The Mechanism Of Tropane Modified Bitopic Agonist Modulating M1 Muscarinic Acetylcholine Receptor

As members of G-protein coupled receptors,muscarinic acetylcholine receptors include five subtypes.M1 muscarinic acetylcholine receptor(M1 mAChR)is mainly distributedinthe central nerve system(e.g.cortex,hippocampus,striatum etc.),participating in the functions of learning and memory.M1 mAChR is closely related with many critical diseases,such as Alzheimer’s disease(AD).However,several M1 mAChR agonists have failed in clinic trials targeting AD,because of the serious peripheral side effects.The poor subtype selectivity was the key problem limiting the clinical use of M1 mAChR drugs.Thehighly conserved orthosteric sites were located in the transmembrane region,displaying high binding affinity.However,the diverse allosteric sites,located in the extracellular area,could produce high subtype selectivity.By simultaneously bindingto both orthosteric and allosteric sites,it has been the hot spots in drug discovery to develop bitopic M1 mAChR agonistswith high affinity and subtype selectivity.VU0184670 was a typical bitopic M1 mAChR agonist.Replacement of the piperidine of VU0184670 with a tropane scaffold produced VU0415371,which reserved the subtype selectivity of M1 mAChR and lost the agonist activity for M2-M5 mAChRs.The different binding mode before and after tropane modification could be caused by interaction changes for a few key resides of binding sites.However,which residues caused such interaction changes and how tropane modificationaffected the binding are still unknown.It is crucial to understand the structure activity relationship of these bitopicagonists and to elucidate the interaction mechanisms.Through homology modeling and docking,the interaction models of M1 mAChR and the biotopic agonists(VU0184670 and VU041537)were constructed and then validated.The key residues participated in the interactions were predictedin molecular dynamic simulations.By using site-directed mutagenesis,the structural basis of the interactions between biotopic agonists and M1 mAChR and the mechanism how tropane modification modulated the interactions were elucidated.The major findings are summarized as follows:(1)The residues,such as Y82,Y85,Q177,Y179,E397,W400,E401,and Y404,were considered as the interaction sites,and K392,E397,E401 and Y404 could contribute to the modulation of M1 mAChR by VU0184670 and VU0415371.(2)The residues of Y82 and Y404 could inhibit the intrinsic activity of M1 mAChR,making the agonist occupied orthosteric sites show poor agonist activity.Because of the existance of K392 and E397,the inhibition was much more limited in M1 mAChR than in M2-M5 mAChR.(3)M1 mAChR could be activated by VU0184670 through the residue of Y85.After tropane modification,the rigid tropane ring could enhance the inhibition of Y82 and Y404,resulting in the loss of agonist activity through Y85 for VU0415371 in mAChRs.Because M1 mAChR can still be activated through specific allosteric site,VU0415371 reserved the subtype selectivity of M1 mAChR and lost the agonist activity for M2-M5 mAChRs.In our study,the mechanism how tropane modification of bitopic agonist modulated M1 mAChR and why the agonist activity for M2-M5 mAChR turned over after tropane modification was elucidated.The findings provide an interaction insight for the development of M1 mAChR bitopic agonists.