Inhibitors and effectors of the glycosidases as therapeutic tools

Motivation: The lysosomal storage disorders (LSDs) conforms a group of over fifty monogenetic diseases characterized by the dysfunction of a lysosomal enzyme, frequently a glycosidase, and the subsequent accumulation of the corresponding substrate, which gives rise to several pathological manifestations. Individually they are rare diseases, but taken together they affect at 1 in 7,000 newborns. For some of these conditions enzyme replacement and/or substrate reduction therapeutic strategies (ERT and SRT, respectively) are available. Yet, most LSDs course with neurological deterioration and are refractory to ERT and SRT, remaining orphan diseases, sice the respective active principles are unable to cros the blood brain barrier (BBB). Ironically, in most of the LSD patients the causative mutation leads to the expression of a mutated ezyme that retains catalytic activity, but it is unable to properly fold at the endoplasmic reticulum (ER) and undertake the secretory pathway to the lysosome. Small molecules capable of promoting the correct folding and restore traficking, termed pharmacological chaperones (PCs), represents therefore a promising therapeutic option that is, in principle, better suited than ERT and SRT to target the neuronopathic forms. Methods: We have conducted research aiming at developing active site-directed pharmacologial chaperones for several LSDs based on glycomimetics (sugar lookaalikes). The molecular design implies a nitrogen-in-the-ring cycle bearing a hydroxylation profile that matches that of the monosaccharide cleave off by the target enzyme (Sánchez-Fernández et al. 2016). Such compounds can sit at the active site of the glycosidase, behaving as competitive inhibitors, subsequently inducing proper folding and trafficking. The chaperones are further elaborated to make them dissociate from the chaperone:enzyme complex at the lysosome, allowing substrate processing (Mena-Barragán et al. 2015).Results: PCs acting as glycosidase effectors in Gaucher, Fabry and GM1 gangliosidosis LSDs have been prepared based on the above concept.Conclusions: The ability of the PCs to cross the (BBB) and revert the accumulus of the substrate in the brain tissue (Takai et al. 2013) supports the promise of pharmacological chaperone therapy for a range of LSDs with neurological implications

Cyclodextrin-based multivalent glycodisplays: Covalent and supramolecular conjugates to assess carbohydrate-protein interactions

Covalent attachment of biorecognizable sugar ligands in several copies at precise positions of cyclomaltooligosaccharide (cyclodextrin, CD) macrocycles has proven to be an extremely flexible strategy to build multivalent conjugates. The commercial availability of the native CDs in three different sizes, their axial symmetry and the possibility of position- and face-selective functionalization allow a strict control of the valency and spatial orientation of the recognition motifs (glycotopes) in low, medium, high and hyperbranched glycoclusters, including glycodendrimer-CD hybrids. >Click-type> ligation chemistries, including copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC), thiol-ene coupling or thiourea-forming reactions, have been implemented to warrant full homogeneity of the adducts. The incorporation of different glycotopes to investigate multivalent interactions in heterogeneous environments has also been accomplished. Not surprisingly, multivalent CD conjugates have been, and continue to be, major actors in studies directed at deciphering the structural features ruling carbohydrate recognition events. Nanometric glycoassemblies endowed with the capability of adapting the inter-saccharide distances and orientations in the presence of a receptor partner or capable of mimicking the fluidity of biological membranes have been conceived by multitopic inclusion complex formation, rotaxanation or self-assembling. Applications in the fields of sensors, site-specific drug and gene delivery or protein stabilization attest for the maturity of the field.Ministerio de Economı´a y Competitividad CTQ2010-15848, SAF2010-1567

Cyclodextrin-scaffolded glycotransporters for gene delivery

Abstract: Conventional drugs consist of a formulation of a bioactive species and a carrier, the former accounting for most of the sophistication of the design. In the case of biomolecular drugs, however, the role of the carrier becomes decisive in enabling the load to reach its target to carry out its designed therapeutic function. Thus, the clinical success of gene therapy, where the active principles are nucleic acids, critically depends on the use of efficient and safe delivery systems. Carbohydrates have proven particularly useful in this regard. Glycocoating, similarly to poly(ethylene)glycol (PEG)-coating (pegylation), can stabilize colloidal aggregates by improving solvation and preventing nonspecific interactions, for example, with serum proteins. Moreover, glycoconjugates can drive specific recognition and receptor-mediated internalization in target cells. Actually, the inherent flexibility of carbohydrate and glycoconjugate chemistry has greatly contributed to enlarging the range of functional materials that can be rationally conceived for gene delivery. Herein, this is illustrated with selected examples that focus on controlling the architectural parameters of the vectors to make them suitable for structure¿activity relationship (SAR) and optimization studies. The members of the cyclomaltooligosaccharide (cyclodextrin, CD) family will be the central actors of the story.Peer Reviewe

