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

Abstract

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.

Integrin-mediated adhesive properties of neutrophils are reduced by hyperbaric oxygen therapy in patients with chronic non-healing wound

“…Adhesion assays on purified neutrophils were performed as previously described [29,33]. Briefly, black 96-well plates were coated overnight at 4˚C with fibronectin (FN) or fibrinogen (Fg) (both 10 μg/mL) to study respectively adhesion mediated by α 4 β 1 and β 2 integrins.…”

“…After three washes, adhered cells were lysed with 0.5% Triton X-100 in 1% BSA (bovine serum albumin) in HBSS (30 min at 4˚C) and fluorescence was measured (Ex485 nm/Em535 nm). To evaluate the ability of ligand 1 [29] (named here RG66) to inhibit neutrophils' adhesion, cells were pre-incubated with various concentrations (10 −4 -10 −10 M) of RG66 or vehicle (methanol) for 30 min at 37˚C before plating cells into coated wells. Neutrophil adhesion assays were also carried out in the presence of an anti-human β 2 or α 4 integrin antibody (both 5 μg/mL; purified mouse anti-human CD18 and purified mouse anti-human CD49d antibody, BD Pharmingen).…”

“…The synthesis of (R)-RG66 has been previously reported [29] via a multistep synthesis starting from an enantiopure (3R,Z)-tert-butyl 3-(allylamino)-2-ethylidene-4-methylpentanoate. The detailed description of the synthetic protocol has been reported in S1 Fig. The biological evaluation showed a strong dependence of the bioactivity on the ring stereochemistry could be detected, since (S)-1 turned out to be completely inactive [29].…”

“…Patient wound area size was measured and pro-inflammatory cytokine levels were evaluated both in neutrophils and in plasma. Furthermore, in vitro cell adhesion assays were performed in the presence of a peptidomimetic integrin antagonist previously developed by our group [29], to investigate if the HBO treatment may influence neutrophil recruitment and adhesion mediated by α 4 β 1 integrin.…”

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

“…The assays were performed as previously described (Tolomelli et al, ; Baiula et al, ). SK‐MEL‐24, K562, D283 and HT‐29 cells were seeded in 96‐well plates coated by passive adsorption with FN (10 μg·mL −1 ) overnight at 4°C.…”