶(receptor tyrosine kinase,RTK)抑制剂的高通量筛选模型。方法:通过基因工程技术表达EGFR-RTK,ELISA法验证其生物学活性;应用表面等离子共振原理筛选与激酶具有结合活性的化合物,ELISA法检测其生物学活性。结果:在原核表达系统中成功表达具有生物学活性的EGFR-RTK蛋白。将蛋白偶联至生物芯片,阳性化合物EI 188与EGFR酪氨酸激酶结合的Kd值为5.00×10-7mol.L-1,IC50为12.37μmol.L-1,与预期结果一致。应用该模型筛选31个待测化合物,发现了6个具有结合活性和酶抑制活性的EGFR-RTK抑制剂。结论:成功建立了基于表面等离子共振原理和ELISA法的高通量EGFR-RTK抑制剂的筛选模型,为发现新型酪氨酸激酶抑制剂奠定了基础。
OBJECTIVE: To establish a high-throughput screening model of epidermal growth factor receptor (EGFR) inhibitor of tyrosine kinase (RTK). Methods: EGFR-RTK was expressed by genetic engineering and its biological activity was verified by ELISA. The compounds with binding activity to kinase were screened by surface plasmon resonance. The biological activity of EGFR-RTK was tested by ELISA. Results: The biologically active EGFR-RTK protein was successfully expressed in prokaryotic expression system. Coupling the protein to the biochip, the positive compound EI 188 bound to EGFR tyrosine kinase with a Kd value of 5.00 x 10-7 mol.L-1 and an IC50 of 12.37 micromol.L-1, consistent with the expected result. Thirty-one compounds tested were screened using this model and six EGFR-RTK inhibitors were found that have both binding activity and enzyme-inhibiting activity. Conclusion: The screening model of high-throughput EGFR-RTK inhibitors based on surface plasmon resonance and ELISA was successfully established, which laid the foundation for the discovery of novel tyrosine kinase inhibitors.