Sialidase Inhibitor Research Articles

New insights into the immunomodulatory potential of sialic acid on monocyte-derived dendritic cells

Sialic acids at the cell surface of dendritic cells (DCs) play an important immunomodulatory role, and their manipulation enhances DC maturation, leading to heightened T cell activation. Particularly, at the molecular level, the increased stability of surface MHC-I molecules in monocyte-derived DCs (MoDCs) underpins an improved DC: T cell interaction. In this study, we focused on the impact of sialic acid remodelling by treatment with Clostridium perfringens sialidase on MoDCs' phenotypic and functional characteristics. Our investigation juxtaposes this novel approach with the conventional cytokine-based maturation regimen commonly employed in clinical settings.Notably, C. perfringens sialidase remarkably increased MHC-I levels compared to other sialidases having different specificities, supporting the idea that higher MHC-I is due to the cleavage of specific sialoglycans on cell surface proteins. Sialidase treatment induced rapid elevated surface expression of MHC-I, MHC-II and CD40 within an hour, a response not fully replicated by 48 h cytokine cocktail treatment. These increases were also observable 48 h post sialidase treatment. While CD86 and PD-L1 showed significant increases after 48 h of cytokine maturation, 48 h post sialidase treatment showed a higher increase in CD86 and shorter increase in PD-L1. CCR-7 expression was significantly increased 48 h after sialidase treatment but not significantly affected by cytokine maturation. Both treatments promoted higher secretion of the IL-12 cytokine. However, the cytokine cocktail induced a more pronounced IL-12 production. SNA lectin staining analysis demonstrated that the sialic acid profile is significantly altered by sialidase treatment, but not by the cytokine cocktail, which causes only slight sialic acid upregulation. Notably, the lipid-presenting molecules CD1a, CD1b and CD1c remained unaffected by sialidase treatment in MoDCs, a finding also further supported by experiments performed on C1R cells. Inhibition of endogenous sialidases Neu1 and Neu3 during MoDC differentiation did not affect surface MHC-I expression and cytokine secretion. Yet, sialidase activity in MoDCs was minimal, suggesting that sialidase inhibition does not significantly alter MHC-I-related functions. Our study highlights the unique maturation profile induced by sialic acid manipulation in MoDCs. These findings provide insights into the potential of sialic acid manipulation as a rapid immunomodulatory strategy, offering promising avenues for targeted interventions in inflammatory contexts.

A “terminal” case of glycan catabolism: Structural and enzymatic characterization of the sialidases of Clostridium perfringens

Sialic acids are commonly found on the terminal ends of biologically important carbohydrates, including intestinal mucin O-linked glycans. Pathogens such as Clostridium perfringens, the causative agent of necrotic enteritis (NE) in poultry and humans, have the ability to degrade host mucins and colonize the mucus layer, which involves removal of the terminal sialic acid by carbohydrate-active enzymes (CAZymes). Here we present the structural and biochemical characterization of the GH33 catalytic domains of the three sialidases of C. perfringens and probe their substrate specificity. The catalytically active domains, which we refer to as NanHGH33, NanJGH33, and NanIGH33, displayed differential activity on various naturally occurring forms of sialic acid. We report the X-ray crystal structures of these domains in complex with relevant sialic acid variants revealing the molecular basis of how each catalytic domain accommodates different sialic acids. NanHGH33 displays a distinct preference for α-2,3-linked sialic acid, but can process α-2,6-linked sialic acid. NanJGH33 and NanIGH33 both exhibit the ability to process α-2,3- and α-2,6-linked sialic acid without any significant apparent preference. All three enzymes were sensitive to generic and commercially available sialidase inhibitors, which impeded sialidase activity in cultures as well as the growth of C. perfringens on sialylated glycans. The knowledge gained in these studies can be applied to in vivo models for C. perfringens growth and metabolism of mucin O-glycans, with a view towards future mitigation of bacterial colonization and infection of intestinal tissues.

Oxazolinone and benzylidene-imidazole derivatives as sialidase inhibitors: Developing novel agents for atherosclerosis treatment

Oxazolinone and benzylidene-imidazole derivatives as sialidase inhibitors: Developing novel agents for atherosclerosis treatment

Novel sialidase inhibitors suppress mumps virus replication and infection.

