组蛋白去乙酰化酶6(HDAC6)抑制剂分子水平高通量筛选模型的建立

旨在建立分子水平HDAC6小分子抑制剂的高通量筛选模型,用于新型HDAC6特异性小分子抑制剂的发现。建立HDAC6的昆虫表达系统,分离纯化HDAC6蛋白,利用底物Boc-Lys(Ac)-AMC对纯化的HDAC6进行测活,并对测活体系进行优化,以SAHA为阳性抑制剂,确定适合高通量筛选的酶及底物浓度,反应时间等。首先构建HDAC6昆虫真核细胞表达载体,转入昆虫细胞中表达,并利用GST亲和柱纯化获得较高纯度的GST-HDAC6融合蛋白;建立体外HDAC6分子测活方法,表明昆虫表达的GST-HDAC6融合蛋白具有去乙酰化酶活性,并通过对多种参数优化使得Z’因子达到0.60,表明分子水平的HDAC6小分子抑制剂高通量筛选体系成功建立。

Establish of Molecular-based High-throughput Screening Model for HDAC6 Inhibitors

It was to establish molecular-based high-throughput screening model for HDAC6 inhibitors.GST-thrombin was subcloned into pFastBac1 vector and then pFastBac1-HDAC6 recombinant plasmid was constructed and transformed into DH10Bac competent cells,generating the recombinant bacmid.Human GST-fusion HDAC6 proteins were expressed in insect Hi5 cells using a baculoviral expression system,and purified using GST sepharose.The deacetylase activity of HDAC6 was assayed with HDAC substrate(Boc-Lys(Ac)-AMC).SAHA was used as the positive control to optimize the activity assay.Results showed the gene segment was confirmed to be the whole coding region of human HDAC6 cDNA by DNA sequencing analysis and the recombinant bacmid was confirmed to have the whole length of HDAC6 cDNA.The GST-HDAC6 fusion protein is expressed in Hi5 insect cells,and purified using GST affinity purification.And the deacetylase activity of HDAC6 was evaluated and confirmed by in vitro assay.Finally,the assay was optimized and the Z’-factor is about 0.60,indicating the molecular-based high-throughput screening model for HDAC6 inhibitors was successfully established.