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Analysis of inhibitor binding in influenza virus neuraminidase

¹ Biomolecular Research Institute, Parkville, Victoria 3052, Australia
² Department of Medicinal Chemistry, Monash University, Parkville, Victoria 3052, Australia

Reprint requests to: Dr. Brian J. Smith, Biomolecular Research Institute, 343 Royal Parade, Parkville, Victoria 3052, Australia; e-mail: brian.smith{at}bioresi.com.au; fax: 61-3-9662-7347.

(RECEIVED October 3, 2000; FINAL REVISION January 2, 2001; ACCEPTED January 2, 2001)

Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.41801.

	Abstract

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) is a transition state analog inhibitor of influenza virus neuraminidase (NA). Replacement of the hydroxyl at the C₉ position in DANA and 4-amino-DANA with an amine group, with the intention of taking advantage of an increased electrostatic interaction with a conserved acidic group in the active site to improve inhibitor binding, significantly reduces the inhibitor activity of both compounds. The three-dimensional X-ray structure of the complexes of these ligands and NA was obtained to 1.4 Å resolution and showed that both ligands bind isosterically to DANA. Analysis of the geometry of the ammonium at the C₄ position indicates that Glu119 may be neutral when these ligands bind. A computational analysis of the binding energies indicates that the substitution is successful in increasing the energy of interaction; however, the gains that are made are not sufficient to overcome the energy that is required to desolvate that part of the ligand that comes in contact with the protein.

Keywords: Influenza virus; neuraminidase inhibitors; electrostatics; X-ray crystal structures; binding energies; molecular modeling

	Introduction

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
Zanamivir has proven to be a safe and effective treatment for influenza infection (Dunn and Goa 1999; Freund et al. 1999; Silagy and Campion 1999). Designed to inhibit the viral neuraminidase (NA) enzyme, its discovery is one of the early examples of the successful application of structure-based drug design (von Itzstein et al. 1993; Wade 1997; Taylor 1998; Varghese 1999). Zanamivir differs from the natural NA inhibitor (Burmeister et al. 1993) 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA, 1) simply by the substitution of the O₄-hydroxyl moiety with a guanidino group.

NA is one of two surface glycoproteins found on influenza virus particles (Colman 1989). Its major function is to cleave terminal sialic acid from cell-surface glycoconjugates, which is the receptor for the other surface protein, hemagglutinin. The relationship between the affinity of hemagglutinin to bind the receptor and the capacity for NA to remove the receptor appears to control replication rates of the virus (McKimm-Breshkin 2000). Inhibition of NA delays the release of progeny virions from the surface of infected cells (Liu et al. 1995), suppressing the viral population and thereby allowing time for the host immune system to eliminate the virus.

The X-ray structure of NA complexed with DANA bound in the active site (Burmeister et al. 1993; Varghese et al. 1998; Fig. 1) shows that it binds with identical interactions as the natural substrate, sialic acid (Varghese et al. 1992). Three arginine residues, 118, 292, and 371, bind the carboxylate. The oxygen and nitrogen atoms of the acetamido form hydrogen bonds with Arg152 and a bound water molecule, respectively, whereas the methyl group lies in a hydrophobic pocket near Ile222 and Trp178. The O₈- and O₉-hydroxyl groups of the glycerol side chain are hydrogen bonded to Glu276. The O₄ hydroxyl sits at the entrance to a pocket formed, in part, by the acidic groups Glu119, Asp151, and Glu227. The 4-guanidino group of zanamivir fits into this pocket and forms stable hydrogen-bonding interactions with the acid residues Asp151 and Glu227 (Varghese et al. 1995).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Schematic of the interactions of the inhibitors with active site residues of neuraminidase (NA;1, X = OH, Y = OH, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid [DANA]; 2, X = NH2, Y = OH, 4-amino-DANA; X = OH, Y = NH2, 3, 9-amino-DANA; 4, X = NH2, Y = NH2, 4,9-diamino-DANA).

Benzoic-acid-based inhibitors were synthesized to overcome the rapid excretion of the carbohydrate-based compounds and therefore provide long-term protection from viral infection (Jedrzejas et al. 1995b). One of these compounds with a guanidino substituent, which is designed to bind in the same pocket as zanamivir, was unexpectedly found to bind with the guanidino group at the position of the glycerol side chain of zanamivir (Singh et al. 1995). Inhibitors of this type can, however, achieve nanomolar inhibition of type-A influenza (Atigadda et al. 1999).

