Selective detection of α4ß1 integrin (VLA-4)-expressing cells using peptide-functionalized nanostructured materials mimicking endothelial surfaces adjacent to inflammatory sites.

Persistent accumulation of immune cells mediated by α4ß1 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 α4ß1 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 α4ß1 integrin peptide ligands, and we analyze the adhesion of model Jurkat cells in comparison to non-α4ß1 integrin-expressing cells. In particular, the peptidomimetic diphenylurea-Leu-Asp-Val-diamine allows significant and selective detection of α4ß1 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.

5-aminomethyloxazolidine-2,4-dione hybrid α/ß-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 α4ß1 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 α4ß1 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.

Synthesis and assay of retro-α4ß1 integrin-targeting motifs.

In recent years, several research groups proposed new peptidomimetic antagonists of integrins αvß3, α5ß1, αIIbß3, αvß6, αvß5, etc. based on retro sequences of the classic integrin-binding motif RGD. The retro strategy is still largely ignored for the non-RGD-binding α4ß1 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 (ß(2)-Pro) as a constrained core, and the amino variant (AMPUMP) of the well-known α4-targeting diphenylurea MPUPA. We discuss α4ß1 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-ß-Pro-AMPUMP (3) and BnCO-isoAsp-ß-Pro-AMPUMP (4) represent promising candidates for designing small molecules as potential anti-inflammatory agents.