Disulfide-Trapping Identifies a New, Effective Chemical Probe for Activating the Nuclear Receptor Human LRH-1 (NR5A2).
Disulfide-Trapping Identifies a New, Effective Chemical Probe for Activating the Nuclear Receptor Human LRH-1 (NR5A2).
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DOI：
10.1371/journal.pone.0159316
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
England PM
中科院分区：
文献类型：
--
作者：
de Jesus Cortez F;Suzawa M;Irvy S;Bruning JM;Sablin E;Jacobson MP;Fletterick RJ;Ingraham HA;England PM
传统的努力依赖于大化合物文库的高通量物理和虚拟筛选，未能为48种人类核受体中的许多人产生高效的化学探针。在这里，我们研究了二硫化物捕获，核受体的一种新方法，是否会提供有效的先导化合物靶向人类肝脏受体同源物1 （hLRH-1, NR5A2）。尽管hLRH-1具有较大的配体结合袋，与磷脂具有较高的亲和力，但现有合成的hLRH-1配体由于溶解度差、疗效低或脱靶效应明显，实用性有限。利用二硫化物捕获技术，我们发现了一种先导化合物，它能以非常高的效率与hLRH-1配体结合域疏水腔内的天然半胱氨酸残基（Cys346）结合。在计算模型和细胞实验的指导下，先导化合物被加工成PME8和PME9配体，它们可以可逆地结合hLRH-1（无半胱氨酸反应性），并增加细胞中hLRH-1的活性。与现有的hLRH-1合成激动剂RJW100相比，PME8和PME9对人HepG2细胞中LRH-1依赖性靶基因CYP24A1的诱导作用相当，早在药物治疗后3小时就开始了。这种诱导是特异性的，因为sirna介导的hLRH-1敲低会使PME8和PME9失效。这些数据表明PME8和PME9是hLRH-1的有效激活剂，并表明随着进一步的开发，该先导系列可能会产生有用的化学探针来控制体内LRH-1的活性。
Conventional efforts relying on high-throughput physical and virtual screening of large compound libraries have failed to yield high-efficiency chemical probes for many of the 48 human nuclear receptors. Here, we investigated whether disulfide-trapping, an approach new to nuclear receptors, would provide effective lead compounds targeting human liver receptor homolog 1 (hLRH-1, NR5A2). Despite the fact that hLRH-1 contains a large ligand binding pocket and binds phospholipids with high affinity, existing synthetic hLRH-1 ligands are of limited utility due to poor solubility, low efficacy or significant off-target effects. Using disulfide-trapping, we identified a lead compound that conjugates with remarkably high-efficiency to a native cysteine residue (Cys346) lining the hydrophobic cavity in the ligand binding domain of hLRH-1. Guided by computational modeling and cellular assays, the lead compound was elaborated into ligands PME8 and PME9 that bind hLRH-1 reversibly (no cysteine reactivity) and increase hLRH-1 activity in cells. When compared with the existing hLRH-1 synthetic agonist RJW100, both PME8 and PME9 showed comparable induction of the LRH-1 dependent target gene CYP24A1 in human HepG2 cells, beginning as early as 3 h after drug treatment. The induction is specific as siRNA-mediated knock-down of hLRH-1 renders both PME8 and PME9 ineffective. These data show that PME8 and PME9 are potent activators of hLRH-1 and suggest that with further development this lead series may yield useful chemical probes for manipulating LRH-1 activity in vivo.