Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.
Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.
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DOI：
10.1016/j.ejphar.2011.01.031
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发表时间：
2011-06-11
影响因子：
5
通讯作者：
Cone, Roger D.
中科院分区：
文献类型：
--
作者：
Pantel, Jacques;Williams, Savannah Y.;Mi, Dehui;Sebag, Julien;Corbin, Jackie D.;Weaver, C. David;Cone, Roger D.
黑皮质素MC 4受体是开发肥胖和恶病质药物的潜在靶点。迄今已知的黑皮质素MC 4受体配体是正构激动剂或拮抗剂，然而，特别是激动剂通常表现出不希望的副作用。对于某些受体，预期变构调节剂可减少副作用特征。为了鉴定黑皮质素MC 4受体的变构调节剂，我们创建了共表达人黑皮质素MC 4受体和修饰的基于腺苷酸酶的cAMP传感器的HEK 293细胞系。监测发光作为实时细胞内cAMP浓度的读数，我们证明该细胞系能够报告黑皮质素激动剂反应，以及对生理AgRP肽的反向激动剂反应。基于MC 4 R-GLO细胞系，我们开发了显示符合HTS标准（Z '= 0.50）的测定。在Microsource Spectrum化合物库（n= 2，000）上进行的中试筛选成功鉴定了62种阳性调节剂。该筛选确定了预测的化合物家族：β2AR激动剂-β2AR在HEK 293细胞中内源性表达-，腺苷酸环化酶激活剂，最后是已充分表征或最近鉴定的磷酸二酯酶（PDE）抑制剂的分布。在这最后一类中，我们鉴定了香豆素衍生化合物的结构家族（欧前胡素、奥司他丁和普瑞药克），沿着地拉考昔，一种具有COX 2抑制特性的兽用药物。后一项发现揭示了地拉昔布作为PDE抑制剂的一种新的脱靶作用机制。总体而言，这些数据是第一次报告的HTS的变构调节剂的Gs蛋白偶联受体。
The melanocortin MC4 receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC4 receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC4 receptor, we created HEK293 cell lines coexpressing the human melanocortin MC4 receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z’=0.50). A pilot screen run on the Microsource Spectrum compound library (n= 2,000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β2AR agonists –the β2AR being endogenously expressed in HEK293 cells-, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of an HTS for allosteric modulators for a Gs protein coupled receptor.
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DOI：
10.1038/nrd2760
发表时间：
2009-01
期刊：
Nature reviews. Drug discovery
影响因子：
--
作者：
通讯作者：
--
影响因子：
1.3
作者：
Case, J. Brad;Fick, Jennifer L.;Rooney, Matthew B.
通讯作者：
Rooney, Matthew B.
影响因子：
5
作者：
He, Jian-Yu;Zhang, Wei;Cao, Yong-Xiao
通讯作者：
Cao, Yong-Xiao
DOI：
10.1073/pnas.96.21.11998
发表时间：
1999-10-12
影响因子：
11.1
作者：
Jin, SLC;Richard, FJ;Conti, M
通讯作者：
Conti, M
影响因子：
158.5
作者：
Greenfield, Jerry R.;Miller, Jeffrey W.;Farooqi, I. Sadaf
通讯作者：
Farooqi, I. Sadaf