Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay
Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay
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DOI：
10.1124/mol.106.028670
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发表时间：
2007-01-01
影响因子：
3.6
通讯作者：
Neubig, Richard R.
中科院分区：
文献类型：
--
作者：
Roman, David L.;Talbot, Jeffery N.;Neubig, Richard R.
G蛋白信号调节因子（Regulators of G-protein signaling，RGS）是通过G蛋白偶联受体（G-protein-coupled receptor，GPCRs）启动的信号转导通路的重要组成部分。RGS蛋白加速G蛋白α亚基（G α）的内在GT3活性，从而缩短时间过程并降低G蛋白α和β γ亚基信号传导的幅度。已经提出抑制RGS作用作为增强GPCR激动剂药物的活性和特异性的手段，但是蛋白质-蛋白质相互作用的药理学靶向通常是困难的。该项目的目的是鉴定RGS 4的抑制剂。使用Luminex 96孔板珠分析仪和一种新的流式细胞术蛋白质相互作用测定，以评估G α-RGS相互作用的高通量筛选，我们确定了第一个小分子抑制剂的RGS蛋白。在筛选的3028种化合物中，1种，N-[（4-氯苯基）磺酰基]-4-硝基苯亚磺酰亚胺酸甲酯（CCG-4986），以3至5 μ M的效力抑制RGS 4/ G α（o）结合。它与RGS 4结合，在体外抑制RGS 4刺激G α（o）GT β活性，并阻止RGS 4调节透化细胞中μ阿片抑制的腺苷酸环化酶活性。此外，CCG-4986对RGS 4具有选择性，并且不抑制RGS 8。因此，我们证明了用小分子靶向RGS/G α蛋白-蛋白相互作用作为调节GPCR介导的信号传导过程的新手段的可行性。
Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein alpha-subunits (G alpha) and thus shorten the time course and reduce the magnitude of G-protein alpha- and beta gamma-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess G alpha-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl) sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/ G alpha(o) binding with 3 to 5 mu M potency. It binds to RGS4, inhibits RGS4 stimulation of G alpha(o) GTPase activity in vitro, and prevents RGS4 regulation of mu-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/G alpha protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.