The interaction of substrate-related ketals with bacterial and viral neuraminidases

Arthrobacter sialophilus neuraminidase catalyzes the hydration of 5-acetamido-2,6-anhydro-3, 5-dideoxy-d-glycero-d-galacto-non-2-enonic acid (2,3-dehydro-AcNeu) with K_m and k_cat values of 8.9 × 10^?4m and 6.40 × 10^?4 s^?1, respectively. The methyl ester of 2,3-dehydro-AcNeu as well as 2,3-dehydro-4-epi-AcNeu are also hydrated by the enzyme. The product resulting from the enzymatic hydration of 2,3-dehydro-AcNeu is N-acetylneuraminic acid. A series of derivatives of 2,3-dehydro-AcNeu (K_I 1.60 × 10^?6m) including 2,3-dehydro-4-epi-AcNeu (2.10 × 10^?4m) and 2,3-dehydro-4-keto-AcNeu (K_I = 6.10 × 10^?5 m) were each competitive inhibitors of the enzyme. The methyl esters of these ketal derivatives were also competitive enzyme inhibitors. Dissociation constants for these ketals were determined independently by fluorescence enzyme titrations which gave values similar to those found kinetically. These six relatives of 2,3-dehydro-AcNeu were also competitive inhibitors for the influenza viral neuraminidases. For the viral neuraminidases, the dissociation constant for 2,3-dehydro-AcNeu and its methyl ester were 2.40 × 10^?6 and 1.17 × 10^?3m, respectively. The interpretation placed upon the K_I values determined for these ketals against the Arthrobacter versus influenza neuraminidases is that the bacterial enzyme has a more flexible glycone binding site.     

Crystal structures of respiratory pathogen neuraminidases

Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7 Å resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.     

Determination of Serratia protease by radioimmunoassay

A specific, highly sensitive radioimmunoassay has been developed for the determination of Serratia protease. The radioimmunoassay (RIA) was based upon competition of the protease with ¹²⁵I-labeled protease for antiprotease, followed by a second antibody to separate bound enzyme from free enzyme. The RIA provided a range of 1 to 10 ng for determining the enzymes under conditions in which the enzymatic activity could not be measured. The assay was completely inhibited in the presence of human plasma. The inhibition resulted from a complex formation of the enzyme with plasma α₂macroglobulin. By treatment of the complex with acetone, however, the RIA could be achieved.     

The synthesis of 4-methylumbelliferyl alpha-ketoside of N-acetylneuraminic acid and its use in a fluorometric assay for neuraminidase

4-Methylumbelliferyl α-ketoside of N-acetylneuraminic acid was synthesized by reacting the sodium salt of 4-methylumbelliferone with the 2-chloro-2-deoxy derivative of peracetylated methyl N-acetylneuraminate, followed by preparative silica gel chromatography, deblocking, and purification by gel filtration on Sephadex G-25. The final product was isolated as either the sodium or ammonium salt, and its suitability as a substrate for neuraminidase was evaluated. The optimal pH values for various neuraminidases were 5.6 in acetate buffer (Arthrobacter ureafaciens), 5.0–5.1 in acetate buffer (Clostridium perfringens), and 4.4 in phosphate-citrate buffer (human fibroblasts). K_m values for these enzymes at the optimal pH were 6 × 10^?4m (Arthrobacter), 1 × 10^?4m (Clostridium), and 3 × 10^?4m (human fibroblasts).     

Bacterial Neuraminidase Rescues Influenza Virus Replication from Inhibition by a Neuraminidase Inhibitor

Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may reduce the efficacy of NA inhibitor drugs during influenza virus infection. (295 words).     

Isolation of Arthrobacter Bacteriophage from Soil

Soil was percolated with water and various nutrient solutions, and then the percolates were analyzed for bacteriophages which produced plaques on various Arthrobacter strains. The water percolates did not contain detectable phage. In contrast, phages for A. globiformis strains ATCC 8010 and 4336, and for several recent Arthrobacter species soil isolates, were easily detected in nutrient broth, soil extract, and cation-complete medium percolates. These percolates did not contain phage that produced plaques on A. oxydans and a recent Arthrobacter species soil isolate. Percolation with a selective nicotine-salts solution was required for demonstrating phage for these bacteria. None of the percolates contained phage for five additional named Arthrobacter species. In addition, phages were not detected for A. crystallopoietes in a 2-hydroxypyridine percolate of soil. Based on their lytic spectra, the phage isolates from this soil were relatively host specific.     

Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidases

A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycone-focused library of 2-difluoromethylphenyl α-sialosides is an effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases. The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-terminated compound library by a click reaction. The focused library showed different inhibition patterns for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and the most potent inhibitors for each neuraminidase were selected. A kinetic analysis of the selected inhibitors demonstrated that the modification of the aglycone moiety improved the K(I) value with little change in the t(1/2) value of the enzyme activity relative to the basic skeleton (2).     

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.     

Applications of a Synthetic Neuraminidase Substrate

A rapid and precise assay for neuraminidase using 2-(3'-methoxyphenyl)-N-acetyl-alpha-neuraminic acid (MPN) is described. It is proposed that this substrate be used for the standardization of activity of neuraminidases from viral, bacterial, and mammalian sources. MPN is also used as a chromogenic substrate to localize influenza and parainfluenza virus foci in tissue culture. This technique permits the recovery of infective virus from these stained "plaques." It has also been demonstrated that immunoprecipitin lines containing neuraminidase complexes with antibody in the Ouchterlony test can be observed by a similar staining procedure. No enzyme inhibition occurs in the presence of anti-neuraminidase antibodies or concanavalin A when MPN is used as a substrate in contrast to the results with high-molecular-weight substrates such as fetuin.     

Effect of normal and specific immune sera on neuraminidase activity]

We have got evidence that there is no antigenic relationship reflecting the structural similarity between neuraminidases synthesized by noncholera vibrios and Arthrobacter nicotianae. The cross-reactions between the enzymes and heterological antisera were not observed. Antibodies against the A. nicotianae neuraminidase inhibited the activity of the enzyme for a glycomacropeptide of milk whey and for components of the blood serum, and had no effect no the neuraminidase from noncholera vibrios. Antibodies against the neuraminidase of noncholera vibrios inhibited only the activity of the homologous enzyme. Upon gel-filtration on Sephadex G-200 the antibodies inhibiting the activity of the enzymes under study were found in the fraction of 7S-gamma-globulins.     

Mechaism of Arthrobacter sialophilus neuraminidase: The binding of substrates and transition-state analogs

2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and its methyl ester are competitive inhibitors of Arthrobacter sialophilus neuraminidase with K_i = 1.4 × 10^?6$\text{M}$ and 4.8 × 10^?5$\text{M}$, respectively. The K_m for the substrate, N-acetylneuraminlactose, is 1.0 × 10^?3$\text{M}$. These data, taken together with the conformation of these compounds, indicate that these compounds are transition-state analogs of the enzyme. These results also suggest that the substrate upon binding to neuraminidase is distorted to a conformation approaching that of a half-chair.     

Investigation of flexibility of neuraminidase 150-loop using tamiflu derivatives in influenza A viruses H1N1 and H5N1

This study focuses on design, synthesis and in vitro evaluation of inhibitory potency of two series of sialylmimetic that target an exosite (“150-cavity”) adjacent to the active site of influenza neuraminidases from A/California/07/2009 (H1N1) pandemic strain and A/chicken/Nakorn-Patom/Thailand/CU-K2-2004 (H5N1). The structure-activity analysis as well as 3-D structure of the complex of parental compound with the pandemic neuraminidase p09N1 revealed high flexibility of the 150-cavity towards various modification of the neuraminidase inhibitors. Furthermore, our comparison of two methods for inhibition constant determination performed at slightly different pH values suggest that the experimental conditions of the measurement could dramatically influence the outcome of the analysis in the compound-dependent manner. Therefore, previously reported K_i values determined at non-physiological pH should be carefully scrutinized.     

Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture

A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1× nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4°C and 10°C while none of the isolates could grow at 37°C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).     