The prevalent human pathogen, mumps virus (MuV; orthorubulavirus parotitidis) causes various complications and serious sequelae, such as meningitis, encephalitis, deafness, and impaired fertility. Direct-acting antivirals (DAAs) targeting MuV which can prevent mumps and mumps-associated complications and sequelae are yet to be developed. Paramyxoviridae family members, such as MuV, possess viral surface hemagglutinin-neuraminidase (HN) protein with sialidase activity which facilitates efficient viral replication. Therefore, to develop DAAs targeting MuV we synthesized MuV sialidase inhibitors. It is proposed that the viral HN has a single functional site for N-acetylneuraminic acid (Neu5Ac) binding and sialidase activity. Further, the known MuV sialidase inhibitor is an analog of Neu5Ac-2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA)-which lacks potency. DANA derivatives with higher MuV sialidase inhibitory potency are lacking. The MuV-HN-Neu5Ac binding site has a hydrophobic cavity adjacent to the C4 position of Neu5Ac. Exploiting this, here, we synthesized DANA derivatives with increasing hydrophobicity at its C4 position and created 3 novel sialidase inhibitors (Compounds 1, 2, and 3) with higher specificity for MuV-HN than DANA; they inhibited MuV replication step to greater extent than DANA. Furthermore, they also inhibited hemagglutination and the MuV infection step. The insight-that these 3 novel DANA derivatives possess linear hydrocarbon groups at the C4-hydroxyl group of DANA-could help develop highly potent sialidase inhibitors with high specificity for MuV sialidase, which may function as direct-acting MuV-specific antivirals.

Complement C1q-mediated microglial synaptic elimination by enhancing desialylation underlies sevoflurane-induced developmental neurotoxicity

BackgroundRepeated neonatal sevoflurane exposures led to neurocognitive disorders in young mice. We aimed to assess the role of microglia and complement C1q in sevoflurane-induced neurotoxicity and explore the underlying mechanisms.MethodsNeonatal mice were treated with sevoflurane on postnatal days 6, 8, and 10, and the Morris water maze was performed to assess cognitive functions. For mechanistic explorations, mice were treated with minocycline, C1q-antibody ANX005, and sialidase-inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA) before sevoflurane exposures. Western blotting, RT-qPCR, Golgi staining, 3D reconstruction and engulfment analysis, immunofluorescence, and microglial morphology analysis were performed. In vitro experiments were conducted in microglial cell line BV2 cells.ResultsRepeated neonatal sevoflurane exposures resulted in deficiencies in learning and cognition of young mice, accompanied by microglial activation and synapse loss. Sevoflurane enhanced microglia-mediated synapse elimination through C1q binding to synapses. Inhibition of microglial activation and phagocytosis with minocycline significantly reduced the loss of synapses. We further revealed the involvement of neuronal sialic acids in this process. The enhanced activity of sialidase by sevoflurane led to the loss of sialic acids, which facilitated C1q binding to synapses. Inhibition of C1q with ANX005 or inhibition of sialidase with NADNA significantly rescued microglia-mediated synapse loss and improved neurocognitive function. Sevoflurane enhanced the engulfment of BV2 cells, which was reversed by ANX005.ConclusionsOur findings demonstrated that C1q-mediated microglial synaptic elimination by enhancing desialylation contributed to sevoflurane-induced developmental neurotoxicity. Inhibition of C1q or sialidase may be a potential therapeutic strategy for this neurotoxicity.

Effective inhibition of bacterial sialidases by phenolic acids and flavonoids

As a pathogenicity factor in some microorganisms, sialidase is a key target for inactivation, as this would have curative and preventive effects on various diseases. Significant results are already achieved with viral sialidase inhibitors, while such studies on bacterial enzymes are scarce. Pure natural compounds representing phenols and flavonoids, were tested for their inhibitory effect on sialidases from Vibrio cholerae non-O1, Arthrobacter nicotianae and Oerskovia paurometabola. All three enzymes were isolated, purified beforehand and stored under suitable conditions. Quinic and gallic acids showed the highest inhibitory activity - 76 to 100% against the three sialidases. Fisetin had a significant inhibitory activity on two of the enzymes. The structurally related thymol and thymoquinone exerted from 80 to 100% inhibition on at least one of the enzymes. Catechin and rutin had significant inhibitory activity, varying from 49 to 100%, on some of the enzymes. Quercetin, known for its inhibitory effect on viral sialidases, had a lesser impact on the studied enzymes. The suppressive effect of quinic acid, rutin and fisetin on bacterial sialidases is observed for the first time.

