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3C protease of enterovirus 68: Structure-based design of Michael acceptor
inhibitors and their broad-spectrum antiviral effects against picornaviruses
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Jinzhi Tana,b,∗, Shyla Georgea,b, Yuri Kusova,b, Markus Perbandta,c, Stefan
Anemüllera,b, Jeroen R. Mestersa,b, Helene Norderd, Bruno Coutarde, Céline
Lacroixf, Pieter Leyssenf, Johan Neytsf, Rolf Hilgenfelda,b,c,g,#
Institute of Biochemistry, Center for Structural and Cell Biology in Medicine,
University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germanya; German
Centre for Infection Research (DZIF), University of Lübeckb; Laboratory for
Structural Biology of Infection and Inflammation, Universities of Lübeck and
Hamburg, Building 22a, c/o DESY, Notkestr. 85, 22603 Hamburg, Germanyc;
Department of Clinical Microbiology, Sahlgrenska Academy, Gothenburg
University, Gothenburg, Swedend; Laboratoire Architecture et Fonction des
Macromolécules Biologiques, UMR 6098, Centre National de la Recherche
Scientifique and Universités d'Aix-Marseille I et II, Case 925, 163 avenue de
Luminy, 13288 Marseille Cedex 9, Francee; Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgiumf;
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu
Chong Zhi Rd., Shanghai 201203, Chinag
# Corresponding author. Mailing address for Rolf Hilgenfeld: Institute of
Biochemistry, Center for Structural and Cell Biology in Medicine, University of
Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.01123-12 JVI Accepts, published online ahead of print on 6 February 2013
Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. Phone: +49-451-500-
4060, fax: +49-451-500-4068. Email:
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∗ Present address: Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zu Chong Zhi Rd., Shanghai 201203, China
Running title: Enterovirus 3Cpro: Broad-spectrum inhibitor design
Word counts:
Title: 18 (150 characters including spaces)
Abstract: 247
Main text: 6661
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Abstract 38
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We have determined the cleavage specificity and the crystal structure of the 3C
protease of enterovirus 68 (EV68 3Cpro). The protease exhibits a typical
chymotrypsin fold with a Cys...His...Glu catalytic triad; its three-dimensional
structure is closely related to that of the 3Cpro of rhinovirus 2 as well as to that of
poliovirus. The phylogenetic position of the EV68 3Cpro between the
corresponding enzymes of rhinoviruses on the one hand and enteroviruses on
the other prompted us to use the crystal structure for the design of irreversible
inhibitors, with the goal of discovering broad-spectrum antiviral compounds. We
synthesized a series of peptidic α,β-unsaturated ethyl esters of increasing length
and for each inhibitor candidate, we determined a crystal structure of its complex
with the EV68 3Cpro, which served as the basis for the next design round. To
exhibit inhibitory activity, compounds must span at least P3 to P1'; the most
potent inhibitors comprise P4 to P1'. Inhibitory activities were found against the
purified 3C protease of EV68 as well as with replicons for poliovirus and EV71
(EC50 = 0.5 μM for the best compound). Antiviral activities were determined using
cell cultures infected with EV71, poliovirus, echovirus 11, and various rhinovirus
serotypes. The most potent inhibitor, SG85, exhibited activity with EC50 values of
≈180 nM against EV71 and ≈60 nM against human rhinovirus 14 in a live virus-
cell-based assay. Even the shorter SG75, spanning only P3 to P1', displayed
significant activity (EC50 = 2 to 5 μM) against various rhinoviruses.
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Introduction 61
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Enteroviruses comprise several pathogens that are implicated in a large variety
of clinical manifestations ranging from mild illnesses to more serious or even life-
threatening diseases, such as meningitis, encephalitis, myocarditis, pancreatitis,
acute paralysis, or neonatal sepsis (30, 32). In recent years, China and several
countries in South East Asia have been hit by outbreaks of Hand-, Foot- and
Mouth Disease (HFMD) caused by enterovirus (EV) 71 or Coxsackievirus A16
(more than 488,000 cases in the 2008 epidemic in China alone (42)).
