Samantha Deianira Dattoli

Development of isoxazoline-containing peptidomimetics as dual αvβ3 and α5β1 integrin ligands.

Isoxazoline‐containing peptidomimetics, designed to be effective αvβ3 and α5β1 integrin ligands, were synthesized through an original procedure involving N,O‐bis(trimethylsilyl)hydroxyamine conjugate addition to alkylidene acetoacetates, followed by intramolecular hemiketalization. To mimic the RGD recognition sequence, basic and acidic terminal appendages were introduced, and the final products were tested in cell adhesion inhibition assays. All the synthesized compounds proved to be excellent ligands for both integrin receptors, and a strong influence on intracellular signaling and phosphorylation pathways was demonstrated by evaluation of fibronectin‐induced phosphorylation of ERK. The molecular basis of the observed inhibitory activity was suggested on the results of docking experiments.

Modulation of αvβ3- and α5β1-integrin-mediated adhesion by dehydro-β-amino acids containing peptidomimetics

A novel class of low molecular weight ligands of αvβ₃ and α₅β₁ integrins, that possess a dehydro-β-amino acid as conformationally constrained core, linked to the pharmacophoric moieties mimicking the RGD recognition sequence, have been synthesized through a very simple protocol. Cell adhesion assays and integrin-mediated signaling activation experiments suggested a good affinity of these compounds toward both integrin receptors. Moreover, further elongation with two glycine units allowed to obtain an excellent dual inhibitor. Structural models for αvβ₃ integrin-ligand binding confirmed that the dehydro-β-amino derivatives are able to act as an electrostatic clamp by establishing several stabilizing interactions with the receptor.

Therapeutic Targeting of Eosinophil Adhesion and Accumulation in Allergic Conjunctivitis

Considerable evidence indicates that eosinophils are important effectors of ocular allergy. Increased worldwide prevalence of allergic eye pathologies has stimulated the identification of novel drug targets, including eosinophils and adhesion molecules. Accumulation of eosinophils in the eye is a key event in the onset and maintenance of allergic inflammation and is mediated by different adhesion molecules. Antihistamines with multiple mechanisms of action can be effective during the early and late phases of allergic conjunctivitis by blocking the interaction between β1 integrins and vascular cell adhesion molecule (VCAM)-1. Small molecule antagonists that target key elements in the process of eosinophil recruitment have been identified and reinforce the validity of α4β1 integrin as a therapeutic target. Glucocorticoids are among the most effective drugs for ocular allergy, but their use is limited by adverse effects. Novel dissociated glucocorticoids can prevent eosinophil accumulation and induce apoptosis of eosinophils, making them promising candidates for ophthalmic drugs. This article reviews recent understanding of the role of adhesion molecules in eosinophil recruitment in the inflamed conjunctiva along with effective treatments for allergic conjunctivitis.

Synthesis and assay of retro-α4β1 integrin-targeting motifs

In recent years, several research groups proposed new peptidomimetic antagonists of integrins αvβ3, α5β1, αIIbβ3, αvβ6, αvβ5, etc. based on retro sequences of the classic integrin-binding motif RGD. The retro strategy is still largely ignored for the non-RGD-binding α4β1 integrin. Herein we present the first examples of retro sequences for targeting this integrin, composed of Asp or isoAsp equipped with an aromatic cap at the N-terminus, (S)-pyrrolidine-3-carboxylic acid (β(2)-Pro) as a constrained core, and the amino variant (AMPUMP) of the well-known α4-targeting diphenylurea MPUPA. We discuss α4β1 receptor affinity (SPA), cell adhesion assays, stability in mouse serum, and conformational analysis. For their significant ability to inhibit cell adhesion and remarkable stability, the retro-peptide mimetics BnCO-Asp-β-Pro-AMPUMP (3) and BnCO-isoAsp-β-Pro-AMPUMP (4) represent promising candidates for designing small molecules as potential anti-inflammatory agents.

Dehydro-β-proline Containing α4β1 Integrin Antagonists: Stereochemical Recognition in Ligand–Receptor Interplay

A novel class of dehydro-β-proline-containing peptidomimetics, designed to be effective as α4β1 integrin ligands, has been developed on the basis of the fundamental requirements for the interactions of these transmembrane receptors with bioactive ligands. Dehydro-β-proline ring has been synthesized through an original pathway, involving ring closing metathesis of a diallylamino derivative. The synthesized products showed to be effective and selective as α4β1 integrin antagonists and displayed IC50 values in the nanomolar range in cell adhesion inhibition assays and in VCAM-1-induced phosphorylation of extracellular-signal-regulated kinases. Significant activity was observed also toward the homologous integrin α4β7, while they did not display any activity toward selected members of β1, β2, and β3 families. A strong dependence on the stereochemistry of the heterocyclic central core could be observed. The great importance of α4β1 integrin in chronic inflammatory and autoimmune diseases suggests a possible exploitation of these ligands as lead compounds for therapeutic tools development.

