The Design And Evaluation Of An Antiangiogenic Peptibody

CADD(computer aided drug design)is an important technical means for design,development and optimization of new drugs through computer simulation,calculation and prediction of the interaction between drug molecules and biological receptors.Peptides,short-chain compounds formed by linking amino acids in the form of peptide bonds,play an important role in the regulation of the body.Peptides,with the advantages of high specificity,high affinity and easy preparation,have been widely used in clinical treatment and drug development in recent years.In order to overcome the shortcomings of peptides,such as poor stability and short half-life,researchers have used a variety of methods to modify the polypeptide.One of the most effective strategies is to combine the polypeptide with Fc(fragment crystallizable)in Ig(immunoglobulin)to construct a fusion protein called a peptibody.Peptibodies can prolong the serum half-life of peptide molecules effectively as well as improve the stability and solubility.The preparation and purification process of peptibodies are comparatively simple,and the cost of drugs can be effectively reduced.The angiogenesis related disease is an important type of ophthalmic diseases caused by vascular malformation in the retina and choroid.Age-related macular degeneration(AMD)is the main cause for the vision decline and even blindness for the elderly over 50 years old.In neovascular AMD,vascular endothelial growth factor(VEGF)is an important factor.VEGF can bind to vascular endothelial growth factor receptor(VEGFR)on cell surface to induce neovascularization,thereby causing related diseases.Presently,the anti-VEGF/VEGFR pathway drugs have a good effect on the treatment of neovascular diseases.However some anti-VEGF drugs still have such problems as low productivity,high cost,poor therapeutic effects and high risks of injection therapy.Therefore,it is quite urgent to develop a new drug which is low-cost,high-performance,non-invasive or minimally invasive for the treatment of fundus neovascular diseases.In response to the development of new anti-VEGF drugs,we screened peptides with anti-angiogenic activity from phage display peptide library by biopanning.We also designed a novel peptibody drug with antiangiogenic function by computer-aided drug design,meanwhile their interactions with VEGFR and immunogenicity were evaluated.The main research contents of the project are as follows:Firstly,a peptide with antiangiogenic activity was obtained by biopanning.The VEGFR-Fc fusion was constructed and used to screen the phage display libraty Ph.D-12.A panel of VEGFR binding peptides were acquired by sequencing,and bioinformatics tools were used to take out the target-unrelated peptides(TUPs).At last,peptide HRH(HRHTKQRHTALH)with VEGFR-Fc fusion binding ability was verified.Secondly,the antiangiogenic ability of HRH peptide was confirmed by biological experiments.HUVEC(human umbilical vein endothelial cells)proliferation assay,chicken chorioallantoic membrane assay and corneal injury in rats were applied in the study.The results of biological experiments indicated that HRH,which competitively prevented the binding of VEGF and VEGFR,could inhibt angiogenesis.Thirdly,a novel antiangiogenic peptibody PbHRH with HRH peptide was designed.The new constructed peptibody was named PbHRH on the base of the template of the first FDA approved peptibody Romiplostim.The AF12505 peptide in the functional domains of Romiplostim was replaced by HRH peptide.Fourthly,PbHRH and its interaction with VEGFR were evaluated.Molecular docking and molecular modeling methods were used to predicate and analyze the structure of PbHRH and the interaction between PbHRH and VEGFR.The results showed that the HRH-Fc fusion would not affect the antiangiogenic activity of HRH peptide but improve its pharmacokinetic properties.Fifthly,the immunogenicity of PbHRH was evaluated and an effective strategy for the evaluation of the immunogenicity of peptibodies was proposed.The prediction of T cell epitope was adopted in this study using Romiplostim as the negative control.IEDB(the immune epitope database)as well as its online prediction and analysis tools were used to predict and analyze the MHC-I and MHC-II binding abilities and the MHC-I,CD4 T cell immunogenicity of PbHRH.The results showed that PbHRH had a similar low immunogenicity with Romiplostim theoretically.Based on this work,an effective procedure for the evaluation of the immunogenicity in the design of peptibody drugs was proposed,and it was verified by the immunogenicity prediction of multiple peptibodies.In summary,a peptide HRH with antiangiogenic activity was extracted by phage display in this study.A novel anti-VEGF peptibody PbHRH was designed and constructed with this functional peptide HRH.The structure of PbHRH and its interaction with VEGFR were predicted and analyzed by molecular docking and molecular dynamics,and the immunogenicity of this peptibody was evaluated.In conclusion,the PbHRH peptibody designed in this study has the potential to treat the angiogenesis related diseases.Providing a basis for the further research and development of this peptibody,our work has important scientific significance and cheerful application prospects.