The Discovery And Lipid Metabolism Regulation Study Of Novel IRW1αInhibitors

The endoplasmic reticulum (ER) is a major compartment in eukaryotic cell, which is responsible for keeping protein homeostasis. Disruption of the balance between protein synthesis and the folding capacity of the ER, which is called ER Stress, could activate a signaling network called the unfolded protein response (UPR) including PERK, ATF6and IRE1α to minimize ER overload and activate ER-associate degradation (ERAD). If the effort to rectify the imbalance is not successful, the UPR may trigger an apoptotic response.IRE1α (inositol-requiring enzyme-1a) is transmembrane'sensor'protein, which consists of an N-terminal, a single-pass transmembrane spanning segment and a cytosolic region subdivided into a Ser/Thr protein kinase domain and a C-terminal endoribonuclease (RNase), directs a key pathway of UPR. IREla triggers the transcription of a series of UPR related gene to alleviate ER Stress via splicing XBP1mRNA; besides, there is a way independent of XBP1to activate JNK to induce apoptosis. The IREla/XBP1pathway participates in a lot of physiological functions:IRE1a kinase activity is required for accumulation of mtHTT, thus aggravate HTT; IRE1α is a key regulator for angiogenesis in tumor; adipogenesis and hepatic lipogenesis is dependent on IREla/XBP1pathway, and so on. But, there is not a clear relationship between IRE1α kinase and RNase.Thus, the discovery of inhibitors of IREla kinase or IRE1α RNase not only lays foundation for the research into signaling pathways and drug related with IRE1α, but also gives clues to the interaction between IREla kinase and IRE1RNase.Successfully, we produced the cytosolic fragment of human IREla as a His-tag protein with high activity of both kinase and RNase in insect cells. After a series of model optimization including concentration of substrate and DMSO, the high-throughput-screening assay for IREla kinase inhibitor was established with Z'-factor about0.58and the coefficients of variation (CV) less than10%. Through high through screening of12683compounds including natural products, we identified20compounds inhibiting kinase activity of IREla effectively We also set up a molecue-based assay for IREla RNase inhibitor by optimizing the concentration of substrate and DMSO, as well as linear time. Finally, we got5direct inhibitors of IREla RNase after screening for810compounds including cellular-based XBP1-DBD-Luciferase inhibitors, NFκB-Luciferase inhibitors, as well as molecue-based IRE1α kinase inhibitors.IRE1α/XBP1pathway plays an important role in lipid metabolism, but there hasn't any compound related to the pathway to regulate it. For the first time, this project evaluated mounts of componds inhibiting cellular-based XBP1splcing on lipid metabolism, and several compounds which could inhibit the differentiation of3T3-L1cell line and decrease TG concent in HepG2cell line were discovered. Taking account of the effect on both IRE1α RNase and XBP1-DBD-Luciferase, we chosed Compound named17which inhibited IRE1α RNase activity with an IC50of14.92μM and significantly impaired XBP1-DBD-Luciferase in293cell line with an IC50of10μM to research into the function of regulating lipid metabolism. As a result, Compound17had a significant effect on suppressing the differentiation of3T3-L1cell line with down-rugulating the expression of FAS, ACC1and SCD1, Compound17also reduced TG content in HepG2cell line with lowering the expression of FAS, ACC1, SCD1and sREBP-c1.In conclusion, this project set up a molecue-based assay for IRE1α RNase inhibitor and a high-throughput-screening assay for IRE1α kinase inhibitor. These inhibitors would improve the basic research into the relationship between IRE1α RNase and kinase and IRE1α signal pathway. Compound17also opens the door for IRE1α/XBP1inhibitors against lipid metabolism dysfunction.