Search

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

10 p.-5 fig.-1 graph. abst., The human macrophage galactose lectin (MGL) is anendocytic type II transmembrane receptor expressed on immaturemonocyte-derived dendritic cells and activated macrophages and playsa role in modulating the immune system in response to infections andcancer. MGL contains an extracellular calcium-dependent (C-type)carbohydrate recognition domain (CRD) that specifically bindsterminalN-acetylgalactosamine glycan residues such as the Tn andsialyl-Tn antigens found on tumor cells, as well as otherN-andO-glycans displayed on certain viruses and parasites. Even though theglycan specificity of MGL is known and several binding glycoproteinshave been identified, the molecular basis for substrate recognition hasremained elusive due to the lack of high-resolution structures. Here wepresent crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactionswith the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like theasialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. Thestructure provides the molecular basis for preferential binding ofN-acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance dataacquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution.Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize LewisXand GalNAc,respectively, This work was supported by funding from Gabrielle’s Angel Foundation for Cancer Research to G.B., grants 12/IA/1398and 16/IA/4419 from Science Foundation Ireland to P.V.M.and GOIPG/2016/858 from the Irish Research Council toA.G. F.M., H.C., and A.D. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding ProjectsIF/00780/2015 and PTDC/BIA-MIB/31028/2017 and UCI-BIO Project UIDB/04378/2020, as well as the Ph.D. grantattributed to A.D. (PD/BD/142847/2018). The NMRspectrometers are part of the National NMR Network(PTNMR) and are partially supported by InfrastructureProject 22161 (co-financed by FEDER through COMPETE2020, POCI, and PORL and FCT through PIDDAC). F.J.C.acknowledges funding from Agencia Estatal de Investigación(Spain) for Grant RTI2018-094751-B-C22 and CIBERES, aninitiative from the Spanish Institute of Health Carlos III. F.C.thanks Agencia Estatal de Investigación (Spain) for Grant RTI2018-099592-B-C2.

Understanding the specific molecular interactions between proteins and β1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the β1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3-1,4-mixed-linked glucans. This is mediated by a conformation-selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked β-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. DATABASE: Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc

COMPETE Programme: SAICTPAC/0019/2015 IF/00780/2015 Project no. 022161 Monocationic and dicationic cholinium ionic liquids (ILs) were synthesized and evaluated as acetylcholinesterase (AChE) inhibitors with in vitro and in silico models, and their cytotoxicity was assessed using human cell lines from skin (CRL-1502) and colon cancer (CaCo-2). The ILs with a longer alkyl chain were stronger AChE inhibitors, the dicationic ILs (DILs) being more active than the monocationic ILs. The best result was obtained for [N1,1,12,2(OH)]2Br2 at a concentration of 0.18 μM by reducing half enzyme activity without affecting the viability of tested cell lines. A saturation-transfer difference NMR (STD-NMR) binding study was carried out, demonstrating that [N1,1,12,2(OH)]2Br2 binds to AChE. STD-NMR competition binding experiments, using galanthamine as a reference ligand, clearly highlight that the IL displaces galanthamine in the AChE binding site pinpointing [N1,1,12,2(OH)]2Br2 inside the deep gorge of AChE. In order to obtain a three-dimensional (3D) view of the molecular recognition process, in silico molecular docking studies on the active site of AChE were carried out. The proposed 3D model of the AChE/DIL complex is in agreement with the STD-derived epitope mapping, which explains the competition with galanthamine and unveils key interactions in both peripheral and catalytic sites of AChE. These interactions seem essential to govern the recognition of DILs by the AChE enzyme. Our study provides a structural and functional platform that can be used for the rational design of choline-based ILs as potent AChE inhibitors. publishersversion published

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcβ1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galβ1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.

Despite the rapidly increasing number of patients suffering from type 2 diabetes, Alzheimer's disease, and diabetes-induced dementia, there are no disease-modifying therapies that are able to prevent or block disease progress. In this work, we investigate the potential of nature-inspired glucosylpolyphenols against relevant targets, including islet amyloid polypeptide, glucosidases, and cholinesterases. Moreover, with the premise of Fyn kinase as a paradigm-shifting target in Alzheimer's drug discovery, we explore glucosylpolyphenols as blockers of Aβ-induced Fyn kinase activation while looking into downstream effects leading to Tau hyperphosphorylation. Several compounds inhibit Aβ-induced Fyn kinase activation and decrease pTau levels at 10 μM concentration, particularly the per-O-methylated glucosylacetophloroglucinol and the 4-glucosylcatechol dibenzoate, the latter inhibiting also butyrylcholinesterase and β-glucosidase. Both compounds are nontoxic with ideal pharmacokinetic properties for further development. This work ultimately highlights the multitarget nature, fine structural tuning capacity, and valuable therapeutic significance of glucosylpolyphenols in the context of these metabolic and neurodegenerative disorders.

