In-Peptide Synthesis of Imidazolidin-2-one Scaffolds, Equippable with Proteinogenic or Taggable/Linkable Side Chains, General Promoters of Unusual Secondary Structures.

Peptidomimetics containing ( S)- or ( R)-imidazolidin-2-one-4-carboxylate (Imi) have been obtained by the expedient in-peptide cyclization of ( S)- or ( R)-α,ß-diaminopropionic acid (Dap) residues. These Imi scaffolds behave as proline analogues characterized by a flat structure and a trans-restricted geometry of the preceding peptide bond and induce well-defined secondary structures in a biomimetic environment. While ( S)-Imi peptides adopted a γ'-turn conformation, ( R)-Imi induced the contemporary formation of a γ-turn and a rare 11-membered H-bonded structure in the 2→4 opposite direction of the sequence, identified as a ε-turn. In order to exploit these Imi scaffolds as general promoters of unusual secondary structures, proteinaceous side chains have been introduced at the N1 position of the five-membered ring, potentially mimicking any residues. Finally, the Imi rings have been equipped with unnatural side chains or with functionalized substituents, which can be utilized as linkers to chemoselectively bind the Imi-peptides onto nanoparticles, biomaterials, or diagnostic probes.

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.

Diagnostic Implementation of Fast and Selective Integrin-Mediated Adhesion of Cancer Cells on Functionalized Zeolite L Monolayers.

The rapid and exact identification and quantification of specific biomarkers is a key technology for always achieving more efficient diagnostic methodologies. We present the first application of a nanostructured device constituted of patterned self-assembled monolayers of disk-shaped zeolite L coated with the cyclic integrin ligand c[RGDfK] via isocyanate linker, to the rapid detection of cancer cells. With its high specificity toward HeLa and Glioma cells and fast adhesion ability, this biocompatible monolayer is a promising platform for implementation in diagnostics and personalized therapy formulation devices.

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.

Synthesis and analysis of the conformational preferences of 5-aminomethyloxazolidine-2,4-dione scaffolds: first examples of ß(2)- and ß(2, 2)-homo-Freidinger lactam analogues.

Constrained peptidomimetic scaffolds are of considerable interest for the design of therapeutically useful analogues of bioactive peptides. We present the single-step cyclization of (S)- or (R)-α-hydroxy-ß(2)- or α-substituted-α-hydroxy-ß(2, 2)-amino acids already incorporated within oligopeptides to 5-aminomethyl-oxazolidine-2,4-dione (Amo) rings. These scaffolds can be regarded as unprecedented ß(2)- or ß(2, 2)-homo-Freidinger lactam analogues, and can be equipped with a proteinogenic side chain at each residue. In a biomimetic environment, Amo rings act as inducers of extended, semi-bent or folded geometries, depending on the relative stereochemistry and the presence of α-substituents.

Easy preparation of dehydroalanine building blocks equipped with oxazolidin-2-one chiral auxiliaries, and applications to the stereoselective synthesis of substituted tryptophans.

Chiral dehydroamino acid building blocks are versatile starting materials for the preparation of optically active unusual amino acids and other compounds of pharmacological interest. Herein we disclose the expedient preparation of dehydroalanines (ΔAla) equipped with oxazolidin-2-one (Oxd) chiral auxiliaries, Ts-Oxd-ΔAla-OMe. These compounds have been obtained in high yields from dipeptides Ts-Ser/Thr/phenylSer-Ser-OMe by the one-pot cyclization-elimination reaction with N,N-disuccinimidyl carbonate and catalytic DIPEA. To test the efficacy of the chiral auxiliaries in controlling asymmetric transformations, the Friedel-Crafts alkylations of indoles carrying diverse substituents were performed in the presence of Lewis and Brønsted acids. The reactions proceeded with good to excellent diastereomeric ratios giving (S)- or (R)-tryptophan derivatives, isolated very conveniently by simple flash chromatography. To verify the utility of this approach, optically pure (S)-2-methyltryptophan and (S)-5-fluorotryptophan were obtained and utilized to prepare analogues of endogenous opioid peptide endomorphin-1, H-Tyr-Pro-Trp-PheNH2.

