Beharry Andrew A, Lacoste Sandrine, O'Connor Timothy R, Kool Eric T
Department of Chemistry, Stanford University , Stanford, California 94305, United States.
Department of Cancer Biology, Beckman Research Institute , Duarte, California 91010, United States.
J Am Chem Soc. 2016 Mar 23;138(11):3647-50. doi: 10.1021/jacs.6b00986. Epub 2016 Mar 15.
The 2-oxoglutarate-dependent iron enzyme ALKBH3 is an antitumor target and a potential diagnostic marker for several tumor types, including prostate cancer. However, there is at present no simple way to measure this enzyme's activity. Here we describe a fluorogenic probe design (MAQ) that is directly responsive to ALKBH3 repair activity. It makes use of the fluorescence-quenching properties of 1-methyladenine; removal of the alkyl group results in a >10-fold light-up signal. The probe is specific for ALKBH3 over its related homologue ALKBH2 and can be used to identify and measure the effectiveness of enzyme inhibitors. Measurements of the enzyme substrate parameters show that MAQ displays Km and kcat values essentially the same as those of the native substrate. Finally, we show that the probe functions efficiently in cells, allowing imaging and quantitation of ALKBH3 activity by microscopy and flow cytometry. We expect that MAQ probes will be broadly useful in the study of the basic biology of ALKBH3 and in clinical cancer applications as well.
依赖2-氧代戊二酸的铁酶ALKBH3是一种抗肿瘤靶点，也是包括前列腺癌在内的多种肿瘤类型的潜在诊断标志物。然而，目前尚无简单方法来测量这种酶的活性。在此，我们描述了一种对ALKBH3修复活性直接有反应的荧光探针设计（MAQ）。它利用了1-甲基腺嘌呤的荧光猝灭特性；烷基的去除会导致信号增强超过10倍。该探针对ALKBH3具有特异性，优于其相关同源物ALKBH2，可用于鉴定和测量酶抑制剂的有效性。酶底物参数测量表明，MAQ的Km和kcat值与天然底物基本相同。最后，我们表明该探针在细胞中能有效发挥作用，可通过显微镜和流式细胞术对ALKBH3活性进行成像和定量。我们预计MAQ探针在ALKBH3基础生物学研究以及临床癌症应用中都将有广泛用途。
