Predicting And Validating Molecular Targets By Celastrol To Block Palmitic Acid-caused NF-κB Activation

We have found that celastol, via inhibiting TLR4/NF-κB signaling, could treat palmitic acid(PA)-induced insulin resistance. In this work, we use bioinformatics analysis in combination with cellular technology, to further identify the targets for celastrol on TLR4/NF-κB pathway. The following two points are achieved. 1. Celastrol could inhibit the key signaling molecules IRAKs on TLR4/NF-κB pathway. One, IRAKs was predicated as potential targets of celastrol, via text-mining tool. Two, AutoDock analysis showed that celastrol could bind the ATP-binding pocket of IRAK4, indicating that it might compete with ATP, thus inhibiting IRAK4 activity. Three, PA could activate IRAK1 and IRAK4, which was blocked by celastrol. Four, knocking down IRAK4 by siRNA could effectively block PA-induced NF-κB activation. 2. Blockage of PA’s entering MD2 might contribute celastrol’s inhibiting PA-induced TLR4/NF-κB activation. Molecular docking indicated that PA could enter a hydrophobic pocket of MD2, and celastrol could also enter this pocket. There are more groups in MD2 which interact with celastrol than with PA, indicating that celastrol combine with MD2 stronger than PA, thus expel the latter to combine. Knocking down MD2 blocked PA-induced NF-κB activation, thus supporting that MD2 plays roles in PA’s action. Coming together, inhibiting IRAK4 activity and blocking the combination between MD2 and PA are novel mechanisms for celastrol to block PA-induced TLR4/ NF-κB activation. This work disclosed new targets for celastrol to reverse PA-caused insulin resistance.