The molecular basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are mostly limited to two residues, allowing it to show some degree of promiscuity. These findings open the door to the chemical design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools. This journal is, Funding from the Agencia Estatal Investigacion of Spain (AEI,RTI-2018-099592-B-C21, CTQ2013-44367-C2-2-P, BFU2016-75633-P,PID2019-105451GB-I00 and PID2019-107476GB-I00), ARAID, the Italian Ministry of Education, University and Research (PRIN2015 contract nr. 2015RNWJAM), the Royal Society (URF\R\180019),the Danish National Research Foundation (DNRF107) and FCTPortugal (iFCT, IF/00624/2015, and Doctoral Studentship SFRH/BD/111556/2015, IF/00780/2015; PTDC/BIA-MIB/31028/2017, UCIBIOUIDB/04378/2020 and NMR Infrastructure project 22161) isacknowledged. I. A. B. and A. A. thank the AECC for predoctoralfellowships. E. J.-M. thanks Universidad de La Rioja for a post-doctoral fellowship. We thank H. Clausen and U. Mandel forproviding us the complete 5E5 antibody.

The glycan structures of the receptor binding domain of the SARS‐CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS‐based analyses. The interaction of the RBD 13C‐labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15N‐labelled galectins (galectins‐3, ‐7 and ‐8 N‐terminal), Siglecs (Siglec‐8, Siglec‐10), and C‐type lectins (DC‐SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed., Unprecedent structural details of the glycans of the RBD of SARS‐CoV‐2 spike glycoprotein have been revealed by NMR spectroscopy. Unexpected and non‐previously reported glycoepitopes have been detected. The interaction of the RBD glycoprotein with diverse human lectins has been scrutinised by exploiting the NMR signature of the 13C‐glycans. Our analysis permitted to identify the corresponding glycan epitopes responsible for the interaction with each lectin.

Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3’s structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions., We thank ARAID, MEC (grant no. CTQ2013-44367-C2-2-P, BFU2016-75633-P and RTI2018-099592-B-C21), the National Institutes of Health (grant no. GM113534 and instrument grant no. GM113534-01S), the Danish National Research Foundation (grant no. DNRF107), the FCT-Portugal (grant no. UID/Multi/04378/2013) and Gobierno de Aragón (grant nos. E34_R17, E35_17R and LMP58_18) with FEDER (grant no. 2014-2020) funds for ‘Building Europe from Aragón’ for financial support. I.C. thanks the Universidad de La Rioja for the FPI grant. F.M. and H.C. thank FCT-Portugal for IF Investigator (IF/00780/2015), PTDC/BIA-MIB/31028/2017 and UID/Multi/04378/2019 projects, and PTNMR (grant no. ROTEIRO/0031/2013 and PINFRA/22161/2016). P.B. acknowledges support from the Labex EpiGenMed, an ‘Investissements d’avenir’ program (grant no. ANR-10-LABX-12-01). The CBS (Montpellier) is a member of France-BioImaging (FBI, ANR-10-INBS-04-01) and the French Infrastructure for Integrated Structural Biology (FRISBI, ANR-10-INBS-05). The research leading to these results has also received funding from the FP7 (2007–2013) under BioStruct-X (grant agreement nos. 283570 and BIOSTRUCTX_5186).

Human macrophage galactose-type lectin (MGL), expressed on macrophages and dendritic cells (DCs), modulates distinct immune cell responses by recognizing GalNAc-containing structures present on pathogens, self-glycoproteins and tumor cells. Herein, we used NMR spectroscopy and molecular dynamics (MD) simulations to investigate the structural preferences of MGL against different GalNAc-containing structures derived from blood group A antigen, Forssman antigen and the GM2 glycolipid. NMR analyses of the MGL carbohydrate recognition domain (MGL-CRD, C181-H316) in the absence and presence of α-methyl GalNAc, a simple monosaccharide, shows that the MGL-CRD is highly dynamic and its structure is strongly altered upon ligand binding. This plasticity of the MGL-CRD structure explains the ability of MGL to accommodate different GalNAc-containing molecules. However, key differences are observed in the recognition process reliant whether the GalNAc is part of the blood group A antigen, the Forssman antigen or GM2-derived structures. These results are in accordance with molecular dynamics simulations that suggest the existence of a distinct MGL binding mechanism depending on the context of GalNAc moiety presentation. These results afford new perspectives for the rational design of GalNAc modifications able to fine tune MGL immune responses in distinct biological contexts, especially in malignancy.