To date, no approved specific antiviral therapy for diseases caused by
enteroviruses is available. There is an urgent need for safe and broad-spectrum
drugs against the existing pathogenic EVs. The same holds true for other
members of the picornavirus family, in particular rhinoviruses, as it is now clear
that some of the latter cause exacerbations of asthma and COPD (30). Also,
drugs against poliovirus are needed to aid in the completion of polio eradication
(9).
The enteroviral genome consists of a single-stranded, positive-sense RNA of
approximately 7,500 bases in length. The coding region of the viral genome is
divided into three primary precursor molecules (P1, P2, P3) containing the four
structural (derived from P1) and 7 nonstructural viral proteins (derived from P2
and P3). The genome gives rise to the viral polyprotein, which is processed co-
and post-translationally through a series of primary and secondary proteolytic
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cleavages by the virus-encoded proteases 2Apro and 3Cpro/3CDpro. 2Apro cleaves
the bond between the P1 and P2 segments of the viral polyprotein, whereas the
3Cpro and its precursor, 3CDpro, are responsible for generating the majority of
precursor and mature proteins (see, e.g., (30)).
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Here, we describe the substrate cleavage specificity and the crystal structure (at
2.4 Å resolution) of the 3C protease of enterovirus 68 (EV68, also called EV-D68
to indicate its membership in the human enterovirus D family). This is the first
crystal structure of a protein from a group-D enterovirus. Like the other members
of the enterovirus family, the EV68 3Cpro is a cysteine protease containing a
Cys...His...Glu catalytic triad and exhibiting a two-domain fold similar to that of
the serine proteases of the chymotrypsin family. A structural comparison of the
3C proteases of known crystal structure revealed an intermediate position of the
EV68 3Cpro between the corresponding enzymes from human rhinovirus 2 (HRV2)
and those from enteroviruses, and prompted us to use the structure for the
design of broad-spectrum antivirals directed against picornaviruses in general,
even though EV68 itself does not play any important role as a pathogen.
Because of their importance in the viral replication cycle and their unique
specificity for glutamine in the P1 position of the substrate (which is not found in
any known host-cell protease), 3C proteases are attractive targets for antiviral
drug discovery (16, 35). However, only one such drug candidate entered clinical
trials so far. Rupintrivir (AG7088) was developed as an inhibitor of the 3Cpro of
human rhinoviruses, but development was stopped after phase II because of
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limited efficacy in the treatment of the common cold (15). Carrying an α,β-
unsaturated alkyl ester moiety, rupintrivir acts as a Michael acceptor for the
nucleophilic Cys residue in the catalytic center of the 3Cpro (25). A series of
studies found α,β-unsaturated alkyl esters to be the most active rhinovirus 3C
protease inhibitors among various electrophilic compounds (11). We and others
have occasionally described α,β-unsaturated esters as inhibitors of coronavirus
or enterovirus proteases (1, 18, 20, 21, 43), but the only systematic study
involving X-ray crystallography and structure-based drug design as well as in-
vivo evaluation deals with the coronavirus main protease as a target (43).
We synthesized a series of peptides of increasing lengths that carry an α,β-
unsaturated ester as a Michael acceptor for the nucleophilic cysteine residue of
the enzyme. A crystal structure of each compound in complex with the EV68
3Cpro revealed details of the interactions occurring in the specificity subsites of
the enzyme and suggested modifications to be applied in the next round of the
design process. We found that the potency of the compounds increased with
length. SG85, which occupied 3Cpro subsites S4 to S1', was the most potent
among these inhibitors, with kobs/[I] = 202200 M-1s-1. Most members of this series
of compounds were not toxic to Vero A, Hela Rh, BGM, RD, and Huh-T7 cells.
