Multivalent inhibition of the Aspergillus fumigatus KDNase.

Aspergillus fumigatus is a saprophytic fungus and opportunistic pathogen often causing fatal infections in immunocompromised patients. Recently AfKDNAse, an exoglycosidase hydrolyzing 3-deoxy-D-galacto-D-glycero-nonulosonic acid (KDN), a rare sugar from the sialic acid family, was identified and characterized. The principal function of AfKDNAse is still unclear, but a study suggests a critical role in fungal cell wall morphology and virulence. Potent AfKDNAse inhibitors are required to better probe the enzyme's biological role and as potential antivirulence factors. In this work, we developed a set of AfKDNAse inhibitors based on enzymatically stable thio-KDN motifs. C2, C9-linked heterodi-KDN were designed to fit into unusually close KDN sugar binding pockets in the protein. A polymeric compound with an average of 54 KDN motifs was also designed by click chemistry. Inhibitory assays performed on recombinant AfKDNAse showed a moderate and strong enzymatic inhibition for the two classes of compounds, respectively. The poly-KDN showed more than a nine hundred fold improved inhibitory activity (IC50 = 1.52 ± 0.37 µM, 17-fold in a KDN molar basis) compared to a monovalent KDN reference, and is to our knowledge, the best synthetic inhibitor described for a KDNase. Multivalency appears to be a relevant strategy for the design of potent KDNase inhibitors. Importantly, poly-KDN was shown to strongly decrease filamentation when co-cultured with A. fumigatus at micromolar concentrations, opening interesting perspectives in the development of antivirulence factors.

ROMP-based Glycopolymers with High Affinity for Mannose-Binding Lectins.

Well-defined, highly reactive poly(norbornenyl azlactone)s of controlled length (number-average degree of polymerization DPn¯ = 10 to 1,000) were made by ring-opening metathesis polymerization (ROMP) of pure exo-norbornenyl azlactone. These were converted into glycopolymers using a facile postpolymerization modification (PPM) strategy based on click aminolysis of azlactone side groups by amino-functionalized glycosides. Pegylated mannoside, heptyl-mannoside, and pegylated glucoside were used in the PPM. Binding inhibition of the resulting glycopolymers was evaluated against a lectin panel (Bc2L-A, FimH, langerin, DC-SIGN, ConA). Inhibition profiles depended on the sugars and the degrees of polymerization. Glycopolymers from pegylated-mannoside-functionalized polynorbornene, with DPn¯ = 100, showed strong binding inhibition, with subnanomolar range inhibitory concentrations (IC50s). Polymers surpassed the inhibitory potential of their monovalent analogues by four to five orders of magnitude thanks to a multivalent (synergistic) effect. Sugar-functionalized poly(norbornenyl azlactone)s are therefore promising tools to study multivalent carbohydrate-lectin interactions and for applications against lectin-promoted bacterial/viral binding to host cells.

Polyvalent Transition-State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases*.

Bacterial sialidases (SA) are validated drug targets expressed by common human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, or Clostridium perfringens. Noncovalent inhibitors of bacterial SA capable of reaching the submicromolar level are rarely reported. In this work, multi- and polyvalent compounds are developed, based on the transition-state analogue 2-deoxy-2,3-didehydro-N-acetylneuraminic (DANA). Poly-DANA inhibits the catalytic activity of SA from S. pneumoniae (NanA) and the symbiotic microorganism B. thetaiotaomicron (BtSA) at the picomolar and low nanomolar levels (expressed in moles of molecules and of DANA, respectively). Each DANA grafted to the polymer surpasses the inhibitory potential of the monovalent analogue by more than four orders of magnitude, which represents the highest multivalent effect reported so far for an enzyme inhibition. The synergistic interaction is shown to operate exclusively in the catalytic domain, and not in the flanked carbohydrate-binding module (CBM). These results offer interesting perspectives for the multivalent inhibition of other SA families lacking a CBM, such as viral, parasitic, or human SA.

Hexavalent thiofucosides to probe the role of the Aspergillus fumigatus lectin FleA in fungal pathogenicity.

