Molecular docking and biochemical validation of (-)-syringaresinol-4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside binding to an allosteric site in monoamine transporters

Abstract

Albizia julibrissin Durazz is one of the most common herbs used for depression and anxiety treatment, but its mechanism of action as an antidepressant or anxiolytic drug have not been fully understood. We previously isolated and identified one lignan glycoside compound from Albizia Julibrissin Durazz, (-)-syringaresinol-4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (SAG), that inhibited all three monoamine transporters with a mechanism of action different from that of the conventional antidepressants. In this study, we generated homology models for human dopamine transporter and human norepinephrine transporter, based on the X-ray structure of Drosophila dopamine transporter, and conducted the molecular docking of SAG to all three human monoamine transporters. Our computational results indicated that SAG binds to an allosteric site (S2) that has been demonstrated to be formed by an aromatic pocket positioned in the scaffold domain in the extracellular vestibule connected to the central site (S1) in these monoamine transporters. In addition, we demonstrated that SAG stabilizes a conformation of serotonin transporter with both the extracellular and cytoplasmic pathways closed. Furthermore, we performed mutagenesis of the residues in both the allosteric and orthosteric sites to biochemically validate SAG binding in all three monoamine transporters. Our results are consistent with the molecular docking calculation and support the association of SAG with the allosteric site. We expect that this herbal molecule could become a lead compound for the development of new therapeutic agents with a novel mechanism of action.

Computational Chemistry in Structure-Based Solute Carrier Transporter Drug Design: Recent Advances and Future Perspectives

“…Many experimental and computational studies have suggested that allosteric sites exist in MATs. ,, In 2016, Coleman et al successfully resolved the crystal structures of SERT bound with escitalopram in its allosteric site (S2 site), providing strong evidence for the allosteric mechanism . The allosteric sites of MATs are usually aromatic and hydrophobic pocket positioned in the extracellular vestibule. , Occupation of S2 site by allosteric modulators has the potential to be a fast-acting and safe new strategy for the treatment of physiological disorders by affecting the binding and dissociation of substrates and the conformational change of protein. , To date, many allosteric modulators of MATs have been designed as potential therapeutic agents. ,,,, …”

“…Since 2005, the first X-ray crystal structure (PDB ID: 2A65) of the bacterial leucine transporter (LeuT) was reported, and the structures of the LeuT, drosophila dopamine transporter (dDAT), and SERT have been used as templates for the construction of structural models of MATs. ,, Fu et al also modeled a large number of structural variabilities of SCLs by homology modeling and added them to the VARIDT database () . Recently, the popular AI protein structure prediction method AlphaFold2 can also be used to accurately predict transporter structures. , These structural models obtained by computational techniques greatly enrich the structural information on SLCs and are widely used in drug design and transport mechanism studies. ,− Nevertheless, the application of computational structures in drug design still faces several challenges; the accuracy of models and diversity of conformations need to be carefully considered . Considering that conformational changes of proteins are closely related to their function, studying the multiple conformations of SLCs is helpful to understand their transport mechanism.…”

“…Many experimental and computational studies have suggested that allosteric sites exist in MATs. 40,99,100 In 2016, Coleman et al successfully resolved the crystal structures of SERT bound with escitalopram in its allosteric site (S2 site), providing strong evidence for the allosteric mechanism. 47 The allosteric sites of MATs are usually aromatic and hydrophobic pocket positioned in the extracellular vestibule.…”

“…47 The allosteric sites of MATs are usually aromatic and hydrophobic pocket positioned in the extracellular vestibule. 100,101 Occupation of S2 site by allosteric modulators has the potential to be a fast-acting and safe new strategy for the treatment of physiological disorders by affecting the binding and dissocia-tion of substrates and the conformational change of protein. 58,87 To date, many allosteric modulators of MATs h a v e b e e n d e s i g n e d a s p o t e n t i a l t h e r a p e u t i c agents.…”

“…58,87 To date, many allosteric modulators of MATs h a v e b e e n d e s i g n e d a s p o t e n t i a l t h e r a p e u t i c agents. 99,100,102,39,103 In addition, SLCs are membrane proteins, and their functions are regulated by the membrane environment. For example, cholesterol, a major component of the synaptic plasma membrane can influence the out-to-inward transition of DAT and the ligand binding to DAT.…”