Dataset Information

Insights into the Binding of Cyclic RGD Peptidomimetics to ?5?1 Integrin by using Live-Cell NMR And Computational Studies.

ABSTRACT: The interaction of a small library of cyclic DKP-RGD peptidomimetics with ?5?1 integrin has been investigated by means of an integrated experimental and computational approach. Bioaffinity NMR techniques, including saturation transfer difference (STD) and transferred NOESY, were applied to the ligands in a suspension of intact MDA-MB-231 breast cancer cells, in which integrin ?5?1 is highly expressed. The NMR data were compared with the docking calculations of the RGD ligands in the crystal structure of the ?5?1 binding site, and were integrated with competitive binding assays to the purified ?5?1 integrin. Ligand binding epitopes involve protons of both the RGD moiety and the DKP scaffold, although the stereochemistry and the functionalization of the DKP scaffold as well as the macrocycle conformation determine a great variability in the interaction. The ligand showing the highest number of STD signals is also the most potent ?5?1 ligand of the series, displaying a nanomolar IC50 value.

SUBMITTER: Guzzetti I

PROVIDER: S-EPMC5288746 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Insights into the Binding of Cyclic RGD Peptidomimetics to α5β1 Integrin by using Live-Cell NMR And Computational Studies.

Guzzetti Ileana I Civera Monica M Vasile Francesca F Arosio Daniela D Tringali Cristina C Piarulli Umberto U Gennari Cesare C Pignataro Luca L Belvisi Laura L Potenza Donatella D

ChemistryOpen 20161209 1

The interaction of a small library of cyclic DKP-RGD peptidomimetics with α5β1 integrin has been investigated by means of an integrated experimental and computational approach. Bioaffinity NMR techniques, including saturation transfer difference (STD) and transferred NOESY, were applied to the ligands in a suspension of intact MDA-MB-231 breast cancer cells, in which integrin α5β1 is highly expressed. The NMR data were compared with the docking calcula ...[more]

PMID: 28168158

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Project description:Integrin alpha(v)beta(3) plays a significant role in tumor angiogenesis and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (RGD) tripeptide sequence. These include vitronectin, fibronectin, fibrinogen, lamin, collagen, Von Willibrand's factor, osteoponin, and adenovirus particles. Integrin alpha(v)beta(3) is expressed at low levels on epithelial cells and mature endothelial cells, but it is overexpressed on the activated endothelial cells of tumor neovasculature and some tumor cells. The restricted expression of integrin alpha(v)beta(3) during tumor growth, invasion, and metastasis presents an interesting molecular target for both early detection and treatment of rapidly growing solid tumors. Over the past decade, many radiolabeled linear and cyclic RGD peptide antagonists have been evaluated as integrin alpha(v)beta(3)-targeted radiotracers. Significant progress has been made on their use for imaging integrin alpha(v)beta(3)-positive tumors by SPECT or PET. Among the radiotracers evaluated in preclinical tumor-bearing models, [18F]Galacto-RGD (2-[18F]fluoropropanamide c(RGDfK(SAA); SAA = 7-amino-L-glyero-L-galacto-2,6-anhydro-7-deoxyheptanamide) and [18F]-AH111585 are currently under clinical investigation for visualization of integrin alpha(v)beta(3) expression in cancer patients. However, their low tumor uptake, high cost, and lack of preparative modules for routine radiosynthesis will limit their continued clinical application. Thus, there is a continuing need for more efficient integrin alpha(v)beta(3)-targeted radiotracers that are readily prepared from a kit formulation without further postlabeling purification. This article will focus on different approaches to maximize the targeting capability of cyclic RGD peptides and to improve the radiotracer excretion kinetics from noncancerous organs. Improvement of tumor uptake and tumor-to-background ratios is important for early detection of integrin alpha(v)beta(3)-positive tumors and/or noninvasive monitoring of therapeutic efficacy of antiangiogenic therapy.

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.

Dataset Information

Insights into the Binding of Cyclic RGD Peptidomimetics to ?5?1 Integrin by using Live-Cell NMR And Computational Studies.

Publications

Insights into the Binding of Cyclic RGD Peptidomimetics to α<sub>5</sub>β<sub>1</sub> Integrin by using Live-Cell NMR And Computational Studies.

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