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Authors: Shanta Bantia, Anita A. Ghate, Sandya L. Ananth, Y. Sudhakar Babu, Gillian M. Air, & Gerald M. Walsh
DOI: https://doi.org/10.1128/AAC.42.4.801•
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Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant khổng lồ NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to lớn essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 lớn select and characterize resistant viruses. BCX-140 binds to lớn the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to lớn Glu-276, whereas in GANA the guanidino group binds khổng lồ Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, & virut resistant to lớn this inhibitor was selected after six passages. The NA of this mutant was still sensitive sầu to lớn inhibition by BCX-140. However, the mutant virut was resistant khổng lồ BCX-140 in plaque và 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virut, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue cthua trận khổng lồ the binding site, while the other change was Arg-132 of HA1 lớn Gln, which in HA1 of serotype H3 is a sialic acid liên hệ (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to lớn that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, & thus, it cannot be changed lớn generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells &, hence, resistant khổng lồ NA inhibitors.

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Influenza is an adễ thương respiratory illness that has afflicted humans since ancient times. Protection can be afforded by annual immunization with a vaccine consisting of inactivated virus. However, limitations in the efficacy of vaccines, the need to lớn reformulate the vaccine every year in response lớn the antigenic drift of circulating influenza viruses, and the possibility of an antigenic shift have sầu stimulated the tìm kiếm for antiviral drugs (15). The currently licensed drugs, amantadine & its analog rimantadine, provide only modest benefit lớn patients because of their limited efficacies (they are active sầu only against influenza A vi khuẩn strains) và adverse side effects và because of the rapid development of drug-resistant strains (2, 5, 7, 13).
Influenza viruses possess two major surface glycoproteins, hemagglutinin (HA) & neuraminidase (NA). The three-dimensional X-ray structure of each has been elucidated for representative sầu strains (3, 24). The function of HA is lớn recognize and bind lớn the cell receptor (4). The main function of NA is the promotion of virus release (12, 17). NA is responsible for removing sialic acid from newly synthesized HAs và NAs which are sialylated by cellular enzymes. In the absence of functional NA, virut release is inhibited và virions are formed but remain attached khổng lồ the cell surface and to lớn each other, forming aggregates on the surfaces of infected cells (17).
NA has been considered a suitable target for antiviral drugs, since it possesses an active site whose amino acid sequence is conserved aao ước all types và subtypes of influenza vi khuẩn (23). 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA) has been described as a highly potent và selective sầu inhibitor of influenza virus NA (22, 25). Recently, it has been shown that the vi khuẩn develops resistance to GANA in cell culture. The mutations responsible for the resistance are seen in both the HA and the NA genes (6, 14, 18, 21).
The aromatic compound BCX-140, 4-(acetylamino)-3-guanidinobenzoic acid (designated BANA-113 by Singh et al. <20>), has been shown lớn inhibit the NA of influenza virut. The two NA inhibitors GANA và BCX-140 bind very differently lớn the active sầu site of NA N9 (A/Tern/Aus/G70c/75) shown in Fig. 1(trăng tròn, 23). In the case of GANA, the terminal two hydroxyl groups of the glycerol moiety interact with Glu-276 & the guanidinyl group interacts with carboxylate groups of Glu-227 and Glu-119. However, in the case of BCX-140 the inhibitor is rotated 180° and the guanidinyl group binds lớn the glycerol binding pocket of GANA và interacts with Glu-276 (11, 20). Since these two compounds bind differently khổng lồ the active sầu site, we have investigated the potential for the development of resistance khổng lồ BCX-140.
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Fig. 1. BCX-140 (pink) & GANA (green) in the active sầu site of NA N9. N2 numbering is used khổng lồ designate the active-site residues.
To determine whether virut exposed lớn BCX-140 may generate resistant variants, virut A/Singapore/1/57 was passaged in Madin-Darby canine kidney (MDCK) cells in the presence of BCX-140. The inhibitor binds entirely within the conserved catalytic site of the enzyme & makes no contacts with residues outside that site (11, 20). Any viral variant with a reduced dependency on NA for release from infected cells or an NA variant that can bind & process substrate but is no longer inhibited by the compound could emerge in the presence of the inhibitor. The aim of the present study was lớn determine if resistant mutants of influenza vi khuẩn can be generated in the presence of BCX-140 in vitro và determine the phenotypic & molecular characteristics of such mutants.
Influenza viruses A/PR/8/34, A/FM/1/47, A/New Jersey/8/76, and B/Allen/45 were obtained from the American Type Culture Collection. A/Singapore/1/57 was obtained from Robert Webster (St. Jude Children’s Hospital, Memphis, Tenn.). It had been adapted khổng lồ MDCK cells & was used as parent virut for the selection of BCX-140-resistant virus. A/Turkey/Mass/76A/Beijing/32/92 & CA/Aichi/2/68-PR8/34 were obtained from Edwin Kilbourne (Department of Microbiology & Immunology, Thủ đô New York Medical College, Valhalla, N.Y.). NWS/G70c was obtained from Graeme Laver (Australian National University, Canberra, Australia).
MDCK cells were obtained from the American Type Culture Collection and were grown in Eagle’s minimal essential medium (Bio-Whitaker) containing 10% fetal bovine serum (FBS; Hyclone Laboratories, Inc.) supplemented with glutamine & antibiotics. NAs from Vibrio cholerae andSalmonella typhimurium were obtained from Sigma. Sheep liver sialidase was partially purified (10) and was used in the NA assay.

