Blevins Melanie A, Kouznetsova Jennifer, Krueger Aaron B, King Rebecca, Griner Lesley Mathews, Hu Xin, Southall Noel, Marugan Juan J, Zhang Qinghong, Ferrer Marc, Zhao Rui
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO, USA.
Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, MD, USA.
J Biomol Screen. 2015 Jun;20(5):663-72. doi: 10.1177/1087057114561400. Epub 2014 Dec 4.
Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor that suppresses multiple proapoptotic and epithelial genes. CtBP is overexpressed in many human cancers, and its overexpression increases stem cell-like features, epithelial-mesenchymal transition, and cancer cell survival. Knockdown of CtBP also increases apoptosis independent of p53 in cell culture. Therefore, targeting CtBP with small molecules that disrupt its interaction with transcription factor partners may be an effective cancer therapy. To elicit its corepressing effect, CtBP binds to a conserved peptide motif in each transcription factor partner. We developed an AlphaScreen high-throughput screening assay to monitor the interaction between CtBP and E1A (which mimics the interaction between CtBP and its transcriptional partners). We screened the LOPAC library of 1280 bioactive compounds and identified NSC95397, which inhibits the CtBP-E1A interaction (IC50 = 2.9 µM). The inhibitory activity of NSC95397 was confirmed using two secondary assays and a counterscreen. NSC95397 also behaved as a weak substrate of CtBP dehydrogenase activity and did not inhibit another dehydrogenase, lactase dehydrogenase. Finally, NSC95397 was able to disrupt CtBP-mediated transcriptional repression of a target gene. These studies present a new possibility for the development of a therapeutic agent targeting tumors through disrupting the CtBP transcriptional complex.
羧基末端结合蛋白（CtBP）是一种转录共抑制因子，可抑制多种促凋亡基因和上皮基因。CtBP在许多人类癌症中过表达，其过表达会增加干细胞样特征、上皮-间质转化和癌细胞存活。在细胞培养中，敲低CtBP也会增加不依赖p53的细胞凋亡。因此，用小分子破坏CtBP与转录因子伴侣之间的相互作用可能是一种有效的癌症治疗方法。为了发挥其共抑制作用，CtBP会与每个转录因子伴侣中的一个保守肽基序结合。我们开发了一种AlphaScreen高通量筛选测定法，以监测CtBP与E1A之间的相互作用（E1A模拟CtBP与其转录伴侣之间的相互作用）。我们筛选了包含1280种生物活性化合物的LOPAC文库，并鉴定出NSC95397，它可抑制CtBP-E1A相互作用（IC50 = 2.9 µM）。使用两种二级测定法和一种反筛选法确认了NSC95397的抑制活性。NSC95397还是CtBP脱氢酶活性的弱底物，并且不抑制另一种脱氢酶——乳酸脱氢酶。最后，NSC95397能够破坏CtBP介导的靶基因转录抑制。这些研究为通过破坏CtBP转录复合物开发靶向肿瘤的治疗药物提供了新的可能性。
