Novel α- and β-amino acid inhibitors of influenza virus neuraminidase

Article abstract of DOI:10.1128/AAC.45.9.2563-2570.2001

In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 α- and β-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited K_i values in the 50 μM range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the α-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the β-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the α- or β-amino groups were replaced with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design.

Full text of DOI:10.1128/AAC.45.9.2563-2570.2001

Novel α- and β-Amino Acid Inhibitors of
Influenza Virus Neuraminidase
Warren M. Kati, Debra Montgomery, Clarence Maring,
Vincent S. Stoll, Vincent Giranda, Xiaoqi Chen, W. Graeme
Laver, William Kohlbrenner and Daniel W. Norbeck
Antimicrob. Agents Chemother. 2001, 45(9):2563. DOI:
10.1128/AAC.45.9.2563-2570.2001.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2001, p. 2563–2570
0066-4804/01/$04.00ϩ0 DOI: 10.1128/AAC.45.9.2563–2570.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 9
Novel ␣- and ␤-Amino Acid Inhibitors of Influenza
Virus Neuraminidase
WARREN M. KATI,1* DEBRA MONTGOMERY,1 CLARENCE MARING,1 VINCENT S. STOLL,1
VINCENT GIRANDA,1 XIAOQI CHEN,1† W. GRAEME LAVER,2 WILLIAM KOHLBRENNER,1
AND DANIEL W. NORBECK1
Discovery Research, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064-6217,1
and John Curtin School of Medical Research, Australian National University, Canberra 2601, Australia2
Received 7 March 2001/Returned for modification 21 May 2001/Accepted 11 June 2001
In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized
that compounds which contain positively charged amino groups in an appropriate position to interact with the
Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 ␣- and ␤-amino acids led
to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited Ki
values in the 50 ␮M range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker
activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series
resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic
acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9
neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to
undergo a small conformational change, but these compounds did not show time-dependent inhibition.
Crystallography also established that the ␣-amino group of the phenylglycine formed hydrogen bonds to the
Asp 152 carboxylate as expected. Likewise, the ␤-amino group of the pyrrolidine forms an interaction with the
Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely
conserved residue. Phenylglycine and pyrrolidine analogs in which the ␣- or ␤-amino groups were replaced
with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the
importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications
for anti-influenza drug design.
The catalytic power of enzymes arises from their ability to
bind the altered substrate in the transition state much more
tightly than the substrate in its unaltered, ground state form
(28, 29). The magnitude of this binding affinity discrimination
can be estimated from a comparison of the rate constants for
the enzymatic and nonenzymatic reactions (29) and appears to
range between 108- and 1017-fold for most enzymes (7, 30).
Any stable compound whose chemical structure resembles that
of the transition state should capture some fraction of this
binding affinity advantage, resulting in a potent and specific
inhibitor of the target enzyme. Compounds believed to mimic
the structure of the transition state or of an intermediate in the
reaction pathway have been described for well over 100 differ-
ent enzymes (29, 31).
Influenza neuraminidase is one enzyme for which a putative
transition state analog has been described (10). This enzyme is
present on the viral surface and functions to cleave terminal
␣-ketosidically linked N-acetylneuraminic acid residues from
glycoproteins, glycolipids, and oligosaccharides in a reaction
which is essential for effective replication of the influenza virus
(20). Enzyme kinetic isotope effect studies (5) have established
that the neuraminidase hydrolytic reaction proceeds through a
planar, oxocarbonium ion intermediate (Fig. 1). Compound 1,
a stable analog of the oxocarbonium ion intermediate, exhibits
a Ki value of 5 ␮M (15), which is four- to eightfold lower than
the Km value for the substrate. However, a comparison of the
neuraminidase enzymatic rate constants (10) with the nonen-
zymatic rate constants for glycoside hydrolysis (1, 32) suggests
that the transition state for this reaction should exhibit a nom-
inal Kd value in the range of 10Ϫ14 to 10Ϫ21 M with neuramin-
idase. Thus, the binding affinity of compound 1 falls far short of
that expected for an ideal transition state analog, even though
the chemical structure of compound 1 contains all of the func-
tional groups and geometrical constraints present in the oxo-
carbonium ion intermediate.
