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.     

Structure-guided design of a novel class of benzyl-sulfonate inhibitors for influenza virus neuraminidase

Influenza NA (neuraminidase) is an antiviral target of high pharmaceutical interest because of its essential role in cleaving sialic acid residues from cell surface glycoproteins and facilitating release of virions from infected cells. The present paper describes the use of structural information in the progressive design from a lead binding ion (a sulfate) to a potent submicromolor inhibitor (K(i) 0.13 microM). Structural information derived from the X-ray structure of an NA complexed with several sulfate ions, in combination with results derived from affinity labelling and molecular modelling studies, was used to guide design of potent sulfonic acid-based inhibitors. These inhibitors are structural fragments of the polysulfonate triazine dye Cibacron Blue 3GA and represent novel lead scaffolds for designing non-carbohydrate inhibitors for influenza neuraminidases.     

Triazole-linked transition state analogs as selective inhibitors against V. cholerae sialidase

Sialidases or neuraminidases are enzymes that catalyze the cleavage of terminal sialic acids from oligosaccharides and glycoconjugates. They play important roles in bacterial and viral infection and have been attractive targets for drug development. Structure-based drug design has led to potent inhibitors against neuraminidases of influenza A viruses that have been used successfully as approved therapeutics. However, selective and effective inhibitors against bacterial and human sialidases are still being actively pursued. Guided by crystal structural analysis, several derivatives of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en or DANA) were designed and synthesized as triazole-linked transition state analogs. Inhibition studies revealed that glycopeptide analog E-(TriazoleNeu5Ac2en)-AKE and compound (TriazoleNeu5Ac2en)-A were selective inhibitors against Vibrio cholerae sialidase, while glycopeptide analog (TriazoleNeu5Ac2en)-AdE selectively inhibited Vibrio cholerae and A. ureafaciens sialidases.     

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).     

Structure-based virtual screening and biological evaluation of potent and selective ADAM12 inhibitors

We describe a series of potent and selective inhibitors of ADAM12 that were discovered using computational screening of a focused virtual library. The initial structure-based virtual screening selected 64 compounds from a 3D database of 67,062 molecules. Being evaluated by a cell-based ADAM12 activity assay, compounds 5, 11, 14, 16 were further identified as the potent and selective inhibitors of ADAM12 with low nanomolar IC₅₀ values. The mechanism underlying the potency and selectivity of a representative compound, 5, was investigated through molecular docking studies.     

Combinatorial design of nonsymmetrical cyclic urea inhibitors of aspartic protease of HIV-1

The aspartic protease (PR) of the human immunodeficiency virus type 1 (HIV-1) is an important target for the design of specific antiviral agents dedicated to treatment of HIV-1 infection. We have employed computer-assisted combinatorial chemistry methods to design a small focused virtual library of nonsymmetrically substituted cyclic urea inhibitors of the PR. Nonsymmetrical compounds with decreased peptidic character were namely found to inhibit the PR with comparable inhibition potencies as their C2-pseudosymmetric counterparts and to possess superior pharmacokinetic properties. To generate the virtual library of fully nonsymmetrical cyclic urea analogs, diverse reagents were selected from databases of available chemicals with characteristics similar to those of the building blocks of known potent PR inhibitors. The X-ray structure of the protease-inhibitor complex PR-XV-638 was used as the receptor model in the structure-based focusing and in silico screening of the virtual library. A target-specific LUDI-type scoring function, parameterized for a QSAR training set of known cyclic urea inhibitors and validated on a set of compounds not included into the training set, was used to predict the inhibition constants (Ki) of the generated analogs toward the HIV-1 PR. The fragments most frequently occurring in the analogs with the highest predicted inhibition potencies (Ki*<10 pM) were then selected to constitute a highly focused library subset containing novel nonsymmetrical cyclic ureas with predicted Ki*s 1 order of magnitude lower than the most potent known cyclic urea inhibitors. ADME properties calculated for the most promising analogs suggested that the cyclic ureas are endowed with a wide range of favorable pharmacokinetic properties, which may favor the discovery of a potent orally administrable antiviral drug.     

