以蛋白激酶B为靶点的新型抗结核药物高通量筛选模型的建立和应用

The establishment and application of a high throughput screening assay for inhibitors of Mycobacterium tuberculosis protein kinase B

摘要:

【目的】建立以结核分枝杆菌蛋白激酶B为靶点的高通量筛选模型，并运用此模型进行化合物的筛选。【方法】克隆和表达结核分枝杆菌蛋白激酶B，并以其为靶酶建立并优化PknB抑制剂高通量筛选模型，利用该模型对化合物样品进行筛选，并对筛选到的阳性化合物进行抗菌和抑酶活性评价。【结果】利用该模型筛选了化合物样品18 000个，得到具有抑酶活性的阳性化合物8个，其中3个化合物具有较好的对结核分枝杆菌、海分枝杆菌、耻垢分枝杆菌的抑菌活性。【结论】建立的以PknB为靶点的抗结核药物高通量筛选模型具有灵敏度高、稳定性强等优点，可成功用于化合物的高效筛选。筛选得到3个在抑酶水平和抗菌方面均具有良好活性的阳性化合物样品，值得进一步研究。

Abstract:

[Objective] To establish and apply an high-throughput assay for screening inhibitors of Mycobacterium tuberculosis protein kinase B (PknB). [Methods] The kinase domain of Mycobacterium tuberculosis PknB was first cloned and expressed in Escherichia coli. The purified protein is utilized for a high-throughput assay for screening PknB inhibitor. [Results] Eight out of 18 000 compounds were identified as exhibiting inhibitory effect upon PknB activity using the screening assay, among which three compounds showed antibacterial activity against Mycobacterium tuberculosis, Mycobacterium marinum and Mycobacterium smegmatis. [Conclusion] The high-throughput screening model has advantages of high sensitivity and stability, which can be used in anti-TB drug screening. Three compounds are worth further study because of their great activities against both PknB and bacterial growth.