One of the features that is present in the chemical structure
of the high energy oxocarbonium ion intermediate but is miss-
ing in the chemical structure of compound 1 is a positive
charge on the ring oxygen. Burmeister and colleagues (4) have
suggested that this positive charge is likely stabilized by an
ionized Tyr 406 phenolate side chain from neuraminidase.
Since this electrostatic interaction does not occur when sub-
strates or products are bound, it seems likely that this interac-
tion is a major contributor to the tight binding of the transition
states and oxocarbonium ion intermediate. It also seems clear
that neuraminidase must either make additional interactions
with the transition states which flank the oxocarbonium ion
intermediate or greatly strengthen an existing interaction, be-
cause the transition state must be the most tightly bound moi-
ety to occur during enzyme catalysis (29). The bond breaking
and bond making that occur in the transition states would be
catalyzed if neuraminidase was able to donate a proton to the
* Corresponding author. Mailing address: Abbott Laboratories, De-
partment 47D, Bldg. AP52, 200 Abbott Park Rd., Abbott Park, IL
60064-6217. Phone: (847) 937-3980. Fax: (847) 938-2756. E-mail: warren
.kati@abbott.com.
† Present address: Tularik Inc., South San Francisco, CA 94080.
2563

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KATI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Possible enzymatic reaction mechanism for influenza virus neuraminidase. Note the presence of partial or full positive charges on the
transition state and oxocarbonium ion structures which may be stabilized by interactions with the Asp 152 and Tyr 406 residues of neuraminidase.
toluene at room temperature to generate the bicyclic product. The benzyl
protecting group and the nitrogen-oxygen bond were cleaved by catalytic hydro-
genation using Pd(OH) and carbon at 1 atm of H2 overnight to yield an enan-
tiomeric mixture of (3R, 4R) and (3S, 4S) 3-amino-4-hydroxymethyl-N-butoxy-
glycosidic oxygen in the bond-breaking step and remove a
proton from the attacking water molecule in the bond-making
step. This acid-base catalytic role could be fulfilled by the Asp
152 side chain (26) or perhaps mediated by Asp 152 via an
intervening water molecule (5). In either case the glycosidic
oxygen might develop a partial positive charge in the transition
states, a feature which is not found in the substrate, product, or
oxocarbonium ion intermediate. It was our hypothesis that the
Tyr 406 and Asp 152 side chains were critical for recognizing
the positively charged features of the transition states and
high-energy intermediate from the ground state substrate and
compound 1. We reasoned that compounds containing posi-
tively charged amino groups in appropriate positions to inter-
act with the Tyr 406 and Asp 152 side chains might be good
transition-state analog inhibitors. This hypothesis led us to test
300 ␣- and ␤-amino acids for inhibition of influenza virus
neuraminidase. Our goal was to identify novel, noncarbohy-
drate neuraminidase inhibitors which could serve as lead struc-
tures for a program to develop an anti-influenza drug therapy.