Inhibitor.

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BCX-140 was synthesized at BioCryst Pharmaceuticals, Inc.

NA assay.


A fluorimetric assay was used lớn measure influenza virut NA activity (19). The substrate 2′-(4-methylumbelliferyl)-alpha-d-acetylneuraminic acid is cleaved by NA khổng lồ yield a fluorescent hàng hóa that can be quantified. The assay mixture contained inhibitor at various concentrations and NA enzyme in 32.5 mM MES <2-(N-morpholino)ethanesulfonic acid> buffer–4 mM calcium chloride (pH 6.5) (total volume, 80 μl). The reaction was started by the addition of 20 μl of the substrate lớn a final concentration of 75 μM. After 10 min at 37°C, 2.4 ml of 0.1 M glycine–NaOH (pH 10.2) was added to lớn 0.1 ml of the reaction mixture to terminate the reaction. A blank was run with the same substrate solution but with no enzyme. Fluorescence was recorded with an Aminco-Bowman fluorescence spectrophotometer (excitation, 360 nm; emission, 450 nm), and readings from the substrate blanks were subtracted from the sample readings. The 1/2 inhibitory concentration (IC50) was calculated by plotting percent inhibition of NA activity versus the inhibitor concentration.
Inhibition of growth of influenza vi khuẩn in MDCK cells by BCX-140 was performed as described by Nasợi et al. (16). MDCK cells were grown in Eagle’s minimal essential medium containing 10% FBS, HEPES, penicillin-streptomycin, và l-glutamine (maintenance medium). Confluent monolayers of MDCK cells in 96-well plates were infected with influenza vi khuẩn A/Singapore/1/57 (H2N2; 16 PFU) in 0.1 ml of infection medium (maintenance medium without FBS) containing tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK) trypsin. The cells were maintained for 30 min at room temperature khổng lồ allow the vi khuẩn khổng lồ adsorb. The vi khuẩn inoculum was then removed, & 200 μl of infection medium containing inhibitor at various concentrations was added. A control incubation was performed without virut infection. The plate was incubated at 37°C for 48 h under 5% carbon dioxide. The medium was then removed và the monolayer was washed with phosphate-buffered saline to remove sầu dead cells resulting from infection with the influenza virus. The number of viable cells was determined by a colorimetric method which is based on the in situ metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by viable cells. The IC50 of BCX-140 was determined as follows: percent survival = <(Ad − A0d)/(Ac −A0d)> × 100, whereAd is the absorbance at a certain drug concentration, A0d is the absorbance with no drug, và Ac is the absorbance with no virus (control sample). The IC50 was calculated by plotting percent survival versus the inhibitor concentration.
Plaque inhibition assays were performed as described by Hayden et al. (8). Confluent monolayers of MDCK cells in six-well plates were washed miễn phí of maintenance medium before use. The cells were infected with influenza vi khuẩn A (H2N2; 30 lớn 50 PFU) in 0.6 ml of infection medium containing 2 μg of TPCK trypsin per ml. The cells were maintained for 30 min at room temperature to lớn allow the virus to lớn adsorb, and the virut inoculum was removed. A 0.5% agar overlay (3 ml) in medium containing trypsin (2 μg/ml) và various concentrations of drug were added to each plate. A control was performed with no drug. The plates were incubated at 37°C under a 5% carbon dioxide atmosphere. After 48 to 72 h the agar was removed và the plates were stained with crystal violet. The IC50 was calculated by plotting plaque numbers as a percentage of that of the control versus the inhibitor concentration.
