Determination of the binding epitope of RGD-peptidomimetics to \u3b1v\u3b23 and \u3b1IIb\u3b23 integrin-rich intact cells by NMR and computational studies

NMR experiments (transferred NOE and Saturation Transfer Difference) were used to shed light on the binding epitope of RGD peptidomimetics 1-3 with integrins \u3b1v\u3b23 and \u3b1IIb\u3b23, expressed on the membrane of ECV304 bladder cancer cells and human platelets, respectively. The NMR results were supported by docking calculations in the active sites of \u3b1v\u3b23 and \u3b1IIb\u3b23 integrin receptors and were compared to the results of competitive \u3b1v\u3b23 receptor binding assays and competitive ECV304 cell adhesion experiments. While cis RGD ligand 1 interacts mainly with the \u3b1 integrin subunit through its basic guanidine group, trans RGD ligands 2 and 3 are able to interact with both the \u3b1 and \u3b2 integrin subunits via an electrostatic clam

Insights into the Binding of Cyclic RGD Peptidomimetics to \u3b15\u3b21 Integrin by using Live-Cell NMR And Computational Studies

The interaction of a small library of cyclic DKP\u2013RGD peptidomimetics with \u3b15\u3b21 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 \u3b15\u3b21 is highly expressed. The NMR data were compared with the docking calculations of the RGD ligands in the crystal structure of the \u3b15\u3b21 binding site, and were integrated with competitive binding assays to the purified \u3b15\u3b21 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 \u3b15\u3b21 ligand of the series, displaying a nanomolar IC50 value

Cyclic isoDGR Peptidomimetics as Low-Nanomolar alphaVbeta3 Integrin Ligands

In the present communication we report a cyclic isoDGR peptidomimetic as lownanomolar αvβ3 integrin ligand. In particular, the new cyclo[(3S,6R)DKP-isoDGR] ligand is ten times more potent in binding integrin αvβ3 than c[GisoDGRphg], developed by Kessler and co-workers, and only two-three times less potent than the powerful RGD ligands c[(3S,6R)DKP-RGD] and c[RGDfV]. Spectroscopic and computational studies show that the isoDGR sequence of cyclo[(3S,6R)DKP-isoDGR] can fit into the RGD-binding pocket of αvβ3 integrin, establishing the same electrostatic clamp as well as additional key interactions

How well does molecular simulation reproduce environment-specific conformations of the intrinsically disordered peptides PLP, TP2 and ONEG?

Understanding the conformational ensembles of intrinsically disordered proteins and peptides (IDPs) in their various biological environments is essential for understanding their mechanisms and functional roles in the proteome, leading to a greater knowledge of, and potential treatments for, a broad range of diseases. To determine whether molecular simulation is able to generate accurate conformational ensembles of IDPs, we explore the structural landscape of the PLP peptide (an intrinsically disordered region of the proteolipid membrane protein) in aqueous and membrane-mimicking solvents, using replica exchange with solute scaling (REST2), and examine the ability of four force fields (ff14SB, ff14IDPSFF, CHARMM36 and CHARMM36m) to reproduce literature circular dichroism (CD) data. Results from variable temperature (VT) 1H and Rotating frame Overhauser Effect SpectroscopY (ROESY) nuclear magnetic resonance (NMR) experiments are also presented and are consistent with the structural observations obtained from the simulations and CD. We also apply the optimum simulation protocol to TP2 and ONEG (a cell-penetrating peptide (CPP) and a negative control peptide, respectively) to gain insight into the structural differences that may account for the observed difference in their membrane-penetrating abilities. Of the tested force fields, we find that CHARMM36 and CHARMM36m are best suited to the study of IDPs, and accurately predict a disordered to helical conformational transition of the PLP peptide accompanying the change from aqueous to membrane-mimicking solvents. We also identify an α-helical structure of TP2 in the membrane-mimicking solvents and provide a discussion of the mechanistic implications of this observation with reference to the previous literature on the peptide. From these results, we recommend the use of CHARMM36m with the REST2 protocol for the study of environment-specific IDP conformations. We believe that the simulation protocol will allow the study of a broad range of IDPs that undergo conformational transitions in different biological environments.</p