Investigation of the Molecular Details of the Interactions of Selenoglycosides and Human Galectin-3.

Human galectin-3 (hGal-3) is involved in a variety of biological processes and is implicated in wide range of diseases. As a result, targeting hGal-3 for clinical applications has become an intense area of research. As a step towards the development of novel hGal-3 inhibitors, we describe a study of the binding of two Se-containing hGal-3 inhibitors, specifically that of di(ß-D-galactopyranosyl)selenide (SeDG), in which two galactose rings are linked by one Se atom and a di(ß-D-galactopyranosyl)diselenide (DSeDG) analogue with a diseleno bond between the two sugar units. The binding affinities of these derivatives to hGal-3 were determined by 15N-1H HSQC NMR spectroscopy and fluorescence anisotropy titrations in solution, indicating a slight decrease in the strength of interaction for SeDG compared to thiodigalactoside (TDG), a well-known inhibitor of hGal-3, while DSeDG displayed a much weaker interaction strength. NMR and FA measurements showed that both seleno derivatives bind to the canonical S face site of hGal-3 and stack against the conserved W181 residue also confirmed by X-ray crystallography, revealing canonical properties of the interaction. The interaction with DSeDG revealed two distinct binding modes in the crystal structure which are in fast exchange on the NMR time scale in solution, explaining a weaker interaction with hGal-3 than SeDG. Using molecular dynamics simulations, we have found that energetic contributions to the binding enthalpies mainly differ in the electrostatic interactions and in polar solvation terms and are responsible for weaker binding of DSeDG compared to SeDG. Selenium-containing carbohydrate inhibitors of hGal-3 showing canonical binding modes offer the potential of becoming novel hydrolytically stable scaffolds for a new class of hGal-3 inhibitors.

Overcoming Microenvironment-Mediated Chemoprotection through Stromal Galectin-3 Inhibition in Acute Lymphoblastic Leukemia.

Environmentally-mediated drug resistance in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) significantly contributes to relapse. Stromal cells in the bone marrow environment protect leukemia cells by secretion of chemokines as cues for BCP-ALL migration towards, and adhesion to, stroma. Stromal cells and BCP-ALL cells communicate through stromal galectin-3. Here, we investigated the significance of stromal galectin-3 to BCP-ALL cells. We used CRISPR/Cas9 genome editing to ablate galectin-3 in stromal cells and found that galectin-3 is dispensable for steady-state BCP-ALL proliferation and viability. However, efficient leukemia migration and adhesion to stromal cells are significantly dependent on stromal galectin-3. Importantly, the loss of stromal galectin-3 production sensitized BCP-ALL cells to conventional chemotherapy. We therefore tested novel carbohydrate-based small molecule compounds (Cpd14 and Cpd17) with high specificity for galectin-3. Consistent with results obtained using galectin-3-knockout stromal cells, treatment of stromal-BCP-ALL co-cultures inhibited BCP-ALL migration and adhesion. Moreover, these compounds induced anti-leukemic responses in BCP-ALL cells, including a dose-dependent reduction of viability and proliferation, the induction of apoptosis and, importantly, the inhibition of drug resistance. Collectively, these findings indicate galectin-3 regulates BCP-ALL cell responses to chemotherapy through the interactions between leukemia cells and the stroma, and show that a combination of galectin-3 inhibition with conventional drugs can sensitize the leukemia cells to chemotherapy.

Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.

Bacterial infection is one of the leading causes of death in young, elderly, and immune-compromised patients. The rapid spread of multi-drug-resistant (MDR) bacteria is a global health emergency and there is a lack of new drugs to control MDR pathogens. We describe a heretofore-unexplored discovery pathway for novel antibiotics that is based on self-targeting, structure-disrupting peptides. We show that a helical peptide, KFF- EcH3, derived from the Escherichia coli methionine aminopeptidase can disrupt secondary and tertiary structure of this essential enzyme, thereby killing the bacterium (including MDR strains). Significantly, no detectable resistance developed against this peptide. Based on a computational analysis, our study predicted that peptide KFF- EcH3 has the strongest interaction with the structural core of the methionine aminopeptidase. We further used our approach to identify peptide KFF- NgH1 to target the same enzyme from Neisseria gonorrhoeae. This peptide inhibited bacterial growth and was able to treat a gonococcal infection in a human cervical epithelial cell model. These findings present an exciting new paradigm in antibiotic discovery using self-derived peptides that can be developed to target the structures of any essential bacterial proteins.-Zhan, J., Jia, H., Semchenko, E. A., Bian, Y., Zhou, A. M., Li, Z., Yang, Y., Wang, J., Sarkar, S., Totsika, M., Blanchard, H., Jen, F. E.-C., Ye, Q., Haselhorst, T., Jennings, M. P., Seib, K. L., Zhou, Y. Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.

Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate-binding domain (VP8*).

The outer capsid spike protein VP4 of rotaviruses is a major determinant of infectivity and serotype specificity. Proteolytic cleavage of VP4 into 2 domains, VP8* and VP5*, enhances rotaviral infectivity. Interactions between the VP4 carbohydrate-binding domain (VP8*) and cell surface glycoconjugates facilitate initial virus-cell attachment and subsequent cell entry. Our saturation transfer difference nuclear magnetic resonance (STD NMR) and isothermal titration calorimetry (ITC) studies demonstrated that VP8*64-224 of canine rotavirus strain K9 interacts with N-acetylneuraminic and N-glycolylneuraminic acid derivatives, exhibiting comparable binding epitopes to VP8* from other neuraminidase-sensitive animal rotaviruses from pigs (CRW-8), cattle (bovine Nebraska calf diarrhoea virus, NCDV), and Rhesus monkeys (Simian rhesus rotavirus, RRV). Importantly, evidence was obtained for a preference by K9 rotavirus for the N-glycolyl- over the N-acetylneuraminic acid derivative. This indicates that a VP4 serotype 5A rotavirus (such as K9) can exhibit a neuraminic acid receptor preference that differs from that of a serotype 5B rotavirus (such as RRV) and the receptor preference of rotaviruses can vary within a particular VP4 genotype.

Role of pentraxin 3 in shaping arthritogenic alphaviral disease: from enhanced viral replication to immunomodulation.

The rising prevalence of arthritogenic alphavirus infections, including chikungunya virus (CHIKV) and Ross River virus (RRV), and the lack of antiviral treatments highlight the potential threat of a global alphavirus pandemic. The immune responses underlying alphavirus virulence remain enigmatic. We found that pentraxin 3 (PTX3) was highly expressed in CHIKV and RRV patients during acute disease. Overt expression of PTX3 in CHIKV patients was associated with increased viral load and disease severity. PTX3-deficient (PTX3(-/-)) mice acutely infected with RRV exhibited delayed disease progression and rapid recovery through diminished inflammatory responses and viral replication. Furthermore, binding of the N-terminal domain of PTX3 to RRV facilitated viral entry and replication. Thus, our study demonstrates the pivotal role of PTX3 in shaping alphavirus-triggered immunity and disease and provides new insights into alphavirus pathogenesis.

Functional and structural characterization of a heparanase.

We report the structural and functional characterization of a novel heparanase (BpHep) from the invasive pathogenic bacterium Burkholderia pseudomallei (Bp), showing ∼24% sequence identity with human heparanase (hHep). Site-directed mutagenesis studies confirmed the active site resi-dues essential for activity, and we found that BpHep has specificity for heparan sulfate. Finally, we describe the first heparanase X-ray crystal structure, which provides new insight into both substrate recognition and inhibitor design.

Galectin-3-Binding Glycomimetics that Strongly Reduce Bleomycin-Induced Lung Fibrosis and Modulate Intracellular Glycan Recognition.

Discovery of glycan-competitive galectin-3-binding compounds that attenuate lung fibrosis in a murine model and that block intracellular galectin-3 accumulation at damaged vesicles, hence revealing galectin-3-glycan interactions involved in fibrosis progression and in intracellular galectin-3 activities, is reported. 3,3'-Bis-(4-aryltriazol-1-yl)thiodigalactosides were synthesized and evaluated as antagonists of galectin-1, -2, -3, and -4 N-terminal, -4 C-terminal, -7 and -8 N-terminal, -9 N-terminal, and -9 C-terminal domains. Compounds displaying low-nanomolar affinities for galectins-1 and -3 were identified in a competitive fluorescence anisotropy assay. X-ray structural analysis of selected compounds in complex with galectin-3, together with galectin-3 mutant binding experiments, revealed that both the aryltriazolyl moieties and fluoro substituents on the compounds are involved in key interactions responsible for exceptional affinities towards galectin-3. The most potent galectin-3 antagonist was demonstrated to act in an assay monitoring galectin-3 accumulation upon amitriptyline-induced vesicle damage, visualizing a biochemically/medically relevant intracellular lectin-carbohydrate binding event and that it can be blocked by a small molecule. The same antagonist administered intratracheally attenuated bleomycin-induced pulmonary fibrosis in a mouse model with a dose/response profile comparing favorably with that of oral administration of the marketed antifibrotic compound pirfenidone.

