Computational Biology Study Of The Mechanism Of Action Of Novel Antidepressants:molecular Simulation And Experimental Validation

Background:Depression is a mental disease with a high incidence rate and complex symptoms.In clinical practice,it is mainly manifested by symptoms such as low mood,reduced interest,and poor sleep quality,seriously affecting people’s physical and mental health.At present,traditional antidepressants such as selective serotonin reuptake inhibitors(SSRIs)have side effects such as slow onset,low efficacy and high toxicity.Therefore,it is urgent to develop new antidepressants with safe and effective effects.Chinese herbal medicine has the characteristics of high efficiency,low toxicity,and overall regulation,which plays a certain advantage in the prevention and treatment of depression.The traditional Chinese medicine ALBIZIAE CORTEX has the effects of relieving depression and calming the nerves.It is one of the most commonly used Chinese medicines in clinical treatment of depression and anxiety disorders.However,its molecular mechanism of antidepressant or anxiolytic effects is not fully understood.The previous research of the research group isolated and identified a natural lignan glycoside compound(-)-syringaresinol-4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside(SAG),the research results show that SAG inhibits the activity of monoamine transporters noncompetitively,so its mechanism of inhibiting serotonin transporter(SERT)is different from traditional antidepressants.Therefore,this topic will further clarify the new mechanism of SAG inhibiting SERT,and provide a basis for the development of new antidepressant drug molecules.Content:1.Investigate the influence of SAG on the cytoplasmic-facing conformation of h SERT.By site-directed mutagenesis at residue 277,which replaced the amino acid with cysteine(S277C)capable of reacting with 2-Aminoethyl methanethiosulfonate hydrobromide(MTSEA)and the conformational changes of the inward-facing state were observed using confocal microscopy.In this study,the S277C/X5C-h SERT expression vector was selected to investigate the effect of SAG on the inward-facing conformation of h SERT.2.Construction of human dopamine transporter and human norepinephrine transporter models.In this study,we first constructed homologous models of human dopamine transporter and human norepinephrine transporter based on the cryogenic electron microscopy(cryo-EM)structure of drosophila dopamine transporter(PDB ID 4XP1)using the homology modeling software MODELLER.From the generated 50 models,select the model with the highest score for follow-up work.3.Molecular docking of SAG with monoamine transporters.Taking the cryo-EM structure of h SERT combined with vilazodone(PDB ID 7LWD)as a reference to set the docking range of SAG,we docked the molecule of SAG with monoamine transporters using Schr?dinger software.4.Immunofluorescence experiments were performed to validate the binding site of SAG with monoamine transporters.Based on the molecular docking results mentioned above,amino acid residues in the S1 central binding site of two human monoamine transporters(SERT and DAT)that bind to substrates and the S2 allosteric sites of three human monoamine transporters that have been widely reported were analyzed.Site-directed mutagenesis and verification of the binding site of SAG to the monoamine transporter by transport and binding experiments.Result:1.Compared with incubation of MTSEA alone,the fluorescence intensity increases when substrate 5-HT is added,while treatment with antidepressant fluoxetine decreases the fluorescence intensity.The results indicate that the addition of SAG can significantly reduce the reaction between S277 C and MTSEA,suggesting that SAG can stabilize the inward-closed conformation of h SERT.Combined with the previous research results of our research group,it is demonstrated that SAG can simultaneously close both the extracellular and intracellular pathways of h SERT,resulting in an occluded conformation state.2.In the molecular docking results,select the complex model with the highest score for further analysis,and found that in h SERT,there are 15 amino acid residues associated with SAG binding;in h DAT,there are 8 amino acid residues associated with SAG binding;in h NET,there are 8 amino acid residues associated with SAG binding.3.Based on immunofluorescence experiments,it was found that the inhibitory ability of SAG on the S2 mutants of the monoamine transporters decreased significantly,while the S1 mutants had no significant effect on the inhibitory ability of SAG.This confirms that SAG binds to the S2 binding site of monoamine transporters and that S1 center binding site mutations do not affect the binding and inhibitory ability of SAG.Conclusion:SAG can simultaneously close both the extracellular and intracellular pathways of h SERT,SAG makes h SERT in the occluded conformational state.Molecular docking results indicate that SAG binds to the S2 allosteric site of three human monoamine transporters.Immunofluorescence experiments confirm SAG binds to S2 allosteric binding site and the ability that SAG inhibit three monoamine transporters is not affected by S1 mutants of the central binding site.In summary,SAG inhibits the transport activity of three monoamine transporters by binding to their S2 sites to achieve antidepressant effects.