They proved also active on the replication of subgenomic replicons and in cells
infected with other picornaviruses such as enterovirus 71 (also called EV-A71 to
indicate its classification as a human enterovirus A (HEV-A) species), poliovirus,
echovirus 11, and rhinoviruses. Thus, our structure-based drug design approach
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underlines the suitability of the picornaviral 3C protease as a target for broad-
spectrum antivirals.
MATERIALS AND METHODS
Synthesis of α,β-unsaturated ester compounds (see Fig. 1A, B, and
Supplemental Material): SG74 was synthesized using a published procedure
(28). To a solution of SG74 (for SG75 and SG81) or SG81 (for SG82, SG83,
SG84, SG85, and SG98) (1 mmol, 1 equiv.) in 1 ml of CH2Cl2, 0.5 ml of TFA was
added at 0°C. The dark brown solution was stirred at room temperature for 2
hours and then concentrated in vacuo. 0.5 ml of diisopropylethylamine was
added to neutralize the residual in situ and the resulting solution was used for the
next step without further purification. Protected amino acid (1 mmol, 1 equiv.),
EDC·HCl (1.1 mmol, 1.1 equiv.), and HOBt (1.1 mmol, 1.1 equiv.) were added to
a solution of the above amine in 5 ml of DMF at 0°C. The resulting solution was
stirred at room temperature for 18 h. Then the reaction mixture was diluted with
ethyl acetate and the organic phase was washed with 10% citric acid, saturated
aqueous NaHCO3 and brine, dried over MgSO4, filtered and concentrated in
vacuo. The resulting white solid was further purified through silica gel
chromatography.
Recombinant protein production. cDNA corresponding to the 3Cpro domain of
EV68 (strain 3799) was amplified prior to cloning into the pOPINE plasmid in
order to produce the EV68 3Cpro in fusion with a lysine residue and a (His)6-tag at
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the C-terminus (4). The coding region was verified by DNA sequencing and the
construct was transformed into Escherichia coli Tuner (DE3) plac I cells for
expression.
The protein was purified using immobilized-metal-affinity chromatography
(Histrap FF column, GE Healthcare), and eluted with 25 mM Tris-HCl, pH 8.0,
300 mM NaCl, and 250 mM imidazole. Further purification was achieved by size-
exclusion chromatography using 25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5 mM
DTT, and a HiLoad 16/60 Superdex 75 prep grade column. Protein concentration
was determined using the Bio-Rad Protein Assay, and the sample was stored at
-70°C.
Crystallization and data collection. The protein was concentrated to 10 mg/ml
for crystallization. Initial crystallization screening was performed using the sitting-
drop vapor-diffusion method in 96-well Intelli-Plates (Dunn Laboratories). Several
commercial kits (Sigma, Jena Bioscience, and Hampton Research) were used for
screening. Using a Phoenix robotic system (Art Robbins), drops were made of
260 nl protein and 260 nl precipitant solution. The optimized crystallization
condition was identified as 0.07 M sodium acetate (pH 4.6), 5 – 15% PEG 4000,
and 10 – 30% glycerol.
The peptidic Michael acceptor compounds (SG74, SG75, SG81, SG82, SG83,
SG84, SG85, and SG98) were dissolved in DMSO. Aliquots of EV68 3C protease
in 25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5 mM DTT were incubated with
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inhibitor solution at a molar ratio of 1:5 for 30 min at room temperature, and then
subjected to co-crystallization using the sitting-drop vapor diffusion method under
the same crystallization conditions as used for the free enzyme.
No cryoprotectant was added for data collection, since the reservoir solutions
contained sufficient amounts of glycerol. All diffraction data were collected at 100
K from a single crystal for each inhibitor complex at beamline BL14.1, BESSY
(Berlin, Germany), using an MX225 CCD detector (Rayonics). Data were
processed with MOSFLM (22), and reduced and scaled using the SCALA (13)
program from the CCP4 suite (8).
