Speeding-up the Determination of Protein-Ligand Affinities by STD NMR: The Reduced Data Set STD NMR Approach (rd-STD NMR).

STD NMR spectroscopy is a powerful ligand-observed NMR tool for screening and characterizing the interactions of small molecules and low molecular weight fragments with a given macromolecule, identifying the main intermolecular contacts in the bound state. It is also a powerful analytical technique for the accurate determination of protein-ligand dissociation constants (KD) of medium-to-weak affinity, of interest in the pharmaceutical industry. However, accurate KD determination and epitope mapping requires a long series of experiments at increasing saturation times to carry out a full analysis using the so-called STD NMR build-up curve approach and apply the "initial slopes approximation". Here, we have developed a new protocol to bypass this important limitation, which allows us to obtain initial slopes by using just two saturation times and, hence, to very quickly determine precise protein-ligand dissociation constants by STD NMR.

Unravelling the Inflammatory Processes in the Early Stages of Diabetic Nephropathy and the Potential Effect of (Ss)-DS-ONJ.

Inflammatory processes play a central role in the pathogenesis of diabetic nephropathy (DN) in the early stages of the disease. The authors demonstrate that the glycolipid mimetic (Ss)-DS-ONJ is able to abolish inflammation via the induction of autophagy flux and provokes the inhibition of inflammasome complex in ex vivo and in vitro models, using adult kidney explants from BB rats. The contribution of (Ss)-DS-ONJ to reducing inflammatory events is mediated by the inhibition of classical stress kinase pathways and the blocking of inflammasome complex activation. The (Ss)-DS-ONJ treatment is able to inhibit the epithelial-to-mesenchymal transition (EMT) progression, but only when the IL18 levels are reduced by the treatment. These findings suggest that (Ss)-DS-ONJ could be a novel, and multifactorial treatment for DN.

Synthesis of sp2-Iminosugar Selenoglycolipids as Multitarget Drug Candidates with Antiproliferative, Leishmanicidal and Anti-Inflammatory Properties.

sp2-Iminosugar glycolipids (sp2-IGLs) represent a consolidated family of glycoconjugate mimetics encompassing a monosaccharide-like glycone moiety with a pseudoamide-type nitrogen replacing the endocyclic oxygen atom of carbohydrates and an axially-oriented lipid chain anchored at the pseudoanomeric position. The combination of these structural features makes them promising candidates for the treatment of a variety of conditions, spanning from cancer and inflammatory disorders to parasite infections. The exacerbated anomeric effect associated to the putative sp2-hybridized N-atom imparts chemical and enzymatic stability to sp2-IGLs and warrants total α-anomeric stereoselectivity in the key glycoconjugation step. A variety of O-, N-, C- and S-pseudoglycosides, differing in glycone configurational patterns and lipid nature, have been previously prepared and evaluated. Here we expand the chemical space of sp2-IGLs by reporting the synthesis of α-d-gluco-configured analogs with a bicyclic (5N,6O-oxomethylidene)nojirimycin (ONJ) core incorporating selenium at the glycosidic position. Structure-activity relationship studies in three different scenarios, namely cancer, Leishmaniasis and inflammation, convey that the therapeutic potential of the sp2-IGLs is highly dependent, not only on the length of the lipid chain (linear aliphatic C12 vs. C8), but also on the nature of the glycosidic atom (nitrogen vs. sulfur vs. selenium). The ensemble of results highlights the α-dodecylseleno-ONJ-glycoside as a promising multitarget drug candidate.

Stereoselective Synthesis of Iminosugar 2-Deoxy(thio)glycosides from Bicyclic Iminoglycal Carbamates Promoted by Cerium(IV) Ammonium Nitrate and Cooperative Brønsted Acid-Type Organocatalysis.

The first examples of iminosugar-type 2-deoxy(thio)glycoside mimetics are reported. The key step is the activation of a bicyclic iminoglycal carbamate to generate a highly reactive acyliminium cation. Cerium(IV) ammonium nitrate efficiently promoted the formation of 2-deoxy S-glycosides in the presence of thiols, probably by in situ generation of catalytic HNO3, with complete α-stereoselectivity. Cooperative phosphoric acid/Schreiner's thiourea organocatalysis proved better suited for generating 2-deoxy O-glycosides, significantly broadening the scope of the approach.

Thiol-ene "Click" Synthesis and Pharmacological Evaluation of C-Glycoside sp2-Iminosugar Glycolipids.