Background Half of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1. Methods and results By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced. Conclusions SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53. General Significance This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status., European Union (FEDER funds through Programa Operacional Factores de Competitividade – COMPETE) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) through the projects UID/QUI/50006/2019, COMPETE 2020 (POCI-01-0145-FEDER-006684/POCI-01-0145-FEDER-007440) and the BioTecNorte operation (NORTE-01-0145-FEDER-000004), (3599-PPCDT) PTDC/DTP-FTO/1981/2014 – POCI-01-0145-FEDER-016581 and UID/QUI/0081/2013; the Italian Association for Cancer Research, AIRC (IG#5506 to G.F.), Compagnia S. Paolo, Turin, Italy (Project 2017.0526 to G.F.) and Ministry of Health, (Project 5 × 1000, 2013 and 2015; Current research 2016). We also thank FCT for the financial support through CEECIND/01772/2017 (M.M.M. Santos), PTDC/QUI-QOR/29664/2017, UID/DTP/04138/2013, IF/01272/2015 (A. Carvalho), IF/00780/2015 (F. Marcelo) and fellowships SFRH/BD/119144/2016 (H. Ramos), PD/BD/114046/2015 (A. S. Gomes), SFRH/BD/128673/2017 (J. B. Loureiro), SFRH/BD/96189/2013 (S. Gomes), SFRH/BPD/110640/2015 (C. Oliveira) and PD/BI/135334/2017 (V. Barcherini), and the Programa Operacional Potencial Humano (POCH), specifically the BiotechHealth Programme (PD/00016/2012), info:eu-repo/semantics/publishedVersion

The dual inhibitory action of the pain related peptide opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH) against neutral endopeptidase (NEP) and aminopeptidase N (AP-N) was further investigated by a SAR study involving minor modifications on the polar side chains of Arg residues and glycosylation with monosaccharides at Ser. None of them exerted dual or individual inhibitory potency superior than opiorphin. However, the correlations deduced offer further proof for the key role of these residues upon the binding and bioactive conformational stabilization of opiorphin. NMR conformational studies on the glycopeptides suggest that they are still very flexible compounds that may attain their respective bioactive conformations.

11 pags, 8 figs, The family of polypeptide N-acetylgalactosamine (GalNAc) transferases (GalNAc-Ts) orchestrates the initiating step of mucin-type protein O-glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc-T isoenzymes are related to different diseases. Recently, it has been demonstrated that an inactive GalNAc-T2 mutant (F104S), which is not located at the active site, induces low levels of high-density lipoprotein cholesterol (HDL-C) in humans. Herein, the molecular basis for F104S mutant inactivation has been deciphered. Saturation transfer difference NMR spectroscopy experiments demonstrate that the mutation induces loss of binding to peptide substrates. Analysis of the crystal structure of the F104S mutant bound to UDP-GalNAc (UDP=uridine diphosphate), combined with molecular dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than that in the wild-type (WT) enzyme. 19F NMR spectroscopy experiments reveal that the WT enzyme only reaches the active state in the presence of UDP-GalNAc, which provides compelling evidence that GalNAc-T2 adopts a UDP-GalNAc-dependent induced-fit mechanism. The F104S mutation precludes the enzyme from achieving the active conformation and concomitantly binding peptide substrates. This study provides new insights into the catalytic mechanism of the large family of GalNAc-Ts and how these enzymes orchestrate protein O-glycosylation., We thank the Ministerio de Economia y Competitividad (CTQ2013-44367-C2-2-P and BFU2016-75633-P to R.H-G., CTQ2015-67727-R to F.C., CTQ2015-64597-C2-1P to J.J-B). F.M. thanks FCT-Portugal for the IF project (IF/00780/2015) and UCIBIO funding UID/Multi/04378/2013 cofinanced by the FEDER (POCI-01-0145-FEDER-007728). The NMR spectrometers are part of PTNMR supported by project no. 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). H.C. thanks the Lundbeck Foundation and the Danish National Research Foundation (DNRF107). E.L-N. acknowledges her postdoctoral EMBO fellowship ALTF 1553-2015 cofunded by the European Commission (LTFCO-FUND2013, GA-2013-609409) and Marie Curie Actions. H.C. and J.J-B. thank the EU for the TOLLerant project. R.H-G. thanks the Agencia Aragonesa para la Investigacion y Desarrollo (ARAID) and the Diputacion General de Aragon (DGA, B89) for financial support. Research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement no. 283570 and BIOSTRUCTX_5186). We thank synchrotron radiation source DIAMOND (Oxford) and beamline I04 (number of experiment MX10121-19). We would also like to thank to Prof. Tom Gerken for his valuable comments on our manuscript.

