Brunell David, Weissbach Herbert, Hodder Peter, Brot Nathan
Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, Florida, USA.
Assay Drug Dev Technol. 2010 Oct;8(5):615-20. doi: 10.1089/adt.2009.0263. Epub 2010 Jun 1.
The methionine sulfoxide reductase (Msr) system has been shown to play an important role in protecting cells against oxidative damage. This family of enzymes can repair damage to proteins resulting from the oxidation of methionine residues to methionine sulfoxide, caused by reactive oxygen species. Previous genetic studies in animals have shown that increased levels of methionine sulfoxide reductase enzyme A (MsrA), an important member of the Msr family, can protect cells against oxidative damage and increase life span. A high-throughput screening (HTS) compatible assay has been developed to search for both activators and inhibitors of MsrA. The assay involves a coupled reaction in which the oxidation of NADPH is measured by either spectrophotometric or fluorometric analysis. Previous studies had shown that MsrA has a broad substrate specificity and can reduce a variety of methyl sulfoxide compounds, including dimethylsulfoxide (DMSO). Since the chemicals in the screening library are dissolved in DMSO, which would compete with any of the standard substrates used for the determination of MsrA activity, an assay has been developed that uses the DMSO that is the solvent for the compounds in the library as the substrate for the MsrA enzyme. A specific activator of MsrA could have important therapeutic value for diseases that involve oxidative damage, especially age-related diseases, whereas a specific inhibitor of MsrA would have value for a variety of research studies.
甲硫氨酸亚砜还原酶（Msr）系统已被证明在保护细胞免受氧化损伤方面发挥着重要作用。该酶家族能够修复因活性氧导致甲硫氨酸残基氧化为甲硫氨酸亚砜而造成的蛋白质损伤。此前在动物身上进行的遗传学研究表明，甲硫氨酸亚砜还原酶A（MsrA）（Msr家族的一个重要成员）水平的提高能够保护细胞免受氧化损伤并延长寿命。现已开发出一种与高通量筛选（HTS）兼容的检测方法，用于寻找MsrA的激活剂和抑制剂。该检测方法涉及一个偶联反应，其中通过分光光度法或荧光分析法测定NADPH的氧化。此前的研究表明，MsrA具有广泛的底物特异性，能够还原多种甲基亚砜化合物，包括二甲基亚砜（DMSO）。由于筛选文库中的化学物质溶解于DMSO中，而DMSO会与用于测定MsrA活性的任何标准底物竞争，因此已开发出一种检测方法，该方法使用作为文库中化合物溶剂的DMSO作为MsrA酶的底物。MsrA的特异性激活剂对于涉及氧化损伤的疾病，尤其是与年龄相关的疾病可能具有重要的治疗价值，而MsrA的特异性抑制剂对于各种研究具有价值。
