Novel carboxylate-based glycolipids: TLR4 antagonism, MD-2 binding and self-assembly properties

New monosaccharide-based lipid A analogues were rationally designed through MD-2 docking studies. A panel of compounds with two carboxylate groups as phosphates bioisosteres, was synthesized with the same glucosamine-bis-succinyl core linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to purified, functional recombinant human MD-2 was studied by four independent methods. All compounds bound to MD-2 with similar affinities and inhibited in a concentration-dependent manner the LPS-stimulated TLR4 signaling in human and murine cells, while being inactive as TLR4 agonists when provided alone. A compound of the panel was tested in vivo and was not able to inhibit the production of proinflammatory cytokines in animals. This lack of activity is probably due to strong binding to serum albumin, as suggested by cell experiments in the presence of the serum. The interesting self-assembly property in solution of this type of compounds was investigated by computational methods and microscopy, and formation of large vesicles was observed by cryo-TEM microscopy.TOLLerant project (H2020-MSC-ETN-642157), the Italian Ministry for Foreign Affairs and International Cooperation (MAECI) and Spanish MINECO (CTQ2014-57141-R and CTQ2017-88353-R grants) are acknowledged. Vesna Hodnik of University of Ljubljana for the help with SPR, Sandra Delgado of CIC BioGUNE for the Cryo-TEM images. RJ was partially funded by the research program P4-0176 by the Slovenian Research Agency

Small Molecules as Toll-like Receptor 4 Modulators Drug and In-House Computational Repurposing

The innate immunity toll-like receptor 4 (TLR4) system is a receptor of paramount importance as a therapeutic target. Virtual screening following a “computer-aided drug repurposing” approach was applied to the discovery of novel TLR4 modulators with a non-lipopolysaccharide-like structure. We screened almost 29,000 approved drugs and drug-like molecules from commercial, public, and in-house academia chemical libraries and, after biological assays, identified several compounds with TLR4 antagonist activity. Our computational protocol showed to be a robust approach for the identification of hits with drug-like scaffolds as possible inhibitors of the TLR4 innate immune pathways. Our collaborative work broadens the chemical diversity for inspiration of new classes of TLR4 modulators.This work was financially supported by the Spanish Ministry for Science and Innovation (grants CTQ2014-57141-R, CTQ2017-88353-R, and PID2020-113588RB-I00 for S.M.S.; grants BES-2012-053653 for L.P.R., BES-2015-071588 for J.G.C. and PID2021-124983OB-I00 for J.C.M.), the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (MINECO/FEDER; SAF2016-75988-R), and the Community of Madrid (S-2010/BMD-2332) for M.F

Synthetic Glycolipids as Molecular Vaccine Adjuvants: Mechanism of Action in Human Cells and In Vivo Activity

Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA

Potential anti-inflammatory, anti-adhesive, anti/estrogenic, and angiotensin-converting enzyme inhibitory activities of anthocyanins and their gut metabolites

Epidemiological studies have indicated a positive association between the intake of foods rich in anthocyanins and the protection against cardiovascular diseases. Some authors have shown that anthocyanins are degraded by the gut microflora giving rise to the formation of other breakdown metabolites, which could also contribute to anthocyanin health effects. The objective of this study was to evaluate the effects of anthocyanins and their breakdown metabolites, protocatechuic, syringic, gallic, and vanillic acids, on different parameters involved in atherosclerosis, including inflammation, cell adhesion, chemotaxis, endothelial function, estrogenic/anti-estrogenic activity, and angiotensin-converting enzyme (ACE) inhibitory activity. From the assayed metabolites, only protocatechuic acid exhibited a slight inhibitory effect on NO production and TNF-α secretion in LPS-INF-γ-induced macrophages. Gallic acid caused a decrease in the secretion of MCP-1, ICAM-1, and VCAM-1 in endothelial cells. All anthocyanins showed an ACE-inhibitory activity. Delphinidin-3-glucoside, pelargonidin-3-glucoside, and gallic acid showed affinity for ERβ and pelargonidin and peonidin-3-glucosides for ERα. The current data suggest that anthocyanins and their breakdown metabolites may partly provide a protective effect against atherosclerosis that is multi-causal and involves different biochemical pathways. However, the concentrations of anthocyanins and their metabolites, as used in the present cell culture and in vitro assays mediating anti-inflammatory, anti-adhesive, anti-estrogenic, and angiotensin-converting enzyme inhibitory activities, were often manifold higher than those physiologically achievable

Development of a new family of conformationally restricted peptides as potent nucleators of β-turns. Design, synthesis, structure, and biological evaluation of a β-lactam peptide analogue of melanostatin