Substantial Receptor-induced Structural Rearrangement of Rotavirus VP8*: Potential Implications for Cross-Species Infection.

Rotavirus-cell binding is the essential first step in rotavirus infection. This binding is a major determinant of rotavirus tropism, as host cell invasion is necessary to initiate infection. Initial rotavirus-cell interactions are mediated by carbohydrate-recognizing domain VP8* of the rotavirus capsid spike protein VP4. Here, we report the first observation of significant structural rearrangement of VP8* from human and animal rotavirus strains upon glycan receptor binding. The structural adaptability of rotavirus VP8* delivers important insights into how human and animal rotaviruses utilize the wider range of cellular glycans identified as VP8* binding partners. Furthermore, our studies on rotaviruses with atypical genetic makeup provide information expected to be critical for understanding the mechanisms of animal rotavirus gene emergence in humans and support implementation of epidemiologic surveillance of animal reservoirs as well as future vaccination schemes.

Relative roles of GM1 ganglioside, N-acylneuraminic acids, and α2ß1 integrin in mediating rotavirus infection.

UNLABELLED: N-acetyl- and N-glycolylneuraminic acids (Sia) and α2ß1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl α-d-N-acetylneuraminide treatment, but did not alter α2ß1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidase-treated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of α2ß1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid. IMPORTANCE: Rotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.

Carbohydrate recognition by rotaviruses.

Rotaviruses are a global health concern causing severe childhood diarrhea. Though the mechanisms mediating rotavirus cell entry require further characterisation, it is acknowledged that an essential and critical step in rotavirus infection is the virus cell attachment via initial recognition of cell surface carbohydrate-containing receptors. This review summarises the current knowledge about recognition of glycan receptors by rotaviruses.

Galectin-1 as a potent target for cancer therapy: role in the tumor microenvironment.

The microenvironment of a tumor is a highly complex milieu, primarily characterized by immunosuppression, abnormal angiogenesis, and hypoxic regions. These features promote tumor progression and metastasis, resulting in poor prognosis and greater resistance to existing cancer therapies. Galectin-1 is a ß-galactoside binding protein that is abundantly secreted by almost all types of malignant tumor cells. The expression of galectin-1 is regulated by hypoxia-inducible factor-1 (HIF-1) and it plays vital pro-tumorigenic roles within the tumor microenvironment. In particular, galectin-1 suppresses T cell-mediated cytotoxic immune responses and promotes tumor angiogenesis. However, since galectin-1 displays many different activities by binding to a number of diverse N- or O-glycan modified target proteins, it has been difficult to fully understand how galectin-1 supports tumor growth and metastasis. This review explores the importance of galectin-1 and glycan expression patterns in the tumor microenvironment and the potential effects of inhibiting galectin-1 as a therapeutic target for cancer treatment.

Investigation into the feasibility of thioditaloside as a novel scaffold for galectin-3-specific inhibitors.

Galectin-3 is extensively involved in metabolic and disease processes, such as cancer metastasis, thus giving impetus for the design of specific inhibitors targeting this ß-galactose-binding protein. Thiodigalactoside (TDG) presents a scaffold for construction of galectin inhibitors, and its inhibition of galectin-1 has already demonstrated beneficial effects as an adjuvant with vaccine immunotherapy, thereby improving the survival outcome of tumour-challenged mice. A novel approach--replacing galactose with its C2 epimer, talose--offers an alternative framework, as extensions at C2 permit exploitation of a galectin-3-specific binding groove, thereby facilitating the design of selective inhibitors. We report the synthesis of thioditaloside (TDT) and crystal structures of the galectin-3 carbohydrate recognition domain in complexes with TDT and TDG. The different abilities of galactose and talose to anchor to the protein correlate with molecular dynamics studies, likely explaining the relative disaccharide binding affinities. The feasibility of a TDT scaffold to enable access to a particular galectin-3 binding groove and the need for modifications to optimise such a scaffold for use in the design of potent and selective inhibitors are assessed.

Structural basis of rotavirus strain preference toward N-acetyl- or N-glycolylneuraminic acid-containing receptors.

