Identification of vasoactive nonpeptidic positive and negative modulators of adrenomedullin using a neutralizing antibody-based screening strategy
Identification of vasoactive nonpeptidic positive and negative modulators of adrenomedullin using a neutralizing antibody-based screening strategy
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DOI：
10.1210/en.2003-1251
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发表时间：
2004-08-01
期刊：
影响因子：
4.8
通讯作者：
Cuttitta, F
中科院分区：
文献类型：
--
作者：
Martínez, A;Julián, M;Cuttitta, F
肾上腺髓质素(adrenomedullin，AM)是一种多肽类激素，参与血压调节和高血压、癌症、糖尿病等重要疾病的病理生理过程。然而，这种肽的非肽调节剂可以用于临床调节其作用，目前还不存在。我们在这里提出了一种有效的新方法来筛选大的小分子文库。将该技术应用于AM函数正、负调制子的识别。开发了一种两层筛选策略，其中第一次筛选需要破坏多肽与中和单抗之间的相互作用。所选化合物的进一步特征是它们能够调节含有特定AM受体的细胞中的第二信使。针对胃泌素释放肽的平行筛选选择了不同的分子亚集，证实了筛选方法的特异性。已确定的AM阳性调节剂在体内降低血压，而AM负性调节剂介导血管收缩，正如AM的血管扩张活性所预测的那样表面等离子体共振分析表明这些小分子与固定化AM结合，K-d值在7.76×10~(-9)~4.14×10~(-6)M之间。AM调节剂的临床前开发可能导致有用的药物来预防和治疗高血压、癌症和糖尿病。
Adrenomedullin (AM) is a peptide hormone implicated in blood pressure regulation and in the pathophysiology of important diseases, such as hypertension, cancer, and diabetes. However, nonpeptidic modulators of this peptide that could be used to clinically regulate its actions are not available. We present here an efficient new method to screen a large library of small molecules. This technology was applied to the identification of positive and negative modulators of AM function. A two-tier screening strategy was developed in which the first screening entails disruption of the interaction between the peptide and a neutralizing monoclonal antibody. Selected compounds were further characterized by their ability to modulate second messengers in cells containing specific AM receptors. A parallel screen against gastrin-releasing peptide selected a different subset of molecules, confirming the specificity of the screening method. Identified AM-positive regulators reduced blood pressure in vivo, whereas AM-negative regulators mediated vasoconstriction, as predicted by the vasodilatory activity of AM. Binding of the small molecules to immobilized AM was demonstrated by surface plasmon resonance assays, with K-d values ranging from 7.76 x 10(-9) to 4.14 x 10(-6) M. Preclinical development of AM modulators may result in useful drugs for the prevention and treatment of hypertension, cancer, and diabetes.