carbonyl pyrrolidine. The 3-amino group was protected with carbobenzoxy
(Cbz)-Cl and triethylamine in THF at 0°C. The hydroxymethyl group was then
oxidized to the carboxylic acid using the Jones reagent in acetone at 0°C. The Cbz
group was removed from the 3-amino group using Pd-C and 1 atm of H2 in
ethanol to afford the (3R, 4R) and (3S, 4S) enantiomeric mixture of compound
3. Compound 4 was synthesized by a multistep procedure beginning with the
condensation of N-Boc-glycine methyl ester and tert-butyl acrylate effected by
potassium t-butoxide to give (Ϯ) 4-oxo-pyrrolidine-1,3-dicarboxylic acid di-tert-
butyl ester. Hydroxylation at the 3 position was effected with m-chloroperoxy-
benzoic acid in dichloromethane at 0°C to room temperature. The resulting
crude hydroxy ketone was reacted with hydroxylamine in ethanol to give the
corresponding 4-oxime. Hydrogenation of the 4-oxime with Raney Nickel at 4
atm in ethanol gave a mixture of two 4-amino diastereomers that were isolated
after protection with N-benzyloxycarbonyloxy-succinimide and separation by
chromatography on silica gel. Selective deprotection of pyrrolidine nitrogen was
accomplished in formic acid (96%) to give 4-benzyloxycarbonylamino-3-hydroxy-
pyrrolidine-3-carboxylic acid tert-butyl ester. Reaction of the pyrrolidine nitrogen
with N,N-isopropylethylcarbamoyl chloride and N,N-diisopropylethylamine in
dichloromethane gave 4-benzyloxycarbonylamino-1-(ethyl-isopropyl-carbamoyl)-
3-hydroxy-pyrrolidine-3-carboxylic acid tert-butyl ester. Simultaneous deprotec-
tion of the 3-carboxyl and 4-amino groups occurred with concentrated HCl at
85°C to give 4-amino-1-(ethyl-isopropyl-carbamoyl)-3-hydroxy-pyrrolidine-3-car-
boxylic acid hydrochloride (compound 4). Compound 6 was obtained in four
steps from the hydroxy ketone described above. Reduction with sodium boro-
hydride in ethanol gave 3,4-dihydroxy-pyrrolidine-1,3-dicarboxylic acid di-tert-
butyl ester. Selective functionalization of the pyrrolidine nitrogen and deprotec-
tion (as previously described) produced compound 6. The stereochemistry of the
4-hydroxyl group was confirmed by X-ray crystallography. Compound 5 was
synthesized from 5-tert-butyl-3-chloro-2-hydroxy-benzaldehyde by reaction with
potassium cyanide in acetic acid to form the cyanohydrin which was subsequently
hydrolyzed to compound 5 with aqueous sodium hydroxide and hydrogen per-
oxide.
MATERIALS AND METHODS
Neuraminidase. The catalytically active head domains were purified from
A/Tokyo/3/67 and B/Memphis/3/89 influenza viruses as described previously
(11). Briefly, purified virus was treated with a protease for a period of time, and
then the solution was ultracentrifuged to pellet the viral cores. The supernatant
was then either chromatographed using a size exclusion column (for the A/
Tokyo/3/67 enzyme) or subjected to sucrose density gradient centrifugation and
dialysis (for the B/Memphis/3/89 enzyme).
Test compounds. Compound 1 was obtained from Boehringer Mannheim.
Compound 2 was synthesized by reacting 2-chloro-4-t-butylphenol with ␣-hy-
droxy hippuric acid in 10% H2SO4–acetic acid overnight at room temperature.
The benzoyl group was removed from the ␣-amino group of the adduct via
treatment with 6 N HCl and heated under vacuum to generate the HCl salt of
compound 2. Compound 3 was synthesized by a six-step procedure which began
by reacting Boc-glycine methyl ester with sodium hydride and allyl bromide in
tetrahydrofuran (THF) at 0°C. The methyl ester of the resulting bis-substituted
amide was reduced to the aldehyde using diisobutylaluminum hydride in meth-
ylene chloride at Ϫ78°C. This material was reacted with a previously formed
solution of N-benzylhydroxyamine hydrochloride and potassium carbonate in
Enzyme inhibition assays. (i) Compounds were initially tested for inhibition of
the influenza virus A/Tokyo/3/67 neuraminidase. Reaction mixtures contained a
final concentration of 30 ␮M 4-methylumbelliferyl sialic acid substrate (Sigma),
0.5 mM test compound in 200 ␮l of freshly prepared 50 mM sodium citrate, 10
mM CaCl2, 0.1 mg of bovine serum albumin/ml, and 5% dimethyl sulfoxide or
ethanol (pH 6.0) buffer. Reactions were started by the addition of neuraminidase
to reaction mixtures which were contained in the wells of white 96-well plates.
Fluorescence measurements were obtained each minute for 30 min using a

VOL. 45, 2001
AMINO ACID INHIBITORS OF NEURAMINIDASE
2565
Fluoroskan II fluorescence plate reader (Titertek Instruments) equipped with
excitation and emission filters of 355 Ϯ 35 nm and 460 Ϯ 25 nm, respectively.