Aspergillus fumigatus is a pathogenic fungus infecting the respiratory system and responsible for a variety of life-threatening lung diseases. A fucose-binding lectin named FleA which has a controversial role in A. fumigatus pathogenesis was recently identified. New chemical probes with high affinity and enzymatic stability are needed to explore the role of FleA in the infection process. In this study, we developed potent FleA antagonists based on optimized and non-hydrolysable thiofucoside ligands. We first synthesized a set of monovalent sugars showing micromolar affinity for FleA by isothermal titration calorimetry. The most potent derivative was co-crystallized with FleA to gain insights into the binding mode in operation. Its chemical multimerization on a cyclodextrin scaffold led to an hexavalent compound with a significantly enhanced binding affinity (Kd = 223 ± 21 nM) thanks to a chelate binding mode. The compound could probe the role of bronchial epithelial cells in a FleA-mediated response to tissue invasion.

Tyrosine Conjugation Methods for Protein Labelling.

Over the last two decades, the development of chemical biology and the need for more defined protein conjugates have fostered active research on new bioconjugation techniques. In particular, a wide range of biorthogonal labelling strategies have been reported to functionalise the phenol side chain of tyrosines (Tyr). Tyr occur at medium frequency and are partially buried at the protein surface, offering interesting opportunities for site-selective labelling of the most reactive residues. Tyr-targeting has proved effective for designing a wide range of important biomolecules including antibody-drug conjugates, fluorescent or radioactive protein probes, glycovaccines, protein aggregates, and PEG conjugates. Innovative methods have also been reported for site-specific labelling with ligand-directed anchors and for the specific affinity capture of proteins. This review will present and discuss these promising alternatives to the conventional labelling of the nucleophilic lysine and cysteine residues.

Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases.

Sialidases (SAs) hydrolyze sialyl residues from glycoconjugates of the eukaryotic cell surface and are virulence factors expressed by pathogenic bacteria, viruses, and parasites. The catalytic domains of SAs are often flanked with carbohydrate-binding module(s) previously shown to bind sialosides and to enhance enzymatic catalytic efficiency. Herein, non-hydrolyzable multivalent thiosialosides were designed as probes and inhibitors of V. cholerae, T. cruzi, and S. pneumoniae (NanA) sialidases. NanA was truncated from the catalytic and lectinic domains (NanA-L and NanA-C) to probe their respective roles upon interacting with sialylated surfaces and the synthetically designed di- and polymeric thiosialosides. The NanA-L domain was shown to fully drive NanA binding, improving affinity for the thiosialylated surface and compounds by more than two orders of magnitude. Importantly, each thiosialoside grafted onto the polymer was also shown to reduce NanA and NanA-C catalytic activity with efficiency that was 3000-fold higher than that of the monovalent thiosialoside reference. These results extend the concept of multivalency for designing potent bacterial and parasitic sialidase inhibitors.

Di- and heptavalent nicotinic analogues to interfere with α7 nicotinic acetylcholine receptors.

In the field of nicotinic acetylcholine receptors (nAChRs), recognized as important therapeutic targets, much effort has been dedicated to the development of nicotinic analogues to agonize or antagonize distinct homo- and heteropentamers nAChR subtypes, selectively. In this work we developed di- and heptavalent nicotinic derivatives based on ethylene glycol (EG) and cyclodextrin cores, respectively. The compounds showed a concentration dependent inhibition of acetylcholine-induced currents on α7 nAChR expressed by Xenopus oocytes. Interesting features were observed with the divalent nicotinic derivatives, acting as antagonists with varied inhibitory concentrations (IC50) in function of the spacer arm length. The best divalent compounds showed a 16-fold lowered IC50 compared to the monovalent reference (12 vs 195 µM). Docking investigations provide guidelines to rationalize these experimental findings.

Electrochemically Promoted Tyrosine-Click-Chemistry for Protein Labeling.

The development of new bio-orthogonal ligation methods for the conjugation of native proteins is of particular importance in the field of chemical biology and biotherapies. In this work, we developed a traceless electrochemical method for protein bioconjugation. The electrochemically promoted tyrosine-click (e-Y-CLICK) allowed the chemoselective Y-modification of peptides and proteins with labeled urazoles. A low potential is applied in an electrochemical cell to activate urazole anchors in situ and on demand, without affecting the electroactive amino acids from the protein. The versatility of the electrosynthetic approach was shown on biologically relevant peptides and proteins such as oxytocin, angiotensin 2, serum bovine albumin, and epratuzumab. The fully conserved enzymatic activity of a glucose oxidase observed after e-Y-CLICK further highlights the softness of the method. The e-Y-CLICK protocols were successfully performed in pure aqueous buffers, without the need for co-solvents, scavenger or oxidizing chemicals, and should therefore significantly broaden the scope of bioconjugation.