Mannose glyco-oligoamide β-D-Man-Py-γ-Py-Ind (β-D-Man, 1) and two new glyco-oligoamides, β-L-Man-Py-γ-Py-Ind (β-L-Man, 2) and 6-deoxy-β-D-Man-Py-γ-Py-Ind (6-deoxy-β-D-Man, 3), have been designed and synthesized to investigate the role of hydrogen-bonding cooperative donor centres of carbohydrates in their recognition by DNA. The free- and bound-state geometries were studied, as were the affinities of the D and L enantiomers of the mannose glyco-oligoamides (1 and 2) for DNA polymers [ct-DNA and poly(dA-dT)2]. TR-NOESY and DF-STD experiments for the diastereomeric complexes formed with DNA allow the asymmetric centres of the sugar residue that are close to the inner and outer regions of the DNA minor grooves to be distinguished. A CN hairpin folding in β-L-Man derivative 2 was observed, with the α face of the sugar close to the indole ring. The C-2 and C-3 centres are orientated towards the inner region of the DNA minor groove. The affinity data for poly(dA-dT)2 indicate that there is a chiral discrimination process, with β-L-Man derivative 2 being the best ligand. 6-Deoxy-β-D-Man derivative 3 forms the least stable complexes with DNA. Molecular dynamics simulations of β-L-Man derivative 2 in complex with a double-strand dodecamer d(AT)12 are in agreement with the experimental NMR spectroscopic data. Thus, the cooperative donor centre 2-OH in the L-mannose enantiomer is a key contributor to the stability of the 2·poly(dA-dT)2 complex.

Protein–glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of 1H15N-HSQC signals. The intensity of the Gal-3 1H15N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of 1H15N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein.

Abnormal glycosylation is a common feature in disease that typically results from misregulation in expression and/or activity of glycosyltransferases (GTs) and glycosidases. Interfering with these enzymes, by developing a prospect of targeted inhibitors, has opened newer avenues to meet the challenge of abnormal glycosylation. Progress in GT inhibitors has been relatively slower in comparison to glycosidases. In case of GTs, their polyspecific nature, structural homology, overlapping specificities, multi-substrate catalytic mechanism and relatively less available 3D-structural data stance a big challenge to explore the whole potential of GT target inhibitors in comparison to glycosidases for therapeutic purposes. In this review, we focus on GT specific inhibitors, which are organised as conventional donor analogues (viz. donor, acceptor and transition state mimetic), glycomimetic, alternative inhibitor chemotype and metabolic chain terminator. Conventional donor analogues are however limited by the poor membrane permeability and chemical instability. Thus, in the last two decades, alternative inhibitor chemotypes caught attention as a promising lead, which are not structurally derived from GT substrates and possess drug like properties offering an alternate non-substrate like inhibitor based strategy for drug development. Although successful cellular GT-targeted molecules are yet to be achieved, recent designing of metabolic inhibitors i.e., dead end substrates are emerging as an impetus to explore the potential of GT families as therapeutic targets.

Glycosylation by simple sugars is a drug discovery alternative that has been explored with varying success for enhancing the potency and bioavailability of opioid peptides. Long ago we described two O-glycosides having either β-Glucose and β-Galactose of (d-Met2, Pro5)-enkephalinamide showing one of the highest antinociceptive activities known. Here, we report the resynthesis of these two analogs and the preparation of three novel neoglycopeptide derivatives (α-Mannose, β-Lactose and β-Cellobiose). Binding studies to cloned zebrafish opioid receptors showed very small differences of affinity between the parent compound and the five glycopeptides thus suggesting that the nature of the carbohydrate moiety plays a minor role in determining the binding mode. Indeed, NMR conformational studies, combined with molecular mechanics calculations, indicated that all glycopeptides present the same major conformation either in solution or membrane-like environment. The evidences provided here highlight the relevance for in vivo activity of the conjugating bond between the peptide and sugar moieties in opioid glycopeptides.

