Design, synthesis, and biological evaluation of human sialidase inhibitors. Part 1: selective inhibitors of lysosomal sialidase (NEU1)

We here report the design and synthesis of selective human lysosomal sialidase (NEU1) inhibitors. A series of amide-linked C9 modified DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) analogues were synthesized and their inhibitory activities against all four human sialidases (NEU1-NEU4) were determined. Structure-based approach was used to investigate the basis of selectivity of the compounds with experimentally observed activity. Results from the present study are found to be informative in a qualitative manner for the further design of isoform selective human sialidase inhibitors for therapeutic value.     

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.     

Molecular cloning and characterization of NEU4, the fourth member of the human sialidase gene family

Several mammalian sialidases have been cloned so far and here we describe the identification and expression of a new member of the human sialidase gene family. The NEU4 gene, identified by searching sequence databases for entries showing homologies to the human cytosolic sialidase NEU2, maps in 2q37 and encodes a 484-residue protein. The polypeptide contains all the typical sialidase amino acid motifs and, apart from an amino acid stretch that appears unique among mammalian sialidases, shows a high degree of homology for NEU2 and the plasma membrane-associated (NEU3) sialidases. RNA dot-blot analysis showed a low but wide expression pattern, with the highest level in liver. Transient transfection in COS7 cells allowed the detection of a sialidase activity toward the artificial substrate 4MU-NeuAc in the acidic range of pH. Immunofluorescence staining and Western blot analysis demonstrated the association of NEU4 with the inner cell membranes.     

NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia: NEU1 restrains endothelial cell migration, whereas NEU3 does not

The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.     

Novel pH-dependent regulation of human cytosolic sialidase 2 (NEU2) activities by siastatin B and structural prediction of NEU2/siastatin B complex

Human cytosolic sialidase (Neuraminidase 2, NEU2) catalyzes the removal of terminal sialic acid residues from glycoconjugates. The effect of siastatin B, known as a sialidase inhibitor, has not been evaluated toward human NEU2 yet. We studied the regulation of NEU2 activity by siastatin B in vitro and predicted the interaction in silico. Inhibitory and stabilizing effects of siastatin B were analyzed in comparison with DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) toward 4-umbelliferyl N-acetylneuraminic acid (4-MU-NANA)- and α2,3-sialyllactose-degrading activities of recombinant NEU2 produced by E. coli GST-fusion gene expression. Siastatin B exhibited to have higher competitive inhibitory activity toward NEU2 than DANA at pH 4.0. We also revealed the stabilizing effect of siastatin B toward NEU2 activity at acidic pH. Docking model was constructed on the basis of the crystal structure of NEU2/DANA complex (PDB code: 1VCU). Molecular docking predicted that electrostatic neutralization of E111 and E218 residues of the active pocket should not prevent siastatin B from binding at pH 4.0. The imino group (¹NH) of siastatin B can also interact with D46, neutralized at pH 4.0. Siastatin B was suggested to have higher affinity to the active pocket of NEU2 than DANA, although it has no C7–9 fragment corresponding to that of DANA. We demonstrated here the pH-dependent affinity of siastatin B toward NEU2 to exhibit potent inhibitory and stabilizing activities. Molecular interaction between siastatin B and NEU2 will be utilized to develop specific inhibitors and stabilizers (chemical chaperones) not only for NEU2 but also the other human sialidases, including NEU1, NEU3 and NEU4, based on homology modeling.     

Neu4, a novel human lysosomal lumen sialidase, confers normal phenotype to sialidosis and galactosialidosis cells

Three different mammalian sialidases have been described as follows: lysosomal (Neu1, gene NEU1), cytoplasmic (Neu2, gene NEU2), and plasma membrane (Neu3, gene NEU3). Because of mutations in the NEU1 gene, the inherited deficiency of Neu1 in humans causes the severe multisystemic neurodegenerative disorder sialidosis. Galactosialidosis, a clinically similar disorder, is caused by the secondary Neu1 deficiency because of genetic defects in cathepsin A that form a complex with Neu1 and activate it. In this study we describe a novel lysosomal lumen sialidase encoded by the NEU4 gene on human chromosome 2. We demonstrate that Neu4 is ubiquitously expressed in human tissues and has broad substrate specificity by being active against sialylated oligosaccharides, glycoproteins, and gangliosides. In contrast to Neu1, Neu4 is targeted to lysosomes by the mannose 6-phosphate receptor and does not require association with other proteins for enzymatic activity. Expression of Neu4 in the cells of sialidosis and galactosialidosis patients results in clearance of storage materials from lysosomes suggesting that Neu4 may be useful for developing new therapies for these conditions.     