The role of sialidases in the pathogenesis of bacterial vaginosis and their use as a promising pharmacological target in bacterial vaginosis.

Bacterial vaginosis (BV) is an infection of the genital tract characterized by disturbance of the normally Lactobacilli-dominated vaginal flora due to the overgrowth of Gardnerella and other anaerobic bacteria. Gardnerella vaginalis, an anaerobic pathogen and the major pathogen of BV, produces sialidases that cleave terminal sialic acid residues off of human glycans. By desialylation, sialidases not only alter the function of sialic acid-containing glycoconjugates but also play a vital role in the attachment, colonization and spread of many other vaginal pathogens. With known pathogenic effects, excellent performance of sialidase-based diagnostic tests, and promising therapeutic potentials of sialidase inhibitors, sialidases could be used as a biomarker of BV. This review explores the sources of sialidases and their role in vaginal dysbiosis, in aims to better understand their participation in the pathogenesis of BV and their value in the diagnosis and treatment of BV.

CD11B/CD18 SIALYLATION REGULATES NEUTROPHIL TRAFFICKING AND INFLAMMATORY FUNCTION IN THE INTESTINE

Abstract Pathogen-triggered neutrophil (PMN) recruitment is critical for innate immunity, but excessive PMN influx and associated mucosal tissue damage is implicated in the pathogenesis of numerous human inflammatory disorders including inflammatory bowel disease (IBD). Critically in both ulcerative colitis and Crohn’s disease, disease flares requiring medical intervention are associated with migration of large numbers of PMNs across colonic epithelium resulting in mucosal injury/ulceration. Recent studies have shown that specific PMN glycans can be targeted to reduce transepithelial migration (TEpM) the critical final step in PMN intestinal trafficking. However the role of Sialic acid (Sia), the most abundant PMN expressed terminal glycan, in regulating PMN migration and inflammatory function in the gut is not well understood. Using sophisticated models of human and murine PMN intestinal migration, we show that blocking sialidase-mediated removal of alpha 2-3 linked sialic acid from CD11b/CD18 inhibits PMN TEpM in vitro and in vivo.Measurement of cytochrome C reduction revealed that inhibition of sialidases reduced bacterial peptide (fMLF) stimulated superoxide release by human and murine PMN. Flow cytometry analyses revealed that sialidase inhibition decreased fMLF mediated PMN degranulation and fMLF mediated CD11b/CD18 conformational activation. Highlighting the functional importance of individual Sia residues, inhibition of alpha 2-3 sialidase activity with 3’ Sialyllactose (3’SL) was found to inhibit PMN intestinal trafficking in vitro and in vivo and reduce PMN degranulation responses. Immunoblotting studies showed that sialidase inhibition deactivates spleen tyrosine kinase (Syk) and p38 MAP kinase, key intracellular mediators that signal downstream of PMN CD11b/CD18. Finally, inhibition of sialidase activity reduced adhesion of PMN to ICAM-1 (a major physiological substrate of CD11b/CD18) demonstrating that endogenous PMN sialidases regulate binding interactions between migrating PMN and intestinal epithelial receptors. Taken together data show that upon activation PMN sialidases remove alpha 2-3 Sia from CD11b/CD18 which leads to integrin activation and subsequent outside in signaling mediated by Syk to drive PMN inflammatory effector functions including migration, degranulation, and superoxide release. As such targeting CD11b/CD18 sialylation represents a promising new therapeutic strategy for reducing dysregulated PMN influx and associated mucosal tissue damage in IBD and other inflammatory disorders.

Enabling Chemoenzymatic Strategies and Enzymes for Synthesizing Sialyl Glycans and Sialyl Glycoconjugates.

