Structure And Function Of Muscarinic Acetylcholine M3 Receptor With Different Ligands

GPCRs,the largest member protein super-family in human,are composed by about800 receptors that primarily detect molecules outside the cell,activate internal pathways for signal transduction and finally trigger cellular responses,which is achieved by coupling G proteins,arrestin and other signaling proteins.GPCRs are involved in a large number of physiological activities,in particular some rhythms regulated by autonomic nerve system（e.g.,visual sense,heartbeat,digestion and breath）.For these reasons,GPCRs are correlated with many diseases and also the key drug target of approximately34%modern medicinal drugs.As one of the crucial components of GPCRs,muscarinic acetylcholine receptors（hereinafter referred as mAChRs）have 5 subtypes of receptor:M1,M2,M3,M4 and M5.mAChRs exist broadly in the body,including CNS,heart,lung as well as grands.Acetylcholine（Ach）,the first discovered neurotransmitter,activates the downstream G protein by binding the mAChRs of postsynaptic membrane,then regulates the concentration of Ca²⁺in cells.M3R is distributed primarily over the smooth muscle of the blood vessels,the lungs and the bladder.As M3R is G_q-coupled,mediating an increase in intracellular calcium,it typically induces the contraction of smooth muscle.M3Rs also exist in glands broadly,which helps to stimulate secretion.Many drugs are developed to treat M3R for its significant physiological function.One of M3R antagonists-tiotropium is also employed for the treatment of COPD and chronic bronchitis and applied in the adjunctive clinical therapy of asthma.However,the design and development of drugs for treating mAChRs now have two emerging challenges to be addressed.The one is selectivity.mAChRs family has 5 subtypes,suggesting that there exist two or more subtypes of mAChRs in the same tissue or organ.The design and development of subtype selective ligands for mAChRs are valuable to screen new therapy drugs,including orthosteric and allosteric ligands.Another strategy for drug design is biased ligands.It is known that GPCRs can bind to the downstream G protein or arrestin protein,forming different signal pathways.Biased ligands are proposed to have reducing side effects in many cases.With the rising number of inactive and active-state GPCR structures determined by X-ray crystallography and Cryo-EM,structural biology will be increasingly significant for drug discovery.For mAChRs,the inactive structures of M1R,M2R,M3R and M4R are obtained by crystallography,as well as the active structures of M2R using nanobody.The antagonists for M2R and M3R are QNB and tiotropium,respectively,which have high affinity with all mAChRs but no selectivity to M2R/M3R.In this study,starting from the published crystal structures of the M2R and M3R,a single amino-acid difference in their orthosteric ligand binding pockets was exploited,together with molecular docking and structure-based design.Finally,the designed selective antagonists were synthesized with about 100-fold selectivity over the M2R in vitro and over 1000-fold selectivity in vivo.The crystal structure of one selective M3R antagonist BS46 in complex with the M3R was determined and closely correlated with the docking-predicted geometry.Darifenacin,one of selective drugs for M3R used to be marketed by Novartis,is a medication used for treating urinary incontinence.Darifenacin works by blocking the M3R,which is primarily responsible for bladder muscle contractions,thereby reducing the urgency to urinate.It is not known what the mechanism of selectivity for M3R over the other subtypes of mAChRs.This study aimed to explain the selectivity mechanism of M3R with crystallography but failed to obtain enough crystals with good diffraction,even though some tiny crystals were found in LCP.Allosteric modulator means that a ligand that can bind to an allosteric site of GPCRs and modulate the binding and/or signaling of an orthosteric ligand.Allosteric modulators,by affecting orthosteric ligand binding affinity or efficiency,or by interacting the G protein binding sites,cause the downstream signal rising or falling.Otherwise,the sites of allosteric modulator binding existed divergences in different receptors,even between several subtypes.As a result,allosteric modulator was suitable for designing subtype-selective ligands as drugs target.Here,one negative allosteric modulator-MK621 was identified for M3R by molecule docking.First,this study tried to crystallize the M3R bound antagonist plus allosteric modulator MK621.However,we failed because PEGs always competitively occupied the allosteric site in crystallography.As a result,MK621position is hard to figure out in M3R.The M3R conformation change after orthosteric ligands and allosteric ligands binding was also studied using NMR.Two labeling strategies,Lys and Met labeling,were used for monitor the molecule dynamics.K522,located in the ECL3 of M3R,was assigned to test M3R conformation change after the addition of various ligands.We found that the peaks of K522 were reduced when adding M3R antagonists,yet they disappeared when antagonists and allosteric modulator MK621 were added,proving the K522involved with MK621.The similar results were found in the Met labeling NMR spectrum,indicating that the MK621 should bind the extracellular region of M3R.