Multiple Independent Binding Sites for Small-Molecule Inhibitors on the Oncoprotein c-Myc
Multiple Independent Binding Sites for Small-Molecule Inhibitors on the Oncoprotein c-Myc
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DOI：
10.1021/ja900616b
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发表时间：
2009-06-03
影响因子：
15
通讯作者：
Metallo, Stevan J.
中科院分区：
文献类型：
--
作者：
Hammoudeh, Dalia I.;Follis, Ariele Viacava;Metallo, Stevan J.
c-Myc 转录因子的失调与许多类型的癌症有关，使这种癌蛋白成为药物发现的有吸引力的目标。抑制其的一种方法是破坏其基本螺旋-环-螺旋亮氨酸拉链 (bHLHZip) 结构域与其二聚化伙伴 Max 上的类似结构域之间形成的二聚复合物。作为单体，bHLHZip 蛋白本质上是无序的 (ID)。之前我们发现两种 c-Myc-Max 抑制剂 10058-F4 和 10074-G5 与单体 c-Myc bHLHZip 结构域的不同 ID 区域结合。在这里，我们使用圆二色性、荧光偏振和 NMR 来证明位于 10058-F4 和 10074-G5 之间存在额外的结合位点。所有七种最初鉴定的 Myc 抑制剂均显示与 c-Myc 的 85 个残基 bHLHZip 结构域内的这三个离散位点之一结合。这些结合位点由短的连续氨基酸组成，可以选择性且独立地结合小分子。抑制剂结合仅诱导局部构象变化，保留 c-Myc 的整体紊乱，并抑制与 Max 的二聚化。 NMR 实验进一步表明，c-Myc 上一个位点的结合既不影响亲和力，也不影响与其他位点结合时发生的结构变化。相反，结合可以同时且独立地发生在三个已识别位点上。我们的结果表明，ID 域内广泛存在易于小分子结合的肽区域。因此，通过靶向 ID 序列，可能可以采用合理且通用的方法来抑制涉及 ID 蛋白的蛋白质-蛋白质相互作用。
Deregulation of the c-Myc transcription factor is involved in many types of cancer, making this oncoprotein an attractive target for drug discovery. One approach to its inhibition has been to disrupt the dimeric complex formed between its basic helix-loop-helix leucine zipper (bHLHZip) domain and a similar domain on its dimerization partner, Max. As monomers, bHLHZip proteins are intrinsically disordered (ID). Previously we showed that two c-Myc-Max inhibitors, 10058-F4 and 10074-G5, bound to distinct ID regions of the monomeric c-Myc bHLHZip domain. Here, we use circular dichroism, fluorescence polarization, and NMR to demonstrate the presence of an additional binding site located between those for 10058-F4 and 10074-G5. All seven of the originally identified Myc inhibitors are shown to bind to one of these three discrete sites within the 85-residue bHLHZip domain of c-Myc. These binding sites are composed of short contiguous stretches of amino acids that can selectively and independently bind small molecules. Inhibitor binding induces only local conformational changes, preserves the overall disorder of c-Myc, and inhibits dimerization with Max. NMR experiments further show that binding at one site on c-Myc affects neither the affinity nor the structural changes taking place upon binding to the other sites. Rather, binding can occur simultaneously and independently on the three identified sites. Our results suggest the widespread existence of peptide regions prone to small-molecule binding within ID domains. A rational and generic approach to the inhibition of protein-protein interactions involving ID proteins may therefore be possible through the targeting of ID sequence.