Karantzelis Nikolaos, Petropoulos Michalis, De Marco Valeria, Egan David A, Fish Alexander, Christodoulou Evangelos, Will David W, Lewis Joe D, Perrakis Anastassis, Lygerou Zoi, Taraviras Stavros
Department of Physiology, Medical School, University of Patras, Patras, Greece.
Department of General Biology, Medical School, University of Patras, Patras, Greece.
Front Pharmacol. 2022 Apr 25;13:860682. doi: 10.3389/fphar.2022.860682. eCollection 2022.
DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis and are also evident in multiple human tumors. In this study, we developed an AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.
DNA复制起始需要在G1期将MCM2 - 7复合物加载到复制起点。复制许可使染色质具备DNA复制能力，其严格调控对于防止异常DNA复制和基因组不稳定至关重要。CDT1是许可过程的关键因子，其活性受多种冗余机制控制，包括Geminin，一种CDT1的蛋白抑制剂。异常的CDT1和Geminin表达已被证明可促进肿瘤发生，并且在多种人类肿瘤中也很明显。在本研究中，我们开发了一种AlphaScreen™高通量筛选（HTS）测定法，用于鉴定靶向CDT1 / Geminin蛋白复合物的小分子抑制剂。最有效化合物AF615的生化特性提供了证据，表明其在体外和细胞内均能特异性、剂量依赖性地抑制Geminin与CDT1的结合。此外，化合物AF615诱导DNA损伤，抑制DNA合成，并选择性降低癌细胞系的活力，且这种作用依赖于CDT1。综上所述，我们的数据表明AF615可能是一种有用的化合物，可用于阐明CDT1 / Geminin蛋白复合物在复制许可和起始点激发中的作用，以及作为进一步药物化学优化的支架。
