Genipin, a natural AKT inhibitor, targets the PH domain to affect downstream signaling and alleviates inflammation.

The iridoid compound genipin (GNP) is a geniposide hydrolysate of ß-glucosidase. GNP has many pharmacological effects, including antioxidant, anti-apoptotic, and anti-inflammation effects. However, its exact target and mechanism of action remain poorly understood. In this study, the binding of GNP to AKT protein was demonstrated via a GNP-modified magnetic microspheres (GNP-MMs) capture and immunofluorescence co-localization test. GNP-MMs fishing coupled with competitive testing and AKT plasma transport experiments indicate that GNP may act on the PH domain of AKT, and affect AKT plasma transport. The specific binding directly inhibits phosphorylation of AKT, affecting the downstream activation, and reducing inflammatory responses. The results indicate that GNP targets the PH domain region of AKT, inhibits the phosphorylation of AKT, and attenuates the transduction of AKT based inflammation signal pathway.

Quantitative Proteomics Combined with Affinity MS Revealed the Molecular Mechanism of Ginsenoside Antitumor Effects.

Ginsenosides have previously been demonstrated to effectively inhibit cancer cell growth and survival in both animal models and cell lines. However, the specific ginsenoside component that is the active ingredient for cancer treatment through interaction with a target protein remains unknown. By an integrated quantitative proteomics approach via affinity mass spectrum (MS) technology, we deciphered the core structure of the ginsenoside active ingredient derived from crude extracts of ginsenosides and progressed toward identifying the target protein that mediates its anticancer activity. The Tandem Mass Tag (TMT) labeling quantitative proteomics technique acquired 55620 MS/MS spectra that identified 5499 proteins and 3045 modified proteins. Of these identified proteins, 224 differentially expressed proteins and modified proteins were significantly altered in nonsmall cell lung cancer cell lines. Bioinformatics tools for comprehensive analysis revealed that the Ras protein played a general regulatory role in many functional pathways and was probably the direct target protein of a compound in ginsenosides. Then, affinity MS screening based on the Ras protein identified 20(s)-protopanaxadiol, 20(s)-Ginsenoside Rh2, and 20(s)-Ginsenoside Rg3 had affinity with Ras protein under different conditions. In particular, 20(s)-protopanaxadiol, whose derivatives are the reported antitumor compounds 20(s)-Ginsenoside Rh2 and 20(s)-Ginsenoside Rg3 that have a higher affinity for Ras via a low KD of 1.22 µM and the mutation sites of G12 and G60, was demonstrated to play a core role in those interactions. Moreover, the molecular mechanism and bioactivity assessment results confirmed the identity of the chemical ligand that was directly acting on the GTP binding pocket of Ras and shown to be effective in cancer cell bioactivity profiles.