An Investigation into the Molecular Interactions and Biological Effect of Natural Stilbenoids at the TRPA1 and TRPV4 Ion Channels

TRPA1 and TRPV4 ion channels are key mediators of various pain and inflammatory conditions, including colitis, pancreatitis, headaches, and chronic cough. Inhibition of these channels, either individually or simultaneously, presents a promising strategy for alleviating pain. Stilbenoids, a class of natural polyphenolic compounds, exhibit a wide range of biological activities. Studies have shown that they also mediate inflammatory and pain signaling pathways through TRP channels. However, their poor solubility, instability, and susceptibility to UV-induced isomerization pose significant challenges to their therapeutic use. In this thesis, we explored molecular binding of natural
stilbenoids as modulators of TRPA1 and TRPV4 ion channels combining molecular modeling and biological studies. Our overall goal was to obtain understanding if the stilbenoid structure could be used for developing more potent and selective TRPA1 or possibly dual TRPV4/TRPA1 inhibitors. In addition, we developed an isolation method of natural stilbenoids and investigated if a nanoparticle formulation can protect the stilbenoids from UV light degradation.

The potential binding modes of the stilbenoids at the ligand binding sites of TRPA1 and TRPV4 were investigated by molecular docking, calculating binding free energies and assessing their stability within the binding sites through molecular dynamics (MD) simulations. The modeling results suggest that stilbenoids exhibit higher affinity for two known agonist binding sites than for an antagonist site of TRPA1. Consistent with this, in vitro results revealed that stilbenoids act as moderate TRPA1 channel agonists and likely inhibit the channel through a desensitization mechanism, rather than function as pure TRPA1 antagonists. On the other hand, our molecular docking studies at hTPRV4 suggested poor binding of the stilbenoids at the known or predicted ligand binding sites of hTRPV4. This was consistent with the in vitro biological activity assay, which showed that none of the compounds had a significant effect on hTRPV4.

Additionally, the impact of natural stilbenoids on NF-κB-mediated inflammation was evaluated in vivo in fruit fly, where stilbenoid oglycones demonstrated an anti-inflammatory effect mediated by the TRPA1 channel. We also computationally predicted the putative binding sites of these compounds at Drosophila melanogaster TRPA1. Selective and high yield methods were developed for isolation of stilbenoid aglycones and glucosides from fresh Scots pine knotwood and Norway spruce inner bark, respectively. Our study revealed several factors that negatively impact the yield of stilbenoids. The drying process particularly reduces the yield of stilbene glucosides. Additionally, prolonged storage and extraction time significantly lower the yield of the stilbenoid aglycones, likely due to degradation or polymerization.

To enhance the therapeutic potential of these compounds, mesoporous silica nanoparticles (MSNs) were employed to improve their photostability under UV exposure. The photostability analysis showed that MSN formulations can effectively protect stilbenoids from the UV light.
Overall, these findings shed light on the molecular interactions and biological activity of natural stilbenoids on TRP ion channels. Moreover, the results give insights into productive isolation and stable formulation of natural stilbenoids. Finally, these results may facilitate the future efforts to (i) exploit the therapeutic effects of natural stilbenoids and to (ii) develop novel TRPA1 and TRPV4 ion channel modulators for the clinic.