A Fluorescent Reporter Enabling High-throughput Screening Of OGT Inhibitors

O-linked β-N-acetylglucosamine(O-GlcNAc)is a ubiquitous post-translational modification of proteins that plays an important regulatory role in many physiological mechanisms of cells.Aberrant O-GlcNAc modification is correlated with the occurrece and development of various cancers and other human diseases.At present,the common detection method of O-GlcNAc modification is immunohistochemistry(IHC)and western-blot based on antibodies.Although such methods are accurate,the procedures are cumbersome and are not suitable for the detection of large numbers of samples and high throughput screening of inhibitors.In addition,some researchers have introduced fluorescence resonance energy transfer(FRET)based on metabolic labeling,which can perform imaging and detection of O-GlcNAcylation.However,some of these methods can only perform extracellular detection,and some need to introduce special compounds,which are prone to cause cytotoxicity.None of these methods have been widely used.Therefore,a method is needed to accurately and efficiently detect O-GlcNAcylation and perform high-throughput screening of related inhibitors.We constructed a fluorescent sensor to detect O-GlcNAc modification while the cells remain physiologically active based on the principle of transcriptional regulation.The sensor mainly consists three elements,including an insulin promoter-driven enhanced green fluorescent protein(eGFP),a CMV promoter-driven red fluorescent protein(RFP)and a human NeuroD1.Here,the fluorescent eGFP is used as a reporter,while RFP is selected as an internal reference to exclude the influence of cells number variation and transfection efficiency in the assay.The transcriptional termination signal and the translational stop codon are inserted between the reporter gene and the reference gene,making the two genes independent of each other.NeuroD1 was ligated downstream of the RFP via P2A(a self-cleaving peptide).Thus,NeuroD1 and RFP share the CMV promoter,but the proteins of the two are separated from each other and function separately.We have verified the function of the reporter(IGCRN),which can accurately detect the down-regulation of O-GlcNAcylation in cells,and can also respond to different concentrations of OGT inhibitor(OSMI-1)and HBP pathway inhibitor(DON).In addition,we used IGCRN to detect the EC50 of OSMI-1 and DON,which were 5.93 μM and 33.08 μM,respectively.We hope that high-throughput screening of OGT and HBP pathway inhibitors can be performed using cell lines that stably expressed IECRN.