Influenza neuraminidase inhibitors: structure-based design of a novel inhibitor series

Combinatorial and structure-based medicinal chemistry strategies were used together to advance a lead compound with an activity of K(i) = 58 microM via a potency enhancement of >70 000-fold to an analogue with an activity of K(i) = 0.8 nM against influenza neuraminidase (A/Tokyo/67). Lead optimization was initiated using molecular modeling and combinatorial chemistry. Protein crystal structures revealed that inconsistent structure-activity relationship (SAR) data resulted from different binding orientations of the inhibitor core five-membered rings from one series to another. Binding modes for a series of compounds showed up to a 180 degrees variation in orientation of the five-membered ring within the active site. Potent analogues were only achieved with chemical series that were observed to bind in the same orientation and yielded consistent SAR. In one series, consistent binding was obtained by an unprecedented occupation of a negatively charged binding pocket by a neutral methyl ester unit. The structural rationale for this novel SAR variation, based on protein crystallographic data, is given.     

In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675

With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.     

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.     

The de-guanidinylated derivative of peramivir remains a potent inhibitor of influenza neuraminidase

The guanidine function in the potent neuraminidase inhibitor peramivir was included early on in the drug design process, and examination of X-ray structural data for the enzyme-inhibitor complex would seem to indicate that the guanidine plays a critical role in promoting binding. However, this functional group may also contribute to the poor oral availability of the drug. Given that the relative stereochemistry on the guanidine-bearing carbon in peramivir is opposite to that in zanamivir (a related neuraminidase inhibitor, for which the guanidine function is known to contribute substantially to the potency), we sought to determine the importance of the guanidine group to peramivir's overall potency. Here we report that the de-guanidinylated analogue of peramivir is only ca. 1-order of magnitude less potent than peramivir itself in two in vitro inhibition assays. This suggests that next-generation inhibitors designed to improve on peramivir's properties might profitably dispense with the guanidine function.     

Chalcones as novel influenza A (H1N1) neuraminidase inhibitors from Glycyrrhiza inflata

The emergence of highly pathogenic influenza A virus strains, such as the new H1N1 swine influenza (novel influenza), represents a serious threat to global human health. During our course of an anti-influenza screening program on natural products, one new licochalcone G (1) and seven known (2-8) chalcones were isolated as active principles from the acetone extract of Glycyrrhiza inflata. Compounds 3 and 6 without prenyl group showed strong inhibitory effects on various neuraminidases from influenza viral strains, H1N1, H9N2, novel H1N1 (WT), and oseltamivir-resistant novel H1N1 (H274Y) expressed in 293T cells. In addition, the efficacy of oseltamivir with the presence of compound 3 (5 μM) was increased against H274Y neuraminidase. This evidence of synergistic effect makes this inhibitor to have a potential possibility for control of pandemic infection by oseltamivir-resistant influenza virus.     

Treatment of influenza with neuraminidase inhibitors: virological implications.

Evaluation of the emergence of influenza virus resistance to neuraminidase inhibitors (NAIs) is now demanded following experience with amantadinamines. Preliminary data have indicated that NAI-resistant virus is unlikely to emerge readily in the clinic and this is consistent with the difficulty experienced in selecting resistant virus in vitro. Resistance mutations can occur in both neuraminidase and haemagglutinin genes. The neuraminidase mutations are viral subtype specific and, therefore, clinically relevant subtypes must be employed for in vitro studies if pre-clinical data are to have predictive value. Haemagglutinin mutations generated in vitro are probably both subtype and cell culture system specific and, therefore, may not be predictive of clinical findings. Analysis of influenza-positive samples from NAI-treated patients in the clinical setting must include samples from late treatment time-points (day 4 and later) in order for resistant virus to be detected as in vitro studies and current clinical experience have indicated that resistant virus is slow to emerge and is transient.     

Oligonucleotide microarray for the subtyping of influenza virus a neuraminidase

A microarray for the subtyping of influenza A neuraminidase is presented. The selection of oligoprobes proceeded in two steps. The first step included the selection of peptides specific for each subtype of neuraminidase. At the second step, the oligoprobes were calculated using the found peptide structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for the determination of each neuraminidase subtype. The microarray testing for 19 samples with the most widespread types (N1 and N2) specifies in an unequivocal definition 18 of them and only 1 isolate has not been identified.     