Radical Oxygen Species, Oxidized Low-Density Lipoproteins, and Lectin-like Oxidized Low-Density Lipoprotein Receptor 1: A Vicious Circle in Atherosclerotic Process.

Atherosclerosis is a complex condition that involves the accumulation of lipids and subsequent plaque formation in the arterial intima. There are various stimuli, cellular receptors, and pathways involved in this process, but oxidative modifications of low-density lipoprotein (ox-LDL) are particularly important in the onset and progression of atherosclerosis. Ox-LDLs promote foam-cell formation, activate proinflammatory pathways, and induce smooth-muscle-cell migration, apoptosis, and cell death. One of the major receptors for ox-LDL is LOX-1, which is upregulated in several cardiovascular diseases, including atherosclerosis. LOX-1 activation in endothelial cells promotes endothelial dysfunction and induces pro-atherogenic signaling, leading to plaque formation. The binding of ox-LDLs to LOX-1 increases the generation of reactive oxygen species (ROS), which can induce LOX-1 expression and oxidize LDLs, contributing to ox-LDL generation and further upregulating LOX-1 expression. This creates a vicious circle that is amplified in pathological conditions characterized by high plasma levels of LDLs. Although LOX-1 has harmful effects, the clinical significance of inhibiting this protein remains unclear. Further studies both in vitro and in vivo are needed to determine whether LOX-1 inhibition could be a potential therapeutic target to counteract the atherosclerotic process.

Immature Surfactant Protein Type B and Surfactant Protein Type D Correlate with Coronary Heart Disease in Patients with Type 2 Diabetes.

BACKGROUND: Different specific surfactant proteins (SPs) have been associated with various pathological conditions, not only of the respiratory system, but also more recently with cardiovascular diseases, such as heart failure. The aim of the present study was to evaluate the role of SP-A, SP-D, and the precursor protein of SP-B (proSP-B) in the pathogenesis of cardiovascular damage in patients affected by type 2 diabetes (T2D). METHODS: The study considered 31 patients with T2D (DN group), 34 patients with both T2D and coronary heart disease (CHD) (DC group), and 30 patients without diabetes but with a diagnosis of CHD (NC group). SP-A, SP-D, and proSP-B concentrations were determined in plasma samples, and were statistically compared using parametric and multivariate methods. RESULTS: Higher plasma concentrations of SP-D and proSP-B were found in patients affected by both T2D and CHD (DC group), and in patients with CHD without diabetes (NC group), in comparison to T2D patients (DN group). A significant correlation, both with linear regression (r = 0.3565, p = 0.001) and Principal Component Analysis (PCA), was found between the plasma levels of SP-D and proSP-B in the overall cohort of patients. No differences in SP-A were observed among the three groups of subjects. CONCLUSION: The present study extends the knowledge on the role of plasma SPs' levels as possible indicators of the risk of CHD being linked to T2D disease progression.

In-peptide synthesis of di-oxazolidinone and dehydroamino acid-oxazolidinone motifs as ß-turn inducers.

Small and easy-to-do mimetics of ß-turns are of great interest to interfere with protein-protein recognition events mediated by ß-turn recognition motifs. We propose a straightforward procedure for constraining the conformation of tetrapeptides lacking a pre-formed scaffold. According to the stereochemistry array, N-Ts tetrapeptides including Thr or PhSer (phenylserine) at the positions 2 or 3 gave rise in a single step to the sequences Oxd(2)-Oxd(3) or ΔAbu(2)-Oxd(3) (Oxd, oxazolidin-2-one; ΔAbu, 2,3-dehydro-2-aminobutyric). These pseudo-Pro residues displayed highly constrained ϕ, ψ, and χ dihedral angles, and induced clear ß-turns or inverse turns of type I or II, as determined by extensive spectroscopic and computational analyses.