Sialic acids are fascinating negatively charged nine-carbon monosaccharides. Sialic acid-containing glycans and glycoconjugates are structurally diverse, functionally important, and synthetically challenging molecules. We have developed highly efficient chemoenzymatic strategies that combine the power of chemical synthesis and enzyme catalysis to make sialic acids, sialyl glycans, sialyl glycoconjugates, and their derivatives more accessible, enabling the efforts to explore their functions and applications. The Account starts with a brief description of the structural diversity and the functional importance of naturally occurring sialic acids and sialosides. The development of one-pot multienzyme (OPME) chemoenzymatic sialylation strategies is then introduced, highlighting its advantages in synthesizing structurally diverse sialosides with a sialyltransferase donor substrate engineering tactic. With the strategy, systematic access to sialosides containing different sialic acid forms with modifications at C3/4/5/7/8/9, various internal glycans, and diverse sialyl linkages is now possible. Also briefly described is the combination of the OPME sialylation strategy with bacterial sialidases for synthesizing sialidase inhibitors. With the goal of simplifying the product purification process for enzymatic glycosylation reactions, glycosphingolipids that contain a naturally existing hydrophobic tag are attractive targets for chemoenzymatic total synthesis. A user-friendly highly efficient chemoenzymatic strategy is developed which involves three main processes, including chemical synthesis of lactosyl sphingosine as a water-soluble hydrophobic tag-containing intermediate, OPME enzymatic extension of its glycan component with a single C18-cartridge purification of the product, followed by a facile chemical acylation reaction. The strategy allows the introduction of different sialic acid forms and diverse fatty acyl chains into the products. Gram-scale synthesis has been demonstrated. OPME sialylation has also been demonstrated for the chemoenzymatic synthesis of sialyl glycopeptides and in vitro enzymatic N-glycan processing for the formation of glycoproteins with disialylated biantennary complex-type N-glycans. For synthesizing human milk oligosaccharides (HMOs) which are glycans with a free reducing end, acceptor substrate engineering and process engineering strategies are developed, which involve the design of a hydrophobic tag that can be easily installed into the acceptor substrate to allow facile purification of the product from enzymatic reactions and can be conveniently removed in the final step to produce target molecules. The process engineering involves heat-inactivation of enzymes in the intermediate steps in multistep OPME reactions for the production of long-chain sialoside targets in a single reaction pot and with a single C18-cartridge purification process. In addition, a chemoenzymatic synthon strategy has been developed. It involves the design of a derivative of the sialyltransferase donor substrate precursor, which is tolerated by enzymes in OPME reactions, introduced to enzymatic products, and then chemically converted to the desired target structures in the final step. The chemoenzymatic synthon approach has been used together with the acceptor substrate engineering method in the synthesis of complex bacterial glycans containing sialic acids, legionaminic acids, and derivatives. The biocatalysts characterized and their engineered mutants developed by the Chen group are described, with highlights on synthetically useful enzymes. We anticipate further development of chemoenzymatic strategies and biocatalysts to enable exploration of the sialic acid space.

Successful treatment with oseltamivir phosphate in children with ITP who failed first-line therapy: a case series report.

Immune thrombocytopenia (ITP) is a common bleeding disorder in children. First-line medicines (glucocorticoids and immunoglobulin) may not be effective for some children, endangering their lives, posing challenges for healthcare facilities, and leading to an unfavorable prognosis. As a sialidase inhibitor, oseltamivir phosphate can reduce the destruction of platelets in liver macrophages by inhibiting the sialylation of platelets, and finally achieve the purpose of increasing platelet count. In this paper, three cases of children with ITP who failed first-line therapy and were cured by oral administration of oseltamivir phosphate granules were reported. The mechanism of action of oseltamivir phosphate granules was clarified.