Carbocyclic compounds more closely resemble the oxo-carbenium transition state intermediate (Smith 1997) than do DANA, and they were expected to bind more tightly than did the DANA derivatives (Kim et al. 1997, 1998). Indeed, the carbocyclic analog of DANA has twice the potency of DANA (Vorwerk and Vasella 1998). The carbocyclic prodrug oseltamivir is an orally active inhibitor of influenza virus NA with inhibitory properties similar to those of zanamivir (Kim et al. 1999).

The design of zanamivir followed characterization of an energetically favorable binding potential for an ammonium group at the O₄ pocket. Replacement of the hydroxyl at this position in DANA with an amino group (4-amino-DANA, 2) was predicted to form a favorable interaction with Glu119 and was subsequently found to produce a potent inhibitor (von Itzstein et al. 1993). In addition to this site, the glycerol-binding site near Glu276 has been identified as an alternative site for binding positively charged groups (Jedrzejas et al. 1995a). A benzoic acid derivative that was designed to take advantage of the negative electrostatic potential at both the O₄ and glycerol sites, however, did not produce the expected increase in viral inhibition, despite binding in the predicted manner (Sudbeck et al. 1997). The effects of conformational strain and desolvation were advanced as possible explanations for the lack of improvement of inhibitor ability.

We consider in this study whether replacement of the O₉ hydroxyl of DANA with an amino group should form a stronger interaction with the active site acid group and improve inhibitor binding. The enzymatic assay of two new derivatives of DANA (von Itzstein et al. 1991), 9-amino (3) and 4,9-diamino-DANA (4), is reported. High resolution X-ray crystallographic structures of DANA and the three derivatives 2–4 are also presented along with a computational analysis of the binding affinities of these ligands.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
The inhibitory activity of compounds 3 and 4 was determined in an NA enzymatic assay described previously (von Itzstein et al. 1993). The inhibition constants (K_i) for these compounds, 4-amino-DANA and DANA are presented in Table 1. Replacement of the O₉ hydroxyl in DANA with an amino group reduced the K_i by almost two orders of magnitude. Further replacement of the O₄ hydroxyl with an amino group was only able to recover what had been lost by the initial amino replacement at the O₉ position. Thus, the 4,9-diamino-DANA compound had similar inhibitor activity as the original DANA. Substitution of an amino group at the O₉ position had effectively reduced inhibitor activity by two orders of magnitude in both DANA and 4-amino-DANA.

Each of the ligands has more than one possible ionization state in which the carboxylate and amino groups can be either neutral or charged. Because the binding of the carboxylate group is identical in all three structures with that of the parent DANA, we have assumed an anionic state. Assignment of the protonation state of the amino group is less clear. Thus the 4-amino and 9-amino derivatives can carry a charge of -1 if the amino group is neutral (4N-DANA, 9N-DANA), or a neutral charge if the amino group is protonated (4N⁺-DANA, 9N⁺-DANA). The 4,9-diamino compound has four possible charge states in which the amino groups are either both neutral (4N,9N-DANA), in which one is charged (4N⁺,9N-DANA, 4N,9N⁺-DANA), or in which both are charged (4N⁺,9N⁺-DANA).

Molecular mechanics calculations were used to minimize the structures of the four ligands that are bound in the active site of NA in each of the possible ionization states. Protein atom coordinates were taken from the individually refined X-ray structures for each ligand. The side chain groups of the ionizable amino acids (Asp, Glu, Arg, and Lys) were charged. Separations between the substituent at the C₄ position of the ligand and the nearest carboxylate oxygen of Glu119 are presented in Table 2. For DANA, significantly better agreement with the X-ray structure is obtained with neutral Glu119 than when it is charged (although the difference between calculated and X-ray separation is still relatively large). The separation calculated for the charged ammonium nitrogen in both the 4-amino- and 4,9-diamino-DANA complexes with charged Glu119 was found to be substantially shorter (2.64 Å–2.69 Å) than that observed in the X-ray structures (2.96 Å–2.94 Å). Considerably better agreement was obtained for the species with a neutral 4-amino group, 2.80 Å–2.87 Å. Neutralization of Glu119 provided structures with the charged 4-ammonium group that provided the best agreement with the X-ray results (2.86 Å–2.92 Å). Also shown in Table 2, the positions of the minimized inhibitor heavy atoms show smaller root –mean square (r.m.s.) deviations from the X-ray positions with Glu119 in the neutral state. These results do not appear to be artefacts of the force field used (CVFF). For example, with the CFF91 force field, a short separation of 2.63 Å is calculated between the Glu119 oxygen and the 4-amino-DANA nitrogen when both groups are charged. However, very good agreement was obtained when Glu119 was neutral (4-amino-DANA nitrogen charged), 2.95 Å.