DeltaSoft II software (Biometallics) controlled the plate reader. Reaction veloc-
ities were calculated from the linear region of the progress curves and compared
to uninhibited control velocities in order to identify inhibitors. Experiments to
test compounds for inhibition of B/Memphis/3/89 neuraminidase were conducted
similarly, except that the substrate concentration was 20 ␮M. (ii) Experiments to
establish the kinetic mechanism of inhibition were conducted with A/Tokyo/3/67
neuraminidase using the plate reader as outlined above, except that both sub-
strate and inhibitor concentrations were varied. (iii) A high-performance liquid
chromatography assay was established to confirm the inhibition observed in the
plate reader assay. Enzyme inhibition reactions were conducted as described
above but were then quenched with 1 mM compound 1 and were immediately
flash frozen in dry ice for subsequent analysis. Immediately after thawing, a
100-␮l aliquot of the quenched reaction mixture was injected onto a 4.6- by
50-mm Lichrosphere reverse-phase C18 column (E. Merck) which had been
equilibrated with 0.1% acetic acid in water with 18% acetonitrile. Reaction
components were separated with a linear gradient from 18 to 32% acetonitrile in
0.1% acetic acid from 0 to 10 min at a flow rate of 1 ml/min. The neuraminidase
reaction product, methylumbelliferone, eluted at about 6 min under these con-
ditions, as judged by UV detection at a wavelength of 352 nm. Methylumbellif-
erone peak areas from compound-treated reaction mixtures were compared to
those of untreated reaction mixtures in order to quantitate inhibition of the
neuraminidase enzymatic reaction. (iv) The Ki values for compounds 4 to 6
versus those of A/Tokyo/3/67 neuraminidase as well as all Ki values versus those
of B/Memphis/3/89 neuraminidase were obtained from initial velocity measure-
ments which were then fit to the following equation (21) by nonlinear regression
using Kaleidagraph software:
FIG. 2. Novel ␣- and ␤-amino acid inhibitors of influenza virus
neuraminidases.
untreated virus control cultures. The cytopathogenic effects were quantitated
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
uptake procedure (17). Briefly, 20 ␮l of a 5-mg/ml solution of MTT in Eagle’s
minimal essential medium and 5% fetal bovine serum was added to each of the
plate wells. The plates were then incubated at 37°C for 6 h, and then 40 ␮l of a
solution containing 30% sodium dodecyl sulfate in 0.02 N HCl was added to each
well. The plates were incubated at 37°C overnight, and then the absorbance from
each well was measured spectrophotometrically at 570 nm. Absorbance values
from compound-treated wells were compared to those obtained from virus and
cell controls to establish the percent reduction in cytopathogenic effects due to
the compound treatment. The 50% effective concentration, which represents the
compound dose that reduced viral cytopathogenic effects by 50%, was calculated
by using a regression analysis program for semilog curve fitting.
1 Ϫ Vi /Vo ϭ [Inhibitor]/{[Inhibitor] ϩ Ki ͑1 ϩ ͓S͔ /Km͒}
where Vi and Vo represent inhibited and uninhibited steady-state reaction veloc-
ities, respectively, and [S] is substrate concentration. Km values of 38 and 17 ␮M
were used in this equation for the calculation of Ki values against A/Tokyo and
B/Memphis neuraminidases, respectively. Ki values for enantiomeric mixtures
were not corrected for enantiomeric purity.
X-ray crystallographic studies. Isolation, purification, and crystallization of
type A N9/tern/Australia/G70c/75 neuraminidase were performed as reported by
Laver et al. (14). Crystals were soaked in a solution containing 0.93 M KH2PO4,
1.0 M K2HPO4, and 3% dimethyl sulfoxide at pH 6.7 containing millimolar
concentrations of test compound. Crystals containing compound 2 were serially
transferred into buffer plus compound 2 containing 0, 10, 20, and 27% glycerol
for 15 min at each step. The crystals were then frozen in a stream of Ϫ140°C
nitrogen. Data were collected using a MAR image plate system on a Rigaku
RU-2000 rotating anode source operating at 100 mA and 50 kV. Data were
processed to 1.9 Å using DENZO (18) and refined in XPLOR (3). Data from
crystals soaked with compound 3 were collected on a Rigaku RTP 300 RC
rotating anode source operating at 100 mA and 50 kV at 160 K using an Oxford
cryosystem. The data were collected using a RAXISII detector with a MAR
image plate and were reduced using the HKL package (19). The model 2BAT
from the Brookhaven protein data bank was used for initial phasing, and the
structures were refined with XPLOR using a combination of simulated annealing
maximum likelihood refinement and individual B-factor refinement. Electron
density maps were inspected on a Silicon Graphics INDIGO2 workstation using
the program package QUANTA 97 from Molecular Simulations.