On the structure-based design of novel inhibitors of H5N1 influenza A virus neuraminidase (NA)

The structure-based design of novel H5N1 neuraminidase inhibitors is currently a research topic of vital importance owing to both a recent pandemic threat by the worldwide spread of H5N1 avian influenza and the high resistance of H5N1 virus to the most widely used commercial drug, oseltamivir-OTV (Tamiflu). A specific criterion used in this work for determining fully acceptable conformations of potential inhibitors is a previous experimental proposal of exploiting potential benefits for drug design offered by the ‘150-cavity’ adjacent to the NA active site. Using the crystal structure of H5N1 NA (PDB ID: 2hty) as the starting point, in a set of 54 inhibitors previously proposed by modifying the side chains of oseltamivir, 4 inhibitors were identified using two different computational strategies (ArgusLab4.0.1, FlexX-E3.0.1) both to lower the binding free energy (BFE) of oseltamivir and to have partially acceptable conformations. These 4 oseltamivr structure-based analogues were found to adopt the most promising conformations by identifying the guanidinium side chain of Arg156 as a prospective partner for making polar contacts, but none of the modified 4-amino groups of oseltamivir in the 4 favorable conformations was found to make polar contacts with the guanidinium side chain of Arg156. Hence, the structures of two additional inhibitors were designed and shown to further lower the binding free energy of OTV relative to the previous 54 inhibitors. These two novel structures clearly suggest that it may be possible for a new substituent to be developed by functional modifications at position of the 4-amino group of oseltamivir in order to make polar contacts with the guanidinium side chain of Arg156, and thereby enhance the binding of a more potent inhibitor. Several standpoints of vital importance for designing novel structures of potentially more effective H5N1 NA inhibitors are established.     

N-(Hydroxyaminocarbonyl)phenylalanine: a novel class of inhibitor for carboxypeptidase A

N-(Hydroxyaminocarbonyl)phenylalanine (1) was designed rationally as a new type of inhibitor for carboxypeptidase A (CPA). The designed inhibitor was readily prepared from phenylalnine benzyl ester in two steps and evaluated to find that rac-1 inhibits CPA in a competitive fashion with the Ki value of 2.09 microM. Surprisingly, inhibitor 1 having the D-configuration is more potent (Ki = 1.54 microM) than its antipode by about 3-fold. A possible explanation for the stereochemistry observed in the inhibition of CPA with 1 is presented.     

Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.     

Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en.

The development of viral resistance to the neuraminidase (NA) inhibitor, 4-guanidino-Neu5Ac2en, of influenza viruses was studied by serial passage of A/Turkey/Minnesota/833/80 (H4N2) in Madin-Darby canine kidney cells in the presence of increasing concentrations of inhibitor. Resistant mutants selected after eight passages, had a 10,000-fold reduction in sensitivity to the inhibitor in plaque assays, but their affinity (1/Kd) to the inhibitor was similar to that of the parental virus. Electron microscopic analysis revealed aggregation of the mutant virus at the cell surface in the presence of the inhibitor. Sequence analysis established that a substitution had occurred in the NA (Arg-249 to Lys) and in the HA2 subunit of the hemagglutinin (Gly-75 to Glu), in the vicinity of the proposed second sialic acid binding site. The change of residue 249 appears to be a chance mutation, for we were unable to reisolate this mutant, whereas subsequent experiments indicate changes in the hemagglutinin. After 13 passages of the parental virus, mutants that were resistant to the high concentrations of inhibitor tested were obtained. These viruses retained their drug-resistant phenotype even after five passages without the inhibitor. Electron microscopic analysis revealed no aggregation of virus on the surface of infected cells in the presence of the inhibitor. Sequence analysis of the NA gene from these drug-resistant mutants revealed an additional substitution of Glu to Ala at the conserved amino acid residue 119. This substitution is responsible for reducing the affinity of the inhibitor to the NA. Our findings suggest that the emergence of mutants resistant to 4-guanidine-Neu5Ac2en is a multistep process requiring prolonged exposure to the inhibitor.     

ZstatFlu®‐II test: a chemiluminescent neuraminidase assay for influenza viral diagnostics

The ZstatFlu®‐II test is a highly sensitive, specific, rapid, point‐of‐care chemiluminescent diagnostic test for influenza infection. Influenza viral neuraminidase‐specific substrate, spiroadamantyl‐1,2‐dioxetane‐4,7‐dimethoxy‐N‐acetyl‐neuraminic acid, is at the core of the ZstatFlu®‐II Test. The enzymatic reaction was carried out at 25°C and neutral pH, representing the optimum assay conditions for influenza types A and B viral neuraminidases. The results were outputted on a Polaroid? High Speed Detector Film. Positive results appeared as a ‘+’‐shaped white film image; negative results produced no image. The ‘glow’ kinetics, facilitated by a unique combination of light enhancers, also ‘tuned’ the wavelength of emission to match the spectral properties of the film. The substrate hydrolysed non‐enzymatically at acid pH or at temperatures above 25°C. In order to minimize false positives, the ZstatFlu®‐II Test was formatted with 0.3–0.4 K_m substrate and freezing the test kit until use. The pH optimization of the ZstatFlu®‐II test is discussed with reference to model compounds of sialyl‐glycosides. A nucleophilic attack or an electrostatic stabilization of a developing carbonium ion under the influence of the adjacent carboxyl group was probably responsible for non‐enzymatic hydrolysis of the substrate. Intramolecular general acid catalysis is proposed as a mechanism for the lability of the O‐glycosidic linkage of the substrate. Copyright © 2003 John Wiley & Sons, Ltd.     