Passaging of the virus in the presence of zanamivir selects for a mutant in which Glu119 is replaced by a glycine (McKim-Breshkin 2000). Structural analysis shows that a water molecule occupies the position of the missing carboxylate (Blick et al. 1995). This mutation, however, shows no significant effect on the binding of 4-amino-DANA. Replacement of a charged carboxylate with a neutral water molecule is unlikely to have such an insignificant effect on binding. It is more likely that the water molecule occupies the position of a neutral carboxyl oxygen. The environment of the carboxylate side chain of Glu119, however, does not indicate that it should be neutral, being flanked by the side chains of Arg118 and Arg156 (with which it forms a salt bridge, Fig. 2). In addition, the ammonium group of 4N⁺-DANA might be expected to confer additional stabilization of charge, although, as a result of binding of the inhibitor, the carboxylate is no longer openly exposed to solvent, thereby removing the solvent stabilization of the charge. In any analysis of binding, there are two important requirements: An accurate geometry (especially of the ligand and its direct environment) and an accurate representation of the atomic charges. The analysis above indicates that a consistent representation of geometry and charges has Glu119 neutral. A neutral Glu119 has therefore been adopted for the binding analysis that follows.

View larger version (82K): [in this window] [in a new window]   Fig. 2. Stereo view of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA)-bound in active site of influenza virus neuraminidase (NA). Only amino acid side chains of active site residues are shown; spheres are water molecules. Residues are overlaid with an electron density isosurface generated at the 1.5 level from the 2Fobs - Fcalc electron density of the refined model. This diagram was generated using CONSCRIPT (Lawrence and Bourke 2000), MOLSCRIPT (Kraulis 1991), and Raster3D (Merritt and Bacon 1997).

We have calculated inhibitor-binding energies through application of the following expression (Gilson and Honig 1988) and solving the Poisson-Boltzman equation to obtain solvation free energies,

(1)

where G_ps is the partial desolvation energy of the ligand by the protein (G_{ps, l}), or partial desolvation energy of the protein by the ligand (G_{ps, p}), and G_ss is the solvent-screened interaction energy of the two molecules. Alternatively, the binding energy can be obtained from the more conventional expression,

(2)

where G_s is the desolvation energy of either the entire ligand (l), protein (p) or ligand-protein complex (c), and G_el is the electrostatic interaction energy between ligand and protein partial charges. The binding energies from either method ought to be equivalent. The disadvantage of equation ii is that the ligand is not generally completely surrounded by the protein, and desolvating the protein or complex is a totally fictitious operation. Interpretation of these energies is therefore troublesome. The components of equation i, in contrast, have a clearer physical interpretation. These methods provide only the electrostatic component to the binding free energy; omission of the contribution of the nonelectrostatic energies may lead to nontrivial errors. In addition, errors in each of the components of the binding energy may not be insignificant. These calculations should, however, provide at least a semiquantitative analysis of the various components of the binding energy.

Binding energies have been calculated for each of the possible ionization states of the amine groups of the ligands. Binding energies, including each of the components in equation i and the ligand solvation energy and the electrostatic interaction energy from equation ii, are presented in Table 3. At neutral pH the ionizable groups of the ligands in solution are likely to be in their standard state (i.e., amines are charged), and the binding energies of the neutral conjugates must be corrected for the reduced concentrations at equilibrium. For the mono-basic inhibitors (4N-DANA and 9N-DANA), the effective binding energy G_eff is given by

(3)

The ionization constants (K_a) for the DANA derivatives are not known. However, the acidity constant for amines does not vary greatly, and the significant separation from the carboxylate group in both cases should allow the use of the standard ionization constant for methylamine (pK_a = 10.62; Albert and Sergeant, 1962) without incurring too large an error. Thus, at the pH in which the assay is performed (Holzer et al. 1993), pH = 5.5, the calculated binding free energy of 4N-DANA and 9N-DANA is reduced by 6.98 kcal mole^-1 to give the effective binding free energy. The same correction can be applied to the 4N⁺,9N-DANA and 4N,9N⁺-DANA derivatives, whereas for 4N,9N-DANA a correction of 13.96 kcal mole^-1 should be applied. The calculated effective binding energies are reported in Table 3 as well as the relative effective binding energies along with a comparison with experiment.