The unique stereoelectronic properties of sp2-iminosugars enable their participation in glycosylation reactions, thereby behaving as true carbohydrate chemical mimics. Among sp2-iminosugar conjugates, the sp2-iminosugar glycolipids (sp2-IGLs) have shown a variety of interesting pharmacological properties ranging from glycosidase inhibition to antiproliferative, antiparasitic, and anti-inflammatory activities. Developing strategies compatible with molecular diversity-oriented strategies for structure-activity relationship studies was therefore highly wanted. Here we show that a reaction sequence consisting in stereoselective C-allylation followed by thiol-ene "click" coupling provides a very convenient access to α-C-glycoside sp2-IGLs. Both the glycone moiety and the aglycone tail can be modified by using sp2-iminosugar precursors with different configurational profiles (d-gluco or d-galacto in this work) and varied thiols, as well as by oxidation of the sulfide adducts (to the corresponding sulfones in this work). A series of derivatives was prepared in this manner and their glycosidase inhibitory, antiproliferative and antileishmanial activities were evaluated in different settings. The results confirm that the inhibition of glycosidases, particularly α-glucosidase, and the antitumor/leishmanicidal activities are unrelated. The data are also consistent with the two later activities arising from the ability of the sp2-IGLs to interfere in the immune system response in a cell line and cell context dependent manner.

sp2 -Iminosugar α-glucosidase inhibitor 1-C-octyl-2-oxa-3-oxocastanospermine specifically affected breast cancer cell migration through Stim1, ß1-integrin, and FAK signaling pathways.

Aberrant glycosylation changes on many glycoproteins are often related to cancer progression and metastasis. sp2 -Iminosugar-type castanospermine analogues, inhibitors of α-glucosidases, have been reported to exhibit antitumor activity. However, their effects on cell migration and the underlying molecular mechanism are not fully understood. Here, we investigated the effect of the pseudo-C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives (CO-OCS) on breast cancer cells (MCF-7 and MDA-MB-231 cells), and MCF-10A mammary normal cell lines. We showed that CO-OCS treatment results in the drastic decrease of breast cancer cell migration without affecting cell proliferation. Furthermore, CO-OCS significantly reduced both the expression of ß1-integrin, which is a crucial interacting partner of Focal Adhesion Kinase (FAK), and the phosphorylation rates of FAK and ERK1/2. CO-OCS also drastically reduced Ca2+ entry through Store Operated Channels (SOC). Orai1 and Stim1, two N-glycosylated proteins, are involved in Store-Operated Calcium Entry (SOCE), and are essential for breast tumor cell migration. Our results showed that CO-OCS decreased the expression, at the protein level, of Stim1 without affecting that of Orai1. Moreover, cell migration and SOCE were attenuated by CO-OCS as well as when Stim1 was silenced. In contrast, in MCF-10A cells, CO-OCS slightly reduced cell migration, but was without effect on gene expression of Stim1, Orai1, ß1-integrin, or FAK and ERK1/2 activation. Our results provide strong evidence for a significant effect of CO-OCS on breast cancer cell migration and support that this effect was associated with ß1-integrin, Stim1, and FAK signaling pathways.

Synthesis of multibranched australine derivatives from reducing castanospermine analogues through the Amadori rearrangement of gem-diamine intermediates: selective inhibitors of ß-glucosidase.

A practical one-pot synthesis of bi- and triantennated australine analogues from a pivotal sp(2)-iminosugar-type reducing castanospermine precursor is reported. The transformation involves a gem-diamine intermediate that undergoes the indolizidine → pyrrolizidine Amadori-type rearrangement and proceeds under strict control of the generalized anomeric effect to afford a single diastereomer. The final compounds behave as selective competitive inhibitors of ß-glucosidase and are promising candidates as pharmacological chaperones for Gaucher disease.

Investigations on the oxygen dependence of a 2-oxoglutarate histone demethylase.

Histone N(ϵ)-methyl lysine demethylases are important in epigenetic regulation. KDM4E (histone lysine demethylase 4E) is a representative member of the large Fe(II)/2-oxoglutarate- dependent family of human histone demethylases. In the present study we report kinetic studies on the reaction of KDM4E with O2. Steady-state assays showed that KDM4E has a graded response to O2 over a physiologically relevant range of O2 concentrations. Pre-steady state assays implied that KDM4E reacts slowly with O2 and that there are variations in the reaction kinetics which are dependent on the methylation status of the substrate. The results demonstrate the potential for histone demethylase activity to be regulated by oxygen availability.

A selenoureido-iminoglycolipid transported by zeolitic-imidazolate framework nanoparticles: a novel antioxidant therapeutic approach.