Computational screening and MM/GBSA-based MD simulation studies reveal the high binding potential of FDA-approved drugs against Cutibacterium acnes sialidase

Cutibacterium acnes is an opportunistic pathogen linked with acne vulgaris, affecting 80–90% of teenagers globally. On the leukocyte (WBCs) cell surface, the cell wall anchored sialidase in C. acnes virulence factor, catalysing the sialoconjugates into sialic acids and nutrients for C. acnes resulting in human skin inflammation. The clinical use of antibiotics for acne treatments has severe adverse effects, including microbial dysbiosis and resistance. Therefore, identifying inhibitors for primary virulence factors (Sialidase) was done using molecular docking of 1030 FDA-approved drugs. Initially, based on binding energies (ΔG), Naloxone (ZINC000000389747), Fenoldopam (ZINC000022116608), Labetalol (ZINC000000403010) and Thalitone (ZINC000000057255) were identified that showed high binding energies as −10.2, −10.1, −9.9 and −9.8 kcal/mol, respectively. In 2D analysis, these drugs also showed considerable structural conformer of hydrogen and hydrophobic interactions. Further, a 100 ns MD simulation study found the lowest deviation and fluctuations with various intermolecular interactions to stabilise the complexes. Out of 4, the Naloxone molecule showed robust, steady, and stable RMSD 0.23 ± 0.18 nm. Further, MMGBSA analysis supports MD results and found strong binding energy (ΔG) −29.71 ± 4.97 kcal/mol. In Comparative studies with Neu5Ac2en (native substrate) revealed naloxone has a higher affinity for sialidase. The PCA analysis showed that Naloxone and Thalitone were actively located on the active site, and other compounds were flickered. Our extensive computational and statistical report demonstrates that these FDA drugs can be validated as potential sialidase inhibitors. Communicated by Ramaswamy H. Sarma

Exploring the inhibition of the soluble lytic transglycosylase Cj0843c of Campylobacter jejuni via targeting different sites with different scaffolds.

Bacterial lytic transglycosylases (LTs) contribute to peptidoglycan cell wall metabolism and are potential drug targets to potentiate β-lactam antibiotics to overcome antibiotic resistance. Since LT inhibitor development is underexplored, we probed fifteen N-acetyl-containing heterocycles in a structure-guided fashion for their ability to inhibit and bind to the Campylobacter jejuni LT Cj0843c. Ten GlcNAc analogs were synthesized with substitutions at the C1 position, with two having an additional modification at the C4 or C6 position. Most of the compounds showed weak inhibition of Cj0843c activity. Compounds with alterations at the C4 position, replacing the -OH with a -NH2 , and C6 position, the addition of a -CH3 , yielded improved inhibitory efficacy. All ten GlcNAc analogs were crystallographically analyzed via soaking experiments using Cj0843c crystals and found to bind to the +1 +2 saccharide subsites with one of them additionally binding to the -2 -1 subsite region. We also probed other N-acetyl-containing heterocycles and found that sialidase inhibitors N-acetyl-2,3-dehydro-2-deoxyneuraminic acid and siastatin B inhibited Cj0843c weakly and crystallographically bound to the -2 -1 subsites. Analogs of the former also showed inhibition and crystallographic binding and included zanamivir amine. This latter set of heterocycles positioned their N-acetyl group in the -2 subsite with additional moieties interacting in the -1 subsite. Overall, these results could provide novel opportunities for LT inhibition via exploring different subsites and novel scaffolds. The results also increased our mechanistic understanding of Cj0843c regarding peptidoglycan GlcNAc subsite binding preferences and ligand-dependent modulation of the protonation state of the catalytic E390. This article is protected by copyright. All rights reserved.

Sialic acid exacerbates gut dysbiosis-associated mastitis through the microbiota-gut-mammary axis by fueling gut microbiota disruption