The calculated binding energies differ from the experimental values by >6 kcal mole^-1. This must be, in part, because of the approximate nature of the calculations. Specifically, no attempt is made to account for the nonelectrostatic component of the binding energy. The salient features found experimentally are, however, reproduced by these calculations, viz., the binding affinity is reduced by the introduction of the 9-amino in both DANA and 4-amino-DANA

Perhaps the most surprising feature of Table 3 is that the electrostatic interaction energy G_el is weaker for 4N⁺-DANA than it is for either 4N-DANA or DANA. In contrast, G_ss for 4N⁺-DANA is stronger than both DANA and 4N-DANA. The solvent-screened interaction energy increases as each of the amine groups is charged in complete contrast to G_el. Moreover, the change in G_el is roughly the same size (but of different sign) to G_ss when an amine is replaced with an ammonium at either the C₄ or the C₉ position. The relative binding energy for 4N⁺-DANA is similar to that obtained previously by an analogous procedure by Taylor and von Itzstein (TvI; Taylor and von Itzstein 1996). However, the individual components of the binding energies are very different. The use of different charges accounts for the absolute differences between the two calculations, but the relative differences also differ. Thus, in the TvI study, in which Glu119 was negatively charged, the electrostatic interaction energy was found to be greater for 4N⁺-DANA than it was for DANA by 40 kcal mole^-1.

The desolvation energy of the ligand (G_s,l) is considerably less for the 4-ammonium derivative than it is for DANA. Analysis of the binding energy using equation ii indicates that this is the major contribution to the stronger binding of 4N⁺-DANA, rather than a stronger interaction energy (G_el) between ligand and protein. This is in stark contrast to the analysis provided by equation i, which shows that improved binding in 4N⁺-DANA is the result of a stronger interaction energy (G_ss), despite the greater partial-desolvation energy (G_ps,l) of the ligand.

A substantial contribution to the difference in binding energies between 4N⁺-DANA and 9N⁺-DANA arises from G_ss, 16 kcal mole^-1, which is mirrored by the difference in G_el. In comparison, however, G_el is 27 kcal mole^-1 smaller for 4N⁺,9N⁺-DANA than for DANA, whereas G_ss is significantly larger (40 kcal mole^-1). It is clear that the major contributing factor to the poorer binding affinity of 4N⁺,9N⁺-DANA is the desolvation energy of the ligand, whether it be partial desolvation or total desolvation.

Replacement of a hydroxyl at either the C₄ or C₉ position with an amine has very little effect on either the partial or total desolvation energies. The partial desolvation energy increases by 11 kcal mole^-1 when either of the amine groups is charged. There is a much larger change when the second amine is charged in both 4N⁺,9N-DANA and 4N,9N⁺-DANA (20 kcal mole^-1). The total desolvation energy does not reflect this type of change. Replacing the amine with an ammonium at the C₄ position reduces the total solvation energy by 15 kcal mole^-1. At the C₉ position, the total solvation energy increases, but only slightly. However, G_s,l is significantly larger with both amines charged than it is when either just one is charged or when both are neutral. The partial solvation energy of the protein does not vary significantly, although its influence on the final relative binding energies is not insignificant.

The calculated binding energy for DANA and 4N⁺-DANA when Glu119 is charged is +7.0 and -20.1 kcal mole^-1, respectively. Thus, the G_bind calculated when Glu119 is charged is -27.1 kcal mole^-1, differing from the experimental value by a massive 24.4 kcal mole^-1.

In addition to hydrogen-bonding Glu276, the substituent at the C₉ position is in close proximity to the positively charged Arg224 (4.06 Å from N in 9-amino-DANA, and 3.90 Å from N in 4,9-diammino-DANA), which must significantly influence the electrostatic potential in the vicinity of the positively charged ammonium moiety. In contrast, the hydroxyl at the C₈ position, which also binds Glu276, is in close proximity to Glu277. Thus, the solvent screened interaction energy of 4N⁺,8N⁺-DANA calculated using the methods described here is -171.2 kcal mole^-1, substantially greater than that of 4N⁺,9N⁺-DANA (-161.2 kcal mole^-1). Despite this, the calculated binding energy is just -4.7 kcal mole^-1, slightly better than 4N⁺,9N⁺-DANA but still less than that of 4N⁺-DANA. The greater solvent-screened interaction energy is offset by the larger ligand partial desolvation energy of 98.0 kcal mole^-1. Thus, totally burying the charge at the C₈ position requires considerably more effort than partial burial of the charge at the C₉ position. In comparison, G_s,l in 4N ⁺,8N⁺- DANA is 160.6 kcal mole^-1, comparable to that calculated for 4N⁺,9N⁺-DANA.

