Screening inhibitors for blocking UHRF1-methylated DNA interaction with capillary electrophoresis

• Published in: Journal of Chromatography A 2021-01, Vol.1636, p.461790, Article 461790
• Main Authors: Lou, Chunli, Ye, Xiongzhen, Chen, Ge, Zhu, Jidong, Kang, Jingwu
• Format: Article
• Language: English
• Subjects: Capillary electrophoresis; DNA methylation; Drug screening; UHRF1 inhibitor
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2020.461790

•A CE method was developed for screening inhibitors of UHRF1–mDNA interaction•Kd and koff of the UHRF1–mDNA complex were determined in one run•Only minute sample and reagent were consumed for screening of inhibitors•Sample pooling strategy can improve the screening throughput Epigenetic inheritance in mammals relies in part on propagation of DNA methylation patterns throughout development. UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) is required for maintenance the methylation pattern. It was reported that UHRF1 is overexpressed in a number of cancer types, and its depletion has been established to inhibit growth and invasion of cancer cells. It has been considered as a new therapeutic target for cancer. In the present work, we described a method for screening inhibitors for blocking the formation of UHRF1–methylated DNA (mDNA) complex by using nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM). A recombinant UHRF1 with the SRA domain (residues 408-643), a fluorescently labeled double strand mDNA (12 mer) and a known inhibitor mitoxantrone were employed for proof of concept. The method allows to measure the dissociation constant (Kd) of the UHRF1–mDNA complex as well as the rate kinetic constant for complex formation (kon) and dissociation (koff). A small chemical library composed of 60 natural compounds were used to validate the method. Sample pooling strategy was employed to improve the screening throughput. The merit of the method was confirmed by the discovery of two natural products proanthocyanidins and baicalein as the new inhibitors for blocking the formation of UHRF1–mDNA complex. Our work demonstrated that CE represents a straightforward and robust technique for studying UHRF1–mDNA interaction and screening of the inhibitors.