Rational Design Of Islet Neogenesis-related Peptide Drugs Based On Improving Structural Stabilit

Diabetes is one of the chronic diseases threatening human health.Insulin injection,pancreas or islet transplantation are common means of diabetes treatment,but insulin can only temporarily relieve the symptoms of diabetes,can not prevent the systemic complications of diabetes,and frequent injection will lead to continuous physical and mental pain of patients.Pancreas or islet transplantation has problems such as immune rejection and lack of donor.The combination of multiple treatments is often required to achieve the maximum therapeutic effect,resulting in a serious financial burden on patients and their families.Islet neogenesis associated protein pentadecapeptide(INGAP-PP)is the 15peptide,derived from Islet neogenesis associated protein(INGAP),which consists of amino acids 104-118,has similar biological function to INGAP,and can stimulate the proliferation of pancreatic ductal cells and induce the generation of new islets.As an endogenous polypeptide,INGAP-PP has the advantages of no immune rejection and little toxic and side effects,so it has a natural advantage in the clinical treatment of diabetes.However,the common problems of polypeptide drugs,such as poor structural stability and short half-life in vivo,limit the clinical application of INGAP-PP to a great extent.The purpose of this study is to improve the structural stability of INGAP-PP,homology modeling,virtual amino acid spurt and molecular dynamics simulation were used in this study,pancreatitis associated protein(PAP-1)was used as a template to modify and screen human pancreatic islet neophytic associated peptides(h INGAP-PP).The stability of the selected h INGAP-PP polypeptide in plasma was tested in vitro.The main results of the study are as follows:(1)Construct and obtain 20 h INGAP models using PAP(PDB ID:1UV0)as template.Using model evaluation methods such as MODELER DOPE,Ramachandran Plot and Profile-3D,the model was graded,sorted and evaluated for rationality.h INGAP.M0011 was selected as the optimal model,and 104～118polypeptide fragments were selected as the initial structure of h INGAP-PP.(2)The amino acid virtual mutation technology was used to design the mutation of h INGAP-PP.Four amino acid sites(Ile104,Gln111,Thr113,Gly117)in h INGAP-PP were identified as the sites to be mutated by ALA-Scanning and multiple sequence scanning.Virtual amino acid mutations were performed at these four sites,and a total of 80 unit point mutations(7 stable mutations),2400 double site mutations(134 stable mutations)and 31397 three-site mutations(1533 stable mutations)were obtained.With the help of score function and preliminary energy optimization,27mutants were selected.(3)Molecular dynamics simulation and locus analysis of 500 ns were performed on 27 mutants,and the calculation results showed that 5 mutants(S7,D3,D8,T1,T3)had good stability.Further analysis of the stability difference between the 5 mutants and wild type h INGAP-PP showed that:the mean hydrogen bond of the mutant S7was 7.96,13.1%more than that of the wild type h INGAP-PP,the mean solvent accessible surface area(SASA)was 15.30 nm~2,7%smaller than the wild-type h INGAP-PP;the change amplitude of RMSD,Rg and RMSF of the mutant S7 was smaller than that of the wild type h INGAP-PP,which indicated that the mutant S7 was more stable.(4)The secondary structure of wild type h INGAP-PP and mutant S7,D3,D8,T1,T3 was compared,and the overall structure of the mutant and wild type h INGAP-PP showed a"U"shape:the N-terminal(amino acid residues 1-2)and C-terminal(amino acid residues 13-15)were coil,the central region(amino acid residues 6-8)was turn,and the two sides of the central region(amino acid residues 3-5 and 9-12)were bend,this indicated that the amino acid changes in the mutant did not affect the overall structure of h INGAP-PP.(5)Covariance matrix analysis found that the positive and negative correlation motion between the main chain atoms of S7 polypeptide was very low,and the flexibility of residues was very weak.The first two main components of S7,PC1 and PC2,accounted for 67.38%of the total movement,and PC1 accounted for almost50%of the total movement,which was 13.06%higher than PC1 of wild type h INGAP-PP,indicating that S7 had a higher degree of coordinated movement.(6)Wild type h INGAP-PP and mutant S7,D3,D8,T1 and T3 were synthesized,and plasma stability was determined in vitro.The results showed that S7 had the highest stability in plasma.After 120 min,the contents of wild type h INGAP-PP and the mutant D3,D8,T1 and T3 decreased significantly in plasma,while the mutant S7remained 60.17%.In conclusion,the h INGAP-PP polypeptide structure model was successfully constructed in this study.The h INGAP-PP polypeptide mutant was designed and screened by amino acid virtual mutation,molecular dynamics simulation and in vitro plasma stability test,and a highly stable islet neovasculation-related polypeptide mutant S7(G117T)was obtained.Temporarily named GT117.After further chemogenic analysis,GT117 peptides can be used as candidate drug molecules for the treatment of diabetes mellitus,which provides theoretical basis and practical reference for rational design and improvement of the structural stability of endogenous peptides.