Cyclohexyl-octahydro-pyrrolo[1,2-a] pyrazine-based inhibitors of human N-myristoyltransferase-1
Cyclohexyl-octahydro-pyrrolo[1,2-a] pyrazine-based inhibitors of human N-myristoyltransferase-1
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DOI：
10.1124/jpet.103.061572
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发表时间：
2004-04-01
影响因子：
3.5
通讯作者：
Smith, CD
中科院分区：
文献类型：
--
作者：
French, KJ;Zhuang, Y;Smith, CD
N-肉豆蔻酰转移酶（NMT）是一种新兴的治疗靶点，其催化肉豆蔻酸与受体蛋白的N末端的连接。我们开发了一种中等通量的检测方法，用于筛选由重组酶、生物素化肽底物和[H-3]肉豆蔻酰辅酶A组成的人NMT-1潜在小分子抑制剂。已经评估了大约16，000种不同的化合物，发现0.8%的化合物对NMT有显著的抑制作用。从这些命中，我们已经确定环己基-八氢吡咯并[1，2-a]吡嗪（COPP）化学型作为抑制人类NMT-1。32个含有该亚结构的化合物抑制NMT-1，IC 50值范围从6 μ M到毫摩尔浓度，并推导出该系列的定量结构-活性关系方程（r（2）= 0.72）。最有效的抑制剂（24，含有9-乙基-9H-咔唑）表现出对NMT-1的肽结合位点的竞争性抑制和对肉豆蔻酰辅酶A位点的非竞争性抑制。使用高度同源的酵母NMT的晶体结构进行的计算对接研究证实，24与酶的肽结合位点具有极好的互补性。为了评价24在完整细胞中抑制NMT活性的能力，用已知的NMT抑制剂或24处理表达N-肉豆蔻酰化绿色荧光蛋白（GFP）融合蛋白的猴CV-1细胞。每种化合物引起GFP从质膜到胞质溶胶的重新分布。此外，24在与抑制蛋白豆蔻酰化的剂量相似的剂量下抑制癌细胞增殖。总之，这些研究建立了一种筛选人NMT抑制剂的有效测定法，并鉴定了一种新型抑制剂家族，该家族在肽结合位点竞争并在完整细胞中具有活性。
N-Myristoyltransferase (NMT) is an emerging therapeutic target that catalyzes the attachment of myristate to the N terminus of an acceptor protein. We have developed a medium-throughput assay for screening potential small molecule inhibitors of human NMT-1 consisting of recombinant enzyme, biotinylated peptide substrate, and [H-3] myristoyl-CoA. Approximately 16,000 diverse compounds have been evaluated, and significant inhibition of NMT was found with 0.8% of the compounds. From these hits, we have identified the cyclohexyl-octahydropyrrolo[ 1,2-a] pyrazine (COPP) chemotype as inhibitory toward human NMT-1. Thirty-two compounds containing this substructure inhibited NMT-1, with IC50 values ranging from 6 muM to millimolar concentrations, and a quantitative structure-activity relationship equation (r(2) = 0.72) was derived for the series. The most potent inhibitor (24, containing 9-ethyl-9H-carbazole) demonstrated competitive inhibition for the peptide-binding site of NMT-1 and noncompetitive inhibition for the myristoyl-CoA site. Computational docking studies using the crystal structure of the highly homologous yeast NMT confirmed that 24 binds with excellent complementarity to the peptide-binding site of the enzyme. To evaluate the ability of 24 to inhibit NMT activity in intact cells, monkey CV-1 cells expressing an N-myristoylated green fluorescent protein (GFP) fusion protein were treated with a known NMT inhibitor or with 24. Each compound caused the redistribution of GFP from the plasma membrane to the cytosol. Furthermore, 24 inhibits cancer cell proliferation at doses similar to those that inhibit protein myristoylation. Overall, these studies establish an efficient assay for screening for inhibitors of human NMT and identify a novel family of inhibitors that compete at the peptide-binding site and have activity in intact cells.