Identification of N10-substituted phenoxazines as potent and specific inhibitors of Akt signaling
Identification of N10-substituted phenoxazines as potent and specific inhibitors of Akt signaling
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DOI：
10.1074/jbc.m507057200
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发表时间：
2005-09-09
影响因子：
4.8
通讯作者：
Houghton, PJ
中科院分区：
文献类型：
--
作者：
Thimmaiah, KN;Easton, JB;Houghton, PJ
合成了30个N-10-取代吩恶嗪类化合物，并筛选出它们作为Akt的潜在抑制剂。在5 mU M的细胞实验中，17个化合物对胰岛素样生长因子1(IGF-I)刺激的Akt(Ser-473)的磷酸化抑制至少50%，但不抑制IGF-I刺激的Erk-1/2(Thr-202/Tyr-204)的磷酸化。与丙基或丁基取代基(7B和14B)相比，具有乙酰基(20B)或吗啉基(12B)侧链的N-10-取代失去了抑制活性。在Rh1、Rh18和Rh30细胞中，Akt磷酸化的抑制与IGF-I对哺乳动物雷帕霉素磷酸化靶(Ser-2448和Ser-2481)、p70 S6激酶(Thr-389)和核糖体蛋白S6(Ser-235/236)的刺激有关。进一步研究了两个最有效的化合物10-[4‘-(N-二乙氨基)丁基]-2-氯苯恶嗪(10B)和10-[4’-[(β-羟乙基)哌嗪]-丁基]2-氯苯恶嗪(15B)(体外IC50类似于1-2微米)。免疫沉淀后，Akt的磷酸化抑制与其激酶活性的抑制相关。AKT抑制剂吩恶嗪不抑制重组磷脂酰肌醇3‘-激酶、PDK1或SGK1的活性，但对重组Akt和缺失Pleckstrin同源域的突变体Akt Delta PH的活性有明显的抑制作用。AKT抑制物可阻断IGF-I刺激的Rh1细胞Akt的核转位，并抑制Rh1、Rh18和Rh30细胞的生长(IC50为2-5mU/M)，而“非活性”的衍生物对细胞生长的抑制作用为原来的1/10。与雷帕霉素类似物相反，Akt抑制药在含血清培养条件下，在与Akt抑制药浓度一致的情况下诱导显著水平的细胞凋亡。因此，对Akt的吩恶嗪抑制剂的细胞反应似乎与雷帕霉素类似物有质的不同。模拟研究表明，抑制性吩恶嗪可能结合在ATP结合部位，尽管ATP竞争研究无法区分竞争性和非竞争性抑制。
A series of 30 N-10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 mu M, 17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity, whereas N-10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents ( 7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481), phosphorylation of p70 S6 kinase (Thr-389), and ribosomal protein S6 (Ser-235/ 236) in Rh1, Rh18, and Rh30 cell lines. The two most potent compounds 10-[4'-(N- diethylamino) butyl]-2-chlorophenoxazine (10B) and 10-[4'-[(beta-hydroxyethyl) piperazino]-butyl]2-chlorophenoxazine (15B) ( in vitro, IC50 similar to 1-2 mu M) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3'-kinase, PDK1, or SGK1 but potently inhibited the kinase activity of recombinant Akt and Akt Delta PH, a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1, Rh18, and Rh30 cells (IC50 2-5 mu M), whereas " inactive" derivatives were >= 10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs, Akt inhibitory phenoxazines induced significant levels of apoptosis under serum-containing culture conditions at concentrations of agent consistent with Akt inhibition. Thus, the cellular responses to phenoxazine inhibitors of Akt appear qualitatively different from the rapamycin analogs. Modeling studies suggest inhibitory phenoxazines may bind in the ATP-binding site, although ATP competition studies were unable to distinguish between competitive and noncompetitive inhibition.