Bioinformatics Transformation, Screening, Identification Of Tumor Inhibitory Peptide Targeting VEGF Receptor

Backgrounds and Objectives:The growth, invasiveness, metastasis of tumors depends on tumor angiogenesis.Anti-angiogenesis has good application prospect. Vascular endothelial growth factor（VEGF） and vascular endothelial growth factor receptor （VEGFR） has become a crucialtarget of anticancer therapy because of their functions of anti-angiogenesis. Vascularendothelial growth factor as the major angiogenesis promoting factor play an importantrole in angiogenesis through combination with VEGF receptor （VEGFR） expressing on thecell membrane. Among all the VEGFR, VEGFR-1（Flt-l） and VEGFR-2（KDR） have higherexpression in tumor cells and new vascular endothelial cells, but have extremely lowexpression or even don’t express in other cells. So VEGFR-1（Flt-l） and VEGFR-2（KDR）are in a core position in tumor angiogenesis, and the growth, invasiveness, metastasis oftumors is closely related to the function of the VEGFR. Therefore, block VEGFR mayinhibit angiogenesis of VEGF to tumor. In conclusion, VEGF/VEGFR is the hotspot targetin current tumor anti-angiogenesis research.VEGF125-136（QKRKRKKSRYKS），a12amino acid peptide encoded by exon6ofVEGF-A, can specifically combine with VEGFR but not activate VEGFR signal pathway.VEGF125-136shut down VEGFR signal pathway, and inhibits angiogenesis of VEGFcompetitively. So VEGF125-136is expected to be the candidate molecule as tumor targetingimaging and radionuclide therapy.Our previous study, supported by Natural Science Foundation ofChina（NSFC,30400114）, indicated that:（1）¹⁸⁸Re-VEGF125-136obviously gathered in tumortissue in tumor bearing nude mouse and inhibited tumor growth;（2） The gatheringquantity of188Re-VEGF125-136significantly increase in the tumor site after beingtransinfected by shortened KDR genes.¹⁸⁸Re-VEGF125-136has a good application prospect in tumor targeting imaging and treatment, but its retention time in the tumor tissue is only3hours.In order to enhance the VEGF125-136tumor imaging quality and antitumor function,improve accumulation quantity （tumor/non-tumor ratio, T/NT） and retention time in thetumor tissue is most important.Improve the affinity between tumor marker and its receptors are the effective way tosolve the problem of T/NT and retention time in tumor. The reversibility is the most basicfeatures between ligand and receptor, and affinity is the important parameters to evaluatethe ability and stability of ligand-receptor. When the affinity is high, there will be muchmore ligand affinying to receptor, and the combination of ligand and receptor is much morestable and not easily to be disaggregated.This study is to investigate the VEGF125-136reforming methods, screen the targetingpeptide. Then seek the optimum radionuclide labeling methods, study the distribution innormal healthy animal and tumor imaging in tumor bearing nude mouse. All the work is tobuild a good experimental basis laid for further tumor targeting imaging and therapy.Methods：1. Reform of VEGF_125-136with bioinformatics: In order to improve the affinity ofreformed VEGF_125-136to VEGFR, we used bioinformatics such as sequence league match,model setup, energy optimization and rational evaluation, molecular docking and so on.2. Test the affinity of reformed peptides to VEGFR receptor competition bindingexperiment In vitro.3. Inhibiting ability identification of reformed peptides to tumor cell proliferationthrough3H-thymidine incorporation experiment.4. Radionuclide labeling of target peptides and identification: Introduce doublefunction chelating agent HYNIC to couple with target peptides, discusses the best labelingconditions. Paper chromatography and HPLC were used to identify specific activity,radiochemical purity and stability in vitro. L-Cysteine displacement experiment were usedto identify chelating ability between99Tcmand target peptides.3H-thymidine incorporationwere used to identify the inhibiting ability of HYNIC-coupled target peptides to tumor cellproliferation. In vitro receptor competition binding experiment were used to identifyaffinity of labeled target peptide to VEGFR. 