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    Ligands for phosphatase binding assay
    8.
    发明授权
    Ligands for phosphatase binding assay 失效
    用于磷酸酶结合测定的配体

    公开(公告)号：US06348572B1

    公开(公告)日：2002-02-19

    申请号：US09069138

    申请日：1998-04-29

    申请人： Sylvie Desmarais ,  Robert Zamboni ,  Richard Friesen ,  Yves LeBlanc ,  Claude Dufresne ,  Robert N. Young ,  Patrick Roy

    发明人： Sylvie Desmarais ,  Robert Zamboni ,  Richard Friesen ,  Yves LeBlanc ,  Claude Dufresne ,  Robert N. Young ,  Patrick Roy

    IPC分类号： C07K508

    CPC分类号： G11B7/244 ,  G11B7/247 ,  G11B7/25

    摘要： Disclosed are new ligands for use in a binding assay for proteases and phosphatases, which contain cysteine in their binding sites or as a necessary structural component for enzymatic binding. The sulfhydryl group of cysteine is the nucleophilic group in the enzyme's mechanistic proteolytic and hydrolytic properties. The assay can be used to determine the ability of new, unknown ligands and mixtures of compounds to competitively bind with the enzyme versus a known binding agent for the enzyme, e.g., a known enzyme inhibitor. By the use of a mutant form of the natural or native wild-type enzyme, in which serine, or another amino acid, e.g., alanine, replaces cysteine, the problem of interference from extraneous oxidizing and alkylating agents in the assay procedure is overcome. The interference arises because of oxidation or alkylation of the sulfhydryl, —SH (or —S−), in the cysteine, which then adversely affects the binding ability of the enzyme. Specifically disclosed is an assay for tyrosine phosphatases and cysteine proteases, including caspases and cathepsins, e.g., Cathepsin K(O2), utilizing scintillation proximity assay (SPA) technology. The assay has important applications in the discovery of compounds for the treatment and study of, for example, diabetes, immunosuppression, cancer, Alzheimer's disease and osteoporosis. The novel feature of the use of a mutant enzyme can be extended to its use in a wide variety of conventional colorimetric, photometric, spectrophotometric, radioimmunoassay and ligand-binding competitive assays.

    摘要翻译： 公开了用于蛋白酶和磷酸酶的结合测定中的新配体，其在其结合位点含有半胱氨酸或作为酶结合的必需结构组分。 半胱氨酸的巯基是酶的机理蛋白水解和水解性质中的亲核基团。 该测定法可用于测定新的未知配体和化合物的混合物与酶竞争性结合的能力，与酶的已知结合剂，例如已知的酶抑制剂。 通过使用天然或天然野生型酶的突变形式，其中丝氨酸或另一氨基酸例如丙氨酸取代半胱氨酸，克服了测定程序中来自外来氧化和烷化剂的干扰问题。 干扰是由于半胱氨酸中巯基-SH（或-S-）的氧化或烷基化而产生的，其然后不利地影响酶的结合能力。 具体公开的是利用闪烁近邻测定（SPA）技术的酪氨酸磷酸酶和半胱氨酸蛋白酶的测定法，包括胱天蛋白酶和组织蛋白酶，例如组织蛋白酶K（O 2）。 该测定在发现用于治疗和研究例如糖尿病，免疫抑制，癌症，阿尔茨海默氏病和骨质疏松症的化合物中具有重要应用。 使用突变酶的新颖特征可以扩展到其在各种常规比色，光度，分光光度，放射免疫测定和配体结合竞争性测定中的用途。