¹⁷O NMR spectroscopy is a powerful technique, which can provide unique information regarding the structure and reactivity of biomolecules. However, the low natural abundance of ¹⁷O (0.04 %) generally requires working with enriched samples, which are not easily accessible. Here, we present simple, fast and cost‐efficient ¹⁷O‐enrichment strategies for amino acids and peptides by using mechanochemistry. First, five unprotected amino acids were enriched under ambient conditions, consuming only microliter amounts of costly labeled water, and producing pure molecules with enrichment levels up to ∼40 %, yields ∼60‐85 %, and no loss of optical purity. Subsequently, ¹⁷O‐enriched Fmoc/tBu‐protected amino acids were produced on a 1 g/day scale with high enrichment levels. Lastly, a site‐selective ¹⁷O‐labeling of carboxylic functions in peptide side‐chains was achieved for RGD and GRGDS peptides, with ∼28 % enrichment level. For all molecules, ¹⁷O ssNMR spectra were recorded at 14.1 T in reasonable times, making this an important step forward for future NMR studies of biomolecules.

Background and Purpose: The activation of 5-HT₄ receptors with agonists has emerged as a valuable therapeutic strategy to treat Alzheimer’s disease (AD) by enhancing the nonamyloidogenic pathway. Here, the potential therapeutic effects of tegaserod, an effective agent for irritable bowel syndrome, were assessed for AD treatment. To envisage its efficient repurposing, tegaserod-loaded nanoemulsions were developed and functionalized by a blood–brain barrier shuttle peptide. Results: The butyrylcholinesterase inhibitory activity of tegaserod and its neuroprotective cellular effects were highlighted, confirming the interest of this pleiotropic drug for AD treatment. In regard to its drugability profile, and in order to limit its peripheral distribution after IV administration, its encapsulation into monodisperse lipid nanoemulsions (Tg-NEs) of about 50 nm, and with neutral zeta potential characteristics, was performed. The stability of the formulation in stock conditions at 4 °C and in blood biomimetic medium was established. The adsorption on Tg-NEs of peptide-22 was realized. The functionalized NEs were characterized by chromatographic methods (SEC and C₁₈/HPLC) and isothermal titration calorimetry, attesting the efficiency of the adsorption. From in vitro assays, these nanocarriers appeared suitable for enabling tegaserod controlled release without hemolytic properties. Conclusion: The developed peptide-22 functionalized Tg-NEs appear as a valuable tool to allow exploration of the repurposed tegaserod in AD treatment in further preclinical studies.

A relevant alternative to enamel matrix derivatives from animal origin could be the use of synthetic amelogenin-derived peptides. This study aimed to assess the effect of a synthetic amelogenin-derived peptide (ADP-5), alone or included in an experimental gellan–xanthan hydrogel, on periodontal cell behavior (gingival fibroblasts, periodontal ligament cells, osteoblasts and cementoblasts). The effect of ADP-5 (50, 100, and 200 µg/mL) on cell metabolic activity was examined using Alamar blue assay, and cell morphology was assessed by confocal imaging. An experimental gellan–xanthan hydrogel was then designed as carrier for ADP-5 and compared to the commercial gel Emdogain^®. Alizarin Red was used to determine the periodontal ligament and cementoblasts cell mineralization. The inflammatory profile of these two cells was also quantified using ELISA (vascular endothelial growth factor A, tumor necrosis factor α, and interleukin 11) mediators. ADP-5 enhanced cell proliferation and remineralization; the 100 µg/mL concentration was more efficient than 50 and 200 µg/mL. The ADP-5 experimental hydrogel exhibited equivalent good biological behavior compared to Emdogain^® in terms of cell colonization, mineralization, and inflammatory profile. These findings revealed relevant insights regarding the ADP-5 biological behavior. From a clinical perspective, these outcomes could instigate the development of novel functionalized scaffold for periodontal regeneration.

Staphylococcus aureus (SA) leukocidin ED (LukED) belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes (red blood cells) and leukocytes (white blood cells). The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2, and CCR5 using a combination of structural, pharmacological, and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining previous and novel experimental data with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.

Tegaserod is a serotonin receptor agonist with potential neuroprotective properties for Alzheimer's disease. Due to its low druggability profile and to avoid detrimental side effects, a brain-targeted formulation is required. Nanocarriers targeting the blood-brain barrier (BBB) with a shuttle peptide, such as peptide-22, or enabling direct nose-to-brain delivery were considered as valuable approaches. This study aimed to compare two types of lipid-based nanocarriers and determine the best formulation for intravenous (IV) or intranasal (IN) administration. Tegaserod-loaded nanoemulsions and liposomes were successfully developed, improving the solubilization of tegaserod in injectable formulations. Their properties were optimized for the IV route, and the two formulations were compared in terms of granulometric properties, stability, stealth properties, and transport across a human model of the human BBB. Based on these evaluations, peptide-22-decorated tegaserod-loaded nanoemulsions were the most promising for IV administration. In addition, tegaserod-loaded nanoemulsions or liposomes were incorporated in a gelling formulation with properties optimized for the IN route, focusing on gelation temperature, osmolarity, and pH. Due to its rheological profile and behavior at room temperature, gel-embedded liposomes emerged as the most suitable formulation for the IN route. The successful development of these nanocarriers will facilitate further preclinical evaluation of tegaserod in Alzheimer's disease.