Pharmacologic efficacy of PU.1 inhibition by heterocyclic dications: a mechanistic analysis.
Pharmacologic efficacy of PU.1 inhibition by heterocyclic dications: a mechanistic analysis.
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DOI：
10.1093/nar/gkw229
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发表时间：
2016-05-19
影响因子：
14.9
通讯作者：
Poon GM
中科院分区：
文献类型：
--
作者：
Stephens DC;Kim HM;Kumar A;Farahat AA;Boykin DW;Poon GM
杂环类适应症作为转录因子的靶向抑制剂正受到越来越多的关注。虽然许多指示物作为纯粹的竞争性抑制剂，但有些不能在预期的药物浓度下有效地取代蛋白质以饱和其DNA目标。为了实现对这些非竞争性效应的机制理解，我们使用了指示的组合，这些指示本质上是荧光和光谱分离的荧光标记DNA，以解剖多组分药物/DNA/蛋白质系统中的复杂相互作用。具体来说，我们研究了转录因子PU.1的位点特异性结合以及DB270对其的扰动，DB270是一种呋喃-双苯并咪唑-二胺，强烈靶向PU.1结合位点，但对PU.1/DNA复合物的抑制作用较差。通过将DB270和/或花氨酸标记的DNA与蛋白质或未标记的DNA进行滴定，并跟踪其荧光偏振的变化，我们发现了DB270与蛋白质结合的直接证据，而不依赖于它们对序列特异性DNA的相互亲和力。这种相互依赖的平衡抵消了DB270的抑制活性，而在降低DB270/PU的条件下，DB270的抑制活性基本恢复。1绑定。与PU.1的结合与DB270体内对PU.1较差的抑制效果一致，而其等位硒烯类似物（DB1976）在体外不与PU.1结合并强烈抑制PU.1/DNA复合物，在体内可完全拮抗PU.1依赖的转激活。
Heterocyclic dications are receiving increasing attention as targeted inhibitors of transcription factors. While many dications act as purely competitive inhibitors, some fail to displace protein efficiently at drug concentrations expected to saturate their DNA target. To achieve a mechanistic understanding of these non-competitive effects, we used a combination of dications, which are intrinsically fluorescent and spectrally-separated fluorescently labeled DNA to dissect complex interactions in multi-component drug/DNA/protein systems. Specifically, we interrogated site-specific binding by the transcription factor PU.1 and its perturbation by DB270, a furan-bisbenzimidazole-diamidine that strongly targets PU.1 binding sites yet poorly inhibits PU.1/DNA complexes. By titrating DB270 and/or cyanine-labeled DNA with protein or unlabeled DNA, and following the changes in their fluorescence polarization, we found direct evidence that DB270 bound protein independently of their mutual affinities for sequence-specific DNA. Each of the three species competed for the other two, and this interplay of mutually dependent equilibria abrogated DB270's inhibitory activity, which was substantively restored under conditions that attenuated DB270/PU.1 binding. PU.1 binding was consistent with DB270's poor inhibitory efficacy of PU.1 in vivo, while its isosteric selenophene analog (DB1976), which did not bind PU.1 and strongly inhibited the PU.1/DNA complex in vitro, fully antagonized PU.1-dependent transactivation in vivo.