BackgroundMastitis is one of the most severe diseases in humans and animals, especially on dairy farms. Mounting evidence indicates that gastrointestinal dysbiosis caused by induction of subacute ruminal acidosis (SARA) by high-grain diet consumption and low in dietary fiber is associated with mastitis initiation and development, however, the underlying mechanism remains unknown.ResultsIn the present study, we found that cows with SARA-associated mastitis have altered metabolic profiles in the rumen, with increased sialic acids level in particular. Consumption of sialic acid (SA) in antibiotic-treated mice, but not healthy mice, induced marked mastitis. SA treatment of antibiotic-treated mice also induced mucosal and systemic inflammatory responses, as evidenced by increased colon and liver injuries and several inflammatory markers. In addition, gut dysbiosis caused by antibiotic impaired gut barrier integrity, which was aggravated by SA treatment. SA potentiated serum LPS level caused by antibiotic treatment, leading to increased activation of the TLR4-NF-κB/NLRP3 pathways in the mammary gland and colon. Moreover, SA facilitated gut dysbiosis caused by antibiotic, and especially enhanced Enterobacteriaceae and Akkermansiaceae, which correlated with mastitis parameters. Fecal microbiota transplantation from SA-antibiotic-treated mice mimicked mastitis in recipient mice. In vitro experiments showed that SA prompted Escherichia coli growth and virulence gene expression, leading to higher proinflammatory cytokine production in macrophages. Targeting the inhibition of Enterobacteriaceae by sodium tungstate or treating with the commensal Lactobacillus reuteri alleviated SA-facilitated mastitis. In addition, SARA cows had distinct ruminal microbial structure by the enrichment of SA-utilizing opportunistic pathogenic Moraxellaceae and the depletion of SA-utilizing commensal Prevotellaceae. Treating mice with the specific sialidase inhibitor zanamivir reduced SA production and Moraxellaceae abundance, and improved mastitis in mice caused by ruminal microbiota transplantation from cows with SARA-associated mastitis.ConclusionsThis study, for the first time, indicates that SA aggravates gut dysbiosis-induced mastitis by promoting gut microbiota disturbance and is regulated by commensal bacteria, indicating the important role of the microbiota-gut-mammary axis in mastitis pathogenesis and suggesting a potential strategy for mastitis intervention based on gut metabolism regulation.-WPXBcu1emhBxPP-wo4QqAVideo

Sialylation regulates neutrophil transepithelial migration, CD11b/CD18 activation, and intestinal mucosal inflammatory function.

Polymorphonuclear neutrophils (PMNs) play a critical role in clearing invading microbes and promoting tissue repair following infection/injury. However, dysregulated PMN trafficking and associated tissue damage is pathognomonic of numerous inflammatory mucosal diseases. The final step in PMN influx into mucosal lined organs (including the lungs, kidneys, skin, and gut) involves transepithelial migration (TEpM). The β2-integrin CD11b/CD18 plays an important role in mediating PMN intestinal trafficking, with recent studies highlighting that terminal fucose and GlcNAc glycans on CD11b/CD18 can be targeted to reduce TEpM. However, the role of the most abundant terminal glycan, sialic acid (Sia), in regulating PMN epithelial influx and mucosal inflammatory function is not well understood. Here we demonstrate that inhibiting sialidase-mediated removal of α2-3-linked Sia from CD11b/CD18 inhibits PMN migration across intestinal epithelium in vitro and in vivo. Sialylation was also found to regulate critical PMN inflammatory effector functions, including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition reduces bacterial peptide-mediated CD11b/CD18 activation in PMN and blocks downstream intracellular signaling mediated by spleen tyrosine kinase (Syk) and p38 MAPK. These findings suggest that sialylated glycans on CD11b/CD18 represent potentially novel targets for ameliorating PMN-mediated tissue destruction in inflammatory mucosal diseases.