5-aminomethyloxazolidine-2,4-dione hybrid α/β-dipeptide scaffolds as inductors of constrained conformations: Applications to the synthesis of integrin antagonists

Peptidomimetics represent an attractive starting point for drug discovery programs; in particular, peptidomimetics that result from the incorporation of a heterocycle may take advantage of increased enzymatic stability and higher ability to reproduce the bioactive conformations of the parent peptides, resulting in enhanced therapeutic potential. Herein, we present mimetics of the α4β1 integrin antagonist BIO1211 (MPUPA‐Leu‐Asp‐Val‐Pro‐OH), containing a aminomethyloxazolidine‐2,4‐dione scaffold (Amo). Interestingly, the retro‐sequences PhCOAsp(OH)‐Amo‐APUMP including either (S)‐ or (R)‐configured Amo displayed significant ability to inhibit the adhesion of α4β1 integrin expressing cells, and remarkable stability in mouse serum. Possibly, the conformational bias exerted by the Amo scaffold determined the affinity for the receptors. These peptidomimetics could be of interest for the development of small‐molecule agents effective against inflammatory processes and correlated autoimmune diseases.

REST is up-regulated by epidermal growth factor in HeLa cells and inhibits apoptosis by influencing histone H3 acetylation.

REST (repressor element 1-silencing transcription factor) is a transcription factor that recruits histone deacetylases to silence gene transcription. REST appears to play a paradoxical role in cancer cells: it exhibits tumor suppressor activity or promotes tumorigenesis, depending upon the setting. The extracellular signaling molecules that control REST gene expression in cancer cells remain poorly understood. In this study, we report that REST expression in HeLa cells is elevated in cells exposed to epidermal growth factor or serum, whereas the rate of cell apoptosis is low. Apoptosis induced by serum withdrawal is significantly increased in HeLa cells treated with an antisense phosphorothioate oligodeoxynucleotide (AS ODN) capable of down-regulating REST expression, whereas in HeLa cells transfected with a REST expressing plasmid, REST overexpression reduces the marked apoptosis caused, in absence of serum, by exposure to an anti-Fas receptor antibody imitating the Fas ligand activity plus PD 98059, a blocker of extracellular signal-regulated kinase 1/2 activation. REST knockdown also reduces mRNA levels of the antiapoptotic protein Bcl-X(L) whereas in HeLa cells overexpressing REST, the reduction of Bcl-X(L) mRNA caused by the anti-Fas receptor antibody plus PD 98059 is significantly decreased. Finally, we report that acetylation of histone H3 is increased in HeLa cells exposed to AS ODN or anti-Fas receptor antibody, whereas it is reduced in cells transfected with the REST expressing plasmid. Our findings indicate that REST is a novel gene regulated by EGF in HeLa cells that potentially contributes to the modulation of apoptosis via epigenetic mechanisms.

DS‐70, a novel and potent α4 integrin antagonist, is an effective treatment for experimental allergic conjunctivitis in guinea pigs

Allergic conjunctivitis is an eye inflammation that involves the infiltration of immune cells into the conjunctiva via cell surface‐adhesion receptors, such as integrin α4β1. These receptors interact with adhesion molecules expressed on the conjunctival endothelium and may be a target to treat this disease. We synthesized DS‐70, a novel α/β‐peptidomimetic α4 integrin antagonist, to prevent the conjunctival infiltration of immune cells and clinical symptoms in a model of allergic conjunctivitis.

Selective detection of α4β1 integrin (VLA-4)-expressing cells using peptide-functionalized nanostructured materials mimicking endothelial surfaces adjacent to inflammatory sites

Persistent accumulation of immune cells mediated by α4β1 integrin (VLA‐4) is a hallmark of the inflammatory diseases and of chronic inflammation observed in the affected tissues of autoimmune diseases. Aiming at exploring new methods for monitoring the course of the inflammatory processes, we designed the first peptide‐functionalized nanostructured devices capable to mimic the high‐density multivalency binding between the α4β1 integrin‐expressing cells and the ligands overexpressed on the endothelial surfaces, in the proximity of the sites of inflammation. Specifically, we describe the first examples of monolayers constituted by dye‐loaded zeolite L crystals, coated with α4β1 integrin peptide ligands, and we analyze the adhesion of model Jurkat cells in comparison to non‐α4β1 integrin‐expressing cells. In particular, the peptidomimetic diphenylurea‐Leu‐Asp‐Val‐diamine allows significant and selective detection of α4β1 integrin‐expressing Jurkat cells, after very rapid incubation time, supporting the possible implementation in a diagnostic device capable to detect the desired cells from biological fluids, obtainable from patients in a noninvasive way.