	Conclusions

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
The design impetus behind 4,9-diamino-DANA was to capture the binding efficiency of 4-amino-DANA and introduce improved binding through the C₉ position. Enzymatic assays show that replacement of the C₉ hydroxyl with an amine in both DANA and 4-amino-DANA, however, reduces the K_i by roughly two orders of magnitude. The X-ray crystal structures of the 4-amino-, 9-amino- and 4,9-diamino-DANA derivatives bound to NA show that they bind isosterically to the parent DANA ligand and that the reduction in inhibition ability is not structural in its origins. Computational analysis of the binding energies of these ligands indicates that the solvent-screened interaction energy is improved by introducing a positive charge at the C₉ position, as predicted, and is significantly larger in 4N⁺,9N⁺-DANA than it is in any of the other ligands studied here. However, the partial desolvation energy of the ligand and protein (i.e., removal of solvent from the region that the other molecule will come to occupy in the complex) is greater than the gains made in the interaction energy.

	Materials and methods

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
X-ray crystallography
Influenza virus N9 NA (A/NWS/G70C) was purified using methods described previously (McKimm-Breshkin et al. 1991) and crystallized by standard protocols (Laver et al. 1984) in 1.9 M phosphate (pH 5.9). The space group is cubic, I432. Inhibitor complexes were prepared by soaking crystals in buffered solutions containing 5 mM concentration of inhibitor for more than 24 hr. The crystals were transferred to 20% glycerol, while maintaining the concentration of phosphate buffer before freezing in a cold stream of nitrogen gas at -166°C. X-ray diffraction data were collected on a Rigaku R-AXIS II imaging plate X-ray detector mounted on a MAC Science SRAM 18XH1 rotating anode X-ray generator, operating at 47 kV and 60 mA with focusing mirrors. Crystals were maintained under cryogenic conditions (liquid nitrogen, -100°C) during data collection. The data were processed using the R-AXIS II software for the DANA complex and with the DENZO and SCALEPACK (Otwinowski 1993) programs for the 4-amino-, 9-amino- and 4,9-diamino-DANA complexes. A model of each of the inhibitors was built into a difference Fourier map using the phases from the refined native structure (Varghese et al. 1995). Each model was then refined using X-PLOR (Brünger 1992) with energy restraints (Engh and Huber 1991). Several cycles of energy and temperature factor refinement followed, with manual intervention using the graphics program O (Jones and Kjeldgaard 1991). Additional water molecules and multiple side-chain conformations were identified during the course of refinement. X-ray data collection and structure refinement statistics are presented in Table 4. The atomic coordinates and structure factors have been deposited with the Protein Data Bank with the following accession codes; DANA, 1F8B; 4-amino-DANA, 1F8C; 9-amino-DANA, 1F8D; 4,9-diamino-DANA, 1F8E.

Solvation electrostatic energies
Electrostatic potential energies were calculated using the DelPhi program (Molecular Simulations Inc.;Nicholls and Honig). Geometries were obtained following the molecular mechanics procedure outlined above. SIP atomic radii and charges (Smith and Hall 1998) were used for all atoms. CHELPG charges (Breneman and Wiberg 1990) were calculated for each inhibitor at the HF/6–31 +G(d) level, on the geometry obtained from the molecular mechanics minimization, using the GAUSSIAN 98 (Gaussian Inc.) quantum mechanical program. For protein residues, charges and radii were taken from the SIP data set for amino acids (Smith 1999). All water molecules were removed in these calculations. The ionic strength was set to zero and dielectric constant values of 1 and 78.54 were assumed for solutes and solvent, respectively. A grid of 201 x 201 x 201 points was used, yielding a grid resolution of 0.5 grid/Å. This maintained a solvent boundary of at least 10.0 Å around the protein.

	Acknowledgments

 
We are grateful to A. van Donkelaar and J. McKimm-Breschkin for their contributions. Thanks also to V. Epa for comments on the manuscript.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

	References

TOP Abstract Introduction Results and Discussion Conclusions Materials and methods References

 
Albert, A. and Serjeant, E.P. 1962. Ionization constants of acids and bases. Methuen, London.

