Modulation Bioinformatic Screening and Assessmen

Authors: Shibin Feng Lingyun Zou Qingshan Ni Xiang Zhang Qianwei Li Lei Zheng Laiping Xie Hongmin Li Dingde Huang

Publish Date: 2014/07/29

Volume: 70, Issue: 3, Pages: 1913-1921

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Abstract

Vascular endothelial growth factor VEGF and VEGF receptor VEGFR are important factors in tumor growth and metastasis Molecular probes or drugs designed to target VEGF/VEGFR interactions are crucial in tumor molecular imaging and targeted therapy Bioinformatic methods enable molecular design based on the structure of biomacromolecules and their interactions This study was aimed to identify tumortargeting smallmolecule peptides with high affinity for VEGFR using bioinformatics screening The VEGFR extracellular immunoglobulinlike modules Ig1–Ig3 were used as the target to systematically alter the primary peptide sequence of VEGF125–136 Molecular docking and surface functional group interaction methods were combined in an in silico screen for polypeptides which in theory would have higher affinities for VEGFR In vitro receptor competition binding assays were used to assess the affinity of the putative VEGFRbinding polypeptides Rhodamineconjugated peptides were used to label and visualize peptidebinding sites on A549 cells Using bioinformatic screening we identified 20 polypeptides with potentially higher affinity for VEGFR The polypeptides were capable of inhibiting the binding of 125IVEGF to VEGFR in a dosedependent manner The IC50 values of QKRKRKKSRKKH and RKRKRKKSRYIVLS 80 and 185 nmol/L respectively were significantly lower than that of VEGF125–136 464 nmol/L thus the affinity of these peptides for VEGFR was 6 and 25fold higher respectively than that of VEGF125–136 Rhodamine labeling of A549 cells revealed peptide binding mainly on the plasma membrane and in the cytoplasm Bioinformatic approaches hold promise for the development of molecular imaging probes Using this approach we designed two peptides that showed higher affinity toward VEGFR These polypeptides may be used as molecular probes or drugs targeting VEGFR which can be utilized in molecular imaging and targeted therapy of certain tumorsIn tumor diagnosis and therapy the discovery of diseaserelevant molecular targets and the construction of molecular probes or targeted drugs with high specificity for these targets are crucial 1 2 Certain molecules including vascular endothelial growth factor receptor VEGFR integrin αvβ3 3 4 somatostatin receptor 5 vasoactive intestinal peptide receptor 6 matrix metalloproteinases 7 Eselectin 8 and CD105 9 are expressed at higher levels in tumor cells and in newly formed vascular endothelial cells Thus these molecules are often used as targets in tumortargeted radionuclide imaging or therapy 10 Among these molecules vascular endothelial growth factor VEGF is the principal factor mediating tumor growth angiogenesis and metastasis Molecular probes or targeted drugs based on VEGF or VEGFR can be widely applied in tumortargeted molecular imaging or therapy 11 12 13 14 15 16 17VEGF125–136 is a 12amino acid peptide QKRKRKKSRYKS encoded by the VEGFA gene exon 6 VEGF125–136 binds specifically to VEGFR but does not activate its signal pathway 18 19 Our previous study demonstrated that VEGF125–136 exhibits good tumortargeting properties and could be used as a highly specific agent for tumor radionuclide imaging and therapy 20 However the previous in vitro study of VEGF125–136 suggested that it has a relatively weak capacity for tumor growth inhibition and exhibits a relatively short retention time in tumor tissues Molecular probes and tumortargeting drugs need to possess high binding affinity as well as a long halflife in the tumor tissues hence we aimed to modify VEGF125–136 to improve its affinity for VEGFRThe current study utilized a combination of bioinformatics and in vitro experimentation to improve VEGF125–136 Two polypeptides with higher VEGFRbinding affinity were selected from the peptide candidates which may be used as molecular probes or targeted drugsScheme of bioinformatic screen for peptides with elevated VEGFRbinding affinity a Peptide sequences are generated by manual design here the peptide “QKRKRKKSRKKH” is used as an example b the initial structure of each peptide is predicted using PEPFOLD software c a stable peptide structure is generated by molecular dynamics simulation using NAMD 5000 steps of energy minimization and 500ps dynamics simulation d a docking procedure to VEGFR is performed for each peptide using AutoDock Vina software and scores assigned according to the output energy here the docking site between VEGR and “QKRKRKKSRKKH” with minimal energy output is shown e subsequences cut from the peptide with a slidingwindow method used to predict binding specificity using the PEPSITE algorithm are scored using Eq 1 and the total binding score S is calculated using Eq 2 the residues of the subsequences from “QKRKRKKSRKKH” are shown as spheres binding to the Ig1–Ig3 domains of VEGRBased on the “lockandkey” principle of the interaction between ligands and receptors molecular docking methods simulate the interaction between a smallmolecule ligand and a macromolecular receptor The extracellular immunoglobulinlike modules Ig1–Ig3 of VEGFR reside in the ligandbinding domain and appear to form a rigid structure whereas the ligand was observed to have a flexible structure In our docking computation we assumed that altered polypeptides based on VEGF125–136 would interact with VEGFR in the same region The binding energy of VEGFR and the polypeptides was calculated using AutoDockVina software where lower binding energy indicates higher affinity interactionsThe initial approximate structure of VEGF125–136 was predicted by PEPFOLD http//bioservrpbsunivparisdiderotfr/PEPFOLD/ and NAMD version 27 http//wwwksuiucedu/Research/namd/ was employed in the molecular dynamics simulation to obtain a refined structure During molecular dynamics simulations all peptide atoms were surrounded with a cubic water box of simple pointcharge water molecules that extended 10 Å from the protein and periodic boundary conditions were applied in all directions The systems were neutralized with Na+ and Cl− counter ions replacing the water molecules and a 5000 step energy minimization was performed followed by a 500ps production molecular dynamics simulation with a timestep of 2 fs at constant pressure 1 atm and temperature 300 K

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