Multivalent Fucosides with Nanomolar Affinity for the Aspergillus fumigatus Lectin FleA Prevent Spore Adhesion to Pneumocytes.

FleA (or AFL), a fucose lectin, was recently identified in the opportunistic mold Aspergillus fumigatus, which causes fatal lung infections in immunocompromised patients. We designed di-, hexa- and octavalent fucosides with various spacer arm lengths to block the hexameric FleA through chelation. Microcalorimetry measurements showed that the ethylene glycol (EG) spacer arm length has a strong influence on the binding affinity of the divalent fucosides. The relationship between the EG length and chelate binding efficiency to FleA was explored according to polymer theory. Hexa- and octavalent compounds based on cyclodextrin and octameric silsesquioxane scaffolds were nanomolar FleA inhibitors, surpassing their monovalent fucose analogue by more than three orders of magnitude. Importantly, some of the fucosides were highly efficient in preventing fungal spore adhesion to bronchoepithelial cells, with half maximal inhibitory concentration values in the micromolar range. We propose that the synergistic antiadhesive effect observed can be ascribed to chelate binding to FleA and to the formation of conidium aggregates, as observed by optical microscopy. These fucosides are promising tools that can be used to better understand the role of FleA in conidia pathogenicity and host defenses against invasive aspergillosis.

A Novel Integrated Way for Deciphering the Glycan Code for the FimH Lectin.

The fimbrial lectin FimH from uro- and enteropathogenic Escherichia coli binds with nanomolar affinity to oligomannose glycans exposing Manα1,3Man dimannosides at their non-reducing end, but only with micromolar affinities to Manα1,2Man dimannosides. These two dimannoses play a significantly distinct role in infection by E. coli. Manα1,2Man has been described early on as shielding the (Manα1,3Man) glycan that is more relevant to strong bacterial adhesion and invasion. We quantified the binding of the two dimannoses (Manα1,2Man and Manα1,3Man to FimH using ELLSA and isothermal microcalorimetry and calculated probabilities of binding modes using molecular dynamics simulations. Our experimentally and computationally determined binding energies confirm a higher affinity of FimH towards the dimannose Manα1,3Man. Manα1,2Man displays a much lower binding enthalpy combined with a high entropic gain. Most remarkably, our molecular dynamics simulations indicate that Manα1,2Man cannot easily take its major conformer from water into the FimH binding site and that FimH is interacting with two very different conformers of Manα1,2Man that occupy 42% and 28% respectively of conformational space. The finding that Manα1,2Man binding to FimH is unstable agrees with the earlier suggestion that E. coli may use the Manα1,2Man epitope for transient tethering along cell surfaces in order to enhance dispersion of the infection.

Multivalency To Inhibit and Discriminate Hexosaminidases.

A set of multivalent polyhydroxylated acetamidoazepanes based on ethylene glycol, glucoside, or cyclodextrin scaffolds was prepared. The compounds were assessed against plant, mammalian, and therapeutically relevant hexosaminidases. Multimerization was shown to improve the inhibitory potency with synergy, and to fine tune the selectivity profile between related hexosaminidases.

The Antiadhesive Strategy in Crohn's Disease: Orally Active Mannosides to Decolonize Pathogenic Escherichia coli from the Gut.

Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent-invasive E. coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10 mg kg(-1) ) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water-soluble and non-cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.

Recent advances in cancer treatment by iron chelators.

The development of new therapeutic alternatives for cancers is a major public health priority. Among the more promising approaches, the iron depletion strategy based on metal chelation in the tumoral environment has been particularly studied in recent decades. After a short description of the importance of iron for cancer cell proliferation, we will review the different iron chelators developed as potential chemotherapeutics. Finally, the recent efforts to vectorize the chelating agents specifically in the microtumoral environment will be discussed in detail.

Polymeric iminosugars improve the activity of carbohydrate-processing enzymes.