The rotavirus spike protein domain VP8* is essential for recognition of cell surface carbohydrate receptors, notably those incorporating N-acylneuraminic acids (members of the sialic acid family). N-Acetylneuraminic acids occur naturally in both animals and humans, whereas N-glycolylneuraminic acids are acquired only through dietary uptake in normal human tissues. The preference of animal rotaviruses for these natural N-acylneuraminic acids has not been comprehensively established, and detailed structural information regarding the interactions of different rotaviruses with N-glycolylneuraminic acids is lacking. In this study, distinct specificities of VP8* for N-acetyl- and N-glycolylneuraminic acids were revealed using biophysical techniques. VP8* protein from the porcine rotavirus CRW-8 and the bovine rotavirus Nebraska calf diarrhea virus (NCDV) showed a preference for N-glycolyl- over N-acetylneuraminic acids, in contrast to results obtained with rhesus rotavirus (RRV). Crystallographic structures of VP8* from CRW-8 and RRV with bound methyl-N-glycolylneuraminide revealed the atomic details of their interactions. We examined the influence of amino acid type at position 157, which is proximal to the ligand's N-acetyl or N-glycolyl moiety and can mutate upon cell culture adaptation. A structure-based hypothesis derived from these results could account for rotavirus discrimination between the N-acylneuraminic acid forms. Infectivity blockade experiments demonstrated that the determined carbohydrate specificities of these VP8* domains directly correlate with those of the corresponding infectious virus. This includes an association between CRW-8 adaption to cell culture, decreased competition by N-glycolylneuraminic acid for CRW-8 infectivity, and a Pro157-to-Ser157 mutation in VP8* that reduces binding affinity for N-glycolylneuraminic acid.

Novel Selective Galectin-3 Antagonists Are Cytotoxic to Acute Lymphoblastic Leukemia.

Galectin-3 is a ß-galactoside-specific, carbohydrate-recognizing protein (lectin) that is strongly implicated in cancer development, metastasis, and drug resistance. Galectin-3 promotes migration and ability to withstand drug treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Due to high amino acid conservation among galectins and the shallow nature of their glycan-binding site, the design of selective potent antagonists targeting galectin-3 is challenging. Herein, we report the design and synthesis of novel taloside-based antagonists of galectin-3 with enhanced affinity and selectivity. The molecules were optimized by in silico docking, selectivity was established against four galectins, and the binding modes were confirmed by elucidation of X-ray crystal structures. Critically, the specific inhibition of galectin-3-induced BCP-ALL cell agglutination was demonstrated. The compounds decreased the viability of ALL cells even when grown in the presence of protective stromal cells. We conclude that these compounds are promising leads for therapeutics, targeting the tumor-supportive activities of galectin-3 in cancer.

Thiodigalactoside inhibits murine cancers by concurrently blocking effects of galectin-1 on immune dysregulation, angiogenesis and protection against oxidative stress.

Cancer cells produce galectin-1 as a tumor promoting protein. Thiodigalactoside (TDG) as a non-metabolised small drug, is shown to suppress tumor growth by inhibiting multiple cancer enhancing activities of galectin-1, including immune cell dysregulation, angiogenesis and protection against oxidative stress. Thus, using B16F10 melanoma and 4T1 orthotopic breast cancer models, intratumoral injection of TDG significantly raised the levels of tumor-infiltrating CD8(+) lymphocytes and reduced CD31(+) endothelial cell content, reducing tumor growth. TDG treatment of tumors in Balb/c nude mice (defective in T cell immunity) reduced angiogenesis and slowed tumor growth by a third less than in immunocompetent mice. Knocking down galectin-1 expression (G1KD) in both cancer cell types significantly impeded tumor growth and the sensitivity of the G1KD tumors to TDG was severely reduced, highlighting a specific role for galectin-1. Endothelial cells were protected by galectin-1 from oxidative stress-induced apoptosis induced by H(2)O(2), but TDG inhibited this antioxidant protective effect of galectin-1 and reduced tube forming activity in angiogenic assays. We show for the first time that the single agent, TDG, concurrently prevents many tumor promoting effects of galectin-1 on angiogenesis, immune dysregulation and protection against oxidative stress, providing a potent and novel small molecule as an anti-cancer drug.

Sialic acid dependence in rotavirus host cell invasion.

