Evaluation of the anti-angiogenic properties of the new selective αVβ3 integrin antagonist RGDechiHCit

Integrins 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.The 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.In 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.Our 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

High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations

The crucial role of integrin in pathological processes such as tumor progression and metastasis formation has inspired intense efforts to design novel pharmaceutical agents modulating integrin functions in order to provide new tools for potential therapies. In the past decade, we have investigated the biological proprieties of the chimeric peptide RGDechi, containing a cyclic RGD motif linked to an echistatin C-terminal fragment, able to specifically recognize αvβ3 without cross reacting with αvβ5 and αIIbβ3 integrin. Additionally, we have demonstrated using two RGDechi-derived peptides, called RGDechi1-14 and ψRGDechi, that chemical modifications introduced in the C-terminal part of the peptide alter or abolish the binding to the αvβ3 integrin. Here, to shed light on the structural and dynamical determinants involved in the integrin recognition mechanism, we investigate the effects of the chemical modifications by exploring the conformational space sampled by RGDechi1-14 and ψRGDechi using an integrated natural-abundance NMR/MD approach. Our data demonstrate that the flexibility of the RGD-containing cycle is driven by the echistatin C-terminal region of the RGDechi peptide through a coupling mechanism between the N- and C-terminal regions

Sviluppo di nuove molecole che modulano l'attività di sistemi coinvolti nell'angiogenesi

[ITALIANO] L’angiogenesi è un processo essenziale per la crescita dei tumori solidi; è stato dimostrato che la soppressione anche di una sola delle fasi che la costituiscono può inibire la formazione di nuovi vasi sanguigni, influenzando la crescita tumorale e la formazione di metastasi. Scopo di questo lavoro è stato lo sviluppo di nuove molecole in grado di modulare l’attività di sistemi coinvolti nel processo angiogenico. A tal fine sono stati scelti due sistemi tra quelli più noti e studiati nell’ambito della regolazione del processo angiogenico: il recettore per le proteine della matrice integrina αVβ3 e il fattore di crescita vascolare endoteliale VEGF. Per quanto riguarda l’integrina αVβ3, è stata progettata e sintetizzata una nuova molecola bifunzionale di natura peptidica, denominata RGDechi, con attività inibitoria nei confronti dell’integrina in esame per la quale si è dimostrata non solo affine ma anche selettiva. Questa molecola potrebbe fungere da lead compound per lo sviluppo di nuovi agenti anti-tumorali e/o di una nuova classe di traccianti non-invasivi da impiegare per il targeting dei recettori αVβ3. Per quanto riguarda il VEGF, sono stati sviluppati antagonisti sintetici di tale proteina mediante impiego di piccole sequenze peptidiche (15 amminoacidi), opportunamente disegnate, che hanno un potenziale impiego per il targeting di cellule tumorali ed eventualmente per la terapia. Tali peptidi potrebbero trovare impiego, opportunamente funzionalizzati, come sonde per il riconoscimento selettivo dei recettori del VEGF overespressi nei fenomeni di angiogenesi patologica. / [ENGLISH] Angiogenesis is an essential process for the growth of solid tumors; it was shown that the suppression of even only one phase of this process may inhibit new vessels formation, thus affecting tumor growth and metastasis generation. Aim of this work was to develop small molecules able to modulate activity of molecular systems involved in the angiogenic process. Two systems that are the most important and showed in the field of regulation of angiogenic process have been select: the receptor for matrix protein integrin αVβ3 and the vascular endothelial growth factor VEGF. About integrin αVβ3, a new chimeric peptide was designed, named RGDechi, that proved a novel and selective inhibitor of this integrin. This molecule may represent the lead compound for the development of novel anticancer drugs and/or new class of diagnostic non-invasive tracers as suitable tools for αVβ3-targeted therapy and imaging. About VEGF, peptidic antagonists of this natural protein were developed using small peptide sequences (15 amino acids), opportunely designed, that have a potential application in tumor cells targeting and therapy. These peptides, opportunely functionalized, may be used as biospecific probes for the selective recognition of VEGF receptors overexpressed in pathological angiogenesis

An Overview of Peptide-Based Molecules as Potential Drug Candidates for Multiple Sclerosis

Multiple sclerosis (MS) belongs to demyelinating diseases, which are progressive and highly debilitating pathologies that imply a high burden both on individual patients and on society. Currently, several treatment strategies differ in the route of administration, adverse events, and possible risks. Side effects associated with multiple sclerosis medications range from mild symptoms, such as flu-like or irritation at the injection site, to serious ones, such as progressive multifocal leukoencephalopathy and other life-threatening events. Moreover, the agents so far available have proved incapable of fully preventing disease progression, mostly during the phases that consist of continuous, accumulating disability. Thus, new treatment strategies, able to halt or even reverse disease progression and specific for targeting solely the pathways that contribute to the disease pathogenesis, are highly desirable. Here, we provide an overview of the recent literature about peptide-based systems tested on experimental autoimmune encephalitis (EAE) models. Since peptides are considered a unique therapeutic niche and important elements in the pharmaceutical landscape, they could open up new therapeutic opportunities for the treatment of MS