Multivalent iminosugars have recently emerged as powerful tools to inhibit the activities of specific glycosidases. In this work, biocompatible dextrans were coated with iminosugars to form linear and ramified polymers with unprecedently high valencies (from 20 to 900) to probe the evolution of the multivalent inhibition as a function of ligand valency. This study led to the discovery that polyvalent iminosugars can also significantly enhance, not only inhibit, the enzymatic activity of specific glycoside-hydrolase, as observed on two galactosidases, a fucosidase, and a bacterial mannoside phosphorylase for which an impressive 70-fold activation was even reached. The concept of glycosidase activation is largely unexplored, with a unique recent example of small-molecules activators of a bacterial O-GlcNAc hydrolase. The possibility of using these polymers as "artificial enzyme effectors" may therefore open up new perspectives in therapeutics and biocatalysis.

Glycopolymers as Antiadhesives of E. coli Strains Inducing Inflammatory Bowel Diseases.

n-Heptyl α-d-mannose (HM) is a nanomolar antagonist of FimH, a virulence factor of E. coli. Herein we report on the construction of multivalent HM-based glycopolymers as potent antiadhesives of type 1 piliated E. coli. We investigate glycopolymer/FimH and glycopolymer/bacteria interactions and show that HM-based glycopolymers efficiently inhibit bacterial adhesion and disrupt established cell-bacteria interactions in vitro at very low concentration (0.1 µM on a mannose unit basis). On a valency-corrected basis, HM-based glycopolymers are, respectively, 10(2) and 10(6) times more potent than HM and d-mannose for their capacity to disrupt the binding of adherent-invasive E. coli to T84 intestinal epithelial cells. Finally, we demonstrate that the antiadhesive capacities of HM-based glycopolymers are preserved ex vivo in the colonic loop of a transgenic mouse model of Crohn's disease. All together, these results underline the promising scope of HM-based macromolecular ligands for the antiadhesive treatment of E. coli induced inflammatory bowel diseases.

Synthesis and biological properties of Quilamines II, new iron chelators with antiproliferative activities.

To selectively target tumor cells expressing an overactive Polyamine Transport System (PTS), we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, derived from the lead compound HQ1-44, which we named Quilamines II. The structures of four new antiproliferative agents were developed. They differ in the size of the linker (HQ0-44 and HQ2-44) or in the nature of the linker (HQCO-44 and HQCS-44) between a hydroxyquinoline moiety (HQ) and a homospermidine (44) chain, the best polyamine vector. The Quilamines II were obtained after 6 to 9 steps by Michael addition, peptide linkage, and reductive amination or by using the Willgerodt-Kindler reaction. The biological evaluation of these second-generation Quilamines showed that modifying the size of the linker increased the selectivity of these compounds for the PTS. In addition, measurement of the toxicity of Quilamines HQ0-44 and HQ2-44 highlighted their marked antiproliferative nature on several cancerous cell lines as well as a differential activity on nontransformed cells (fibroblasts). In contrast, Quilamines HQCO-44 and HQCS-44 presented low selectivity for the PTS, probably due to a loss of electrostatic interaction. We also demonstrated that the HCT116 cell line, originating from a human colon adenocarcinoma, was the most responsive to the various Quilamines. As deduced from the calcein and HVA assays, the higher iron chelating capacity of HQ1-44 could explain its higher antiproliferative efficiency.

Multivalent inhibitors for carbohydrate-processing enzymes: beyond the "lock-and-key" concept.

During the last decades, tremendous chemical efforts have been dedicated to design monovalent inhibitors of carbohydrate-processing enzymes, with comparatively few rewards in terms of marketed drugs. Recently, an alternative to the traditional "lock and key" approach has emerged. Multivalency, a widely used strategy for lectin inhibition, has been successfully applied to specific glycosidases and glycosyltransferases.

Simple but precise engineering of functional nanocapsules through nanoprecipitation.

A general, rapid, and undemanding method to generate at will functional oil-filled nanocapsules through nanoprecipitation is reported. On the basis of polymer and hexadecane/water/acetone phase diagrams, the composition can be set so that polymer chains preferentially stick at the interface of the oil droplets to create nanocapsules. The nanocapsules can be decorated with biorelevant molecules (biotin, fluorescent tags, metal nanoparticles) within the shell and loaded with hydrophobic molecules in a simple one-pot procedure.

Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina.

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across different animal species, such as rodents, non-human primates, and humans. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.