表皮生长因子受体酪氨酸激酶抑制剂高通量筛选模型的建立

Establishment of high throughput screening model for epidermal growth factor receptor tyrosine kinase inhibitor

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（HTS） model for epidermal growth factor（EGF） receptor tyrosine kinase（RTK） inhibitor.Methods： The bioactive EGFR-RTK was expressed by genetic engineering technology.The binding activity and bioactivity of the compounds were examined by surface plasmon resonance and ELISA.Results： The bioactive EGFR-RTK protein was successfully expressed in the prokaryotic expression system and was immobilized on the sensor chip.The equilibrium constants（Kd） between TKI（EI 188） and RTK was 5.00×10^-7 mol·L^-1,and the IC50 was 12.37 μmol·L^-1,which was consistent with expected.With this model we screened 31 compounds and found that 6 compounds had binding activity and inhibitory activity.Conclusion： A novel HTS model of EGFR-TKI has been successfully established using surface plasmon resonance biosensor and ELISA,which lays a foundation for discovery of new TKIs.