Confluent monolayers of MDCK cells in six-well plates were washed không lấy phí of maintenance medium before use. Two sets of cells were infected with influenza vi khuẩn A (H2N2; 30 to lớn 50 PFU) in 0.6 ml of infection medium containing 2 μg of TPCK trypsin per ml. The cells were maintained for 30 min at room temperature to allow the virus to lớn adsorb, & then the medium containing unadsorbed virus was removed. The first mix of cells was incubated in the presence of BCX-140 (500 μM), & the second set was used as an untreated control. The plates were incubated at 37°C under a 5% carbon dioxide atmosphere for 48 h. The cells were centrifuged out, & the supernatant was used to lớn infect the next batch of cells (second passage). Again, the first set of cells was incubated in the presence of BCX-140 (500 μM) and the second set was used as an untreated control. After each passage both MTT và plaque assays were performed to lớn look for the development of resistant strains. The resistant stoông chồng obtained after six passages in the presence of BCX-140 was designated BCX-140RM, và vi khuẩn passaged six times in the absence of the drug is designated C6A. BCX-140RM was grown in MDCK cells in the absence of the drug before its use in the enzyme assays. Both the plaque and the MTT assays showed that BCX-140RM remains resistant to lớn the drug even after it is grown in the absence of the drug. A second selection was done in the same way but with 250 μM BCX-140, & the resulting resistant stock was passaged twice at limiting dilution in the presence of the inhibitor.
The resistant viruses BCX-140RM & the wild-type virus were grown in MDCK cells and were purified with sucrose gradients. The purified viruses were disrupted with sodium dodecyl sulfate & were digested with proteinase K at 56°C for đôi mươi min. The RNA was extracted with hot phenol, followed by phenol-chlorosize extraction và ethanol precipitation (1). Full-length NA and HA cDNAs were synthesized from virion RNA with avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim). These were then amplified by PCR (94°C for 1 min, 34°C for 1 min, & 72°C for 2 min, for 34 cycles và then 72°C for 8 min). The PCR fragments were gel purified and extracted with the Wizard kit (Promega). The sequences of these PCR fragments were determined with the ABI PRISM dye terminator cycle sequencing kit (Perkin-Elmer, Applied Biosystems Inc.).
Hemagglutination assays were performed in microtiter U-bottom plates with 50 μl of virut and 50 μl of washed 1% chicken or human erythrocytes (RBCs) suspended in 0.9% saline. The plates were incubated at 4°C for approximately 1 h. To assay elution by the viral NA, 8 hemagglutination units of virut was preincubated for 30 min at room temperature either without inhibitor or with twofold serial dilutions of BCX-140 starting with a concentration of 12 μM in the first well. The vi khuẩn was allowed lớn agglutinate chicken or human RBCs at 4°C for 1 h. Elution of the virus was monitored at 37°C by looking for the appearance of the RBC button.

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The results of the NA inhibition assays are presented in Table1. BCX-140 inhibited the enzyme activities of influenza virut type A subtypes N1, N2, & N9 and effectively inhibited influenza virut type B. The IC50s ranged from 2 to 55 μM.
Virus IC50 (μM)a
A/PR/8/34 (H1N1)55 ± 8
A1/FM/1/47 (H1N1)15 ± 1
A/New Jersey/8/76 (H1N1)22.0 ± 0.3
A/Singapore/1/57 (H2N2)5.3 ± 0.3
CA/Aichi/68(HA)PR8/34(NA)(H3N2)5.5b
A/Turkey/Mass/76(HA)/Beijing/32/92(NA) (H6N2)5.1 ± 0.6
A/NWS/G70C (H1N9)2.5 ± 0.5
B/Allen/4524 ± 1


Chuyên mục: Tin Tức