A selenium-containing metal-organic framework with remarkable antioxidant capacity and ROS-scavenging activity was constructed by a controlled de novo encapsulation approach of a glycoconjugate mimetic, specifically a sp2-iminoglycolipid bearing a selenoureido fragment (DSeU), within a zeolitic-imidazolate framework exoskeleton. Biocompatible and homogeneous nanosized particles of ∼70 nm (DSeU@ZIF8) were obtained, which could be efficiently internalized in cells, overcoming the poor solubility in biological media and limited bioavailability of glycolipids. The ZIF-particle served as nanocarrier for the intracellular delivery of the selenocompound to cells, promoted by the acidic pH inside endosomes/lysosomes. As demonstrated by in vitro studies, the designed DSeU@ZIF8 nanoparticles displayed a high antioxidant activity at low doses; lower intracellular ROS levels were observed upon the uptake of DSeU@ZIF8 by human endothelial cells. Even more interesting was the finding that these DSeU@ZIF8 particles were able to reverse to a certain level the oxidative stress induced in cells by pre-treatment with an oxidizing agent. This possibility of modulating the oxidative stress in living cells may have important implications in the treatment of diverse pathological complications that are generally accompanied with elevated ROS levels.

Fluoro-labelled sp2-iminoglycolipids with immunomodulatory properties.

The unique electronic properties of the fluorine atom make its strategic incorporation into a bioactive compound a very useful tool in the design of drugs with optimized pharmacological properties. In the field of the carbohydrates, its selective installation at C2 position has proven particularly interesting, some 2-deoxy-2-fluorosugar derivatives being currently in the market. We have now transferred this feature into immunoregulatory glycolipid mimetics that contain a sp2-iminosugar moiety, namely sp2-iminoglycolipids (sp2-IGLs). The synthesis of two epimeric series of 2-deoxy-2-fluoro-sp2-IGLs, structurally related to nojirimycin and mannonojirimycin, has been accomplished by sequential Selectfluor-mediated fluorination and thioglycosidation of sp2-iminoglycals. Exclusively the α-anomer is obtained regardless of the configurational profile of the sp2-IGL (d-gluco or d-manno), highlighting the overwhelming anomeric effect in these prototypes. Notably, the combination of a fluorine atom at C2 and an α-oriented sulfonyl dodecyl lipid moiety in compound 11 led to remarkable anti-proliferative properties, featuring similar GI50 values than the chemotherapy drug Cisplatin against several tumor cell lines and better selectivity. The biochemical data further evidence a strong reduction of the number of tumor cell colonies and apoptosis induction. Mechanistic investigations revealed that this fluoro-sp2-IGL induces the non-canonical activation mode of the mitogen-activated protein kinase signaling pathway, causing p38α autoactivation under an inflammatory context.

sp2-Iminosugar O-, S-, and N-glycosides as conformational mimics of α-linked disaccharides; implications for glycosidase inhibition.

The synthesis of mimics of the α(1→6)- and α(1→4)-linked disaccharides isomaltose and maltose featuring a bicyclic sp(2)-iminosugar nonreducing moiety O-, S-, or N-linked to a glucopyranoside residue is reported. The strong generalized anomeric effect operating in sp(2)-iminosugars determines the α-stereochemical outcome of the glycosylation reactions, independent of the presence or not of participating protecting groups and of the nature of the heteroatom. It also imparts chemical stability to the resulting aminoacetal, aminothioacetal, or gem-diamine functionalities. All the three isomaltose mimics behave as potent and very selective inhibitors of isomaltase and maltase, two α-glucosidases that bind the parent disaccharides either as substrate or inhibitor. In contrast, large differences in the inhibitory properties were observed among the maltose mimics, with the O-linked derivative being a more potent inhibitor than the N-linked analogue; the S-linked pseudodisaccharide did not inhibit either of the two target enzymes. A comparative conformational analysis based on NMR and molecular modelling revealed remarkable differences in the flexibility about the glycosidic linkage as a function of the nature of the linking atom in this series. Thus, the N-pseudodisaccharide is more rigid than the O-linked derivative, which exhibits conformational properties very similar to those of the natural maltose. The analogous pseudothiomaltoside is much more flexible than the N- or O-linked derivatives, and can access a broader area of the conformational space, which probably implies a strong entropic penalty upon binding to the enzymes. Together, the present results illustrate the importance of taking conformational aspects into consideration in the design of functional oligosaccharide mimetics.

Mannobioside biomimetics that trigger DC-SIGN binding selectivity.

