Cyclization Modification And Evalμation Of Pathological Endocytosis Blocking Polypeptide GluA2-3Y Of AMPA Receptor

PurposeIn this study,the peptide Glu A2-3Y with the highest affinity with Brag2 to block AMPA receptor pathological endocytosis was identified and modified through cyclization strategy,thereby improving the transmembrane efficiency,in vitro plasma stability and neuroprotective activity of peptide drugs.MethodBiomacromolecule interaction detection:In this study,a series of polypeptides were designed based on the Glu A2-3Y sequence,and surface plasmon resonance technology was used to screen the key amino acids that bind to Brag2 protein and the sequence with the highest affinity,and the molecular docking technology was used to interpret the surface plasmon resonance results.Polypeptide synthesis method:The desired peptide was synthesized in solid phase by Fmoc method,and the solid-phase cyclization of the peptide was completed on the resin.Method for evaluating the in vitro permeability of peptide drugs:MDCK-MDRI cell transmembrane transport model and PAMPA transmembrane transport model were used to evaluate the ability of polypeptide drugs to penetrate biomembrane,and the apparent permeability coefficient of each drug group was calculated.Peptide cell viability evaluation method:each group of polypeptide gradient concentrations of 10,50,100,500,and 1000μM were incubated with PC-12 cells in a highly differentiated state for 30 min,and 30 m M glutamic acid was added,and incubated with cells for 24 h.CCK8 was added to detect the absorbance of cells at 450 nm wavelength with a microplate reader,and the cell viability was calculated.The method for evaluating the stability of peptides in vitro in plasma:each group of peptides was prepared at a concentration of 10 g/L,40 u L was added to 160μL rat plasma,and incubated at 37°C for 0,0.5,1,2,4,6,12,24 h.The samples were treated with 5%TFA/acetonitrile to extract the polypeptides,and the content was detected by RP-HPLC,and the enzymatic degradation rate of each group of polypeptides was calculated.Peptide drug safety evaluation method:The peptides with better activity were screened by the cell activity test and formulated into concentrations of 1,10,20,50,100,500,1000μM,and each group of peptides was incubated with PC-12 cells for 3 h,CCK8was added,and the absorbance of cells was detected by a microplate reader at a wavelength of 450 nm,and the cell viability was calculated.The peptides with better activity were screened out by the cell viability test and formulated into final concentrations of 1,10,20,50,100,500,1000μg/m L,incubated with 6%rat erythrocytes for 3 hours,and then used a microplate reader at 450 nm.The absorbance of red blood cells was detected at the wavelength,and the hemolysis rate was calculated.Animal activity evaluation method:C57 mice were used to construct the MCAO model,and the positive control drug Tat-Glu A2-3Y and the experimental group drugs were injected intraperitoneally.Each drug was compared at the dose of 2 mg/kg,4 mg/kg and 8 mg/kg.Brain infarct size and neurological score in mice.ResultsGlu A2-3Y binds to Brag2 protein through stronger van der Waals force,hydrogen bond and hydrophobic interaction,and has the highest affinity.In molecular docking,the binding free energy of Glu A2-3Y is also the highest of 143.692 kcal/mol.The molecular docking results are consistent with the surface plasmon resonance results.Through the cyclization strategy,the 8 cyclic peptides required in the study were synthesized,and after cyclization,it was found that CMT-3Y1,CMT-3Y2,MT-C3Y and CMT-C3Y transmembrane transmembrane efficiency in the MDCK-MDR1 cell transmembrane transport model and PAMPA.The Papp value of the amphiphilic CPP-modified cyclic peptide is 3-4 times higher than that of Tat-Glu A2-3Y.Among all cyclic peptides,CMT-C3Y has the best in vitro permeability,and its EC₅₀value was 29.82μM.In terms of stability,the plasma stability of all cyclic peptides was higher than that of the linear peptide Tat-Glu A2-3Y,of which CMT-C3Y has the best plasma stability.The cytotoxicity of CMT-3Y1,CMT-3Y2,MT-C3Y and CMT-C3Y was not significantly different from that of Tat-Glu A2-3Y(F_group=0.153,P>0.05)and was within the safe range.In the evaluation of hemolysis,except for the hemolysis rate of MT-C3Y at a concentration of 1000μg/m L（14.64±0.65）%>10%,mild hemolysis occurred,other cyclic peptides and Tat-Glu A2-The hemolysis rate of 3Y at different concentrations was less than 10%Compared with the positive control Tat-Glu A2,the neurological score and cerebral infarct size were significantly different when the drug dose was 4 mg/kg and 8mg/kg（I-J=0.80～8.66,P<0.05）.ConclusionWithin the nine amino acid sequence of Glu A2-3Y,the more amino acids the polypeptide contains,the higher the negative binding free energy,and the more stable the energy system between the polypeptide and the Brag2 protein.After the peptide is modified by cyclization,the increase in lipid solubility helps to penetrate the biological membrane;through the modification strategies such as conformational restriction,D-amino acids and unnatural amino acids,the hydrolysis rate of the peptide in plasma can be reduced and the stability of the peptide can be improved;The peptide modification strategy restricts the conformation of the peptide,reduces the entropy loss of the linear peptide,and improves the activity of the peptide.In addition,CMT-C3Y had the best effect on stroke treatment.