Development of a High-Throughput Screening Paradigm for the Discovery of Small-Molecule Modulators of Adenylyl Cyclase: Identification of an Adenylyl Cyclase 2 Inhibitor
Development of a High-Throughput Screening Paradigm for the Discovery of Small-Molecule Modulators of Adenylyl Cyclase: Identification of an Adenylyl Cyclase 2 Inhibitor
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DOI：
10.1124/jpet.113.207449
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发表时间：
2013-11-01
影响因子：
3.5
通讯作者：
Watts, Val J.
中科院分区：
文献类型：
--
作者：
Conley, Jason M.;Brand, Cameron S.;Watts, Val J.
腺苷酸环化酶（AC）亚型涉及几种生理过程和疾病状态，但AC亚型的治疗靶向进展受到缺乏有效的和亚型选择性小分子调节剂的限制。AC亚型选择性小分子的发现有望促进AC亚型作为治疗靶点的验证，并增强AC亚型在体内功能的研究。AC 2的化学探针的鉴定是特别重要的，因为没有发表的遗传缺失研究和一些小分子调节剂。本报告描述了开发和实施的完整的细胞，小分子筛选方法和随后的验证范式的发现AC 2抑制剂。使用PMA刺激的cAMP积累作为功能读数，筛选NIH临床收集I和II的AC 2活性抑制剂。随后使用正交和反筛选试验确认并验证活性化合物为直接AC 2抑制剂。筛选工作确定SKF-83566 [8-溴-2，3，4，5-四氢-3-甲基-5-苯基-1H-3-苯并氮杂卓-7-醇氢溴酸盐]是一种选择性AC 2抑制剂，与目前可用的AC抑制剂相比，具有选择性调节AC 2的上级药理学特性。在C2 C12小鼠骨骼肌细胞和人支气管平滑肌细胞中证明了SKF-83566作为小分子探针研究内源性AC功能的实用性。
Adenylyl cyclase (AC) isoforms are implicated in several physiologic processes and disease states, but advancements in the therapeutic targeting of AC isoforms have been limited by the lack of potent and isoform-selective small-molecule modulators. The discovery of AC isoform-selective small molecules is expected to facilitate the validation of AC isoforms as therapeutic targets and augment the study of AC isoform function in vivo. Identification of chemical probes for AC2 is particularly important because there are no published genetic deletion studies and few small-molecule modulators. The present report describes the development and implementation of an intact-cell, small-molecule screening approach and subsequent validation paradigm for the discovery of AC2 inhibitors. The NIH clinical collections I and II were screened for inhibitors of AC2 activity using PMA-stimulated cAMP accumulation as a functional readout. Active compounds were subsequently confirmed and validated as direct AC2 inhibitors using orthogonal and counterscreening assays. The screening effort identified SKF-83566 [8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide] as a selective AC2 inhibitor with superior pharmacological properties for selective modulation of AC2 compared with currently available AC inhibitors. The utility of SKF-83566 as a small-molecule probe to study the function of endogenous ACs was demonstrated in C2C12 mouse skeletal muscle cells and human bronchial smooth muscle cells.