Rodriguez Alice L, Tamrazi Anobel, Collins Margaret L, Katzenellenbogen John A
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
J Med Chem. 2004 Jan 29;47(3):600-11. doi: 10.1021/jm030404c.
Nuclear receptors (NRs) complexed with agonist ligands activate transcription by recruiting coactivator protein complexes. In principle, one should be able to inhibit the transcriptional activity of the NRs by blocking this transcriptionally critical receptor-coactivator interaction directly, using an appropriately designed coactivator binding inhibitor (CBI). To guide our design of various classes of CBIs, we have used the crystal structure of an agonist-bound estrogen receptor (ER) ligand binding domain (LBD) complexed with a coactivator peptide containing the LXXLL signature motif bound to a hydrophobic groove on the surface of the LBD. One set of CBIs, based on an outside-in design approach, has various heterocyclic cores (triazenes, pyrimidines, trithianes, cyclohexanes) that mimic the tether sites of the three leucines on the peptide helix, onto which are appended leucine residue-like substituents. The other set, based on an inside-out approach, has a naphthalene core that mimics the two most deeply buried leucines, with substituents extending outward to mimic other features of the coactivator helical peptide. A fluorescence anisotropy-based coactivator competition assay was developed to measure the specific binding of these CBIs to the groove site on the ER-agonist complex with which coactivators interact; control ligand-binding assays assured that their interaction was not with the ligand binding pocket. The most effective CBIs were those from the pyrimidine family, the best binding with K(i) values of ca. 30 microM. The trithiane- and cyclohexane-based CBIs appear to be poor structural mimics, because of equatorial vs axial conformational constraints, and the triazene-based CBIs are also conformationally constrained by amine-substituent-to-ring resonance overlap, which is not the case with the higher affinity alkyl-substituted pyrimidines. The pyrimidine-based CBIs appear to be the first small molecule inhibitors of NR coactivator binding.
与激动剂配体复合的核受体（NRs）通过招募共激活蛋白复合物来激活转录。原则上，人们应该能够通过使用适当设计的共激活剂结合抑制剂（CBI）直接阻断这种对转录至关重要的受体 - 共激活剂相互作用，来抑制NRs的转录活性。为了指导我们设计各类CBI，我们利用了与激动剂结合的雌激素受体（ER）配体结合域（LBD）的晶体结构，该结构与一个包含LXXLL特征基序的共激活剂肽复合，该肽与LBD表面的疏水凹槽结合。基于由外而内设计方法的一组CBI具有各种杂环核心（三氮烯、嘧啶、三硫烷、环己烷），它们模拟肽螺旋上三个亮氨酸的连接位点，并在其上连接类似亮氨酸残基的取代基。另一组基于由内而外方法的CBI具有一个萘核心，它模拟两个埋藏最深的亮氨酸，取代基向外延伸以模拟共激活剂螺旋肽的其他特征。开发了一种基于荧光各向异性的共激活剂竞争测定法，以测量这些CBI与共激活剂相互作用的ER - 激动剂复合物上的凹槽位点的特异性结合；对照配体结合测定确保它们的相互作用不是与配体结合口袋。最有效的CBI来自嘧啶家族，结合效果最佳，K(i)值约为30 microM。基于三硫烷和环己烷的CBI似乎是较差的结构模拟物，这是由于赤道与轴向构象限制，而基于三氮烯的CBI也受到胺取代基与环共振重叠的构象限制，而具有较高亲和力的烷基取代嘧啶则不存在这种情况。基于嘧啶的CBI似乎是NR共激活剂结合方面的首批小分子抑制剂。
