Integrins moderate diverse important functions in the human body and are promising targets in cancer therapy. Hence, the selective inhibition of specific integrins is of great medicinal interest. Here, we report the optimization of a grafted lasso peptide, yielding MccJ25(RGDF), which is a highly potent and selective αvβ3 integrin inhibitor. Furthermore, its NMR structure was elucidated and employed in a molecular dynamics approach, revealing information about the integrin binding mode and selectivity profile of MccJ25(RGDF).

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BackgroundIntegrins are heterodimeric receptors that play a critical role in cell-cell and cell-matrix adhesion processes. Among them, αVβ3 integrin, that recognizes the aminoacidic RGD triad, is reported to be involved in angiogenesis, tissue repair and tumor growth. We have recently synthesized a new and selective ligand of αVβ3 receptor, referred to as RGDechiHCit, that contains a cyclic RGD motif and two echistatin moieties.MethodsThe aim of this study is to evaluate in vitro and in vivo the effects of RGDechiHCit. Therefore, we assessed its properties in cellular (endothelial cells [EC], and vascular smooth muscle cells [VSMC]) and animal models (Wistar Kyoto rats and c57Bl/6 mice) of angiogenesis.ResultsIn EC, but not VSMC, RGDechiHCit inhibits intracellular mitogenic signaling and cell proliferation. Furthermore, RGDechiHCit blocks the ability of EC to form tubes on Matrigel. In vivo, wound healing is delayed in presence of RGDechiHCit. Similarly, Matrigel plugs demonstrate an antiangiogenic effect of RGDechiHCit.ConclusionsOur data indicate the importance of RGDechiHCit in the selective inhibition of endothelial αVβ3 integrin in vitro and in vivo. Such inhibition opens new fields of investigation on the mechanisms of angiogenesis, offering clinical implications for treatment of pathophysiological conditions such as cancer, proliferative retinopathy and inflammatory disease.

Neonatal changes in cardiac morphology were explained by late-trimester maternal body mass index; myocardial glucose overexposure seen in minipigs can justify early human findings. Longer term effects in minipigs consisted of myocardial insulin resistance, enzymatic alterations, and hyperdynamic systolic function.

Gold nanoparticles were obtained by reduction of a tetrachloroaurate aqueous solution in the presence of a RGD-(GC)(2) peptide as stabilizer. As comparison, the behavior of the (GC)(2) peptide has been studied. The (GC)(2) and RGD-(GC)(2) peptides were prepared ad hoc by Fmoc synthesis. The colloidal systems have been characterized by UV-visible, TGA, ATR-FTIR, mono and bidimensional NMR techniques, confocal and transmission (TEM) microscopy, ζ-potential, and light scattering measurements. The efficient cellular uptake of Au-RGD-(GC)(2) and Au-(GC)(2) stabilized gold nanoparticles into U87 cells (human glioblastoma cells) were investigated by confocal microscopy and compared with the behavior of (GC)(2) capped gold nanoparticles. A quantitative determination of the nanoparticles taken up has been carried out by measuring the pixel brightness of the images, a measure that highlighted the importance of the RGD termination of the peptide. Insight in the cellular uptake mechanism was investigated by TEM microscopy. Various important evidences indicated the selective uptake of RGD-(GC)(2) gold nanoparticles into the nucleus.

Malfunctions in transcriptional regulation are associated with a number of critical human diseases. As a result, there is considerable interest in designing artificial transcription activators (ATAs) that specifically control genes linked to human diseases. Like native transcriptional activator proteins, an ATA must minimally contain a DNA-binding domain (DBD) and a transactivation domain (TAD) and, although there are several reliable methods for designing artificial DBDs, designing artificial TADs has proven difficult. In this manuscript, we present a structure-based strategy for designing short peptides containing natural amino acids that function as artificial TADs. Using a segment of the TAD of p53 as the scaffolding, modifications are introduced to increase the helical propensity of the peptides. The most active artificial TAD, termed E-Cap-(LL), is a 13-mer peptide that contains four key residues from p53, an N-capping motif and a dileucine hydrophobic bridge. In vitro analysis demonstrates that E-Cap-(LL) interacts with several known p53 target proteins, while in vivo studies in a yeast model system show that it is a 20-fold more potent transcriptional activator than the native p53-13 peptide. These results demonstrate that structure-based design represents a promising approach for developing artificial TADs that can be combined with artificial DBDs to create potent and specific ATAs.

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