Selective DC-SIGN targeting vs. langerin might lead to anti-infective agents, given their counteracting effects upon infection by some pathogens. Here we show that multivalent sp2-iminosugar-containing mannobioside analogs can achieve total DC-SIGN selectivity by levering the canonic binding mode towards high-mannose oligosaccharide ligands, behaving as factual biomimics.

Anti-Inflammatory (M2) Response Is Induced by a sp2-Iminosugar Glycolipid Sulfoxide in Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the most common complications of Diabetes Mellitus (DM) and is directly associated with inflammatory processes. Currently, neuro-inflammation is considered an early event in DR and proceeds via microglia polarization. A hallmark of DR is the presence of retinal reactive gliosis. Here we report the beneficial effect of (SS,1R)-1-docecylsulfiny-5N,6O-oxomethylidenenojirimycin ((Ss)-DS-ONJ), a member of the sp2-iminosugar glycolipid (sp2-IGL) family, by decreasing iNOS and inflammasome activation in Bv.2 microglial cells exposed to pro-inflammatory stimuli. Moreover, pretreatment with (Ss)-DS-ONJ increased Heme-oxygenase (HO)-1 as well as interleukin 10 (IL10) expression in LPS-stimulated microglial cells, thereby promoting M2 (anti-inflammatory) response by the induction of Arginase-1. The results strongly suggest that this is the likely molecular mechanism involved in the anti-inflammatory effects of (SS)-DS-ONJ in microglia. (SS)-DS-ONJ further reduced gliosis in retinal explants from type 1 diabetic BB rats, which is consistent with the enhanced M2 response. In conclusion, targeting microglia polarization dynamics in M2 status by compounds with anti-inflammatory activities offers promising therapeutic interventions at early stages of DR.

Amplified Detection of Breast Cancer Autoantibodies Using MUC1-Based Tn Antigen Mimics.

In many human carcinomas, mucin-1 (MUC1) is overexpressed and aberrantly glycosylated, resulting in the exposure of previously hidden antigens. This generates new patient antibody profiles that can be used in cancer diagnosis. In the present study, we focused on the MUC1-associated Tn antigen (α-O-GalNAc-Ser/Thr) and substituted the GalNAc monosaccharide by a glycomimic to identify MUC1-based glycopeptides with increased antigenicity. Two different glycopeptide libraries presenting the natural Tn antigen or the sp2-iminosugar analogue were synthesized and evaluated with anti-MUC1 monoclonal antibodies in a microarray platform. The most promising candidates were tested with healthy and breast cancer sera aiming for potential autoantibody-based biomarkers. The suitability of sp2-iminosugar glycopeptides to detect anti-MUC1 antibodies was demonstrated, and serological experiments showed stage I breast cancer autoantibodies binding with a specific unnatural glycopeptide with almost no healthy serum interaction. These results will promote further studies on their capabilities as early cancer biomarkers.

Synthesis, conformational analysis and in vivo assays of an anti-cancer vaccine that features an unnatural antigen based on an sp2-iminosugar fragment.

The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp2-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.

sp2-Iminosugar glycolipids as inhibitors of lipopolysaccharide-mediated human dendritic cell activation in vitro and of acute inflammation in mice in vivo.

Glycolipid mimetics consisting of a bicyclic polyhydroxypiperidine-cyclic carbamate core and a pseudoanomeric hydrophobic tail, termed sp2-iminosugar glycolipids (sp2-IGLs), target microglia during neuroinflammatory processes. Here we have synthesized and investigated new variants of sp2-IGLs for their ability to suppress the activation of human monocyte-derived dendritic cells (DCs) by lipopolysaccharide (LPS) signaling through Toll-like receptor 4. We report that the best lead was (1R)-1-dodecylsulfonyl-5N,6O-oxomethylidenenojirimycin (DSO2-ONJ), able to inhibit LPS-induced TNFα production and maturation of DCs. Immunovisualization experiments, using a mannoside glycolipid conjugate (MGC) that also suppress LPS-mediated DC activation as control, evidenced a distinct mode of action for the sp2-IGLs: unlike MGCs, DSO2-ONJ did not elicit internalization of the LPS co-receptor CD14 or induce its co-localization with the Toll-like receptor 4. In a mouse model of LPS-induced acute inflammation, DSO2-ONJ demonstrated anti-inflammatory activity by inhibiting the production of the pro-inflammatory interleukin-6. The ensemble of the data highlights sp2-IGLs as a promising new class of molecules against inflammation by interfering in Toll-like receptor intracellular signaling.

Synthesis of polyfluoroalkyl sp2-iminosugar glycolipids and evaluation of their immunomodulatory properties towards anti-tumor, anti-leishmanial and anti-inflammatory therapies.

