Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Eur J Pharmacol. 2011 Jun 11;660(1):139-47. doi: 10.1016/j.ejphar.2011.01.031. Epub 2011 Feb 4.
The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) 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 MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that 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=2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β(2)AR agonists - the β(2)AR 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 a HTS for allosteric modulators for a Gs protein coupled receptor.
黑皮质素 MC(4) 受体是开发肥胖症和恶病质治疗药物的潜在靶点。迄今为止已知的黑皮质素 MC(4) 受体配体是变构激动剂或拮抗剂，然而，这些激动剂通常表现出不受欢迎的副作用。对于一些受体，变构调节剂有望降低副作用谱。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) 受体和改良基于荧光素酶的 cAMP 传感器的 HEK293 细胞系。通过监测发光作为实时细胞内 cAMP 浓度的读出，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 MC4R-GLO 细胞系，我们开发了一种测定法，该测定法被证明符合高通量筛选 (HTS) 标准 (Z'=0.50)。在 Microsource Spectrum 化合物库上进行的先导筛选 (n=2000) 成功鉴定了 62 种阳性调节剂。该筛选鉴定了预测的化合物家族：β(2)AR 激动剂 - β(2)AR 在 HEK293 细胞中内源性表达，一种腺苷酸环化酶激活剂，最后是磷酸二酯酶 (PDE) 抑制剂的分布特征良好或最近鉴定。在最后一类中，我们鉴定了一组香豆素衍生的化合物（茴芹素、蛇床子素和前莱汀）和 deracoxib，后者是兽医中用于 COX2 抑制特性的药物。这一发现揭示了 deracoxib 作为 PDE 抑制剂的新的非靶标作用机制。总体而言，这些数据是高通量筛选 Gs 蛋白偶联受体变构调节剂的首次报道。
