High-throughput Screening Of Compounds By Targeting Cell Surface N-glycans And Its Anti-tumor Mechamism

Glycosylation of cell surface proteins is a common way of post-translational modification of proteins.The glycoprotein produced is a glycoconjugate formed by covalently linking a polypeptide chain to a branched oligosaccharide chain,which is rich in living organisms.And the glycoprotein is involved in many important biological behaviors such as communication recognition,signal transduction,and intercellular adhesion.On the surface of tumor cells,N-glycans on membrane proteins are generally overexpressed and exhibit a multi-antenna complex glycan structure.This increase in complex glycan branching reduces the adhesion between cells and enhances the transfer and invasion ability of tumor cells.Plant lectins are a kind of plant-derived proteins that reversibly uniquely recognize glycoconjugate ligands of a particular structure.Using this feature,lectins can be used to detect complex glycosylation changes during the development and progression of tumor cells,as well as differences in the expression of different complex glycans in different tumor cells.In this paper,a high-throughput screening and detection method for N-glycan structure types targeting live cell surfaces was established using phytolectins with fluorophores coupled.Through this method,compounds that can specifically affect the surface protein N-glycosylation of tumor cells were screened from small molecular compound libraries,and the anti-tumor mechanism was discussed.The research results are as follows:1、The abundance of the corresponding glycan type was examined in various tumor cell lines using the four fluorophore-containing phytolectins WGA,L-PHA,Con A and Lc H.The results showed there ware significant differences in the content and connection of N-glycans in different B lymphoma cells.2、A high-throughput method for targeting cell surface N-glycan structures was established.And this method was used to screen and rescreen more than 1,500FDA-approved small molecule compounds.Five candidates were obtained with significantly increasing activity,and one which significantly inhibited the sugar chain recognized by WGA on the surface of tumor cells.3、Penfluridol can change the structure of the glycan on the cell surface,and it can affect the glycan chain modification spectrum of the glycoprotein PD-L1 at the cellular immune checkpoint in a dose-dependent manner.The mechanism of action is significantly different from the positive compound Tunicamycin.4、Penfluridol has good anti-tumor activity with IC₅₀of 0.4μM and 1.1μM in Raji cells and MDA-MB-231 cells respectively.Further mechanistic studies found that penfluridol can induce endoplasmic reticulum stress（ER Stress）by phosphorylating e IF2ɑto activate related signaling pathways.In addition,the formation of stress granules can be observed in U2OS cell lines.In summary,this paper uses a fluorescently labeled phytohemagglutinin to establish a high-throughput screening method for small molecule compounds targeting cell surface N-glycan structure by flow cytometry.A compound,penfluridol,which successfully inhibits the sugar chain structure of WGA on the surface of tumor cells,was successfully screened.This dissertation demonstrates that this compound can cause endoplasmic reticulum stress and up-regulation of Bi P protein expression.And this result suggests that penfluridol is likely to exert anti-tumor activity by inhibiting the formation of three or four antennas of the N-glycan.This provides new ideas and theoretical data for screening and researching of the anti-tumor metastasis drugs by inhibiting N-glycosylation on the surface of tumor cells.