Itoh Yukihiro, Suzuki Miki, Matsui Taiji, Ota Yosuke, Hui Zi, Tsubaki Kazunori, Suzuki Takayoshi
Graduate School of Medical Science, Kyoto Prefectural University of Medicine.
Chem Pharm Bull (Tokyo). 2016;64(8):1124-8. doi: 10.1248/cpb.c16-00123.
Fluorescence assays are useful tools for estimating enzymatic activity. Their simplicity and manageability make them suitable for screening enzyme inhibitors in drug discovery studies. However, researchers need to pay attention to compounds that show auto-fluorescence and quench fluorescence, because such compounds lower the accuracy of the fluorescence assay systems by producing false-positive or negative results. In this study, we found that aurone compound 7, which has been reported as a histone deacetylase (HDAC) inhibitor, gave false-positive results. Although compound 7 was identified by an in vitro HDAC fluorescence assay, it did not show HDAC inhibitory activity in a cell-based assay, leading us to suspect its in vitro HDAC inhibitory activity. As a result of verification experiments, we found that compound 7 interferes with the HDAC fluorescence assay by quenching the HDAC fluorescence signal. Our findings underscore the faults of fluorescence assays and call attention to careless interpretation.
荧光测定法是评估酶活性的有用工具。其简单性和可操作性使其适用于药物发现研究中的酶抑制剂筛选。然而，研究人员需要注意那些显示自发荧光和淬灭荧光的化合物，因为此类化合物会产生假阳性或阴性结果，从而降低荧光测定系统的准确性。在本研究中，我们发现已报道为组蛋白脱乙酰酶（HDAC）抑制剂的橙酮化合物7给出了假阳性结果。尽管化合物7通过体外HDAC荧光测定法得以鉴定，但它在基于细胞的测定中并未显示出HDAC抑制活性，这使我们怀疑其体外HDAC抑制活性。经过验证实验，我们发现化合物7通过淬灭HDAC荧光信号干扰了HDAC荧光测定。我们的研究结果强调了荧光测定法的缺陷，并提醒注意草率的解读。
