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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
李林1△,李国2△,陈莉莉3,罗海斌3,陈静3,沈旭3,梅长林1*
0
(1.第二军医大学长征医院肾内科,上海 200003;2.海军411医院骨科,上海 200081;3.中国科学院上海药物研究所,上海 201203)
摘要:
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5.00×10-7 mol·L-1,IC50为12.37 μmol·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。
关键词:  表皮生长因子受体  酪氨酸激酶抑制剂  模型  高通量筛选  表面等离子共振
DOI:10.3724/SP.J.1008.2009.01349
投稿时间:2009-09-04修订日期:2009-11-27
基金项目:国家自然科学基金重点项目(30330640),国家自然科学基金(30170901),国家科技部重大科技专项(2002AA2Z3130).
Establishment of high throughput screening model for epidermal growth factor receptor tyrosine kinase inhibitor
LI Lin1Δ,LI Guo2Δ,CHEN Li-li3,LUO Hai-bin3,CHEN Jing3,SHEN Xu3,MEI Chang-lin1*
(1.Department of Nephrology,Changzheng Hospital,Second Military Medical University,Shanghai 200003,China;2.Department of Orthopaedics,No. 411 Hospital of PLA,Shanghai 200081;3.Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 201203)
Abstract:
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.
Key words:  epidermal growth factor receptor  tyrosine kinase inhibitor  model  high throughout screening  surface plasmon resonance