Anti-influenza activity in cell culture. The anti-influenza activity assays were
conducted by Southern Research Institute, Birmingham, Ala. The assay proce-
dure tests six concentrations of each compound in triplicate against influenza
virus using Madin-Darby canine kidney (MDCK) cells grown in monolayers in a
96-well microtiter plate format. The wells were initially seeded with 4 ϫ 105
cells/well, and then the cells were grown for 3 days in order to reach confluent
state. The antiviral efficacy for test compounds was assessed from wells that
contained MDCK cells, 0.1 ml of the test compound solution, and 0.1 ml of an
influenza virus stock solution. The titer of the influenza virus inoculum was 126
cell culture 50% infectious doses (CCID50) per ml for the A/Victoria/3/75
(H3N2) virus and 1,000 CCID50/ml for the B/Hong Kong/5/72 influenza virus. In
addition, each plate contained cell controls that contained medium only, virus-
infected cell controls that contained medium and virus, compound cytotoxicity
controls that contained medium and each compound concentration, reagent
controls that contained compound and medium but no cells, and compound
colorimetric controls that contained compound and medium but no cells. The
plates were incubated at 37°C in a humidified atmosphere containing 5% carbon
dioxide for 3 days until maximum cytopathogenic effects were observed in the
RESULTS
Discovery of lead compounds 2 and 3. We tested approxi-
mately 300 ␣- and ␤-amino acids as potential inhibitors of the
neuraminidase catalytic domain from A/N2/Tokyo3/67 influ-
enza virus. We chose to test this group of compounds because
we felt that the positively charged amino group could mimic
the partial positive charge which we hypothesize occurs on the
glycosidic oxygen in the transition state during hydrolysis of
N-acetylneuraminic acid substrates by neuraminidase. Al-
though several amino acid inhibitors were identified using this
approach, the two most potent compounds were a phenylgly-
cine (compound 2) and a pyrrolidine (compound 3) (Fig. 2).
The results from studies to establish the kinetic mechanism
of inhibition for compounds 2 and 3 are shown in Fig. 3A and
B and 4A and B, respectively. Both compounds appear to be
competitive inhibitors relative to the substrate, with Ki values
of 41 ␮M for compound 2 and 59 ␮M for compound 3. The KiЈ
values, derived from replots of 1/Vmax versus inhibitor concen-
tration, were at least 10-fold larger than the Ki values (data not
shown) which identified these compounds as competitive in-
hibitors. No evidence for time-dependent inhibition, some-
times called slow binding inhibition (16), was observed.
The X-ray crystal structures were solved for compounds 2
and 3 bound to the neuraminidase catalytic domain from A/N9
influenza virus. As shown in Fig. 5, the carboxylate of inhibitor

2566
KATI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
hydrogen bonds to Glu 120 and Tyr 406. The carboxylate side
chain of Asp 152 is 3.7 Å from the ␤-amino group and also is
at a poor angle for a productive hydrogen bond. However, we
cannot rule out a small contribution to binding affinity from an
electrostatic interaction between these two oppositely charged
groups. Once again, the side chain of Glu 278 appears in its
induced conformation, exposing the methylene carbons to
form hydrophobic interactions with the t-butyl side chain of
inhibitor 3.
In vitro evaluation of compound 4. The crystal structure of
pyrrolidine compound 3 suggested that a compound containing
a hydroxyl group in an ␣ position in relation to the carboxylate
might form good hydrogen bonds with the Asp 152 side chain.