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.     

Novel linear polymers bearing thiosialosides as pendant-type epitopes for influenza neuraminidase inhibitors

A conventional synthesis of alpha-thioglycoside of sialic acid as a glycomonomer was accomplished. Radical copolymerization of the glycomonomer with vinyl acetate proceeded smoothly to afford a new class of glycopolymers having thiosialoside residues, in which all protection was removed by a combination of transesterification and saponification to provide a water-soluble thiosialoside cluster. The results of a preliminary study on biological responses against influenza virus neuraminidases using the thiosialoside polymer as a candidate for a neuraminidase inhibitor showed that the glycopolymer has potent inhibitory activity against the neuraminidases.     

Discovery of novel purine derivatives with potent and selective inhibitory activity against c-Src tyrosine kinase

We report here the discovery of novel purine derivatives with potent and selective inhibitory activity against c-Src tyrosine kinase by adopting a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay. Thirty two compounds were selected and synthesized. All compounds showed potent inhibitory activity against c-Src kinase with IC₅₀ values ranging from 3.14 μM to 0.02 μM. Compound 5i was identified as one of the most potent agent with an IC₅₀ 120 times lower than those of the hits. The high hit rate (100%) and the potency of the new Src kinase inhibitors demonstrated the efficiency of the strategy for the focused library design and virtual screening. The novel active chemical entities reported here should be good leads for further development of purine-based anticancer drugs targeting Src tyrosine kinase.     

Analogue inhibitors by modifying oseltamivir based on the crystal neuraminidase structure for treating drug-resistant H5N1 virus

The worldwide spread of H5N1 avian influenza and the increasing reports about its resistance to the existing drugs have made a priority for the development of the new anti-influenza molecules. The crystal structure of H5N1 avian influenza neuraminidase reported recently by Russell et al. [R.J. Russell, L.F. Haire, D.J. Stevens, P.J. Collins, Y. P. Lin, G.M. Blackburn, A.J. Hay, S.J. Gamblin, J.J. Skehel, The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design, Nature 443 (2006) 45-49] have provided new opportunities for drug design in this regard. It is revealed through the structure that the active sites of the group-1 neuraminidases, which contain the N1 subtype, have a very different three-dimensional structure from those of group-2 neuraminidases. The key difference is in the 150-loop cavity adjacent to the conserved active site in neuraminidase. Based on these findings and by modifying oseltamivir, six analog inhibitors were proposed as candidates for developing inhibitors against H5N1 virus, particularly against the oseltamivir-resistant H5N1 virus strain.     

Identification of selective covalent inhibitors of platelet activating factor acetylhydrolase 1B2 from the screening of an oxadiazolone-capped peptoid–azapeptoid hybrid library

A potent and selective inhibitor of platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is described. The compound was derived by improvement of a modest affinity primary hit isolated from the screening of a bead-displayed peptoid–azapeptoid hybrid library tethered to an oxadiazolone ‘warhead’. The oxadiazolone moiety of the inhibitors was found to react covalently with the active site serine residue of PAFAH1B2. This screening strategy may be useful for the identification of many selective, covalent inhibitors of serine hydrolases.     

Nucleotide sequence of the influenza virus A/USSR/90/77 neuraminidase gene

The complete nucleotide sequence of the N1 neuraminidase gene of influenza virus A/USSR/90/77 was determined. Comparison of its predicted amino acid sequence with other N1 and N2 neuraminidases indicates that the N1 neuraminidases share most of the antigenic determinants mapped on the N2 neuraminidase but display at least one additional potentially antigenic region probably as a result of intersubtypic differences in glycosylation.     

Enzymatic properties of neuraminidases from Arthrobacter ureafaciens.

Neuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51,000 and 39,000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with N-acetylneuraminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53 degrees C, were stable between pH 6.0 and 9.0, and were thermostable up to 50 degrees C. They did not require Ca2+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by p-chloromercuribenzoic acid of Hg2+. Both neuraminidases I and II were able to hydrolyze the alpha-ketosidic linkage of N-glycolylneuraminic acid as well as that of N-acetylneuraminic acid, and were able to liberate substantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid. Remarkable differences were observed between neuraminidases I and II as regards substrate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neuraminidase II 143-fold. Although neuraminidases I and II were able to hydrolyze (alpha,2-3), (alpha,2-6), and (alpha,2-8) linkages, the initial rate of hydrolysis of N-acetylneuraminosyl-alpha,2-6-lactose was greater than that of the alpha,2-3-isomer.     

Characterization of Influenza Virus Neuraminidases: Peptide Changes Associated with Antigenic Divergence Between Early and Late N2 Neuraminidases

Neuraminidases (EC 3.2.1.18) of 1957, 1960, and 1969 influenza virus strains were isolated after proteolytic digestion of viral hemagglutinin. Each neuraminidase was recovered with a final yield of about 15% and had similar specific activities. Immunization of rabbits with the neuraminidases elicited monospecific neuraminidase antibodies, with no antibodies to viral hemagglutinin. Further evidence of purity was the existence of only a single component, about 50,000 daltons in size, when reduced neuraminidase preparations were examined by sodium dodecyl sulfate acrylamide gel electrophoresis. However, storage of neuraminidase in solution resulted in the appearance of slightly smaller degradation products. Preparations of each neuraminidase were denatured under reducing conditions, and exposed sulfhydryl residues were blocked by reaction with (14)C-iodoacetamide. After tryptic digestion, peptide maps were prepared for the neuraminidases, and the (14)C-labeled cysteinyl peptides were then identified by autoradiography. About 20 peptides were present, in agreement with the number predicted from amino acid analysis for neuraminidase subunits of only one type. The 1957 and 1960 neuraminidases exhibited a small antigenic divergence from each other, and maps of their cysteinyl peptides appeared to be identical. The 1969 neuraminidase exhibited considerable antigenic divergence from the other two neuraminidases, and maps of 1969 neuraminidase peptides revealed two major and several minor differences from the other maps. Thus, antigenic divergence between the neuraminidases of Asian and Hong Kong influenza viruses is associated with a small number of changes in the primary structure of the neuraminidase subunit.     

A two stage click-based library of protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction pathways. Potent and selective PTP inhibitors are useful for probing these pathways and also may serve as drugs for the treatment of a variety of diseases including type 2 diabetes and infection by the bacterium Yersinia pestis. In this report Cu(I)-catalyzed 'click' cycloaddition reactions between azides and alkynes were employed to generate two sequential libraries of PTP inhibitors. In the first round library methyl 4-azidobenzoylformate was reacted with 56 mono- and diynes. After hydrolysis of the methyl esters, the resulting alpha-ketocarboxylic acids were assayed in crude form against the Yersinia PTP and PTP1B. Four compounds were selected for further evaluation, and one compound was chosen as the lead for generation of the second round library. This lead compound was modified by conversion of an alcohol into an azide group, and the resulting azide was reacted with the same 56 mono- and diynes that were used in the first generation library. After screening the crude inhibitors against the Yersinia PTP and PTP1B, four compounds were selected and evaluated in pure form against the Yersinia PTP, PTP1B, TCPTP, LAR, and CD45. The best bis(alpha-ketocarboxylic acid) inhibitor 34 had an IC(50) value of 550nM against the Yersinia PTP and an IC(50) value of 710nM against TCPTP. The most potent inhibitor containing a single alpha-ketocarboxylic acid group 32 had IC(50) values of 2.1, 5.7, and 2.6 microM against the Yersinia PTP, PTP1B, and TCPTP, respectively.     

