Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.
Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.
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DOI：
10.1371/journal.pone.0010479
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发表时间：
2010-05-05
期刊：
影响因子：
3.7
通讯作者：
Koide S
中科院分区：
文献类型：
--
作者：
Shemon AN;Heil GL;Granovsky AE;Clark MM;McElheny D;Chimon A;Rosner MR;Koide S
Raf激酶抑制蛋白（RKIP），也称为磷脂酰乙醇胺结合蛋白（PEBP），已显示可抑制Raf，从而通过Raf/MAP激酶途径负调节生长因子信号传导。RKIP还显示出抑制转移。我们以前已经证明，RKIP/Raf相互作用是由两种机制：磷酸化的RKIP在Ser-153，和占领的RKIP的保守的配体结合域与磷脂（2-二己酰-sn-甘油-3-磷酸乙醇胺; DHPE）。除了磷脂，其他配体已被报道结合这个域，但其结合特性仍然没有得到表征。在这项研究中，我们使用高分辨率的异频NMR光谱来筛选一个化学库，并测定一些潜在的RKIP配体与蛋白质的结合。令人惊讶的是，许多以前假定为RKIP配体的化合物在接近生理溶液条件下甚至在毫摩尔浓度下也没有显示出可检测的结合。相比之下，我们发现了三种新的RKIP配体，特异性结合RKIP口袋。有趣的是，与磷脂DHPE不同，这些新鉴定的配体不影响RKIP与Raf-1或RKIP磷酸化的结合。三种配体中有一种表现出脱靶生物学效应，损害EGF诱导的MAPK和代谢活性。这项工作定义了RKIP配体在接近生理条件下的结合特性，建立了RKIP对疏水配体的亲和力以及庞大的脂肪链对抑制其功能的重要性。这些化合物的共同结构元素定义了RKIP结合的最低要求，因此它们可以用作未来设计具有治疗潜力的RKIP配体的先导化合物。
Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.
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DOI：
10.1016/0167-4838(86)90128-7
发表时间：
1986-05-12
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
BERNIER, I;TRESCA, JP;JOLLES, P
通讯作者：
JOLLES, P
影响因子：
3.6
作者：
CORNWALL, GA;VREEBURG, JT;ORGEBINCRIST, MC
通讯作者：
ORGEBINCRIST, MC
影响因子：
64.8
作者：
Lorenz, K;Lohse, MJ;Quitterer, U
通讯作者：
Quitterer, U
影响因子：
16
作者：
Eves, Eva M.;Shapiro, Paul;Rich Rosner, Marsha
通讯作者：
Rich Rosner, Marsha
影响因子：
1.6
作者：
Nallamsetty, S;Kapust, RB;Waugh, DS
通讯作者：
Waugh, DS