Chebulagic Acid

The Src Homology 2 (SH2) domain, the most conserved region of STAT5a/b (aa 573– 712), is crucial for receptor-specific recruitment and STAT5 dimerization, making it a therapeutic target in prostate cancer (PCa).

This study explored the SH2 domain of STAT5a and carried out the identification of natural STAT5a inhibitors.

Using template-based homology modeling, we constructed the structure of human STAT5a (VP1P) and compared it with its 3D crystal of the STAT5a protein obtained from the RCSB database and the model generated by the AlphaFold database. In this study, we carried out molecular docking studies using AutoDock Vina on the top 500 natural compounds identified through a pharmacophore search of the ZINC database using ZINCPharmer. Furthermore, the top ten compounds with the highest binding energies were evaluated for their drug-likeness and ADMET properties using SWISS ADME and ProTox-II, followed by 100 ns molecular dynamics (MD) simulations using the Desmond module of the Schrodinger suite.

Docking studies revealed Pedunculagin (-10.5 kcal/mol), Folic acid (-10.1 kcal/mol), Chebulinic acid (- 10.0 kcal/mol), Chebulagic acid (-9.8 kcal/mol), and Oleandrin (-9.8 kcal/mol) as the top candidates, compared to the STAT5 inhibitor (Phase-II Clinical Trial) (-8.5 kcal/mol). ADMET analysis confirmed their safety profiles. MD simulations showed stable protein-ligand complexes, with all compounds interacting with the conserved Arg638 residue at the active site, similar to the STAT5 inhibitor.

Pedunculagin demonstrated the strongest binding energy and stability, making it a promising candidate for further development as a novel lead compound to disrupt STAT5a/b dimerization in PCa therapy.

The highly conserved N-terminal domain of polymerase acidic endonuclease (PA_N) makes it an attractive target for the development of anti-influenza virus drugs. This study developed an innovative multi-detector based at-line nanofractionation platform by coupling an HPLC system containing triple-detector (diode array detector (DAD), charged aerosol detector (CAD) and MS) with fluorescence resonance energy transfer (FRET)-based bioassays to systematically identify potential PA_N inhibitors from traditional Chinese medicine (TCM). A preliminary screening of 230 TCM extracts revealed an exceptional PA_N inhibition by Terminalia chebula Retz. (IC₅₀ = 0.88 μg/mL). A time-resolved FRET bioassay in 384 well-plates with parallelized MS/MS analysis enabled real-time acquisition of both biological activity profiles and chemical information from Terminalia chebula Retz. extracts. HPLC-DAD-CAD analysis further elucidated the correlation between the content and bioactivity of the identified potential PA_N-inhibitory compounds, addressing the limitations of traditional single UV-based detection. Remarkably, 31 bioactive compounds (including gallotannins, ellagitannins, and phenolic acids) were identified through this approach, with 75.9% exhibiting low abundance (CAD response < 1 pA·min), highlighting the unique capability of this platform in discovering low-abundant bioactive entities. Five verified compounds (ellagic acid, chebulinic acid, punicalagin, chebulagic acid, and 1,3,6-tri-O-galloyl-β-D-glucose) exhibited superior PA_N inhibition (IC₅₀ = 0.52-1.00 μM) in comparison to baloxavir marboxil (IC₅₀ = 10.83 ± 1.46 μM). A molecular docking study elucidated their crucial PA_N-binding interactions and fundamental mechanisms. Overall, this study not only identified several potential PA_N inhibitors from Terminalia chebula Retz., but also offered a scalable at-line nanofractionation strategy to reveal the content-activity correlation of complex TCM extracts.