Synthesis and evaluation of a new series of substituted acyl(thio)urea and thiadiazolo [2,3-a] pyrimidine derivatives as potent inhibitors of influenza virus neuraminidase

A series of substituted acyl(thio)urea and 2H-1,2,4-thiadiazolo [2,3-a] pyrimidine derivatives were prepared and both of their cell culture and enzymatic activity toward influenza virus were tested. Their in vitro neuraminidase inhibitory activities were in good agreement with the corresponding activities in cultured cells and they were evaluated as potent neuraminidase inhibitors. Of the analogues that demonstrated IC₅₀s < 0.1 μM, 16 and 60 were further investigated as candidates with the most potential for future development. The molecular docking work of the representative compound was described to provide more insight into their mechanism of action and further rationalize the observations of this new series herein, which represents a novel class of highly potent and selective inhibitors of influenza virus.     

4,5-dialkylsubstituted 2-imino-1,3-thiazolidine derivatives as potent inducible nitric oxide synthase inhibitors

In the course of our search for selective iNOS inhibitors, we have previously reported that 2-imino-1,3-oxazolidine derivatives (1) and 2-aminothiazole derivatives (2) are selective iNOS inhibitors. In order to find more potent iNOS inhibitors, we focused our efforts on the synthesis and evaluation of the inhibitory activity against iNOS and selectivity for iNOS both in vitro and in vivo of a series of 2-imino-1,3-thiazolidine derivatives (3), which are analogues of 1 and 2. Our results show that among the compounds synthesized (4R,5R)-5-ethyl-2-imino-4-methyl-1,3-thiazolidine [(4R,5R)-14a: ES-1537] exhibited potent inhibitory activity and selectivity for iNOS. In addition, ES-1537 had good pharmacokinetic profile in rats with BA value of 80%. It is therefore expected that ES-1537 may be therapeutically useful for the treatment of diseases related to excess production of NO.     

Antigenic relationship of influenza-virus neuraminidases from Asian,Hong Kong,and Equi-2 strains

Two Asian influenza strains, the Hong Kong strain and an Equi-2 influenza strain have been investigated. Adsorption isotherms of purified neuraminidases from these strains with isolated antibodies from homologous antisera are given. Cross testing by neuraminidase inhibition and haemagglutination inhibition are reported. Hong Kong neuraminidase appears to be indistinguishable from the earlier Asian neuraminidases, and not related to Equi-2 influenza neuraminidase. The haemagglutinin from the Hong Kong strain is related to earlier Asian strains as well as to the equine strain.In collaboration with the Chief Medical Officer of Health and Epidemiology, Department of Public Health, Den Haag, The Netherlands.It is a pleasure to express our thanks to Mr. J. Jacobs, N. V. Philips Duphar, Weesp (The Netherlands) for his interesting discussions and suggestions; and to Mr. H. de Jonge, Department of Medical Statistics, University of Leiden (The Netherlands) for his advice.     

Membrane-bound neuraminidases of rat liver. Neuraminidase activity in Golgi apparatus.

The bulk (60 to 65%) of the neuraminidase activity present in rat liver homogenates was found in the M + L (mitochondria plus lysosomes) fraction, The patterns of subcellular distribution were essentially identical whether disialogangliosides or neuramin-lactose (2 yields 3') were utilized as substrates. A new neuraminidase, which hydrolyzes sialyl trisaccharides but which does not act upon glycoproteins and gangliosides, was detected in Golgi apparatus. Unlike the other particulate neuraminidases of rat liver, the Golgi enzyme is stimulated by prior incubation and by the addition of Ca2+ or Zn2+ at 1 mM concentration. Although plasma membrane-rich fractions are often contaminated by Golgi membranes the marked differences in their enzymic properties allowed a clear distinction between the neuraminidases present in these two types of membranes.     

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.