Life Sciences Institute (R.A.B., H.V.W.), Departments of Medicinal Chemistry (H.V.W., S.D.L.) and Pharmacology (R.A.B., Z.Y.W., M.A.P.), and Vahlteich Medicinal Chemistry Core, College of Pharmacy (H.V.W., S.D.L.), University of Michigan, Ann Arbor, Michigan; and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology (Y.-C.Y., J.J.G.T.), Purdue University, West Lafayette, Indiana.
Life Sciences Institute (R.A.B., H.V.W.), Departments of Medicinal Chemistry (H.V.W., S.D.L.) and Pharmacology (R.A.B., Z.Y.W., M.A.P.), and Vahlteich Medicinal Chemistry Core, College of Pharmacy (H.V.W., S.D.L.), University of Michigan, Ann Arbor, Michigan; and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology (Y.-C.Y., J.J.G.T.), Purdue University, West Lafayette, Indiana
Mol Pharmacol. 2020 Jun;97(6):392-401. doi: 10.1124/mol.119.118661. Epub 2020 Mar 31.
G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or scaffold. A new inhibitor, , which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. and the closely related compound also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or scaffold to block internalization of the -opioid receptor (MOR). showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on , which explains why -based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, , which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that blocked the internalization of the -opioid receptor most efficaciously because it has the ability to cross cell membranes.
G 蛋白偶联受体激酶 (GRK) 在终止激动剂结合 G 蛋白偶联受体引发的信号中发挥关键作用。然而，G 蛋白偶联受体的慢性刺激，如心力衰竭期间发生的刺激，会导致 GRK 的过度表达和细胞表面 G 蛋白偶联受体的适应性下调。我们之前报道了基于帕罗西汀或 骨架发现的有效且选择性的 GRK 抑制剂家族。一种新的抑制剂 ，基于帕罗西汀，对 GRK2 亚家族表现出更高的效力，并在小鼠中表现出有利的药代动力学参数。 和密切相关的化合物 也在 104 种激酶的激酶组面板中对 GRK2 亚家族表现出高选择性。我们开发了一种基于细胞的测定法，以筛选 、 以及其他 10 种具有不同 GRK 亚家族选择性且基于帕罗西汀或 骨架的抑制剂抑制 -阿片受体 (MOR)内化的能力。 在测试的化合物中，显示出阻断 MOR 内化的最佳功效。此外，我们表明基于帕罗西汀的化合物比基于 的化合物具有更好的细胞通透性，这解释了为什么基于 的抑制剂尽管在体外有效，但并不总是在基于细胞的测定中显示出功效。这项研究验证了帕罗西汀骨架作为在活细胞中抑制 GRK 最有效的骨架，证实 GRK2 主要驱动我们测试的细胞系中 MOR 的内化，并强调了高分辨率基于细胞的测定法在评估化合物功效方面的实用性。 意义声明：G 蛋白偶联受体激酶 (GRK) 是开发心力衰竭治疗药物的有吸引力的靶点。我们合成了一种新的 GRK2 亚家族选择性抑制剂 ，对 GRK2 具有纳摩尔效力，对其他激酶具有优异的选择性。使用活细胞受体内化测定法测试了 GRK2 抑制剂赋予细胞中 GRK 依赖性过程功效的能力。我们的数据表明 最有效地阻断了 -阿片受体的内化，因为它具有穿过细胞膜的能力。