5. Pharmacokinetic Study of labeled target peptides:8Japanese rabbits were used,every rabbit were injected with37MBq/200μl labeled targets peptide through left ear vein,Took blood from the right ear respectively in1.5,3,5,10,30,60,120,240,480min afterthe injection, measured blood margin radioactive concentration. Best compartment modeland pharmacokinetic parameters were analyzed by pharmacokinetic analysis software （DAS3.0）.6. Biodistribution study of labeled target peptides in normal Kunming mice:15Kunming mice were used in this study. Every Kunming mouse was injected with7.4MBq/200μl labeled targets peptide. Collected blood in the30min,1h,2h respectively,then killed the mice through cervical dislocated method, collected the heart, liver, spleen,lung, kidney, brain, stomach, intestine, thyroid, bone, muscle. Draw the body radioactivebiodistribution figure.7. Imaging study of Japan’s white rabbit: The white rabbit was injected with18.5MBq/200μl labeled targets peptide. Image Acquisition with one frame per1min in thefirst60minutes, then image Acquisition at90,120,180,240min with one frame per5min,observe dynamic radioactive distribution of the organs.8. Planar imaging and competitive imaging of labeled target peptides in tumor bearingnude mice: As the tumor was1.5cm3in volume, injected11.1MBq/150μl labeled targetspeptide into tumor bearing nude mice through caudal vein. Anterior SPECT imaging wasobserved at0.5h,1h,1.5h,2h,3h,4h after the injection. In the study of competitiveimaging,1mg peptide was injected into through caudal vein tumor bearing micerespectively,1h later,11.1MBq/150μl labeled peptide was injected. Anterior SPECTimaging was observed at0.5h,1h after the injection, observe the binding specificity invivo between peptide and tumor VEGFR.Results：1. We got two target peptide which have higher affinity to VEGFR than VEGF125-136and can significantly inhibited tumor cell proliferation through bioinformatics reformingand the experiments in vitro, peptide sequence is QKRKRKKSRKKH,RKRKRKKSRYIVLS.2. Radionuclide labeling and identification of target peptide: The optimized labelingconditions as follows:2ml cells freeze-stored tube,20μg target peptide, HYNIC-QKRKRKKSRKKH50mg Tricine, HYNIC-RKRKRKKSRYIVLS80mg Tricine,5mg TPPTS,100μL0.25mmol/L succinic acid buffer solution （pH=5）,100μL Na⁹⁹Tc^mO4185MBq, total volume is200μL. The reaction was performed under nitrogen atmosphereand100°C in water bath for20min.The labeling rate of HYNIC-RKRKRKKSRYIVLS and HYNIC-QKRKRKKSRKKHis94.13%±1.77%,93.79%±3.53%separately., The radiochemical purity is still more than90%after24h at room temperature. The inhibiting ability of HYNIC-QKRKRKKSRKKHand HYNIC-RKRKRKKSRYIVLS is same as QKRKRKKSRKKH andRKRKRKKSRYIVLS separately. In vitro competitive binding assay also demonstrated thatthe labeled target peptide kept a good affinity to VEGFR.3. Tracer kinetics analysis in vivo of⁹⁹Tcmlabeled peptide in healthy rabbits:99Tcm-HYNIC-QKRKRKKSRKKH pharmacokinetics in rabbits was in line with a threecompartment model.⁹⁹Tc^m-HYNIC-RKRKRKKSRYIVLS pharmacokinetics in rabbitswas in line with a two compartment model.4. Tissue distribution and imaging study of⁹⁹Tcmlabeled peptide in healthy animals:Two target peptide is hydrophilic, excrete mainly through the urinary system, is cleard fastin blood flow, have little biodistribution in heart, liver, spleen, lung, brain, stomach,intestines, thyroid, bone, muscle tissue.5. Tissue distribution and imaging study of⁹⁹Tc^mlabeled peptide in tumor bearing nudemice: At0.5h after caudal vein intravenous injection, labeled target peptide have asignificant radioactivity accumulation was viewed at the tumor site in the right anteriorlimb, and the image was clear. Double kidneys and bladder have radioactivity accumulation,whereas, there is little radioactivity accumulation in heart, liver, spleen, lung, brain,stomach, intestines, thyroid, bone, muscle tissue. The radioactivity accumulation in tumorcompetitive binding imaging is significant less than tumor bearing nude mice imaging.Conclusions：The research had successfully reformed VEGF^125-136through bioinformaticstransformation and experiment in Vitro. The screened target peptide is successfully labeledwith⁹⁹Tc^m. The labeled target peptide marker kept a good stability and affinity to VEGFR.⁹⁹Tc^m-HYNIC-QKRKRKKSRKKH,⁹⁹Tc^m-HYNIC-RKRKRKKSRYIVLS had a highradioactivity accumulation in tumor tissue in tumor bearing nude mouse. The study had laid a good experimental basis for further research in VEGFR targeting tumor imaging andtherapy.