Atigadda, V.R., Brouillette, W.J., Duarte, F., Ali, S.M., Babu, Y.S., Banta, S., Chand, P., Chu, N., Montgomery, J.A., Walsh, D.A., et al. 1999. Potent inhibition of influenza sialidase by a benzoic acid containing a 2-pyrrolidinone substituent. J. Med. Chem. 42: 2332–2343.[CrossRef][Medline]

Bamford, M.J., Pichel, J.C., Husman, W., Patel, B., Storer, R., and Weir, N.G. 1995. Synthesis of 6-, 7- and 8-carbon sugar analogues of potent anti-influenza 2,3-didehydro-2,3-dideoxy-N-acetylneuraminic acid derivatives. J. Chem. Soc. Perkin Trans. 1: 1181–1187.

Blick, T.J., Tiong, T., Sahasrabudhe, A., Varghese, J.N., Colman, P.M., Hart, G.J., Bethell, R.C., and McKimm-Breshkin, J.L. 1995. Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac. Virology 214: 475–484.[CrossRef][Medline]

Breneman, C.M. and Wiberg, K.B. 1990. Determining atom-centered monopoles from molecular electrostatic potentials. The need for high sampling density in formamide conformational analysis. J. Comput. Chem. 11: 361–373.[CrossRef]

Brünger, A.T. 1992. X-PLOR, Version 3.1. A system for X-ray crystallography and NMR. Yale University Press, New Haven, CT.

Burmeister, W.P., Henrissa, B., Bosso, C., Cusack, S., and Ruigrok, R.W.H. 1993. Influenza B can synthesize its own inhibitor. Structure 1: 19–26.[Medline]

Chandler, M., Bamford, M.J., Conroy, R., Lamont, B., Patel, B., Patel, V.K., Steeples, I.P., Storer, R., and Weir, N.G. 1995. Synthesis of the potent influenza neuraminidase inhibitor 4-gaunidino Neu5Ac2en. X-ray molecular structure of 5-acetamido-4-amino-2,6-anhydro-3,4,5-trideoxy-D-erythroL-gluconononic acid. J. Chem. Soc. Perkin Trans. 1: 1173–1180.

Colman, P.M. 1989. Influenza virus neuraminidase - Enzyme and antigen. In The influenza viruses (ed. R.M. Krug), pp. 175–218. New York, Plenum..

Dunn, C.J. and Goa, K.L. 1999. Zanamivir: a review of its use in influenza. Drugs 58 : 761–784.

Engh, R.A. and Huber, R. 1991. Accurate bond and angle parameters for X-ray protein-structure refinement. Acta Crystalogr. A. 47: 392–400.[CrossRef]

Freund, B., Gravenstein, S., Elliott, M., and Miller, I. 1999. Zanamivir: a review of clinical safety. Drug Saf. 21: 267–281.[Medline]

Gaussian 98, 1998. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Zakrzewski VG, Montgomery Jr. JA, Stratmann RE, Burant JC, Dapprich S, Millam JM, Daniels AD, Kudin KN, Strain MC, Farkas O, Tomasi J, Barone V, Cossi M, Cammi R, Mennucci B, Pomelli C, Adamo C, Clifford S, Ochterski J, Petersson GA, Ayala PY, Cui Q, Morokuma K, Malick DK, Rabuck AD, Raghavachari K, Foresman JB, Cioslowski J, Ortiz JV, Stefanov BB, Liu G, Liashenko A, Piskorz P, Komaromi I, Gomperts R, Martin RL, Fox DJ, Keith T, Al-Laham,MA, Peng CY, Nanayakkara,A, Gonzalez C, Challacombe M, Gill PMW, Johnson B, Chen W, Wong MW, Andres JL, Gonzalez C, Head-Gordon M, Replogle ES, Pople JA. Revision A.6, Gaussian, Inc., Pittsburgh PA.

Gilson, M.K. and Honig, B. 1988. Calculation of the total electrostatic energy of a macromolecular system: Solvation energies, binding energies, and conformational analysis. Proteins 4: 7–18.[CrossRef][Medline]

Holzer, C.T., von Itzstein, M., Jin, B., Pegg, M.S., Stewart, W.P., and Wu, W.-Y. 1993. Inhibition of sialidases from viral, bacterial and mammalian sources by analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid modified at the C-4 position. Glycoconjug. J. 10: 40–44.