Pharmacological chaperone therapy for Gaucher disease: A patent review

Introduction: Mutations in the gene encoding for acid β-glucosidase (β-glucocerebrosidase, GlcCerase) are seen in Gaucher disease (GD), which give rise to significant protein misfolding effects and result in progressive accumulation of glucosyl ceramide. The main treatment for GD is enzyme replacement therapy (ERT). The iminosugar glycosidase inhibitor N-(n-butyl)-1-deoxynojirimycin (miglustat, Zavesca™) is used in a second treatment modality known as substrate reduction therapy. At the beginning of the 21st century, a third therapeutic paradigm was launched, namely, pharmacological chaperone therapy (PCT). This therapeutic strategy relies on the capability of such inhibitors to promote the correct folding and stabilize mutant forms of lysosomal enzymes, such as GlcCerase, as they pass through the secretory pathway. Areas covered: This review summarizes the different approaches used to implement the concept of PCT for GD. It discusses the relevant research, patents and patent applications filed in the last decade. Expert opinion: While the significance of PCT remains a matter of debate, the great interest gathered regarding it in a relatively few years reflects its broad potential scope, well beyond GD. The fact that pharmacological chaperones can be designed to cross the blood brain barrier (BBB) make them candidates for the treatment of neuronopathic forms of GD that are not responsive to ERT. Combined therapies offer even broader possibilities that deserve to be fully explored.Ministerio de Ciencia e Innovación CTQ2007-61180/PPQ, SAF2010-15670Junta de Andalucía P08-FQM-0371

Glycomimetic-based pharmacological chaperones for lysosomal storage disorders: lessons from Gaucher, GM1-gangliosidosis and Fabry diseases

Lysosomal storage disorders (LSDs) are often caused by mutations that destabilize native folding and impair the trafficking of enzymes, leading to premature endoplasmic reticulum (ER)-associated degradation, deficiencies of specific hydrolytic functions and aberrant storage of metabolites in the lysosomes. Enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) are available for a few of these conditions, but most remain orphan. A main difficulty is that virtually all LSDs involve neurological decline and neither proteins nor the current SRT drugs can cross the blood–brain barrier. Twenty years ago a new therapeutic paradigm better suited for neuropathic LSDs was launched, namely pharmacological chaperone (PC) therapy. PCs are small molecules capable of binding to the mutant protein at the ER, inducing proper folding, restoring trafficking and increasing enzyme activity and substrate processing in the lysosome. In many LSDs the mutated protein is a glycosidase and the accumulated substrate is an oligo- or polysaccharide or a glycoconjugate, e.g. a glycosphingolipid. Although it might appear counterintuitive, substrate analogues (glycomimetics) behaving as competitive glycosidase inhibitors are good candidates to perform PC tasks. The advancements in the knowledge of the molecular basis of LSDs, including enzyme structures, binding modes, trafficking pathways and substrate processing mechanisms, have been put forward to optimize PC selectivity and efficacy. Moreover, the chemical versatility of glycomimetics and the variety of structures at hand allow simultaneous optimization of chaperone and pharmacokinetic properties. In this Feature Article we review the advancements made in this field in the last few years and the future outlook through the lessons taught by three archetypical LSDs: Gaucher disease, GM1-gangliosidosis and Fabry disease.The Spanish Ministerio de Economía y Competitividad (MINECO, contract numbers SAF2013-44021-R and CTQ2015-64425-C2-1-R), the Junta de Andalucía (contract number FQM-1467), and the European Union Seventh Framework Programme (FP7-People-2012-CIG), grant agreement number 333594 (to E. M. S. F., Marie Curie Reintegration Grant) are acknowledged. Cofinancing from the European Regional Development Funds (FEDER and FSE) is also thanked.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Harmonized tuning of nucleic acid and lectin binding properties with multivalent cyclodextrins for macrophage-selective gene delivery

Polycationic amphiphilic cyclodextrins (paCDs) have been shown to behave as efficient non-viral gene carriers paralleling the efficacy of commercial vectors towards a variety of cell lines. Their molecular framework and modular design allow the installation of saccharidic antennae to promote specific carbohydrate–protein interactions, thus potentially endowing them with selective targeting abilities. Yet, the presence of these additional functionalities onto the polycationic cluster may hamper paCD self-assembly and nucleic acid condensation. In this report we describe the influence of paCD mannosylation extent on paCD-pDNA nanocomplex stability as well as the consequences of varying glycotope density on mannose-specific lectin recognition and gene delivery capabilities. The work aims at exploring the potential of this approach to optimize both properties in order to modulate cell transfection selectivity.Ministerio de Economía y Competitividad SAF2013-44021-RJunta de Andalucía FQM-146