FtsZ is the key protein of bacterial cell-division and target for new antibiotics. Selective inhibition of FtsZ polymerization without impairing the assembly of the eukaryotic homologue tubulin was demonstrated with C8-substituted guanine nucleotides. By combining NMR techniques with biochemical and molecular modeling procedures, we have investigated the molecular recognition of C8-substituted-nucleotides by FtsZ from Methanococcus jannaschii (Mj-FtsZ) and Bacillus subtilis (Bs-FtsZ). STD epitope mapping and trNOESY bioactive conformation analysis of each nucleotide were employed to deduce differences in their recognition mode by each FtsZ species. GMP binds in the same anti conformation as GTP, whereas 8-pyrrolidino-GMP binds in the syn conformation. However, the anti conformation of 8-morpholino-GMP is selected by Bs-FtsZ, while Mj-FtsZ binds both anti- and syn-geometries. The inhibitory potencies of the C8-modified-nucleotides on the assembly of Bs-FtsZ, but not of Mj-FtsZ, correlate with their binding affinities. Thus, MorphGTP behaves as a nonhydrolyzable analog whose binding induces formation of Mj-FtsZ curved filaments, resembling polymers formed by the inactive forms of this protein. NMR data, combined with molecular modeling protocols, permit explanation of the mechanism of FtsZ assembly impairment by C8-substituted GTP analogs. The presence of the C8-substituent induces electrostatic remodeling and small structural displacements at the association interface between FtsZ monomers to form filaments, leading to complete assembly inhibition or to formation of abnormal FtsZ polymers. The inhibition of bacterial Bs-FtsZ assembly may be simply explained by steric clashes of the C8-GTP-analogs with the incoming FtsZ monomer. This information may facilitate the design of antibacterial FtsZ inhibitors replacing GTP. © 2013 American Chemical Society., MINECO; Comunidad de Madrid, Fundación Severo Ochoa; European Comission (BM1003, CM1102 COST)

Two carbene-based ligands have been attached to perbenzylated and permethylated cyclodextrins. Their palladium complexes were synthesized, characterized and used as catalysts in Suzuki–Miyaura coupling reactions both in ethanol and water.

Acetylcholinesterase (AChE) inhibition is one of the most currently available therapies for the management of Alzheimer's disease (AD) symptoms. In this context, NMR spectroscopy binding studies were accomplished to explain the inhibition of AChE activity by Salvia sclareoides extracts. HPLC-MS analyses of the acetone, butanol and water extracts eluted with methanol and acidified water showed that rosmarinic acid is present in all the studied samples and is a major constituent of butanol and water extracts. Moreover, luteolin 4'-O-glucoside, luteolin 3',7-di-O-glucoside and luteolin 7-O-(6''-O-acetylglucoside) were identified by MS(2) and MS(3) data acquired during the LC-MS(n) runs. Quantification of rosmarinic acid by HPLC with diode-array detection (DAD) showed that the butanol extract is the richest one in this component (134 μg mg(-1) extract). Saturation transfer difference (STD) NMR spectroscopy binding experiments of S. sclareoides crude extracts in the presence of AChE in buffer solution determined rosmarinic acid as the only explicit binder for AChE. Furthermore, the binding epitope and the AChE-bound conformation of rosmarinic acid were further elucidated by STD and transferred NOE effect (trNOESY) experiments. As a control, NMR spectroscopy binding experiments were also carried out with pure rosmarinic acid, thus confirming the specific interaction and inhibition of this compound against AChE. The binding site of AChE for rosmarinic acid was also investigated by STD-based competition binding experiments using Donepezil, a drug currently used to treat AD, as a reference. These competition experiments demonstrated that rosmarinic acid does not compete with Donepezil for the same binding site. A 3D model of the molecular complex has been proposed. Therefore, the combination of the NMR spectroscopy based data with molecular modelling has permitted us to detect a new binding site in AChE, which could be used for future drug development.