Molecular dynamics simulations of viral neuraminidase inhibitors with the human neuraminidase enzymes: Insights into isoenzyme selectivity

Inhibitors of viral neuraminidase enzymes have been previously developed as therapeutics. Humans can express multiple forms of neuraminidase enzymes (NEU1, NEU2, NEU3, NEU4) that share a similar active site and enzymatic mechanism with their viral counterparts. Using a panel of purified human neuraminidase enzymes, we tested the inhibitory activity of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir, oseltamivir, and peramivir against each of the human isoenzymes. We find that, with the exceptions of DANA and zanamivir, these compounds show generally poor activity against the human neuraminidase enzymes. To provide insight into the interactions of viral inhibitors with human neuraminidases, we conducted molecular dynamics simulations using homology models based on coordinates reported for NEU2. Simulations revealed that an organized water is displaced by zanamivir in binding to NEU2 and NEU3 and confirmed the critical importance of engaging the binding pocket of the C7–C9 glycerol sidechain. Our results suggest that compounds designed to target the human neuraminidases should provide more selective tools for interrogating these enzymes. Furthermore, they emphasize a need for additional structural data to enable structure-based drug design in these systems.     

Differential effect of various inhibitors on four types of rat sialidase

The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid andN-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu²⁺ (1mm) and thiol-modifying 4-hydroxymercuribenzoate (50 µm) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac-s-(2-6)Gal(1-4)Glc(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-dehydro-N-acetylneuraminic acid - 4MU-Neu5Ac 4-methylumbelliferyl--N-acetyl-d-neuraminic acid     

New insights on the sialidase protein family revealed by a phylogenetic analysis in metazoa

Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis), early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals). We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may be involved in the evolution of specific properties of sialidases.     

Sialidases on Mammalian Sperm Mediate Deciduous Sialylation during Capacitation

Sialic acids (Sias) mediate many biological functions, including molecular recognition during development, immune response, and fertilization. A Sia-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their Sias. We quantified shed Sia monosaccharides released from capacitated sperm and measured sperm sialidase activity. We report the presence of two sialidases (neuraminidases Neu1 and Neu3) on mammalian sperm. These are themselves shed from sperm during capacitation. Inhibiting sialidase activity interferes with sperm binding to the zona pellucida of the ovum. A survey of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases in sperm of some male idiopathic infertility cases. The results contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization.     

Expression of a novel human sialidase encoded by the NEU2 gene

Sialidases (E.C.3.2.1.18) belong to a group of glycohydrolytic enzymes, widely distributed in nature, which remove sialic acid residues from glycoproteins and glycolipids. All of the sialidase so far characterized at the molecular level share an Asp block, repeated three to five times in the primary structure, and an F/YRIP sequence motif which is part of the active site. Using a sequence homology-based approach, we previously identified a human gene, named NEU2, mapping to chromosome 2q37. NEU2 encoded protein is a polypeptide of 380 amino acids with two Asp block consensuses and the YRIP sequence in the amino terminal part of the primary structure. Here we demonstrate that NEU2 encodes a functional sialidase. NEU2 was expressed in COS7 cells, giving rise to a dramatic increase in the sialidase activity measured in cell extracts with the artificial substrate 4-MU-NANA. Using a rabbit polyclonal antiserum, on Western blots a protein band with a molecular weight of about 42 kDa was detectable, and its cytosolic localization was demonstrated with cell fractionation experiments. These results were confirmed using immunohistochemical techniques. NEU2 expression in E.coli cells allowed purification of the recombinant protein. As already observed in the enzyme expressed in COS7 cells, NEU2 pH optimum corresponds to 5.6 and the polypeptide showed a K(m)for 4-MU-NANA of 0.07 mM. In addition, based on the detectable similarities between the NEU2 amino acid sequence and bacterial sialidases, a prediction of the three-dimensional structure of the enzyme was carried out using a protein homology modeling approach.     

