Data for 'The development of DUAL BRD4 and CBP/p300 degraders from ISOX-DUAL' thesis by Anthony Edmonds (2022)

ESI related to AE Edmonds' thesis entitled 'The Development of DUAL BRD4 and CBP/p300 Degraders From ISOX-DUAL' (published 21.03.2022)
Contains 1H, 13C and other NMR spectra, HRMS, HPLC data and also scans of the original data.
AbstractThe development of Heterobifunctional molecules in the field of targeted degradation is a hot topic, with various modalities appearing over the past 20 years, with examples such as; PROTACs, SNIPERs, dTAGs, HaloTags, AUTACS, ATTECs and LYTACs. In 2019 alone there were 107 PROTAC publications, an increase of 55 from the previous year. ISOX-DUAL is an inhibitor of both BRD4 (IC50 = 1.5 µM) and CBP/p300 (IC50 = 0.65 µM) bromodomains and, as such, is a useful chemical probe for research into epigenetics.2 The published and our in-house protocols toward this target molecule were poor yielding and not amenable to scale-up. Here, synthetic routes towards the title compound were re- investigated, and now achieves an overall yield of 42%, compared to the literature published 1%. Using literature co-crystal structures in the bromodomains of BRD4 and CBP/p300 of the, structurally similar, inhibitor BDOIA383, two solvent exposed exit vectors were discovered for potential linkage to E3 recruiters. ISOX-DUAL was then re-designed with the optimised synthetic route to afford two degrader precursors (3.07) and (3.27) which were designed through replacement of the N,N-dimethylpropylamine to a propyl carboxylic acid (3.07) and the replacement of the morpholine moiety to a piperazine (3.27). Degrader mimics (3.09, 3.29) were synthesised from these compounds and showed no loss in binding affinities to the bromodomains of BRD4 or CBP/p300. A small library of 20 ISOX-DUAL based degraders were synthesised, guided by predicted physiochemical properties. Select degraders (4.69-4.72) were subjected to cell-free ubiquitination assays, to which, confirmed induction of ubiquitination of the target. The 20 synthesised degraders were initially treated in HeLa cells and 4.68 caused a reduction in BRD4 (75%) and CBP (73%), the most potent degrader in this assay. The investigation goes on to highlight the importance of cell lines when assessing these compounds and describes a series of future experiments, which, should be performed.

本数据集关联AE Edmonds于2022年3月21日发表的题为《基于ISOX-DUAL开发DUAL BRD4与CBP/p300降解剂》的学位论文的补充信息（ESI）。数据集涵盖1H核磁共振谱、13C核磁共振谱及其他类型核磁共振（NMR）谱图，高分辨质谱（HRMS）、高效液相色谱（HPLC）数据，以及原始实验数据的扫描件。

摘要：靶向降解领域中双功能小分子的开发是当前研究热点，近二十年来涌现出多种技术范式，例如蛋白水解靶向嵌合体（PROTACs）、SNIPERs、dTAGs、HaloTags、AUTACS、ATTECs以及LYTACs等。仅2019年，PROTAC相关研究论文就达107篇，较上年增长55篇。ISOX-DUAL是BRD4与CBP/p300溴结构域的双重抑制剂，其对二者的半最大抑制浓度（IC50）分别为1.5 μM与0.65 μM，因此是表观遗传学研究中极具价值的化学探针[2]。已发表文献及本课题组内部针对该目标分子的合成方案不仅产率低下，且难以实现规模化放大。本研究重新优化了目标化合物的合成路线，最终总产率可达42%，相较已发表文献中的1%产率有显著提升。借助已发表的、与结构类似抑制剂BDOIA383复合的BRD4与CBP/p300溴结构域共晶结构，本研究发现了两个可暴露于溶剂的连接位点，可用于连接E3连接酶招募剂。基于优化后的合成路线，本研究对ISOX-DUAL进行重新设计，得到两款降解剂前体3.07与3.27：其中3.07通过将N,N-二甲基丙胺替换为丙基羧酸获得，3.27则通过将吗啉基团替换为哌嗪获得。基于上述前体合成了降解剂模拟物3.09与3.29，二者对BRD4及CBP/p300溴结构域的结合亲和力未出现明显下降。基于预测的理化性质，本研究合成了包含20款基于ISOX-DUAL的降解剂的小型化合物库。选取部分降解剂（4.69~4.72）进行无细胞泛素化实验，结果证实其可诱导靶蛋白发生泛素化修饰。初步在HeLa细胞中测试了全部20款合成降解剂的活性，其中4.68可分别使BRD4与CBP的表达水平降低75%与73%，是本次实验中活性最强的降解剂。本研究进一步阐明了细胞系在化合物活性评价中的重要性，并提出了一系列有待开展的后续实验方向。