A21 ROLE OF SIALIC ACID O-ACETYLATION IN MAINTAINING MUCUS INTEGRITY AND HOMEOSTASIS OF THE COLONIC MUCOSA

Abstract Background The mucus network provides innate immune defense to protect our gastrointestinal tract from pathogens, and promote homeostasis with our resident microbiota. This network is constituted by the mucin MUC2 (Muc2 in mouse), which is ~80% complex O-linked glycans by weight. Sialic acid (Sia) is a key capping monosaccharide on complex O-glycans which has recently been linked to preserving mucus integrity. Sia can undergo enzymatic modifications including the addition of O-acetyl groups. The 9-O-acetyltransferase CasD1 is responsible for the 9-OAc Sia variants. Functionally, the OAc-modification is known to inhibit microbial sialidase activities which may preserve Sia’s protective roles on mucins. However, the extent of these OAc modifications in human and murine Mucin-2, and how they influence mucus function is unclear. Purpose: To determine whether and how Sia O-acetylation on colonic mucus regulates mucus integrity, host-microbe interactions, and colitis susceptibility. Method We used viral-derived probes that target specific OAc-Sia analogues on mucus on sections from human feces and mouse feces and colon tissues to visualize their spatial arrangement and microbial interaction in situ. For glycomics, OAc-Sia analogues were quantitated on purified human MUC2 and mouse Muc2 by HPLC-MS after derivatization with 4,5-dimethyl-1,2-diaminobenzamine (DMBA). O-glycans were released via non-reductive ammonia-catalyzed β-elimination and analyzed by mass spectrometry. For in vivo work, we generated intestinal epithelial cell-specific Casd1 KO mice (Casd1flox/flox;VillinCre or IEC Casd1-/- mice) and analyzed their mucins. Sialidase activities were quantified in the supernatants of colon fecal materials from WT and IEC Casd1-/- mutants mice using a fluorogenic substrate 4-MU-NeuNAc. Colitis susceptibility was monitored using 1.5% w/v Dextran Sodium Sulfate (DSS). Result(s) We found Sias on both human MUC2 and murine Muc2 were heavily O-acetylated, with ~75% and ~45% of Sias having 9-OAc-based modification in humans and mice respectively, and were distributed throughout the niche and barrier layers of mucus in situ. IEC Casd1-/- mice were viable and healthy with knockdown confirmed by 9-OAc staining, western blot of protein lysates and mucins, and sialylomics. The mucus encapsulation appeared overall intact regardless of OAc status. However, IEC Casd1-/- mice showed heightened susceptibility to 1.5% DSS colitis, linked to thinning of the mucus in IEC Casd1-/- vs WT littermates after challenge. Consistent with the role of OAc Sia in sialidase inhibition, loss of OAc Sia was associated with increased sialidase activities as assessed by heightened 4 MU signal in fecal supernatants in WT vs littermate IEC Casd1-/- mice. O-glycomics also showed reduction in the number of sialylated O-glycan structures upon loss of 9-OAc Sia. Conclusion(s) Sia O-acetylation appears important in maintaining key aspects of Sia-dependent mucus function and protecting from inflammatory insult. Please acknowledge all funding agencies by checking the applicable boxes below: CCC Disclosure of Interest None Declared

Inhibitors of the Sialidase NEU3 as Potential Therapeutics for Fibrosis.

Fibrosing diseases are a major medical problem, and are associated with more deaths per year than cancer in the US. Sialidases are enzymes that remove the sugar sialic acid from glycoconjugates. In this review, we describe efforts to inhibit fibrosis by inhibiting sialidases, and describe the following rationale for considering sialidases to be a potential target to inhibit fibrosis. First, sialidases are upregulated in fibrotic lesions in humans and in a mouse model of pulmonary fibrosis. Second, the extracellular sialidase NEU3 appears to be both necessary and sufficient for pulmonary fibrosis in mice. Third, there exist at least three mechanistic ways in which NEU3 potentiates fibrosis, with two of them being positive feedback loops where a profibrotic cytokine upregulates NEU3, and the upregulated NEU3 then upregulates the profibrotic cytokine. Fourth, a variety of NEU3 inhibitors block pulmonary fibrosis in a mouse model. Finally, the high sialidase levels in a fibrotic lesion cause an easily observed desialylation of serum proteins, and in a mouse model, sialidase inhibitors that stop fibrosis reverse the serum protein desialylation. This then indicates that serum protein sialylation is a potential surrogate biomarker for the effect of sialidase inhibitors, which would facilitate clinical trials to test the exciting possibility that sialidase inhibitors could be used as therapeutics for fibrosis.

Modification of the C3-Position of 2,3-Dehydro-2-deoxy-N-acetylneuraminic Acid with An Acetic Acid Equivalent

Direct modification at the C-3 position of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) derivatives with malonates has been achieved by means of Mn(OAc)3-mediated oxidative coupling reaction, giving C3-modified DANA derivatives in good yield with high diastereo-selectivity. We also designed and synthesized 3-CH2COOH-DANA as a candidate sialidase inhibitor, but found that its inhibitory activity towards several sialidases was weak, possibly because the acidic functional group is located too close to the DANA skeleton.

Sialidase Inhibitors with Different Mechanisms.