Mammalian sialidases: physiological and pathological roles in cellular functions

Sialic acids are terminal acidic monosaccharides, which influence the chemical and biological features of glycoconjugates. Their removal catalyzed by a sialidase modulates various biological processes through change in conformation and creation or loss of binding sites of functional molecules. Sialidases exist widely in vertebrates and also in a variety of microorganisms. Recent research on mammalian sialidases has provided evidence for great importance of these enzymes in various cellular functions, including lysosomal catabolism, whereas microbial sialidases appear to play roles limited to nutrition and pathogenesis. Four types of mammalian sialidases have been identified and characterized to date, designated as NEU1, NEU2, NEU3 and NEU4. They are encoded by different genes and differ in major subcellular localization and enzymatic properties including substrate specificity, and each has been found to play a unique role depending on its particular properties. This review is an attempt to concisely summarize current knowledge concerning mammalian sialidases, with a special focus on their properties and physiological and pathological roles in cellular functions.     

Novel inhibitors of influenza sialidases related to GG167 structure-activity, crystallographic and Molecular dynamics studies with 4H-pyran-2-carboxylic acid 6-carboxamides

The structure-activity relationships of a series of 4-amino and guanidino-4H-pyran-2-carboxylic acid 6-carboxamides are described. These compounds represent a new class of inhibitor of influenza sialidases and are particularly active against influenza A sialidase. The binding of the N-phenethyl-N-propylamide 41 to influenza A and B sialidases has been investigated using X-ray crystallography and molecular dynamics simulations. Our results suggest that formation of a hitherto unobserved intramolecular salt bridge within the enzymes may account for the observed activity and selectivity of the series.     

Selective and slow-binding inhibition of shikonin derivatives isolated from Lithospermum erythrorhizon on glycosyl hydrolase 33 and 34 sialidases

Sialidases are enzymes that catalyze the hydrolysis of sialic acid residues from various glycoconjugates, which are widely found in a number of viral and microbial pathogens. In this study, we investigated the biological evaluation of isolated six shikonins (1-6) and three shikonofurans (7-9) from Lithospermum erythrorhizon. The nine isolated compounds 1-9 showed strong and selective inhibition of glycosyl hydrolase (GH) 33 and -34 sialidases activities. In GH33 bacterial-sialidase inhibition assay, the inhibitory activities against GH33 siadliase of all shikonofuran derivatives (7-9) were greater than shikonin derivatives (1-6). Shikonofuran E (8) exhibited the most potent inhibitory activity toward GH33 sialidases (IC(50)=0.24μM). Moreover, our detailed kinetic analysis of these species unveiled that they are all competitive and simple reversible slow-binding inhibitors. Otherwise, they showed different inhibitory capacities and kinetic modes to GH34 viral-sialidase activity. All the naphthoquinone derivatives (1-6) were of almost equal efficiency with IC(50) value of 40μM and shikonofurans (7-9) did not show the significant inhibitory effect to GH34 sialidase. Kinetic analyses indicated that naphthoquinones acted via a noncompetitive mechanism.     

Inhibitor selectivity of a new class of oseltamivir analogs against viral neuraminidase over human neuraminidase enzymes

The viral neuraminidase enzyme is an established target for anti-influenza pharmaceuticals. However, viral neuraminidase inhibitors could have off-target effects due to interactions with native human neuraminidase enzymes. We report the activity of a series of known inhibitors of the influenza group-1 neuraminidase enzyme (N1 subtype) against recombinant forms of the human neuraminidase enzymes NEU3 and NEU4. These inhibitors were designed to take advantage of an additional enzyme pocket (known as the 150-cavity) near the catalytic site of certain viral neuraminidase subtypes (N1, N4 and N8). We find that these modified derivatives have minimal activity against the human enzymes, NEU3 and NEU4. Two compounds show moderate activity against NEU3, possibly due to alternative binding modes available to these structures. Our results reinforce that recognition of the glycerol side-chain is distinct between the viral and human NEU enzymes, and provide experimental support for improving the selectivity of viral neuraminidase inhibitors by exploiting the 150-cavity found in certain subtypes of viral neuraminidases.     

