Molecular Simulation Study On The Interaction Mechanism Of Amylin Receptors And Their Agonists And Drug Design

Obesity is a chronic disease caused by excessive accumulation of fat in the body and is the main factor leading to Type 2 Diabetes Mellitus（T2DM）.T2DM is a chronic disease caused by insulin resistance or relative insufficiency of insulin secretion.Studies have shown that obesity and T2DM are closely related:on the one hand,obesity is the main factor leading to T2DM;on the other hand,T2DM will aggravate the degree of obesity and increase the risk of obesity complications.Complications of T2DM and obesity seriously affect the quality of life and longevity of modern people.How to prevent and treat these two diseases has attracted great attention all over the world.Existing treatments cannot effectively reduce morbidity and mortality.Therefore,it is of great significance to discover new drug targets related to obesity and T2DM for the prevention and treatment of these two diseases.Amylin receptors（AMYRs）exhibit the physiological effects of glucose-lowering and weight loss simultaneously,showing promising performance in the treatment of obesity and T2DM.It has been found that amylin and salmon calcitonin（s CT）can act on AMYRs to reduce blood glucose and weight,and existing drugs such as cagrilintide and KBP-042 are modified based on the above two,respectively.Therefore,exploring the mechanism between AMYRs and peptides（r Amy and s CT）is of great value to drugs for the treatment of T2DM and weight loss.Molecular dynamics simulation has been successfully used to predict the mechanism of action of drugs and targets and to evaluate their activity.In this work,we investigated the mechanism and key residues of rat amylin（r Amy）and s CT with AMY₃R from the aspects of thermodynamics and dynamics,and optimized the peptides accordingly.This paper mainly includes three aspects of work:Part1,in order to explore the thermodynamic mechanism of the interaction of AMY₃R with peptide agitators（r Amy and s CT）,we first used molecular dynamics simulation,residue energy decomposition and alanine scanning mutation to identify the key residues of peptides（r Amy and s CT）with AMY₃R.The results showed that W79,S129,N135,Y146,L291,V293,E294,W361,H377,etc.of AMY₃R,A8,Q10,R11,L12,A13,N14,L16,R18,etc.of r Amy,V8,L9,L12 Q14,E15,L16,H17,L19,etc.of s CT are key residues.Then,principal component analysis,static network analysis and hydrogen bond occupancy analysis were used to explore interaction mode.The results showed that peptides（r Amy and s CT）and AMY₃R can form extensive hydrogen bonds,stable salt bridge andπ-πstacking.For example,Q10^{r Amy}-L291 and Q14^{s CT}-V293/E294formed hydrogen bonds,K11^{s CT}-E294 and R18^{r Amy}-E294 formed salt bridge,as well as Y37^{r Amy}-W79 and Y22^{s CT}-Y41 formedπ-πstacking.The identification of these key residues and interactions provides a good structural basis for drug screening targeting AMYRs and peptide sequence optimization based on peptides（r Amy and s CT）templates.Part2,to investigate the kinetic mechanism of the interaction between AMY₃R and peptides（r Amy and s CT）,we used random accelerated molecular dynamics simulation to predict the dissociation paths of peptides（r Amy and s CT）.The results showed that they left the binding pocket between TM1 and TM2 to the extramembrane.Then,we used steer molecular dynamics simulation to investigate key residues and mechanism of the intermediate state during the dissociation of peptides（r Amy and s CT）as well as the average force potential during the dissociation process.The results showed that W79,S129,N135,Y146,L291,V293,E294,W361,H377,etc.of AMY₃R,A8,Q10,R11,L12,F15,L16,V17,R18,etc.of r Amy,and V8,L9,G10,K11,L12,L16,H17,L19,etc.of s CT are key residues in the dissociation process,and hinder the dissociation of peptides（r Amy and s CT）from AMY₃R by forming hydrogen bonds,salt Bridges andπ-πstacking.For example,N14^{r Amy}-E294 and V8^{s CT}-H377 formed hydrogen bonds.R18^{r Amy}-D97,K11^{s CT}-E294 and E15^{s CT}-K141 formed salt bridge,as well as Y37^{r Amy}-W79 and Y22^{s CT}-Y41 formedπ-πstacking.Part3,due to the low activity,poor stability and short half-life of r Amy,its application was limited.Therefore,random mutation and molecular dynamics simulation methods were used to optimize the sequence of r Amy,in order to obtain a peptide agonist with high stability and strong affinity.Firstly,theα-helical region in the middle of r Amy was randomly mutated,and then the mutation energy was calculated and the toxicity was predicted,and 27 mutant peptides were initially screened.Then,these 27 peptides were evaluated by 100ns molecular dynamics simulation and simulation locus analysis to evaluate their stability,and 8 peptides with high stability were selected.Finally,300ns molecular dynamics simulation was performed for these8 peptides and the binding free energy was calculated.The results showed that the affinity of these 8 peptides was stronger than that of wild-type r Amy at the calculated level.Eight peptides,A8L,Q10D,Q10N,A13D,A13M,A13Y,N14F and L16I,were selected by evaluating their stability and affinity.In summary,this paper explored the mechanism of action and key residues between peptides（r Amy and s CT）and AMY₃R from the perspective of combining thermodynamics and dissociation kinetics,and optimized the sequence of r Amy,providing a theoretical basis for drug development of obesity and T2DM.