Smith Anthony M, Chan John, Oksenberg Donna, Urfer Roman, Wexler David S, Ow Arnie, Gao Liping, McAlorum Alanna, Huang Shu-Gui
Lead Discovery Department, AGY Therapeutics Inc, South San Francisco, CA 94080, USA.
J Biomol Screen. 2004 Oct;9(7):614-20. doi: 10.1177/1087057104265292.
Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.
蛋白质二硫键异构酶（PDI）在真核细胞中底物蛋白合成后催化二硫键重排，在蛋白质折叠过程中起关键作用。除了在内质网中对分泌蛋白的加工和成熟起主要作用外，这种酶及其同源物还参与多种重要的细胞过程；然而，它们尚未成为治疗药物开发的靶点。作者开发了一种在384孔微孔板中针对PDI及其同源酶的高通量筛选测定法。该方法基于在二硫苏糖醇存在下酶催化胰岛素的还原，并在650nm处测量还原胰岛素链的聚集。通过使用过氧化氢作为终止试剂，将这种动力学测定法转化为终点测定法。在初步筛选中证明了这种高通量测定法用于筛选化学文库的可行性。作者表明，这种均相比浊测定法稳健且具有成本效益，可用于从化学文库中鉴定PDI抑制剂，为这类酶的治疗探索开辟了道路。
