Small molecule inhibitors can be used not only as effective drug leaders for the development of new therapies for related diseases, but also as chemical probes for studying the biological functions of targets in cells. In the 1970s, researchers isolated the natural product Tunicamycin (Tunicamycin) from Streptomyces and found that it can globally inhibit the N-glycosylation post-translational modification of intracellular proteins. The research system using this inhibitor revealed The function of N-glycosylation in protein folding and transport. At present, tunicamycin has become an indispensable tool for studying protein N-glycosylation. For another widespread post-translational modification of O-glycosylation in cells, there is currently no active inhibitor that can target the level of global O-glycosylation in cells.
In recent years, researchers have discovered that the GalNAc-Ts enzyme responsible for O-glycosylation can catalyze the O-glycosylation of a variety of important substrate proteins, such as angiopoietin-like 3 (ANGPTL3), fibroblast growth factor 23 (FGF23), amyloid beta precursor protein (APP) and apoC-III, etc. In addition, antibodies targeting secreted ANGPTL3, FGF23, and Aβ(the cleavage product of APP) have been approved by the FDA for the treatment of family hypercholesterolemia (Regeneron Evkeeza® monoclonal antibody) and hypophosphatemia rickets, respectively (Ultragenyx Pharmaceutical Crysvita® monoclonal antibody) and Alzheimer's disease (Bojian and Eisai jointly developed Aducanumab® monoclonal antibody).
Therefore, the discovery of specific small molecule inhibitors targeting GalNAc-Ts can not only provide pharmacological tools for the study of intracellular O-glycosylation function, but also provide new clues and new targets for related disease research.
Schematic diagram of O-glycosylation inhibitor discovery
In response to this scientific problem, Fang Wu’s research group from the Institute of System Biomedicine at Shanghai Jiaotong University used a specific GalNAc-T2 activity reporting tool developed by Professor Linstedt of Carnegie Mellon University in the United States, through high-throughput screening at the cellular level for about 7000 An FDA-approved drug or natural product small molecule discovered that a class of quinic acid derivatives with a novel chemical skeleton derived from the traditional Chinese medicine Coltsfoot can specifically inhibit the activity of purified GalNAc-T2 in cells or in vitro. Importantly, this type of inhibitor has no cytotoxicity and avoids the problems of non-specificity and cytotoxicity faced by the development of pharmacological inhibitors. Furthermore, the gene editing tool CRISPR/Cas9 was used to knock out COSMC, the key protein for O-sugar chain extension, and found that these inhibitors can still inhibit the content of O-sugar chain, indicating that it can target GalNAc, which is responsible for the initiation of O-sugar chain. -T2 is not a glycosyltransferase that inhibits the extension of other sugar chains.
In addition, in vitro structure-activity relationship studies have shown that the two caffeic acids on the quinic acid backbone of these inhibitors are key pharmacophores necessary for their inhibitory activity; molecular mutation experiments further prove that their binding sites are located in the catalytic domain of GalNAc-T2 ; Surface plasmon resonance technology found that the affinity constant KD of the inhibitor and GalNAc-T2 can reach 9 μM and 1.3 μM, indicating that the compound has a strong direct binding to GalNAc-T2.
In summary, the novel O-glycosylation natural product inhibitors discovered in this study can specifically inhibit GalNAc-T2 activity and O-glycosylation in cells. These findings can provide biologically active and specific pharmacological probe tools for studying the functions of GalNAc-T2 and O-glycosylation, as well as drug leads and new targets for the research of related diseases.
The article entitled "Novel Quinic Acid Glycerates from Tussilago farfara Inhibit Polypeptide GalNAc-Transferase. https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/cbic.202100539)" was published on December 1, 2021 Published online in ChemBioChem, an international journal of chemical biology. Doctoral student Feng Juan of Shanghai Jiaotong University and master student Li Yupeng of Jinan University are the co-first authors. Professor Zhang Hua of Jinan University and researcher Wu Fang of Shanghai Jiaotong University are the corresponding authors. This work was supported by the National Natural Science Foundation of China and Shandong Natural Science Foundation. Funding.