恶性转化中的代谢重连通常伴随着代谢同工酶表达的改变。磷酸烯醇丙酮酸羧激酶-2 (PCK2) 催化糖异生的限速步骤，是包括三阴性乳腺癌 (TNBC) 在内的许多癌症的主要亚型。我们的目标是鉴定 PCK2 酶活性的小分子抑制剂。我们在体外评估了 shRNA 下调 PCK2 对 TNBC 细胞生长的影响，并使用 AtomNet® 深度卷积神经网络软件鉴定了基于 PCK2 结构的潜在小分子抑制剂。我们在体外 PCK-2 酶测定中反复测试了候选化合物。还评估了顶部命中对代谢通量和细胞活力的影响。PCK2 下调减少了 BT-549 和 MDA-MB-231 细胞的生长，并减少了通过丙酮酸羧化酶的代谢通量。首次 AtomNet® 计算机结构筛选 700 万种化合物产生了 86 个结构，并在体外进行了 PCK2 酶测定测试。顶级命中 (IC50 = 2.4 µM) 用于完善第二轮计算机筛选，产生 82 个候选物进行体外测试，其中 45 个分子的抑制率 > 20%。在第二次体外筛选中，我们还加入了 3-(3,4-二羟基苯基)-2-羟基丙酸酯，之前根据结构认为它是 PCK2 抑制剂，它成为热门产品。该化合物的特异性在 PCK1 和 PCK2 酶测定中进行了测试，结果显示，PCK1 和 PCK2 的 IC50 分别为 500 nM 和 3.5-27 nM。3-(3,4-二羟基苯基)-2-羟基丙酸酯是一种高亲和力 PCK2 酶抑制剂，在体外乳腺细胞系中也具有显着的生长抑制活性，是一种潜在的治疗先导化合物。© 2024。作者获得 Springer Science Business Media, LLC（Springer Nature 旗下公司）的独家许可。

Metabolic rewiring in malignant transformation is often accompanied by altered expression of metabolic isozymes. Phosphoenolpyruvate carboxykinase-2 (PCK2) catalyzes the rate-limiting step of gluconeogenesis and is the dominant isoform in many cancers including triple-negative breast cancer (TNBC). Our goal was to identify small molecule inhibitors of PCK2 enzyme activity.We assessed the impact of PCK2 down regulation with shRNA on TNBC cell growth in vitro and used AtomNet® deep convolutional neural network software to identify potential small molecule inhibitors of PCK2-based structure. We iteratively tested candidate compounds in an in vitro PCK-2 enzyme assay. The impact of the top hit on metabolic flux and cell viability was also assessed.PCK2 downregulation decreased growth of BT-549 and MDA-MB-231 cells and reduced metabolic flux through pyruvate carboxylase. The first AtomNet® in silico structural screen of 7 million compounds yielded 86 structures that were tested in PCK2 enzyme assay in vitro. The top hit (IC50 = 2.4 µM) was used to refine a second round of in silico screen that yielded 82 candidates to be tested in vitro, which resulted in 45 molecules with inhibition > 20%. In the second in vitro screen we also included 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, previously suggested to be PCK2 inhibitor based on structure, which emerged as the top hit. The specificity of this compound was tested in PCK1 and PCK2 enzymatic assays and showed IC50 of 500 nM and 3.5-27 nM for PCK1 and PCK2, respectively.3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is a high affinity PCK2 enzyme inhibitor that also has significant growth inhibitory activity in breast cell lines in vitro and represents a potential therapeutic lead compound.© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.