Design of benzoic acid inhibitors of influenza neuraminidase containing a cyclic substitution for the N-acetyl grouping.

A 2-pyrrolidinone ring containing a single hydroxymethyl side chain effectively replaces the N-acetylamino group of 4-(N-acetylamino)-3-guanidinobenzoic acid, a low micromolar inhibitor of influenza neuraminidase. This novel structural template affords new opportunities to evolve more potent benzoic acid inhibitors.     

Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) substrate.

This report describes the preparation of a sodium (4-methylumbelliferyl-α-d-N-acetylneuraminate) substrate and its use in a sensitive fluorometric assay of neuraminidase (EC 3.2.1.18) from Vibrio cholerae, cultured fibroblasts, and human leucocytes. V. cholerae neuraminidase showed maximum activity at pH 4.6 and an apparent K_m of 1.5 mm and was activated by CaCl₂ and inhibited by ethylenediaminetetraacetate, NaCl, and N-acetylneuraminic acid. The inhibition by N-acetylneuraminic acid was competitive (K_i = 6.1 mm). Cultured fibroblast and leucocyte neuraminidases showed maximum activity between pH 4.2 and 4.4 and apparent K_m values of 0.13 and 0.22 mm, respectively. Neuraminidase activity was considerably reduced in cultured fibroblasts of patients with mucolipidosis types I, II, and III.     

Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6

Background

The quaternary structure of eukaryotic NADH:ubiquinone oxidoreductase (complex I), the largest complex of the oxidative phosphorylation, is still mostly unresolved. Furthermore, it is unknown where transiently bound assembly factors interact with complex I. We therefore asked whether the evolution of complex I contains information about its 3D topology and the binding positions of its assembly factors. We approached these questions by correlating the evolutionary rates of eukaryotic complex I subunits using the mirror-tree method and mapping the results into a 3D representation by multidimensional scaling.

Results

More than 60% of the evolutionary correlation among the conserved seven subunits of the complex I matrix arm can be explained by the physical distance between the subunits. The three-dimensional evolutionary model of the eukaryotic conserved matrix arm has a striking similarity to the matrix arm quaternary structure in the bacterium Thermus thermophilus (rmsd=19 ?) and supports the previous finding that in eukaryotes the N-module is turned relative to the Q-module when compared to bacteria. By contrast, the evolutionary rates contained little information about the structure of the membrane arm. A large evolutionary model of 45 subunits and assembly factors allows to predict subunit positions and interactions (rmsd = 52.6 ?). The model supports an interaction of NDUFAF3, C8orf38 and C2orf56 during the assembly of the proximal matrix arm and the membrane arm. The model further suggests a tight relationship between the assembly factor NUBPL and NDUFA2, which both have been linked to iron-sulfur cluster assembly, as well as between NDUFA12 and its paralog, the assembly factor NDUFAF2.

Conclusions

The physical distance between subunits of complex I is a major correlate of the rate of protein evolution in the complex I matrix arm and is sufficient to infer parts of the complex??s structure with high accuracy. The resulting evolutionary model predicts the positions of a number of subunits and assembly factors.     

Synthesis of a carbocyclic sialic acid analogue for the inhibition of influenza virus neuraminidase

The influenza virus neuraminidase (NA) is essential for viral infection and offers a potential target for antiviral drug development. We prepared a carbocyclic sialic acid analogue, potentially able to inhibit NA. Its structure is an analogue of the transition-state of the reaction catalysed by NA. As starting material, quinic acid was selected owing to its ready availability and its stereochemical feature suitable for the target structure. The quinic acid was first converted in the shikimic acid; then two of the three hydroxyl functions of this product were selectively functionalised to obtain the target molecule (3R,4S,5R)-4-acetamido-3-guanidino-5-hydroxycyclohex-1-ene-1-carboxylic acid.     

Stereospecific synthesis of a GS 4104 metabolite: determination of absolute stereochemistry and influenza neuraminidase inhibitory activity.

The total synthesis for the determination of the absolute stereochemistry of a GS 4104 metabolite 3 is described. In addition, the influenza neuraminidase inhibitory activity of 3 and related intermediates are reported.