We used NMR spectroscopy, molecular modeling and infectivity competition assays to investigate the key interactions between the spike protein (VP8(*)) from 'sialidase-insensitive' human Wa and 'sialidase-sensitive' porcine CRW-8 rotaviruses and the glycans of gangliosides G(M1) and G(D1a). Our data provide strong evidence that N-acetylneuraminic acid is a key determinant for binding of these rotaviruses. This is in contrast to the widely accepted paradigm that sialic acids are irrelevant in host cell recognition by sialidase-insensitive rotaviruses.

CuAAC synthesis of resorcin[4]arene-based glycoclusters as multivalent ligands of lectins.

Synthetic multivalent glycoclusters show promise as anti-adhesives for the treatment of bacterial infections. Here we report the synthesis of a family of tetravalent galactose and lactose functionalised macrocycles based on the resorcin[4]arene core. The development of diastereoselective synthetic routes for the formation of lower-rim propargylated resorcin[4]arenes and their functionalistion via Cu-catalyzed azide-alkyne click chemistry is described. ELLA binding studies confirm that galactose sugar clusters are effective ligands for the PA-IL bacterial lectin of Pseudomonas aeruginosa while poor binding for the lactose-based monovalent probe and no binding could be measured for the multivalent glycoclusters was observed for the human galectin-1.

Crystallization and preliminary X-ray crystallographic analysis of zebrafish prototype galectin Drgal1-L2.

Zebrafish (Danio rerio) are an important developmental and embryological model given the optical clarity of the embryos and larvae, which permits real-time viewing of developing pathologies. More recently, a broader scope for these vertebrates to model a range of human diseases, including some cancers, has been indicated. Zebrafish Drgal1-L2 has been identified as an orthologue of mammalian galectin-1, which is is a carbohydrate-binding protein that exhibits ß-galactoside-binding specificity and which is overexpressed by many aggressive human cancers. This study describes the cloning, expression in Escherichia coli, purification and crystallization of recombinant Drgal1-L2 protein in the presence of lactose (ligand). X-ray diffraction data from these novel crystals of zebrafish Drgal1-L2 were collected to a resolution of 1.5 Šusing a synchrotron-radiation source, enabling their characterization.

Linear triazole-linked pseudo oligogalactosides as scaffolds for galectin inhibitor development.

Galectins play key roles in numerous biological processes. Their mode of action depends on their localization which can be extracellular, cytoplasmic, or nuclear and is partly mediated through interactions with ß-galactose containing glycans. Galectins have emerged as novel therapeutic targets notably for the treatment of inflammatory disorders and cancers. This has stimulated the design of carbohydrate-based inhibitors targeting the carbohydrate recognition domains (CRDs) of the galectins. Pursuing this approach, we reasoned that linear oligogalactosides obtained by straightforward iterative click chemistry could mimic poly-lactosamine motifs expressed at eukaryote cell surfaces which the extracellular form of galectin-3, a prominent member of the galectin family, specifically recognizes. Affinities toward galectin-3 consistently increased with the length of the representative oligogalactosides but without reaching that of oligo-lactosamines. Elucidation of the X-ray crystal structures of the galectin-3 CRD in complex with a synthesized di- and tri-galactoside confirmed that the compounds bind within the carbohydrate-binding site. The atomic structures revealed that binding interactions mainly occur with the galactose moiety at the non-reducing end, primarily with subsites C and D of the CRD, differing from oligo-lactosamine which bind more consistently across the whole groove formed by the five subsites (A-E) of the galectin-3 CRD.

Rational Design and Synthesis of Methyl-ß-d-galactomalonyl Phenyl Esters as Potent Galectin-8N Antagonists.

Galectin-8 is a ß-galactoside-recognizing protein having an important role in the regulation of bone remodeling and cancer progression and metastasis. Methyl ß-d-galactopyranoside malonyl aromatic esters have been designed to target and engage with particular amino acid residues of the galectin-8N extended carbohydrate-binding site. The chemically synthesized compounds had in vitro binding affinity toward galectin-8N in the range of 5-33 µM, as evaluated by isothermal titration calorimetry. This affinity directly correlated with the compounds' ability to inhibit galectin-8-induced expression of chemokines and proinflammatory cytokines in the SUM159 breast cancer cell line. X-ray crystallographic structure determination revealed that these monosaccharide-based compounds bind galectin-8N by engaging its unique arginine (Arg59) and simultaneously cross-linking to another arginine (Arg45) located across the carbohydrate-binding site. This structure-based drug design approach has led to the discovery of novel monosaccharide galactose-based antagonists, with the strongest-binding compound (Kd 5.72 µM) holding 7-fold tighter than the disaccharide lactose.