Sialidases, or neuraminidases, are enzymes that catalyze the hydrolysis of sialic acid (Sia)-containing molecules, mostly removal of the terminal Sia (desialylation). By desialylation, sialidase can modulate the functionality of the target compound and is thus often involved in biological pathways. Inhibition of sialidases with inhibitors is an important approach for understanding sialidase function and the underlying mechanisms and could serve as a therapeutic approach as well. Transition-state analogues, such as anti-influenza drugs oseltamivir and zanamivir, are major sialidase inhibitors. In addition, difluoro-sialic acids were developed as mechanism-based sialidase inhibitors. Further, fluorinated quinone methide-based suicide substrates were reported. Sialidase product analogue inhibitors were also explored. Finally, natural products have shown competitive inhibiton against viral, bacterial, and human sialidases. This Perspective describes sialidase inhibitors with different mechanisms and their activities and future potential, which include transition-state analogue inhibitors, mechanism-based inhibitors, suicide substrate inhibitors, product analogue inhibitors, and natural product inhibitors.

B Initio and DFT Investigation of Effect of Substituent at the C7 Position of 4-Amino-DANA Sialidase Inhibitor

Sialic acid is the active site of neuraminidase protein, eventually it cleaves form its substrate via sialsyl cation intermediate and proliferates the viral infection to other cells. On account of weak binding affinity between substrate and receptor, the viral infection communicates to other cells and leads to mortality of humans. DANA is the first sialidase inhibitor formed by the dehydration of the C2 hydroxyl group of sialic acid. The replacement of hydroxyl group at C4 position of DANA by an amino group drastically increases the binding affinity and results 4-amino-DANA inhibitor, which is potent than parent DANA. Crystal structure of DANA shows that several binding sites remain free and it should be explored for more powerful sialidase inhibitors. The current study systematically investigates the effect of substituent on the C7 position of 4-amino-DANA in gas phase and solvent phase as well. X-Ray crystallographic study reveals that the C7 of glycerol side chain remains free. Hence, substituent effect at C7 analysis is carried in search of potent sialidase inhibitors. The abinitio and DFT investigation reveals that guanidinoand methyl group at C7 position drastically increases the binding affinity between substrate and receptor. Hence further investigation of methyl and guanidine derivatives of the 4-amino-DANA could act as a promising candidate for the design and development of sialidase inhibitors. Vaccination for the H1N1 is not effective due to the new viral mutagenic strains and hence, it cannot contain the viral infection.Therefore antiviral drugs will address the limitation of vaccination. The current finding of sialidase antiviral inhibitors will effectively contain the viral infection and prevent the morbidity.

Ab Initio and DFT Investigation of Effect of Substituent at the C7 Position of 4 Amino Sialidase Inhibitor

Sialic acid is the active site of neuraminidase protein, eventually it cleaves form its substrate via sialsyl cation intermediate and proliferates the viral infection to other cells. On account of weak binding affinity between substrate and receptor, the viral infection communicates to other cells and leads to mortality of humans. DANA is the first sialidase inhibitor formed by the dehydration of the C2 hydroxyl group of sialic acid. The replacement of hydroxyl group at C4 position of DANA by an amino group drastically increases the binding affinity and results 4-amino-DANA inhibitor, which is potent than parent DANA. Crystal structure of DANA shows that several binding sites remain free and it should be explored for more powerful sialidase inhibitors. The current study systematically investigates the effect of substituent on the C7 position of 4-amino-DANA in gas phase and solvent phase as well. X-Ray crystallographic study reveals that the C7 of glycerol side chain remains free. Hence, substituent effect at C7 analysis is carried in search of potent sialidase inhibitors. The ab initio and DFT investigation reveals that guanidino and methyl group at C7 position drastically increases the binding affinity between substrate and receptor. Hence further investigation of methyl and guanidine derivatives of the 4-amino-DANA could act as a promising candidate for the design and development of sialidase inhibitors. Vaccination for the H1N1 is not effective due to the new viral mutagenic strains and hence, it cannot contain the viral infection.Therefore antiviral drugs will address the limitation of vaccination. The current finding of sialidase antiviral inhibitors will effectively contain the viral infection and prevent the morbidity