Azobenzene-based Photoswitches For Reversible Regulation Of TRPA1 Channel With High Spatiotemporal Resolution

Background and Objective:Transient receptor potential ankyrin 1（TRPA1）channel is a nonselective cation channel that plays an important role in inflammatory and neuropathic pain,itch,and respiratory diseases.The traditional pharmacological molecular tools of TRPA1 channel lack good temporal and spatial resolution,which limits their use.Although optogenetics provides a tool for neuropharmacological research by imparting photosensitivity to relevant neurons or protein targets,optogenetics requires the introduction of two exogenous components,chemical ligands and genes encoding the target protein,which is technically difficult.Different from the genetic modification of proteins required by optogenetics,photopharmacology directly endows ion channels with photosensitivity and precisely activates or inhibits TRPA1 channels through photoisomerization or ligand opening/closing,so as to achieve in vitro or in vivo studies of TRPA1 channels with high spatial and temporal accuracy.This study aimed to design and develop photoswitchable molecules for reversible photoregulation of human TRPA1channels with high spatiotemporal resolution.The activation and inactivation of human TRPA1 channels are regulated by light switching of different wavelengths,and the effects and applications of these photoswitchable molecules on TRPA1 channels have been explored at the cellular or animal level.Methods and Results:A suitable optically-controlled molecular binding pocket was found in the extracellular region of the channel by structural analysis and intelligent design of the TRPA1 channel.New azobenzene photoswitches TRDPA23-1 to TRDPA23-4 were designed and synthesized using azobenzene as the photoisomerization group and the TRPA1 specific agonist TRDPA23-0 as the recognition group.The chemical and optical properties of the novel azobenzene photoswitches were evaluated in vitro,and it was found that cis-trans isomerization could occur under light irradiation at different wavelengths.Among them,TRDPA23-2 and TRDPA23-4 have excellent reversibility and photocycling stability in vitro.The activity of the compounds was then evaluated on CHO cells transfected with human TRPA1 using electrophysiological patch-clamp technique.The results showed that compared with TRDPA23-0,the new azobenzene photoswitches introduced with azo groups significantly reduced the EC₅₀ value of TRPA1 channels,indicating that their agonistic activity was prominently improved.Meanwhile,TRDPA23-2 and TRDPA23-4 exhibited stable,reversible and opposite photoregulatory effects on the TRPA1 channel in the light switching between dark and blue light.The following molecular mechanism exploration and verification experiments proved that the novel azobenzene photoswitchs had an agonistic effect on TRPA1 channel through the hydrogen bond interaction between its secondary amine group and the Tyr926 residue of TRPA1 channel.Cell activity experiments demonstrate that the novel azobenzene photoswitchs with low toxicity can be applied to cells and animal models and relieve pain through desensitization when light-controlled TRPA1 channels.Conclusions and Significance:Our results demonstrate that TRDPA23-2 and TRDPA23-4 can perform cis-trans isomerization under different wavelengths of light,and TRDPA23-2 and TRDPA23-4 can reversibly regulate TRPA1 channel when switching between dark and blue light,and the photocontrol effect is opposite and stable.The mechanism of action is that TRDPA23-2 and TRDPA23-4 produce reversible photoregulation through hydrogen bonding between secondary amine groups and Tyr926site of TRPA1 channel.TRDPA23-2 and TRDPA23-4 demonstrated their photoanalgesic effects on TRPA1 channel in animal level,providing an important photopharmacological tool for studying the role of transient changes in TRPA1 channel in biological signal transduction.To the best of our knowledge,this is the first reversible and reproducible photoregulatory tool for human TRPA1 channels based on the photoisomerization of azobenzene.