Sialidase NEU3 is a peripheral membrane protein localized on the cell surface and in endosomal structures

Sialidase NEU3 is also known as the plasma-membrane-associated form of mammalian sialidases, exhibiting a high substrate specificity towards gangliosides. In this respect, sialidase NEU3 modulates cell-surface biological events and plays a pivotal role in different cellular processes, including cell adhesion, recognition and differentiation. At the moment, no detailed studies concerning the subcellular localization of NEU3 are available, and the mechanism of its association with cellular membranes is still unknown. In the present study, we have demonstrated that sialidase NEU3, besides its localization at the plasma membrane, is present in intracellular structures at least partially represented by a subset of the endosomal compartment. Moreover, we have shown that NEU3 present at the plasma membrane is internalized and locates then to the recycling endosomal compartment. The enzyme is associated with the outer leaflet of the plasma membrane, as shown by selective cell-surface protein biotinylation. This evidence is in agreement with the ability of NEU3 to degrade gangliosides inserted into the plasma membrane of adjacent cells. Moreover, the mechanism of the protein association with the lipid bilayer was elucidated by carbonate extraction. Under these experimental conditions, we have succeeded in solubilizing NEU3, thus demonstrating that the enzyme is a peripheral membrane protein. In addition, Triton X-114 phase separation demonstrates further the hydrophilic nature of the protein. Overall, these results provide important information about the biology of NEU3, the most studied member of the mammalian sialidase family.     

Synthesis,biological evaluation and molecular docking studies of amide-coupled benzoic nitrogen mustard derivatives as potential antitumor agents

A series of amide-coupled benzoic nitrogen mustard derivatives as potential EGFR and HER-2 kinase inhibitors were synthesized and reported for the first time. Some of them exhibited significant EGFR and HER-2 inhibitory activity. Of all the studied compounds, compounds 5b and 5t exhibited the most potent inhibitory activity, which was comparable to the positive control erlotinib. Docking simulation was performed to position compounds 5b and 5t into the EGFR active site to determine the probable binding model. Antiproliferative assay results indicated that some of the benzoic nitrogen mustard derivatives possessed high antiproliferative activity against MCF-7. In particular, compounds 5b and 5t with potent inhibitory activity in tumor growth inhibition may function as potential antitumor agents.     

New ganglioside analogs that inhibit influenza virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac alpha(2-S-6)Glc beta(1-1)Ceramide (1) and the GM3 analog Neu5Ac alpha(2-S-6)Gal beta(1-4)Glc beta(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent Ki value of 2.8 x 10(-6) and 1.5 x 10(-5) M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac alpha(2-S-6)Gal beta(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (Ki = 1.0 x 10(-4) M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were non-hydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases from Clostridium perfringens and Arthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.     

Secretary sialidase activity and GM3 ganglioside

The sialidase activities with GM3 ganglioside and sialyllactitol were demonstrated in the conditioned medium of human fibroblasts. pH versus activity profiles of conditioned medium with GM3 as substrate suggested the presence of two sialidases with optimal activities at pH 4.5 and pH 6.5. The GM3 sialidase activity at pH 6.5 was suppressed in the medium of contact-inhibited cells. This sialidase may function in the metabolism of cell surface GM3 since there was a selective loss of labeled sialic acid from GM3 at different times of incubation after pulse-labeling with a radioactive sialic acid precursor ([3H]N-acetyl-mannosamine) and a radioactive ceramide precursor ([14C]serine). In addition, a sialidase inhibitor, 2-deoxy-2, 3-dehydro-N-acetyl-neuraminic acid (NeuAc-2-en) resulted in a reversible growth inhibitory effect and the suppression of the sialidase activity in the medium. We have speculated that GM3 hydrolysis on the cell surface by the sialidase may be coordinated with the cell cycle and may be at its maximum during early in the G1 phase.