Amyloid peptides, Aβ1-40 and Aβ1-42, represent major molecular targets to develop potential drugs and diagnostic tools for Alzheimer's Disease (AD). In fact, oligomeric and fibrillar aggregates generated by these peptides are amongst the principal components of amyloid plaques found post mortem in patients suffering from AD. Rosmarinic acid has been demonstrated to be effective in preventing the aggregation of amyloid peptides in vitro and to delay the progression of the disease in animal models. Nevertheless, no information is available about its molecular mechanism of action. Herein, we report the NMR characterization of the interaction of Salvia sclareoides extract and that of its major component, rosmarinic acid, with Aβ1-42 peptide, whose oligomers have been described as the most toxic Aβ species in vivo. Our data shed light on the structural determinants of rosmarinic acid-Aβ1-42 oligomers interaction, thus allowing the elucidation of its mechanism of action. They also provide important information for the rational design of new compounds with higher affinity for Aβ peptides to generate new anti-amyloidogenic molecules and/or molecular tools for the specific targeting of amyloid aggregates in vivo. In addition, we identified methyl caffeate, another natural compound present in different plants and human diet, as a good ligand of Aβ1-42 oligomers, which also shows anti-amyloidogenic activity. Finally, we demonstrated the possibility to exploit STD-NMR and trNOESY experiments to screen extracts from natural sources for the presence of Aβ peptide ligands.

International audience; The synthesis of three conformationally locked D-glucopyranoside analogs displaying the hydroxyl pattern of the parent sugar is described. A two carbon bridge connecting the C-2 and C-5 atoms of the pyranose ring allows a torsion of the sugar ring toward a B-2,B-5 conformation as confirmed by conformational analysis. This conformation is strongly believed to be adopted by the oxacarbenium ion-like transition state of several inverting glucosidases.

Two bicyclic iminosugars have been synthesized from polyhydroxylated azepanes by ring isomerization followed by debenzylative cyclization. Their conformations have been studied by NMR and molecular modeling and their glycosidase inhibition profiles were determined. They both adopt a chair conformation for the piperidine ring and display weak inhibition on α-glucosidases.

9 páginas, 4 figuras, 2 tablas, 2 esquemas -- PAGS nros. 128-136, A D-fructose moiety in which a D-proline ring has been engineered with a spiranic junction in the glycidic scaffold has been used as a proline mimetic. The bicyclic structure, which possesses high structural rigidity, was then included in model peptides to explore their prolyl cis/trans amide rotamer populations. Model peptide conformations were studied by molecular dynamics and NMR experiments, We gratefully acknowledge financial support by Regione Lombardia (Programma Operativo Regione Lombardia Ob. 3 Fondo Sociale Europeo 2000–2006), Sovvenzione Globale Progetto Ingenio A0001127/2007, Consorzio Interuniversitario Nazionale “Metodologie e Processi Innovativi di Sintesi” (C.I.N.M.P.I.S.) and the Fondo di Ateneo per la Ricerca FAR 2008. The Fundação para Ciência e Tecnologia (FCT) Portugal is also thanked for post-doctoral research (grant SFRH/BDP/65462/2009)

The total synthesis of the epimer at C-6' of the core of the complex nucleoside antibiotic miharamycin B is reported. Optimization of the N-glycosylation key step involving the atypical 2-aminopurine nucleobase and a bicyclic glycosyl donor enabled to access the N 9 -nucleoside in reasonable yield which was further decorated. The stereochemistry at C-6' has been secured by X-ray crystallography.

8 páginas, 1 figura, 1 tabla, 6 esquemas -- PAGS nros. 10066-10073, The relative configuration at C-6′ of nucleoside antibiotic miharamycin A has been elucidated by NMR spectroscopy and proved to be S. The total synthesis of miharamycin B has also been investigated, which has led to the unprecedented construction of its core. The bicyclic sugar moiety has been elaborated by means of a SmI2-based keto–alkyne coupling. Elongation of its C-6 position towards a bicyclic sugar amino acid and conversion into a suitable glycosyl donor enabled efficient N-glycosylation with 2-aminopurine to take place to afford the nucleosidic part of miharamycin B. Final peptide coupling with arginine afforded the skeleton of miharamycin B. Unfortunately, attempts to deprotect this scaffold failed to afford the complex nucleoside antibiotic, This work was supported by the Fundação para a Ciência e Tecnologia (FCT, project POCI/QUI/59672/2004, Portugal). F.M. thanks the FCT for a research grant (SFRH/BD/17775/2004