Synthesis of sugar oxazolines by intramolecular Ritter-like reaction of D-fructose precursors

Treatment of D-fructopyranose and D-fructofuranosc 1,2-O-acetonide derivatives with triflic acid in the presence of a variety of nitriles results in the formation of fused or spiro 2-oxazolines. The reaction implies (i) activation of the anomeric centre with simultaneous isopropylidene cleavage, (ii) nucleophilic addition of the nitrile to the corresponding oxocarbenium cation intermediate and (iii) subsequent trapping of the resulting nitrilium ion species by the remaining hydroxyl group in an intramolecular Ritter-like reaction

Conformationally-Locked C-Glycosides: Tuning Aglycone Interactions for Optimal Cheperone Behaviour in Gaucher Fibroblasts

A series of conformationally locked C-glycosides based on the 3-aminopyrano[3,2-b]pyrrol-2(1H)-one (APP) scaffold has been synthesized. The key step involved a totally stereocontrolled C-Michael addition of a serine-equivalent C-nucleophile to tri-O-benzyl-2-nitro-D-galactal, previously published by the authors. Stereoselective transformations of the Michael adduct allowed us the synthesis of compounds with mono- or diantennated aglycone moieties and different topologies. In vitro screening showed highly selective inhibition of bovine liver β-glucosidase/β-galactosidase and specific inhibition of human β-glucocerebrosidase among lysosomal glycosidases for compounds bearing palmitoyl chains in the aglycone, with a marked dependence of the inhibition potency upon their number and location. Molecular dynamics simulations highlighted the paramount importance of an optimal orientation of the hydrophobic substituent to warrant efficient non-glycone interactions, which are critical for the binding affinity. The results provide a rationale for the strong decrease of the inhibition potency of APP compounds on going from neutral to acidic pH. The best candidate was found to behave as pharmacological chaperone in Gaucher fibroblasts with homozygous N370S and F213I mutations, with enzyme activity enhancements similar to those encountered for the reference compound AmbroxolMinisterio de Economía y Competitividad CTQ2012-36365, SAF2013-44021-RJunta de Andalucía FQM-1467European Union Seventh Framework Programme FP7-People-2012-CI

Targeted gene delivery by new folate-polycationic amphiphilic cyclodextrin-DNA nanocomplexes in vitro and in vivo

Aim Development and evaluation of a new targeted gene delivery system by first preforming self-assembled nanocomplexes from a polycationic amphiphilic cyclodextrin (paCD) and pDNA and then decorating the surface of the nanoparticles with folic acid (FA). Experimental section The cyclodextrin derivative (T2) is a tetradecacationic structure incorporating 14 primary amino groups and 7 thioureido groups at the primary face of a cyclomaltoheptaose (β-CD) core and 14 hexanoyl chains at the secondary face. Results and conclusions T2 complexed and protected pDNA (luciferase-encoding plasmid DNA, pCMVLuc) and efficiently mediated transfection in vitro and in vivo with no associated toxicity. The combination of folic acid with CDplexes afforded ternary nanocomplexes (Fol-CDplexes) that enhanced significantly the transfection activity of pCMVLuc in human cervix adenocarcinoma HeLa cells, especially when formulated with 1 μg FA/μg DNA. The observed transfection enhancement was associated to specific folate receptor (FR)-mediated internalization of Fol-CDplexes, as corroborated by employing a receptor-deficient cell line (HepG2) and an excess of free folic acid. The in vivo studies, including luciferase reporter gene expression and biodistribution, indicated that 24 h after intravenous administration of the T2-pDNA nanocomplexes, transfection takes part mainly in the liver and partially in the lung. Interestingly, the corresponding Fol-CDplexes lead to an increase in the transfection activity in the lung and the liver compared to non-targeted CDplexes. Folate-CDplexes developed in this study have improved transfection efficiency and although various methods have been used for the preparation of ligand–DNA-complexes, covalent binding is usually needed and insoluble aggregates are formed unless the concentration of the components is minimized. However, the complexes developed by first time in this work were prepared by simple mixing. The synthetic nature of this formulation provides the potential of flexibility in terms of composition and the capability of inexpensive and large-scale production of the complexes. These nanovectors may be an adequate alternative to viral vectors for gene therapy in the future.Ministerio de Economía y Competitividad, CTQ2010 - 15848, SAF2010 - 1567