在这里,我们报告了与[1,2,4]三唑并[4,3-a]基于喹喔啉的化合物相关的详细结构-活性关系(SAR)研究,该化合物靶向含溴结构域蛋白9(BRD9)的读取器模块。基于3D结构的药效团模型,之前由我们介绍,被用来评估第二代化合物,探索杂环核上不同的取代模式。从我们先前确定的基于[1,2,4]三唑并[4,3-a]喹喔啉的化合物1-4获得的有希望的数据开始,化学合成,生物物理和体外试验导致了一组新的衍生物的鉴定,选择用于彻底探索溴结构域结合位点的化学空间。更多细节,对C-4位不同接头的研究强调了胺间隔基是与蛋白质对应物结合的强制性要求,以及C-1上的烷基取代基对增加对BRD9的选择性的关键作用。此外,氢键供体基团的重要性,锚定ZA区的关键和与Ile53残基相互作用所需的,是从我们收集的结果分析中推断出来的。在这里,我们还提出了一个优化和更新我们以前报道的“pharm-druglike2”基于3D结构的药效团模型,将其介绍为“pharm-druglike2.1”。化合物24-26、32、34和36被鉴定为新的有价值的BRD9结合剂,其特征在于IC50值在低微摩尔范围内。其中,图24和36显示了对BRD9的优异选择性和对一组白血病模型的良好抗增殖作用。特别是对CCRF-CEM细胞系,对健康细胞没有细胞毒性。值得注意的是,24和36与溴结构域和含PHD指状蛋白1(BRPF1)的相互作用也出现了,揭示它们是这两种高度参与白血病的蛋白质的新的和未开发的双重抑制剂。这些发现凸显了识别新的有吸引力的双重表皮的潜力,以及开发具有抗癌活性的化学降解剂的有希望的起点。
Here we report a detailed structure-activity relationship (SAR) study related to [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the reader module of bromodomain containing-protein 9 (BRD9). 3D structure-based pharmacophore models, previously introduced by us, were here employed to evaluate a second generation of compounds, exploring different substitution patterns on the heterocyclic core. Starting from the promising data obtained from our previously identified [1,2,4]triazolo[4,3-a]quinoxaline-based compounds 1-4, the combination of in silico studies, chemical synthesis, biophysical and in vitro assays led to the identification of a new set of derivatives, selected for thoroughly exploring the chemical space of the bromodomain binding site. In more details, the investigation of different linkers at C-4 position highlighted the amine spacer as mandatory for the binding with the protein counterpart and the crucial role of the alkyl substituents at C-1 for increasing the selectivity toward BRD9. Additionally, the importance of a hydrogen bond donor group, critical to anchor the ZA region and required for the interaction with Ile53 residue, was inferred from the analysis of our collected results. Herein we also propose an optimization and an update of our previously reported \"pharm-druglike2\" 3D structure-based pharmacophore model, introducing it as \"pharm-druglike2.1\". Compounds 24-26, 32, 34 and 36 were identified as new valuable BRD9 binders featuring IC50 values in the low micromolar range. Among them, 24 and 36 displayed an excellent selectivity towards BRD9 and a good antiproliferative effect on a panel of leukemia models, especially toward CCRF-CEM cell line, with no cytotoxicity on healthy cells. Notably, the interaction of 24 and 36 with the bromodomain and PHD finger-containing protein 1 (BRPF1) also emerged, disclosing them as new and unexplored dual inhibitors for these two proteins highly involved in leukemia. These findings highlight the potential for the identification of new attractive dual epidrugs as well as a promising starting point for the development of chemical degraders endowed with anticancer activities.