A functional food oil, rich in fatty acids and antioxidants, coloured with pigments (carotenoids) extracted with supercritical CO2 from the microalga Chlorella vulgaris, was produced, having in view its use in food industry (namely for derived seafood). The supercritical fluid extraction (SFE) was carried out in order to study the effect of several modifiers (oil mixed with the microalga and ethanol with the supercritical CO2), the degree of crushing of the microalga and the supercritical fluid flow rate, at a pressure of 300 bar and temperature of 40 °C. Moreover, the microalga pigments were also extracted with acetone and with vegetable oil at room and high temperature. The recovery of carotenoids was 100% with oil at room temperature for 17 h, 70% with oil at 100 °C for 30 min, 69% with supercritical CO2 at 40 °C and 300 bar. In SFE the degree of crushing strongly influenced the extraction recovery and higher pigment recoveries were obtained with well crushed biomass. The stability of soybean oil containing the extracted pigments was also evaluated (light protected) over six weeks, in terms of total carotenoid content and peroxide value. Carotenoids exhibited good preservation over the time, practically without loss. Peroxide values were stable for all extraction systems, showing only a slight increase over time.

The identification of MUC1 tumor-associated Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.

© 2015 American Chemical Society. FtsZ is the organizer of cell division in most bacteria and a target in the quest for new antibiotics. FtsZ is a tubulin-like GTPase, in which the active site is completed at the interface with the next subunit in an assembled FtsZ filament. Fluorescent mant-GTP has been extensively used for competitive binding studies of nucleotide analogs and synthetic GTP-replacing inhibitors possessing antibacterial activity. However, its mode of binding and whether the mant tag interferes with FtsZ assembly function were unknown. Mant-GTP exists in equilibrium as a mixture of C2′- and C3′-substituted isomers. We have unraveled the molecular recognition process of mant-GTP by FtsZ monomers. Both isomers bind in the anti glycosidic bond conformation: 2′-mant-GTP in two ribose puckering conformations and 3′-mant-GTP in the preferred C2′ endo conformation. In each case, the mant tag strongly interacts with FtsZ at an extension of the GTP binding site, which is also supported by molecular dynamics simulations. Importantly, mant-GTP binding induces archaeal FtsZ polymerization into inactive curved filaments that cannot hydrolyze the nucleotide, rather than straight GTP-hydrolyzing assemblies, and also inhibits normal assembly of FtsZ from the Gram-negative bacterium Escherichia coli but is hydrolyzed by FtsZ from Gram-positive Bacillus subtilis. Thus, the specific interactions provided by the fluorescent mant tag indicate a new way to search for synthetic FtsZ inhibitors that selectively suppress the cell division of bacterial pathogens., BFU2011-23416 and BFU2014-51823-R (J.M.A), CTQ2012-32065 (J.J.B.), CM 2010/BMD-2353 (J.J.B. and J.M.A.), FCT SFRH/BPD/65462/2009 and UID/Multi/04378/2013 (F.M.) and a FPI fellowship (L.B.R.A).

Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from Haematococcus pluvialis was carried out, for several experimental conditions, using a semi-continuous apparatus. The microalga was previously freeze-dried and ground with a ball mill. The effects of pressure (200 and 300 bar), temperature (40 and 60 °C), degree of crushing, as well as the use of ethanol as a co-solvent (10%) on the extraction efficiency were assessed. Organic solvent extractions, using acetone, were also carried out in a vortex, on ground cells mixed with very small glass beads. Supercritical extraction from the completely crushed alga was compared with acetone and the highest recovery of carotenoids (92%) was obtained at the pressure of 300 bar and the temperature of 60 °C, using ethanol as a co-solvent. The extraction recovery increased with the pressure at 60 °C. On the other hand, the increase in temperature, at 300 bar, led to a slight improvement. The main carotenoid of Haematococcus pluvialis is the esterified astaxanthin (about 75%). Other carotenoids present are lutein, astaxanthin (free), β-carotene and canthaxanthin. All of them were recovered through supercritical fluid extraction with values higher than 90%, with the exception of canthaxanthin (about 85%), at a pressure of 300 bar and a temperature of 60 °C.

Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is observed when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues, Arg and Asp, present in the PDTR sequence establish noteworthy fundamental interactions with the lectin surface as well as fix the conformation of the peptide backbone, favoring the presentation of the sugar moiety toward the lectin. These results may help to better understand glycopeptide–lectin interactions and may contribute to engineer new binding sites, allowing novel glycosensors for Tn antigen detection to be designed.

The human macrophage galactose-type lectin (MGL) is a key physiological receptor for the carcinoma-associated Tn antigen (GalNAc-α-1-O-Ser/Thr) in mucins. NMR and modeling-based data on the molecular recognition features of synthetic Tn-bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non-glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca(2+) . NMR data were complemented with molecular dynamics simulations and Corcema-ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn-presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH-π contacts involving exclusively the NHAc moiety.

