High-Throughput Screening For The Complex Of MTORC2 Specific Small Molecular Inhibitor

Objective:To establish the cellular and molecular models for phosphorylation-AKT (Ser473) which is the specific downstream target of mTORC2, cooperating with North China Pharmaceutical Group (NCPC) New Drug Research and Development Co.Ltd, and screening specific small molecule inhibitor of mTORC2 from the microbial strain metabolism library;Methods:We used phosphorylation-AKT (Ser473) which was the specific downstream target of mTORC2 as the screening targets of first round. Using DMSO, Rapamycin, PI-103, HRP as negative control, activation positive control, suppression positive control and substrate blank control respectively, we conducted Cytoblot screening in normal bronchial epithelial cell line BEAS-2B. Eight concentration gradients were designed in each sample,3-fold dilution between two adjacent gradients. The first test samples were 1693 kinds of monomeric compounds owned by North China Pharmaceutical Group (NCPC) Center for New Drug Screening;Results:When the signal value of a compound was lower than the value of PI-103 or equivalent, and presenting preferable reproducibility and concentration-dependent manner, we took further Western identification, and finally obtained three kinds of small molecule inhibitors which had suppressive activity of phosphorylation-AKT (Ser473); When the signal value of a compound was higher than the value Rapamycin or equivalent, and also presenting preferable reproducibility and concentration-dependent manner, we took further Western identification, and finally obtained one kind of small molecule agonist which had activation on phosphorylation-AKT (Ser473).Conclusions:Phosphorylation-AKT (Ser473) Cytoblot screening method, is based on the whole living cells, which is simple and fast, having high sensitivity and specificity. It's could be used as a preliminary screening model to screen specific inhibitors of mTORC2; Objective: To explore molecular mechanism of the phenomenon that small moleculecompounds HA induced Rictor protein degradation selectivly, and providing a novel threadfor screening specific inhibitors of mTORC2 complex;Methods: We further tested the effects of HA induced Rictor protein degradation in avariety of tumor cell lines by western blot; in HA-induced Rictor protein degradation model,when we added the proteasome inhibitor MG-132, which could inhibit the proteasomedegradation pathway, Rictor protein degradation could be reversed; and then we dividedRictor into five clips, for the Rictor protein is too large, to reconstruct GST fusion proteinsrespectively, established In vitro Ubiquitination model, further confirmed Rictor proteincould be ubiquitylated;Results: HA could selectively induce Rictor protein degradation occurring on MM cellline in ubiquitin-proteasome pathway; at the same time, HA could not induce mTORprotein and Raptor protein degradation, both of which are the important constituents of thecomplex of mTORC1; in HA-induced Rictor protein degradation model, we could see thatRictor protein was degraded, mTORC2 function was inhibited, the phosphorylation level ofits specific substrate p-AKT (Ser473) sites reduced; when combining with proteaseinhibitor, the Rictor protein degradation was reversed, the phosphorylation level of p-AKT(Ser473) sites was recovered;Conclusion: Based on the induced Rictor degradation model by the compound of HA, bythorough research the molecular mechanism of this selective degradation, we will get apromising strategy to screen specific inhibitors of mTORC2.