Jedrzejas, M.J., Singh, S., Brouillette, W.J., Air, G.W., and Luo, M. 1995a. A strategy for theoretical binding constant, K_i, calculations for neuraminidase aromatic inhibitors designed on the basis of the active site structure of influenza virus neuraminidase. Proteins 23:264–277.[CrossRef][Medline]

Jedrzejas, M.J., Singh, S., Brouillette, W.J., Laver, W.G., Air, G.M., and Luo, M. 1995b. Structures of aromatic inhibitors of influenza virus neuraminidase. Biochemistry 34: 3144–3151.[CrossRef][Medline]

Jones, T.A. and Kjeldgaard, M. 1991. Improved methods for the building of protein models in electron density maps and the location of errors in these models. Acta Cystallogr. A. 47: 110–119.[CrossRef]

Kim, C.U., Chen X., and Mendel, D.B. 1999. Neuraminidase inhibitors as anti-influenza virus agents. Antivir. Chem. Chemother. 10: 141–154.[Medline]

Kim, C.U., Lew, W., Williams, M.A., Liu, H., Zhang, L., Swaminathan, S., Bischofberger, N., Chen, M.S., Mendel, D.B., Tai C.Y., et al. 1997. Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity. J. Am. Chem. Soc. 119: 681–690.[CrossRef][Medline]

Kim, C.U., Lew, W., Williams, M.A., Wu, H., Zhang, L., Chen, X., Escarpe, P.A., Mendel, D.B., Laver, W.G., and Stevens, R.C. 1998. Structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors. J. Med. Chem. 41: 2451–2460.[CrossRef][Medline]

Kraulis, P.J. 1991. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. Appl. Cryst. 24: 946–950.[CrossRef]

Laver, W.G., Colman, P.M., Webster, R.G., Hinshaw, V.S., and Air, G.M. 1984. Influenza virus neuraminidase with haemagglutinin activity. Virology 137: 314–323.[CrossRef][Medline]

Lawrence, M.C. and Bourke, P. 2000. CONSCRIPT: a program for generating electron density isosurfaces for presentation in protein crystallography. J. Appl. Cryst. 33: 990–991.

Liu, C., Eichelberger, M.C., Compans, R.W., and Air, G.W. 1995. Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly or budding. J. Virol. 69: 1099–1106.[Abstract]

McKimm-Breschkin, J.L. 2000. Resistance of influenza viruses to neuraminidase inhibitors - a review. Antiviral Research 47: 1–17.[CrossRef][Medline]

McKimm-Breschkin, J.L., Caldwell, J.B., Guthrie, R.E., and Kortt, A.A. 1991. A new method for the purification of influenza A virus. J. Virol. Methods 32: 121–124.[CrossRef][Medline]

Merritt, E.A. and Bacon, D.J. 1997. Raster3D: Photorealistic molecular graphics. Methods Enzymol. 277: 505–524.[Medline]

Nicholls, A. and Honig, B. 1991. A rapid finite difference algorithm utilising successive over-relaxation to solve the Poisson-Boltzmann equation. J. Comput. Chem. 12: 435–445.[CrossRef]

Otwinowski, Z. 1993. Oscillation data reduction program. In Proceedings of the CCP4 Study Weekend: Data Collection and Processing (ed. L. Sawyer et al.), pp. 56–62. SERC Daresbury Laboratory, Warrington, UK.

Silagy, C.A. and Campion, K. 1999. Effectiveness and role of zanamivir in the treatment of influenza infection. Ann. Med. 31: 313–317.[Medline]

Singh, S., Jedrzejas, M.J., Air, G.M., Luo, M., Laver, W.G., and Brouillette, W.J. 1995. Structure-based inhibitors of influenza virus sialidase. A benzoic acid lead with novel interaction. J. Med. Chem. 38: 3217–3225.[CrossRef][Medline]

Smith, B.J. 1997. A conformational study of 2-oxanol: insight into the role of ring distortion on enzyme-catalyzed glycosidic bond cleavage. J. Am. Chem. Soc. 119: 2699–2706.[CrossRef]

Smith, B.J. 1999. Solvation parameters for amino acids. J. Comput. Chem. 20: 428–442.[CrossRef]

Smith, B.J. and Hall, N.E. 1998. Atomic radii: Incorporation of solvation effects. J. Comput. Chem. 19: 1482–1493.[CrossRef]

Smith, P.W., Sollis, S.L., Howes, P.D., Cherry, P.C., Starkey, I.D., Cobley, K.N., Weston, H., Scicinski, I.J., Merritt, A., Whittington, A. et al. 1998. Dihydropyrancarboxamides related to zanamivir; A new series of inhibitors of influenza virus sialidases. 1. Discovery, synthesis, biological activity, and structure-activity relationships of 4-guanidino- and 4-amino-4H-pyran-6-carboxamides. J. Med. Chem. 41: 787–797.[CrossRef][Medline]