2,3-dehydro-4-epi-N-acetylneuraminic acid; a neuraminidase inhibitor

Treatment of N-acetylneuraminic acid methyl ester with sulfuric acid and acetic anhydride at 50° followed by deacetylation gave 2,3-dehydro-2-deoxy-N-acetylneuraminic acid methyl ester and methyl 5-acetamido-2,6-anhydro-2,3,5-trideoxy-d-glycero-d-talo-non-2-enonate (2,3-dehydro-4-epi-NeuAc methyl ester) in equal yields (～40% each). The structure of the latter was ascertained primarily from analysis of its mass spectrum and ¹H- and ¹³C-nuclear magnetic resonance spectra. The relative proportions of these two glycals in the foregoing reaction was dependent on temperature, as at 0°, the yield of 2,3-dehydro-4-epi-NeuAc was markedly diminished. A minor by-product of this acetylation reaction was 2-methyl-(methyl 7,8,9- tri-O-acetyl-2,6-anhydro-2,3,5-trideoxy-d-glycero-d-talo-non-2-enonate)-[4,5-d]-2-oxazoline. Based upon this finding and additional interconversion experiments, a mechanism involving the intermediacy of the latter oxazoline to account for the epimerization is proposed. These glycals and their methyl esters are competitive inhibitors of Arthrobacter sialophilus, neuraminidase, suggesting that the 4-hydroxyl group must be equatorially oriented for maximal enzyme inhibition.     

Prediction of selective inhibition of neuraminidase from various influenza virus strains by potential inhibitors

A universal model of inhibition of neuraminidases from various influenza virus strains by a particular inhibitor has been developed. It is based on known 3D structures for neuraminidases from three influenza virus strains (A/Tokyo/3/67, A/tern/Australia/G70C/75, B/Lee/40) and modeling of the 3D structure of neuraminidases from other strains (A/PR/8/34 and A/Aichi/2/68). Using docking and molecular dynamics, we have modeled 235 enzyme-ligand complexes for 185 compounds with known IC₅₀ values. Selection of final variants among three intermediate results obtained for each enzyme-ligand pair and calculation of independent variables for generation of linear regression equations were performed using MM-PBSA/MM-GBSA. This resulted in the set of equations for individual strains and the equations pooling all the data. Thus using this approach it is possible to predict inhibition for neuraminidase from each the considered strains by a particular inhibitor and to predict the range of its action on neuraminidases from various influenza virus strains.     

Assay of proteolytic enzymes by the fluorescence polarization technique.

Neuraminidase substrates of high specific activity (>300 μCi/μmol) were prepared by reduction of sialyllactose with NaB³H₄, followed by separation of the 2 → 3 and 2 → 6 isomers of [³H]sialyllactitol by paper chromatography. Hydrolysis of sialyllactitol by neuraminidase was monitored by measuring the radioactivity in the neutral reaction product, which was separated from the charged substrate by passage over a small anion exchange column. The assay was applied to the neuraminidase activity of cultured human skin fibroblasts. The K_m was found to be 1.1 mm for both substrates; the pH optimum, 4.0; the 2 → 3 isomer was hydrolyzed twice as fast as the 2 → 6. In several genetic disorders associated with neuraminidase deficiency, the activity toward both isomers was reduced almost completely (mucolipidoses I and II; Goldberg syndrome), or only partially (mucolipidosis III; adult myoclonus syndrome); however, the relative activity towards the two isomers remained approximately the same in all cases.     

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.     

Neuraminidase-triggered activation of prodrug-type substrate of 4-nitroaniline

Artificial substrates for probing neuraminidase activity are powerful tools for studying the physiological and pathological roles of neuraminidases. Most of the substrates are α-O-linked sialosides involving hydroxyl-containing reporters for visualization, and neuraminidase-catalyzed cleavage of the sialic acid residues directly activates the reporters. However, the use of amine-containing reporters has been avoided because α-N-linked sialosides are marginal substrates for neuraminidases. To expand the applicability of reporters to amine-containing compounds, we have focused on prodrug design. Herein we describe the synthesis and enzymatic study of a model substrate involving 4-nitroaniline as an amine-containing chromogenic reporter. The substrate can respond to neuraminidase from Clostridium perfringens. Neuraminidase-mediated hydrolysis of the sialic acid moiety of the substrate initiates self-immolative elimination of the linker moiety, leading the liberation of yellow-colored reporter 4-nitroaniline. The elimination process involves generation of quinone methide intermediate, which causes to neutralize neuraminidase. The substrate, thus, works as not only a chromogenic substrate but also a suicide inactivator.