Selective Targeting of αvβ5 Integrin in HepG2 Cell Line by RGDechi15D Peptide

Recently, the research community has become increasingly concerned with the receptor αvβ5, a member of the well-known integrin family. Different ongoing studies have evidenced that αvβ5 integrin regulates not only physiological processes but also a wide array of pathological events, suggesting the receptor as a valuable biomarker to specifically target for therapeutic/diagnostic purposes. Remarkably, in some tumors the involvement of the receptor in cell proliferation, tumor dissemination and angiogenesis is well-documented. In this scenario, the availability of a selective αvβ5 antagonist without 'off-target' protein effects may improve survival rate in patients with highly aggressive tumors, such as hepatocellular carcinoma. We recently reported a cyclic peptide, RGDechi15D, obtained by structure-activity studies. To our knowledge it represents the first peptide-based molecule reported in the literature able to specifically bind αvβ5 integrin and not cross react with αvβ3. Here we demonstrated the ability of the peptide to diminish both adhesion and invasion of HepG2 cells, an in vitro model system for hepatocellular carcinoma, to reduce the cell proliferation through an apoptotic process, and to interfere with the PI3K pathway. The peptide, also decreases the formation of new vessels in endothelial cells. Taken together these results indicate that the peptide can be considered a promising molecule with properties suited to be assessed in the future for its validation as a selective therapeutic/diagnostic weapon in hepatocarcinoma

Straightforward Entry to <i>S</i>‑Glycosylated Fmoc-Amino Acids and Their Application to Solid Phase Synthesis of Glycopeptides and Glycopeptidomimetics

Streamlined access to <i>S</i>-glycosylated Fmoc-amino acids was developed. The process provides diverse glycosylated modified amino acids in high yield and stereoselectivity taking advantage of the <i>in situ</i> generation of a glycosylthiolate obtained from carbohydrate acetates in a few steps. Mild basic conditions make the conjugation reaction compatible with Fmoc-iodo-amino acids. To validate the strategy the glycosylated building blocks were used for SPPS and the unprecedented incorporation of a long thio-oligosaccharide to the peptide chain was demonstrated

A selective αvβ5 integrin antagonist hidden into the anophelin family protein cE5 from the malaria vector Anopheles gambiae

A RGD motif was identified in the N-terminal region of cE5, a potent salivary thrombin inhibitor from the African malaria vector Anopheles gambiae. A peptide (APQ30) encompassing the first 30 amino acids residues of the protein and including the RGD tripeptide was tested in cell adhesion assays and found to inhibit avb3 and avb5 mediated adhesion. A shorter peptide (APQ16), strongly conserved among members of the A. gambiae species complex and including only the first 16 residues, retained adhesion inhibitory properties, however with enhanced specificity toward avb5. In addition, migration and invasion assays showed its capacity to inhibit the invasiveness of the malignant cell lines HepG2 and MDA-MB231. Altogether our data point to APQ16 as a new promising candidate as theranostic agent

Therapeutic Potential of a Novel α_vβ₃ Antagonist to Hamper the Aggressiveness of Mesenchymal Triple Negative Breast Cancer Sub-Type

The mesenchymal sub-type of triple negative breast cancer (MES-TNBC) has a highly aggressive behavior and worse prognosis, due to its invasive and stem-like features, that correlate with metastatic dissemination and resistance to therapies. Furthermore, MES-TNBC is characterized by the expression of molecular markers related to the epithelial-to-mesenchymal transition (EMT) program and cancer stem cells (CSCs). The altered expression of &#945;v&#946;3 integrin has been well established as a driver of cancer progression, stemness, and metastasis. Here, we showed that the high levels of &#945;v&#946;3 are associated with MES-TNBC and therefore exploited the possibility to target this integrin to reduce the aggressiveness of this carcinoma. To this aim, MES-TNBC cells were treated with a novel peptide, named &#968;RGDechi, that we recently developed and characterized for its ability to selectively bind and inhibit &#945;v&#946;3 integrin. Notably, &#968;RGDechi was able to hamper adhesion, migration, and invasion of MES-TNBC cells, as well as the capability of these cells to form vascular-like structures and mammospheres. In addition, this peptide reversed EMT program inhibits mesenchymal markers. These findings show that targeting &#945;v&#946;3 integrin by &#968;RGDechi, it is possible to inhibit some of the malignant properties of MES-TNBC phenotype

Evaluation of the anti-angiogenic properties of the new selective α_Vβ₃integrin antagonist RGDechiHCit

Abstract Background Integrins 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. Methods The 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. Results In 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. Conclusions Our 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.</p