Smith, P.W., Starkey, I.D., Howes, P.D., Sollis, S.L., Keelong, S.P., Cherry, P.C., von Itzstein, M., Wu, W.-Y., and Jin, B. 1996. Synthesis and influenza virus sialidiase inhibitory activity of analogues 4-gaunidino-Neu5Ac2en (GG167) with modified 5-substituents. Eur. J. Med. Chem. 31: 143–150.[CrossRef]

Starkey, I.D., Mahmoudian, M., Noble, D., Smith, P.W., Cherry, P.C., Howes, P.D., and Sollis, S.L. 1995. Synthesis and influenza virus sialidase inhibitory activity of the 5-desacetamido analogue of 2,3-didehydro-2,4-dideoxy-4-gaunidinyl-N-acetylneuraminic acid (GG167). Tetrahedron Lett. 299–302.

Sudbeck, E.A., Jedrzejas, M.J., Singh, S., Brouilette, W.J., Air, G.M., Laver, W.G., Babu, Y.S., Bantia, S., Chand, P., Chu, N., et al. 1997. Guanidinobenzoic acid inhibitors of influenza virus neuraminidase. J. Mol. Biol. 267: 584–594.[CrossRef][Medline]

Taylor, N.R. 1998. Inhibition of sialidase. In Methods and Principles in Medicinal Chemistry, Vol. 6, (eds. R. Mannhold) Wiley-VCH. pp. 105–118.

Taylor, N.R., and von Itzstein, M. 1994. Molecular Modeling studies on ligand binding to sialidase from influenza virus and the mechanism of catalysis. J. Med. Chem. 37: 616–624.[CrossRef][Medline]

———. 1996. A structural and energetics analysis of the binding of a series of N-acetylneuraminic-acid-based inhibitors to influenza virus sialidase. J. Comput-Aided Mol. Des. 10: 233–246.

Taylor, N.R., Cleasby, A., Singh, O., Skarzynski, T., Wonacott, A.J., Smith, P.W., Sollis, S.L., Howes, P.D., Cherry, P.C., Bethell, R., et al. 1998. Dihydropyrancarboxamides related to zanamivir: A new series of inhibitors of influenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran-6-carboxamides and sialidase from influenza virus types A and B. J. Med. Chem. 41: 798–807.[CrossRef][Medline]

Varghese, J.N. 1999. Development of neuraminidase inhibitors as anti-influenza virus drugs. Drug Develop. Res. 46: 176–196.[CrossRef]

Varghese, J.N., Epa, V.C., and Colman, P.M. 1995. Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase. Protein Science 4: 1081–1087.[Abstract]

Varghese, J.N., McKimm-Breshkin, J.L., Caldwell, J.B., Kortt, A.A., and Colman, P.M. 1992. The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor. Proteins 14: 327–332.[CrossRef][Medline]

Varghese, J.N., Smith, P.W., Sollis, S.L., Blick, T.J., Sahasrabudhe, A., Mc-Kimm-Breschin, J.L., and Colman, P.M. 1998. Drug design against a shifting target: A structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase. Structure 6: 735–746.[Medline]

von Itzstein, M., Wu, W.-Y., and Jin, B. 1994. The synthesis of 2,3-didehydro-2,4-dideoxy-4-guanidinyl-N-acetylneuraminic acid - a potent influenza virus sialidase inhibitor. Carbohydrate Res. 259: 301–305.[CrossRef][Medline]

von Itzstein, M., Wu, W.-Y., Kok, G.B., Pegg, M.S., Dyason, J.C., Jin, B., Phan, T.V., Smythe, M.L., White, H.F., Oliver, S.W., et al. 1993. Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363: 418–423.[CrossRef][Medline]

von Itzstein, M., Wu, W.-Y., Phan, T.V., Danylec, B., and Jin, B. 1991. International Patent App. WO911632-A91.10.31 (9147) CA 117:49151y.

Vorwerk, S. and Vasella, A. 1998. Carbocyclic analogues of N-acetyl-2,3-didehydro-2-deoxy-D-neuraminic acid (Neu5Ac2en, DANA): Synthesis and inhibition of viral and bacterial neuraminidases. Angew Chem. Int. d. 37: 1732–1734.[CrossRef]

Wade, R.C. 1997. `Flu' and structure-based drug design. Structure 15: 1139–1145.

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