Compound 4 differs from compound 3 in that compound 4
contains the desired hydroxyl group as well as an N-ethyl-N-
isopropyl urea linkage to the pyrrolidine nitrogen. Compound
4 exhibited a Ki value of 0.36 ␮M against A/Tokyo neuramin-
idase, which is a 160-fold improvement in binding affinity rel-
ative to that of compound 3. The crystal structure of compound
4 bound to N9 neuraminidase (not shown) is essentially iden-
tical to that shown in Fig. 6 for compound 3, except that the
FIG. 3. Kinetics of inhibition of A/Tokyo/3/67 influenza virus neur-
aminidase by compound 2. (A) Double reciprocal plot showing com-
petitive inhibition of the reaction by compound 2 final concentrations
of 0 (F), 35 (E), 70 (f), 140 (Ⅺ), and 210 (}) ␮M. Fluor., fluores-
cence. (B) Replot of the slopes from panel A against inhibitor con-
centration to determine the Ki value of 41 ␮M from the abscissa
intercept.
2 was well stabilized through the formation of two hydrogen
bonds with the Arg 372 residue and single hydrogen bonds with
residues Arg 294 and Arg 119. The ␣-amino group of inhibitor
2 forms two hydrogen bonds with Asp 152, which is the residue
believed to interact with the glycosidic oxygen in the transition
state. The hydroxyl and chloro groups on the phenyl ring of
inhibitor 2 make no apparent interactions with the protein.
Two of the methyl carbons present on the t-butyl group of
inhibitor 2 appear to make van der Waals contacts with the
beta and gamma methylene carbons of the Glu 278 side chain.
Significantly, the Glu 278 residue adopts a new conformation
in order to accommodate the t-butyl group of inhibitor 2. This
induced conformation permits the carboxylate oxygens of the
Glu 278 side chain to form two new hydrogen bonds to the side
chain of Arg 226.
FIG. 4. Kinetics of inhibition of A/Tokyo/3/67 influenza virus neur-
aminidase by compound 3. (A) Double reciprocal plot showing com-
petitive inhibition of the reaction by compound 3 final concentrations
of 0 (F), 35 (E), 70 (f), 140 (Ⅺ), 210 (}), and 280 ({) ␮M. (B) Replot
of the slopes from panel A against inhibitor concentration to deter-
mine the Ki value of 59 ␮M from the abscissa intercept.
The structure of inhibitor 3 bound to N9 neuraminidase is
shown in Fig. 6. The carboxylate oxygens of inhibitor 3 form
hydrogen bonds with arginine residues 294, 372, and 119 in a
fashion which is similar to the interactions of inhibitor 2 with
the enzyme. The ␤-amino group is also well satisfied with

VOL. 45, 2001
AMINO ACID INHIBITORS OF NEURAMINIDASE
2567
FIG. 5. X-ray crystal structure of compound 2 bound to A/N9 neuraminidase. The conformation of the Glu 278 side chain of the native enzyme
is shown in thin lines.
␣-hydroxyl group of compound 4 is centrally positioned be-
tween the two oxygen atoms of the Asp 152 carboxyl group,
with distances of 3.2 and 3.4 Å. Thus, compound 4 was found
to bind to the enzyme in a manner which was predicted by
computer modeling. The results for the in vitro cell culture
evaluation of compound 4 are shown in Table 1. Compound 4
shows a clear antiviral effect against the A/Victoria/3/75 influ-
enza virus and appears to be about 24-fold more active than
FIG. 6. X-ray crystal structure of compound 3 bound to A/N9 neuraminidase. The conformation of the Glu 278 side chain of the native enzyme
is shown in thin lines.

2568
KATI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Inhibition of influenza virus replication
by test compoundsa
suggested to act as a general acid during catalysis by either
protonating the glycosidic oxygen directly or through an inter-
vening water molecule (5). In either case, the glycosidic oxygen
would likely develop a partial positive charge in the transition
state. In principle, this partial positive charge could contribute
several kilocalories/mole in selective binding affinity of the
transition state relative to the substrate and planar intermedi-
ate. For example, biological specificity studies have shown that
hydrogen bonds in which one partner is charged have bond
strengths of 3.5 to 4.5 kcal/mol, whereas hydrogen bonds in-
volving uncharged partners have bond strengths only in the 0.5
to 1.5 kcal/mol range (6). It seemed likely that compounds
containing a positively charged amino group rather than a
hydroxyl group might be stable analogs of this putative transi-
tion state structure for the neuraminidase reaction. Screening
of a limited number of ␣- and ␤-amino acids led to the discov-
ery of the phenylglycine compound 2 and pyrrolidine com-
pound 3 as neuraminidase inhibitors. These two compounds
represent the first influenza virus neuraminidase inhibitors to
contain an amino group functionality in an ␣ or ␤ position in
relation to the carboxylic acid group. For reference, the potent
neuraminidase inhibitors GG167 (27), GS4071 (12), and BCX-
1812 (2) contain their basic functional groups in a ␥ position
relative to the carboxylic acid group.