Albumin Thiolation and Oxidative Stress Status in Patients with Aortic Valve Stenosis.

Recent evidence indicates that reactive oxygen species play an important causative role in the onset and progression of valvular diseases. Here, we analyzed the oxidative modifications of albumin (HSA) occurring on Cysteine 34 and the antioxidant capacity of the serum in 44 patients with severe aortic stenosis (36 patients underwent aortic valve replacement and 8 underwent a second aortic valve substitution due to a degenerated bioprosthetic valve), and in 10 healthy donors (controls). Before surgical intervention, patients showed an increase in the oxidized form of albumin (HSA-Cys), a decrease in the native reduced form (HSA-SH), and a significant reduction in serum free sulfhydryl groups and in the total serum antioxidant activity. Patients undergoing a second valve replacement showed levels of HSA-Cys, free sulfhydryl groups, and total antioxidant activity similar to those of controls. In vitro incubation of whole blood with aspirin (ASA) significantly increased the free sulfhydryl groups, suggesting that the in vivo treatment with ASA may contribute to reducing oxidative stress. We also found that N-acetylcysteine and its amide derivative were able to regenerate HSA-SH. In conclusion, the systemic oxidative stress reflected by high levels of HSA-Cys is increased in patients with aortic valve stenosis. Thiol-disulfide breaking agents regenerate HSA-SH, thus paving the way to the use these compounds to mitigate the oxidative stress occurring in the disease.

Oxidative Stress and Arginine/Nitric Oxide Pathway in Red Blood Cells Derived from Patients with Prediabetes.

The effects of the oral glucose tolerance test (OGTT) on red blood cells (RBCs) have not been thoroughly investigated, although it is known that the ingestion of 75 g of glucose during OGTT results in a systemic state of inflammation and oxidative stress. Therefore, we evaluated the effect of OGTT on oxidative stress and L-arginine/Nitric Oxide (L-Arg/NO) metabolic pathway in RBCs obtained from patients with prediabetes. Blood samples were collected from all participants before (T0) and at 10 (T1), 20 (T2), 30 (T3), 60 (T4), 90 (T5), 120 (T6), 150 (T7), and 180 (T8) minutes after glucose loading. Results showed a significant increase in oxidative stress status characterized by a rise in the GSSG/GSH ratio at T4 and T6 that increased in parallel with a reduction of NO production in RBCs. In addition, in this time frame, increased exposure of phosphatidylserine on RBCs membrane was observed. These metabolic modifications were rescued at T8, together with an increase in activated RBC NO synthase expression. These findings provide a possible explanation of the phenomena occurring after glucose loading and suggest that, even in the early stages of diabetes, it may be important to avoid acute variations in glycemia in order to prevent diabetic complications.

Heterocyclic Scaffolds in the Design of Peptidomimetic Integrin Ligands: Synthetic Strategies, Structural Aspects, and Biological Activity.

The integrin receptors represent valuable targets for therapeutic interventions; being overexpressed in many pathological states, their inhibition can be effective to treat a number of severe diseases. Since integrin functions are mediated by interactions with ECM protein ligands, the inhibition can be achieved by interfering with such interactions using small mimetics of the integrin-ligand recognition motifs (e.g. RGD, LDV, etc.). In this review, we focus on the antagonists with peptideheterocycle hybrid structures. The introduction of well-designed scaffolds has met considerable success in the rational design of highly stable, bioavailable, and conformationally defined antagonists. Two main approaches are discussed herein. The first approach is the use of scaffolds external to the main recognition motifs, aimed at improving conformational definition. In the second approach, heterocyclic cores are introduced within the recognition motifs, giving access to libraries of 3D diverse candidate antagonists.

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.