A strategy to create cooperative hydrogen-bonding centers by using strong and directional intramolecular hydrogen-bonding motifs that can survive in aqueous media is presented. In particular, glyco-oligoamides, a family of DNA minor groove binders, with cooperative and non-cooperative hydrogen-bonding donor centers in the carbohydrate residues have been designed, synthesized, and studied by means of NMR spectroscopy and molecular modeling methods. Indeed, two different sugar moieties, namely, β-D-Man-Py-γ-Py-Ind (1; Ind = indole, Man = mannose, Py= pyrrole) and β-D-Tal-Py-γ-Py-Ind (2; Tal = talose), were chosen according to our design. These sugar molecules should present oneor two-directional intramolecular hydrogen bonds. The challenge has been to study the conformation of the glyco-oligoamides at low temperature in physiological media by detecting the exchangeable protons (amide NH and OH resonances) by means of NMR spectroscopic analysis. In addition, two more glyco-oligoamides with non-cooperative hydrogen-bonding centers, that is, β-D-Glc-Py-γ-Py-Ind (3; Glc = glucose), β-D-Gal-Py-γ-Py-Ind (4; Gal = galac-tose), and the model compounds β-D-Man-Py-NHAc (5) and β-D-Tal-Py-NHAc (6) were synthesized and studied for comparison. We have demonstrated the existence of directional intramolecular hydrogen bonds in 1 and 2 in aqueous media. The unexpected differences in terms of stabilization of the intramolecular hydrogen bonds in 1 and 2 relative to 5 and 6 promoted us to evaluate the influence of CH-π interactions on the establishment of intramolecular hydrogen bonds by using computational methods. Initial binding studies of 1 and 2 with calf-thymus DNA and poly(dA-dT)2 by NMR spectroscopic analysis and molecular dynamics simulations were also carried out. Both new sugar-oligoamides are bound in the minor groove of DNA, thus keeping a stable hairpin structure, as in the free state, in which both intramolecular hydrogen-bonding and CH-π interactions are present.

8-β-d-Glucopyranosylgenistein (1), the major component of Genista tenera, was synthesized and showed an extensive therapeutical impact in the treatment of STZ-induced diabetic rats, producing normalization of fasting hyperglycemia and amelioration of excessive postprandial glucose excursions and and increasing β-cell sensitivity, insulin secretion, and circulating insulin within 7 days at a dose of 4 (mg/kg bw)/day. Suppression of islet amyloid polypeptide (IAPP) fibril formation by compound 1 was demonstrated by thioflavin T fluorescence and atomic force microscopy. Molecular recognition studies with IAPP and Aβ1-42 employing saturation transfer difference (STD) confirmed the same binding mode for both amyloid peptides as suggested by their deduced epitope. Insights into the preferred conformation in the bound state and conformers' geometry resulting from interaction with Aβ1-42 were also given by STD, trNOESY, and MM calculations. These studies strongly support 8-β-d-glucopyranosylgenistein as a promising molecular entity for intervention in amyloid events of both diabetes and the frequently associated Alzheimer's disease.

3 páginas 3 figuras, 1 tabla -- PAGS nros. 5390-5392, Here we report the synthesis of a series of polyhydroxylated 3- and 5-acetamido azepanes and detail the molecular basis of their inhibition of family 84 glycoside hydrolases. These family 84 enzymes include human O-GlcNAcase, an enzyme involved in post-translational processing of intracellular proteins modified by O-linked β-N-acetylglucosamine residues. Detailed structural analysis of the binding of these azepanes to BtGH84, a bacterial homologue of O-GlcNAcase, highlights their conformational flexibility. Molecular mechanics and molecular dynamics calculations reveal that binding to the enzyme involves significant conformational distortion of these inhibitors from their preferred solution conformations. The binding of these azepanes provides structural insight into substrate distortion that likely occurs along the reaction coordinate followed by O-GlcNAcase during glycoside hydrolysis. This class of inhibitors may prove to be useful probes for evaluating the conformational itineraries of glycosidases and aid the development of more potent and specific glycosidase inhibitors, Support for this research was provided by the Fundação para a Ciência e Tecnologia (FCT, Portugal), the Biotechnology and Biological Sciences Research Council (U.K.), and the Natural Sciences and Engineering Research Council (Canada)and the Canada Research Chair in Chemical Glycobiology

International audience; no abstract