X-ray crystallography has shown that the amino group of
compound 2 interacts directly with the Asp 152 side chain. This
␣-amino–Asp 152 side chain interaction is an important con-
tributor to the binding, as shown by the observation that the
␣-amino compound 2 is a 365-fold more potent inhibitor than
the ␣-hydroxyl-substituted compound 4. This improvement is
slightly better than the 100-fold difference reported for com-
pound 1 versus its 4-amino counterpart (27). We note that the
hydrogen bond lengths between the ␣-amino group of com-
pound 2 and Asp 152 are 2.8 and 3.1 Å, whereas hydrogen
bond lengths from Asp 152 and Glu 120 to the 4-position of
compound 1 are 3.1 and 3.4 Å, respectively (26). The closer
hydrogen bond distances to the ␣-amino group of compound 2
are a possible explanation for the larger amino group effect
observed for compound 2 relative to the 4-amino counterpart
of compound 1.
Pyrrolidine compound 3 is the first neuraminidase inhibitor
reported to date to make a hydrogen bond to the hydroxyl
group of the strictly conserved Tyr 406 residue. The Tyr 406
hydroxyl group has been proposed to ionize during enzymatic
turnover to stabilize the positively charged oxocarbonium ion
intermediate compound 1 (4). Site-directed mutagenesis stud-
ies provide further support for this critical role, since the
Tyr406Phe mutant is devoid of enzymatic activity (8). The
2.9-Å distance between the Tyr 406 hydroxyl group and the
␤-amino group of compound 3 or 4 is identical to that observed
between the Tyr 406 hydroxyl group and the ␣-carbon of com-
pound 1 and, by extension, the presumed oxocarbonium ion
intermediate shown in Fig. 1. Our studies suggest that the
electrostatic interaction between Tyr 406 and the oxocarbo-
nium ion intermediate may be significant because replacement
of the positively charged amino group of compound 4 with a
neutral hydroxyl group of compound 5 leads to a 2,600-fold
loss of binding affinity. The interpretation of these results is
complicated somewhat by the fact that the amino group of
compound 4 also makes an interaction with Glu 120. Thus, the
A/Victoria/3/75 (H3N2)
B/Hong Kong/5/72
Compound
EC50
TC25
EC50
TC25
4
1
2.0 Ϯ 1.8
48 Ϯ 26
5.9 Ϯ 2.7
Ͼ1,000
Ͼ3,200
Ͼ130
305 Ϯ 15
650 Ϯ 320
6.3 Ϯ 3.6
Ͼ1,000
Ͼ3,200
Ͼ130
Ribavirin
a
EC50, the concentration (␮M) of compound that reduced virus-induced
cytopathogenic effects by 50% in MDCK cells. Values are means Ϯ standard
deviations of triplicate measurements. TC25, the concentration (␮M) of com-
pound that resulted in the death of 25% of the MDCK cells.
the reference neuraminidase inhibitor 1. However, compound
4 is only about twofold more active than inhibitor 1 against the
B/Hong Kong/5/72 virus. It should be noted that the cytopatho-
genicity assay used here provides no information about the
ability of the compounds in Table 1 to reduce plaque size or
plaque number. Compound 4 shows no detectable cytotoxicity
at concentrations of as high as 1 mM.
Inhibitory activity of compounds 2 to 4 against B-strain
neuraminidase. Compounds 2 to 4 were tested for their inhib-
itory activity against the neuraminidase catalytic domain from
B/Memphis/3/89 influenza virus, and the results are shown in
Table 2. Compounds 2 to 4 were found to be 17- to 580-fold
weaker inhibitors of the B-strain neuraminidase than of the
A/N2 strain enzyme.
