Small molecule inhibition of the TNF family cytokine CD40 ligand through a subunit fracture mechanism.
Small molecule inhibition of the TNF family cytokine CD40 ligand through a subunit fracture mechanism.
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DOI：
10.1021/cb2000346
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发表时间：
2011-06
影响因子：
4
通讯作者：
L. Silvian;Jessica E. Friedman;K. Strauch;T. Cachero;E. Day;Fang Qian;B. Cunningham;Amy D. Fung;Lihong Sun;G. Shipps;Lihe Su;Zhongli Zheng;G. Kumaravel;A. Whitty
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文献类型：
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作者：
L. Silvian;Jessica E. Friedman;K. Strauch;T. Cachero;E. Day;Fang Qian;B. Cunningham;Amy D. Fung;Lihong Sun;G. Shipps;Lihe Su;Zhongli Zheng;G. Kumaravel;A. Whitty
BIO 8898是最近报道的几种合成有机分子之一，可抑制组成性三聚体肿瘤坏死因子（TNF）家族细胞因子CD 40配体（CD 40 L，又名CD 154）的受体结合和功能。蛋白质-蛋白质界面的小分子抑制剂相对罕见，并且它们的发现通常非常具有挑战性。因此，为了了解BIO 8898如何实现这一壮举，我们使用生化测定和X射线晶体学来表征其作用机制。在细胞试验中，BIO 8898抑制可溶性CD 40 L与CD 40-IG结合的效价为IC（50）= 25 μM，并抑制CD 40 L依赖性细胞凋亡。BIO 8898与CD 40 L的共晶体结构显示，每个蛋白质三聚体结合一个抑制剂分子。令人惊讶的是，该化合物不是在蛋白质的表面结合，而是通过深深插入同源三聚体细胞因子的两个亚基之间，破坏组成性蛋白质-蛋白质界面并破坏蛋白质的3重对称性。该化合物与蛋白质形成几个氢键，在疏水结合口袋内。除了翻译分裂的三聚体，结合的BIO 8898伴随着额外的本地和较长范围的构象扰动的蛋白质，无论是在核心和表面环。BIO 8898的结合是可逆的，所得复合物是稳定的，不会导致蛋白质三聚体的可检测解离。我们的研究结果表明，一组核心的芳香族残基是保守的一个子集的TNF家族细胞因子可能代表一个通用的热点诱导适合结合的三聚体破坏小分子。
BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, aka CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging. Therefore, to understand how BIO8898 achieves this feat, we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC(50) = 25 μM and inhibited CD40L-dependent apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein's 3-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.