Dataset Information

Investigating the Interaction of Cyclic RGD Peptidomimetics with ?V?? Integrin by Biochemical and Molecular Docking Studies.

ABSTRACT: The interaction of a small library of cyclic RGD (Arg-Gly-Asp) peptidomimetics with ?V?? integrin has been investigated by means of competitive solid phase binding assays to the isolated receptor and docking calculations in the crystal structure of the ?V?? binding site. To this aim, a rigid receptor-flexible ligand docking protocol has been set up and then applied to predict the binding mode of the cyclic RGD peptidomimetics to ?V?? integrin. Although the RGD interaction with ?V?? recapitulates the RGD binding mode observed in ?V??, differences between the integrin binding pockets can strongly affect the ligand binding ability. In general, the peptidomimetics exhibited IC50 values for integrin ?V?? (i.e., the concentration of compound required for 50% inhibition of biotinylated fibronectin binding to isolated ?V?? integrin) in the nanomolar range (77-345 nM), about 10-100 times higher than those for the related ?V?? receptor, with a single notable ligand displaying a low nanomolar IC50 value (2.3 nM). Insights from the properties of the binding pocket combined with the analysis of the docking poses provided a rationale for ligand recognition and selectivity.

SUBMITTER: Civera M

PROVIDER: S-EPMC5664067 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Investigating the Interaction of Cyclic RGD Peptidomimetics with αVβ₆ Integrin by Biochemical and Molecular Docking Studies.

Civera Monica M Arosio Daniela D Bonato Francesca F Manzoni Leonardo L Pignataro Luca L Zanella Simone S Gennari Cesare C Piarulli Umberto U Belvisi Laura L

Cancers 20170921 10

The interaction of a small library of cyclic RGD (Arg-Gly-Asp) peptidomimetics with αVβ₆ integrin has been investigated by means of competitive solid phase binding assays to the isolated receptor and docking calculations in the crystal structure of the αVβ₆ binding site. To this aim, a rigid receptor-flexible ligand docking protocol has been set up and then applied to predict the binding mode of the cyclic RGD peptidomimetics to αVβ₆ integrin. Although the RGD in ...[more]

PMID: 28934103

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Project description:Cadherins are homophilic cell-cell adhesion molecules whose aberrant expression has often been shown to correlate with different stages of tumor progression. In this work, we investigate the interaction of two peptidomimetic ligands with the extracellular portion of human E-cadherin using a combination of NMR and computational techniques. Both ligands have been previously developed as mimics of the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 of the cadherin adhesion arm, and have been shown to inhibit E-cadherin-mediated adhesion in epithelial ovarian cancer cells with millimolar potency. To sample a set of possible interactions of these ligands with the E-cadherin extracellular portion, STD-NMR experiments in the presence of two slightly different constructs, the wild type E-cadherin-EC1-EC2 fragment and the truncated E-cadherin-(Val3)-EC1-EC2 fragment, were carried out at three temperatures. Depending on the protein construct, a different binding epitope of the ligand and also a different temperature effect on STD signals were observed, both suggesting an involvement of the Asp1-Trp2 protein sequence among all the possible binding events. To interpret the experimental results at the atomic level and to probe the role of the cadherin adhesion arm in the dynamic interaction with the peptidomimetic ligand, a computational protocol based on docking calculations and molecular dynamics simulations was applied. In agreement with NMR data, the simulations at different temperatures unveil high variability/dynamism in ligand-cadherin binding, thus explaining the differences in ligand binding epitopes. In particular, the modulation of the signals seems to be dependent on the protein flexibility, especially at the level of the adhesive arm, which appears to participate in the interaction with the ligand. Overall, these results will help the design of novel cadherin inhibitors that might prevent the swap dimer formation by targeting both the Trp2 binding pocket and the adhesive arm residues.

Project description:This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1-0.3 g. Tumors were harvested for integrin αvβ3/αvβ5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvβ3/αvβ5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvβ3/αvβ5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin β3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD2 (an integrin αvβ3/αvβ5-targeted radiotracer) and the relative integrin αvβ3/αvβ5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvβ3/αvβ5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvβ3/αvβ5 expression levels in tumor tissues.

Dataset Information

Investigating the Interaction of Cyclic RGD Peptidomimetics with ?V?? Integrin by Biochemical and Molecular Docking Studies.

Publications

Investigating the Interaction of Cyclic RGD Peptidomimetics with α<sub>V</sub>β₆ Integrin by Biochemical and Molecular Docking Studies.

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