Replacement of the amino groups of compounds 2 and 4
with a hydroxyl group. Compound 5 has a chemical structure
similar to that of compound 2, except that compound 5 con-
tains a hydroxyl group instead of an amino group at the ␣
position in relation to the carboxylate. Compound 5 was syn-
thesized in order to understand the importance of the amino
group for the binding of compound 2. Compound 5 showed
10% inhibition at 2.5 mM, which extrapolates to an estimated
Ki value of 15 mM, or a nearly 365-fold weaker binding affinity
than that of compound 2 against the A/N2/Tokyo neuramini-
dase. Likewise, compound 6 is similar to compound 4, except
that the ␤-amino group of compound 4 has been replaced with
a hydroxyl group in compound 6. A comparison of the binding
affinities of compounds 4 and 6 in Table 2 shows that the
binding affinity of compound 6 is 2,600-fold weaker than that of
compound 4.
DISCUSSION
The hypothetical reaction scheme shown in Fig. 1 was
adapted from the X-ray structural analysis of neuraminidase
bound to sialic acid (26). The Asp 152 side chain has been
TABLE 2. Inhibition of influenza virus neuraminidases
Ki (␮M) for:
Compound
A/Tokyo/3/67
B/Memphis/3/89
1
2
3
4
5
6
1.5
1.4
690
41
59
2,500
0.36
15,000 (estimated)
940
210
Not tested
Not tested

VOL. 45, 2001
AMINO ACID INHIBITORS OF NEURAMINIDASE
2569
2,600-fold replacement effect results from altered interactions
with both active-site amino acids. Nevertheless, the replace-
ment effect is quite large and underscores the importance of
charged interactions between the Tyr 406 and Glu 120 side
chains and bound ligands.
principle, lead to the discovery of novel, potent neuraminidase
inhibitors.
In conclusion, we have used enzyme mechanistic informa-
tion to form a hypothesis about specific interactions which
might be important for the tight binding of ligands to the active
site of influenza neuraminidase. We used that hypothesis to
test a limited number of compounds with the appropriate
chemical substructure and identified two classes of compounds
with modest inhibitory potency. Using analogs, we demon-
strated that interactions with Asp 152 and Tyr 406 and Glu 120
are important contributors to the binding affinities of these
compounds. The Asp 152 and Tyr 406 residues are particularly
attractive for targeted inhibitor design, because they are strictly
conserved and lab-generated mutant enzymes at these posi-
tions exhibit poor enzymatic activity (8). Thus, drug resistance
may be less likely to develop for compounds which interact
with these residues. The phenylglycine and pyrrolidine core
structures will serve as the foundation of our program to dis-
cover effective anti-influenza drugs.
It has been suggested that the time-dependent inhibition of
influenza virus neuraminidase by GS4071 results from the side
chain of Glu 278 undergoing a slow conformational change
upon binding of this inhibitor (25). However, compounds 2 to
4 also induce the Glu 278 side chain conformational change
but do not show time-dependent inhibition. These results in-
dicate that the Glu 278 conformational change does not nec-
essarily lead to time-dependent inhibition. We have previously
suggested that the time-dependent inhibition phenomenon as-
sociated with GS4071 and GG167 (9) results from the low rate
of dissociation (koff values in the 10Ϫ4 sϪ1 range) of these very
potent compounds from the enzyme (11). The koff values for
compounds 2 to 4 are too fast to be measured using our routine
assays, but we estimate that the values must be larger than
0.005 sϪ1. Thus, the koff values for compounds 2 to 4 are at
least 20-fold faster than those measured for GS4071 and
GG167, and so one might not expect compounds 2 to 4 to
exhibit slow binding kinetics if this phenomenon resulted from
low off rates.
ACKNOWLEDGMENTS
We gratefully acknowledge Louise Westbrook and Lois Allen of
Southern Research Institute for their anti-influenza cell culture test
results.
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Compounds 2 to 4 were 17- to 580-fold weaker inhibitors of
B-strain neuraminidase than of A-strain enzyme. These results
are consistent with previous reports that compounds which
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