Repurposing a Histamine Detection Platform for High-Throughput Screening of Histidine Decarboxylase
Repurposing a Histamine Detection Platform for High-Throughput Screening of Histidine Decarboxylase
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DOI：
10.1177/2472555218778053
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发表时间：
2018-10-01
期刊：
影响因子：
3.1
通讯作者：
Partridge, Anthony William
中科院分区：
文献类型：
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作者：
Juang, Yu-Chi;Fradera, Xavier;Partridge, Anthony William
组氨酸脱羧酶（HDC）是催化组氨酸转化为组胺的主要酶。HDC参与许多生理反应，如组胺在过敏反应、神经反应、胃酸分泌、细胞增殖分化等方面发挥重要作用。HDC的小分子调节代表了一系列组胺相关疾病的潜在治疗策略，包括炎症性疾病、神经系统疾病、胃溃疡和某些癌症。目前用于测量HDC活性的高通量筛选（HTS）方法有限。在这里，我们报告了一种时间分辨荧光共振能量转移（TR-FRET）测定监测HDC活性的发展。该分析是基于hdc生成的组胺和荧光团标记的组胺与隐酸铕（EuK）标记的抗组胺抗体结合的竞争。我们证明了该检测方法高度敏感且易于开发。采用小体积384孔板进行分析验证实验，得到良好的统计参数。试验性HTS屏幕的Z′得分为bb0.5，命中率为1.1%，从而确定了一个有效的命中系列。总的来说，通过作为一种适用于大规模HTS和随后对化合物结构-活性关系的询问的新工具，所提出的分析应该有助于发现治疗性HDC抑制剂。
Histidine decarboxylase (HDC) is the primary enzyme that catalyzes the conversion of histidine to histamine. HDC contributes to many physiological responses as histamine plays important roles in allergic reaction, neurological response, gastric acid secretion, and cell proliferation and differentiation. Small-molecule modulation of HDC represents a potential therapeutic strategy for a range of histamine-associated diseases, including inflammatory disease, neurological disorders, gastric ulcers, and select cancers. High-throughput screening (HTS) methods for measuring HDC activity are currently limited. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring HDC activity. The assay is based on competition between HDC-generated histamine and fluorophore-labeled histamine for binding to a Europium cryptate (EuK)-labeled anti-histamine antibody. We demonstrated that the assay is highly sensitive and simple to develop. Assay validation experiments were performed using low-volume 384-well plates and resulted in good statistical parameters. A pilot HTS screen gave a Z' score > 0.5 and a hit rate of 1.1%, and led to the identification of a validated hit series. Overall, the presented assay should facilitate the discovery of therapeutic HDC inhibitors by acting as a novel tool suitable for large-scale HTS and subsequent interrogation of compound structure-activity relationships.