Immunomodulatory glycolipids, among which α-galactosylceramide (KRN7000) is an iconic example, have shown strong therapeutic potential in a variety of conditions ranging from cancer and infection to autoimmune or neurodegenerative diseases. A main difficulty for those channels is that they often provoke a cytokine storm comprising both pro- and anti-inflammatory mediators that antagonize each other and negatively affect the immune response. The synthesis of analogues with narrower cytokine secretion-inducing capabilities is hampered by the intrinsic difficulty at controlling the stereochemical outcome in glycosidation reactions, particularly if targeting the α-anomer, which seriously hampers drug optimization strategies. Here we show that replacing the monosaccharide glycone by a sp2-iminosugar glycomimetic moiety allows accessing N-linked sp2-iminosugar glycolipids (sp2-IGLs) with total α-stereocontrol in a single step with no need of protecting groups or glycosidation promotors. The lipid tail has been then readily tailored by incorporating polyfluoroalkyl segments of varied lengths in view of favouring binding to the lipid binding site of the master p38 mitogen activated protein kinase (p38 MAPK), thereby polarizing the immune response in a cell-context dependent manner. The compounds have been evaluated for their antiproliferative, anti-leishmanial and anti-inflammatory activities in different cell assays. The size of the fluorous segment was found to be critical for the biological activity, probably by regulating the aggregation and membrane-crossing properties, whereas the hydroxylation profile (gluco or galacto-like) was less relevant. Biochemical and computational data further support a mechanism of action implying binding to the allosteric lipid binding site of p38 MAPK and subsequent activation of the noncanonical autophosphorylation route. The ensemble of results provide a proof of concept of the potential of sp2-IGLs as immunoregulators.

The sp2-iminosugar glycolipid 1-dodecylsulfonyl-5N,6O-oxomethylidenenojirimycin (DSO2-ONJ) as selective anti-inflammatory agent by modulation of hemeoxygenase-1 in Bv.2 microglial cells and retinal explants.

Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have reported the beneficial effects of 1R-DSO-ONJ, a member of the sp2-iminosugar glycolipid (sp2-IGL) family, in targeting microglia and reducing gliosis in diabetic db/db mice. Herein, we analyzed the effect of DSO2-ONJ, another family compound incorporating a sulfone group that better mimics the phosphate group of phosphatidylinositol ether lipid analogues (PIAs), in Bv.2 microglial cells treated with bacterial lipopolysaccaride (LPS) and in retinal explants from db/db mice. In addition to decreasing iNOS and inflammasome activation, the anti-inflammatory effect of DSO2-ONJ was mediated by direct p38α MAPK activation. Computational docking experiments demonstrated that DSO2-ONJ binds to p38α MAPK at the same site where PIAs and the alkyl phospholipid perifosine activators do, suggesting similar mechanism of action. Moreover, treatment of microglial cells with DSO2-ONJ increased both heme-oxygenase (HO)-1 and Il10 expression regardless the presence of LPS. In retinal explants from db/db mice, DSO2-ONJ also induced HO-1 and reduced gliosis. Since IL-10-mediated induction of HO-1 expression is mediated by p38α MAPK activation, our results suggest that this molecular mechanism is involved in the anti-inflammatory effects of DSO2-ONJ in microglia.

Fluorinated Chaperone-ß-Cyclodextrin Formulations for ß-Glucocerebrosidase Activity Enhancement in Neuronopathic Gaucher Disease.

Amphiphilic glycomimetics encompassing a rigid, undistortable nortropane skeleton based on 1,6-anhydro-l-idonojirimycin and a polyfluorinated antenna, when formulated as the corresponding inclusion complexes with ß-cyclodextrin (ßCD), have been shown to behave as pharmacological chaperones (PCs) that efficiently rescue lysosomal ß-glucocerebrosidase mutants associated with the neuronopathic variants of Gaucher disease (GD), including the highly refractory L444P/L444P and L444P/P415R single nucleotide polymorphs, in patient fibroblasts. The body of work here presented includes the design criteria for the PC prototype, the synthesis of a series of candidates, the characterization of the PC:ßCD complexes, the determination of the selectivity profiles toward a panel of commercial and human lysosomal glycosidases, the evaluation of the chaperoning activity in type 1 (non-neuronopathic), type 2 (acute neuronopathic), and type 3 (adult neuronopathic) GD fibroblasts, the confirmation of the rescuing mechanism by immunolabeling, and the analysis of the PC:GCase binding mode by docking experiments.