Novel enantiopure (i)-(β-lactam)-(Gly)-(i+3) peptide models, defined by the presence of a central α-alkyl-α-amino-β-lactam ring placed as the (i+1) residue, have been synthesized in a totally stereocontrolled way by α-alkylation of suitable N-[bis(trimethylsilyl)methyl]-β-lactams. The structural properties of these β-lactam pseudopeptides have been studied by X-ray crystallography, Molecular Dynamics simulation, and NOESY-restrained NMR simulated annealing techniques, showing a strong tendency to form stable type II or type II‘ β-turns either in the solid state or in highly coordinating DMSO solutions. Tetrapeptide models containing syn- or anti-α,β-dialkyl-α-amino-β-lactam rings have also been synthesized and their conformations analyzed, revealing that α-alkyl substitution is essential for β-turn stabilization. A β-lactam analogue of melanostatin (PLG amide) has also been prepared, characterized as a type-II β-turn in DMSO-d6 solution, and tested by competitive binding assay as a dopaminergic D2 modulator in rat neuron cultured cells, displaying moderate agonist activity in the micromolar concentration range. On the basis of these results, a novel peptidomimetic design concept, based on the separation of constraint and recognition elements, is proposed.We thank The University of the Basque Country and Ministerio de Ciencia y Tecnologia (Spain) for financial support (Project No. BQU 2002-01737). Grants to A.B. from Gobierno Vasco and to R.M. . from European Commissions (Marie Curie HMPT-CT-2000-00173) are acknowledged.Peer reviewe

Development of a new family of conformationally restricted peptides as potent nucleators of β-turns. Design, synthesis, structure, and biological evaluation of a β-lactam peptide analogue of melanostatin. Supporting Information

Analytical and spectral characterization data of all new compounds, NMR/NOESY interprotonic distances, and crystallographic data (CIF).Peer reviewe

Amphiphilic Guanidino­calixarenes Inhibit Lipopolysaccharide (LPS)- and Lectin-Stimulated Toll-like Receptor 4 (TLR4) Signaling

We recently reported on the activity of cationic amphiphiles in inhibiting TLR4 activation and subsequent production of inflammatory cytokines in cells and in animal models. Starting from the assumption that opportunely designed cationic amphiphiles can behave as CD14/MD-2 ligands and therefore modulate the TLR4 signaling, we present here a panel of amphiphilic guanidino­calixarenes whose structure was computationally optimized to dock into MD-2 and CD14 binding sites. Some of these calixarenes were active in inhibiting, in a dose-dependent way, the LPS-stimulated TLR4 activation and TLR4-dependent cytokine production in human and mouse cells. Moreover, guanidino­calixarenes also inhibited TLR4 signaling when TLR4 was activated by a non-LPS stimulus, the plant lectin PHA. While the activity of guanidino­calixarenes in inhibiting LPS toxic action has previously been related to their capacity to bind LPS, we suggest a direct antagonist effect of calixarenes on TLR4/MD-2 dimerization, pointing at the calixarene moiety as a potential scaffold for the development of new TLR4-directed therapeutics

Conformational Plasticity in Glycomimetics: Fluorocarbamethyl-L-idopyranosides Mimic the Intrinsic Dynamic Behaviour of Natural Idose Rings

International audienceSugar function, structure and dynamics are intricately correlated. Ring flexibility is intrinsically related to biological activity; actually plasticity in L-iduronic rings modulates their interactions with biological receptors. However, the access to the experimental values of the energy barriers and free-energy difference for conformer interconversion in water solution has been elusive. Here, a new generation of fluorine-containing glycomimetics is presented. We have applied a combination of organic synthesis, NMR spectroscopy and computational methods to investigate the conformational behaviour of idose- and glucose-like rings. We have used low-temperature NMR spectroscopic experiments to slow down the conformational exchange of the idose-like rings. Under these conditions, the exchange rate becomes slow in the (FNMR)-F-19 spectroscopic chemical shift timescale and allows shedding light on the thermodynamic and kinetic features of the equilibrium. Despite the minimal structural differences between these compounds, a remarkable difference in their dynamic behaviour indeed occurs. The importance of introducing fluorine atoms in these sugars mimics is also highlighted. Only the use of (FNMR)-F-19 spectroscopic experiments has permitted the unveiling of key features of the conformational equilibrium that would have otherwise remained unobserved

Enhancing Potency and Selectivity of a DC‐SIGN Glycomimetic Ligand by Fragment‐Based Design: Structural Basis

International audienceChemical modification of pseudo-dimannoside ligands guided by fragment-based design allowed for the exploitation of an ammonium-binding region in the vicinity of the mannose-binding site of DC-SIGN, leading to the synthesis of a glycomimetic antagonist (compound 16) of unprecedented affinity and selectivity against the related lectin langerin. Here, the computational design of pseudo-dimannoside derivatives as DC-SIGN ligands, their synthesis, their evaluation as DC-SIGN selective antagonists, the biophysical characterization of the DC-SIGN/16 complex, and the structural basis for the ligand activity are presented. On the way to the characterization of this ligand, an unusual bridging interaction within the crystals shed light on the plasticity and potential secondary binding sites within the DC-SIGN carbohydrate recognition domain