Side chain effect in the modulation of αvß3/α5ß1 integrin activity via clickable isoxazoline-RGD-mimetics: development of molecular delivery systems.

Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose αvß3 and α5ß1 integrins' potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as "shuttles" for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards α5ß1 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated α5ß1 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application.

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

BACKGROUND AND PURPOSE: Allergic conjunctivitis is an eye inflammation that involves the infiltration of immune cells into the conjunctiva via cell surface-adhesion receptors, such as integrin α4 ß1 . 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/ß-peptidomimetic α4 integrin antagonist, to prevent the conjunctival infiltration of immune cells and clinical symptoms in a model of allergic conjunctivitis. EXPERIMENTAL APPROACH: In vitro, DS-70 was pharmacologically characterized using a scintillation proximity procedure to measure its affinity for α4 ß1 integrin, and its effect on cell adhesion mediated by different integrins was also evaluated. The effects of DS-70 on vascular cell adhesion molecule-1 (VCAM-1)-mediated degranulation of a human mast cell line and an eosinophilic cell line, which both express α4 ß1 , and on VCAM-1-mediated phosphorylation of ERK 1/2 in Jurkat E6.1 cells were investigated. Effects of DS-70 administered in the conjunctival fornix of ovalbumin-sensitized guinea pigs were evaluated. KEY RESULTS: DS-70 bound to integrin α4 ß1 with nanomolar affinity, prevented the adhesion of α4 integrin-expressing cells, antagonized VCAM-1-mediated degranulation of mast cells and eosinophils and ERK 1/2 phosphorylation. Only 20% was degraded after an 8 h incubation with serum. DS-70 dose-dependently reduced the clinical symptoms of allergic conjunctivitis, conjunctival α4 integrin expression and conjunctival levels of chemokines and cytokines in ovalbumin-sensitized guinea pigs. CONCLUSIONS AND IMPLICATIONS: These findings highlight the role of α4 integrin in allergic conjunctivitis and suggest that DS-70 is a potential treatment for this condition.

New ß-Lactam Derivatives Modulate Cell Adhesion and Signaling Mediated by RGD-Binding and Leukocyte Integrins.

A novel series of ß-lactam derivatives that was designed and synthesized to target RGD-binding and leukocyte integrins is reported. The compound library was evaluated by investigating the effects on integrin-mediated cell adhesion and cell signaling in cell lines expressing αvß3, αvß5, αvß6, α5ß1, αIIbß3, α4ß1, and αLß2 integrins. SAR analysis of the new series of azetidinones enabled the recognition of structural elements associated with integrin selectivity. We obtained selective and potent agonists that could induce cell adhesion and promote cell signaling mediated by αvß3, αvß5, α5ß1, or α4ß1 integrin, and antagonists for the integrins αvß3 and α5ß1, as well as α4ß1 and αLß2, preventing the effects elicited by the respective endogenous agonists.

Dehydro-ß-proline Containing α4ß1 Integrin Antagonists: Stereochemical Recognition in Ligand-Receptor Interplay.

A novel class of dehydro-ß-proline-containing peptidomimetics, designed to be effective as α4ß1 integrin ligands, has been developed on the basis of the fundamental requirements for the interactions of these transmembrane receptors with bioactive ligands. Dehydro-ß-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 α4ß1 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 α4ß7, while they did not display any activity toward selected members of ß1, ß2, and ß3 families. A strong dependence on the stereochemistry of the heterocyclic central core could be observed. The great importance of α4ß1 integrin in chronic inflammatory and autoimmune diseases suggests a possible exploitation of these ligands as lead compounds for therapeutic tools development.

Targeting integrins αvß3 and α5ß1 with new ß-lactam derivatives.

The αvß3 and α5ß1 integrins are widely expressed in different cancer types and recognize the tripeptide Arg-Gly-Asp (RGD) motif present in several extracellular matrix proteins. We report here the design, synthesis and biological activity of some new ß-lactam derivatives specifically designed to target integrins. The new molecules contain the azetidinone as the only cyclic framework armed with carboxylic acid and amine terminals spaced from 9 to 14 atoms to switch on recognition by integrins. All tested molecules showed a concentration-dependent enhancement in fibronectin-mediated adhesion of K562 and SK-MEL-24 cells; in particular 1, expressed a higher affinity towards α5ß1 integrin (EC50 of 12 nM) and 2 was more selective for integrin αvß3 (EC50 of 11 nM).

Modulation of αvß3- and α5ß1-integrin-mediated adhesion by dehydro-ß-amino acids containing peptidomimetics.

A novel class of low molecular weight ligands of αvß3 and α5ß1 integrins, that possess a dehydro-ß-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 αvß3 integrin-ligand binding confirmed that the dehydro-ß-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 ß(1) 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 α(4)ß(1) 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.

Development of isoxazoline-containing peptidomimetics as dual αvß3 and α5ß1 integrin ligands.

Isoxazoline-containing peptidomimetics, designed to be effective α(v)ß(3) and α(5)ß(1) 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.