Structure determination. The crystal structure of the EV68 3C protease was
determined by molecular replacement (33) using MOLREP (37) and the atomic
coordinates of Human Rhinovirus-2 3Cpro (25) as the search model. The initial
model was built using Coot (12), and the program Refmac5 (8, 29) was used for
model refinement. The complex structures were determined by molecular
replacement using the structure of the free enzyme and refined as mentioned
above, to resolutions between 1.80 and 2.65 Å. Refinement statistics are
summarized in Table 1.
The stereochemistry of the structures was assessed using PROCHECK (19). The
quality of the structure-factor data and their agreement with the atomic model
was evaluated with the program SFCHECK (38). The atomic coordinates and
structure factors for all structures have been deposited with the RCSB Protein
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Data Bank; the PDB accession codes are listed in Table 1. All figures were
generated using PyMOL (Schroedinger).
Determination of substrate specificity and enzyme kinetics. Eight
dodecapeptide substrates (see Fig. 2) for the HPLC-based cleavage assay were
purchased from GL Biochem (Shanghai) Ltd. These substrates represent the
eight putative cleavage sites of the 3Cpro in the EV68 polyprotein. At their N- and
C-termini, the peptides carried tryptophan residues for easy detection by UV
spectroscopy, as described earlier for peptide substrates of the SARS-
coronavirus main protease (36). To determine the substrate specificity of the
protease, 50 μM of each peptide (dissolved in water) was incubated with 1 μM
protease in buffer A containing 20 mM Tris (pH 7.3), 100 mM NaCl, 1 mM EDTA
at room temperature for 24 hours. Reactions were terminated by adding 0.1%
trifluoroacetic acid, and the samples were analyzed using a reverse-phase HPLC
C12 column (Jupiter 4u Proteo 90Å; Phenomenex) with detection at 280 nm. The
peak areas of the products were integrated to calculate the reaction rate of each
substrate under the catalysis of the enzyme. The optimum substrate with the
fastest reaction rate was selected for determination of the enzyme kinetics using
fluorescence spectrophotometry. The FRET peptide used as the substrate,
Dabcyl-KEALFQ↓GPPQFE-Edans amide (95% purity, Biosyntan), contained the
2C/3A cleavage site (indicated by the arrow).
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Kinetic measurements were performed in the same buffer as for the HPLC test.
The enhanced fluorescence due to the cleavage of this substrate as catalyzed by
the enzyme was monitored at 490 nm with excitation at 340 nm, using a Cary
Eclipse fluorescence spectrophotometer. The experiments were performed in a
fluorescence cuvette (volume: 1 ml) with a light-path of 1 cm for excitation and a
light-path of about 2 mm for emission, at a 90-degree angle. Slit widths were 5
nm in both cases. The photomultiplier voltage was 600 V, and the assay volume
was 400 µl. The enzyme concentration for measuring Km and kcat was 0.2 μM,
and the concentrations of the FRET peptide were varied from 2 to 20 μM. The
initial rate, within 10% of the substrate consumption, was used to calculate the
kinetic parameters using Michaelis-Menten equation fitting with the Origin
program (OriginLab). Owing to solubility limitations, the FRET substrate could not
be used at concentrations higher than 20 μM; the Km value was therefore
estimated by non-linear regression analysis according to ref. 41. By using only
relatively low substrate concentrations, we also avoided potential inner-filter
effects caused by the substrate.
Enzyme inhibition assay. The FRET-based assay was also used for the
determination of the inhibition constants of the α,β-unsaturated esters. At the
outset, we experimentally characterized the inhibitors with respect to possible
inner-filter effects. The absorption spectra of all of them were measured at a
concentration of 100 µM in DMSO in the range from 260 to 600 nm. Only
compound SG84 displayed a significant absorption above 260 nm, and up to 310
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nm. However, even from the latter compound, no absorption was registered
around 340 nm, the excitation wavelength used in the fluorimetric assay. Also, at
the emission wavelength used for the fluorimetric assay (i.e., 490 nm), none of
the compounds showed significant absorption. Finally, fluorescence emission
spectra were also recorded for all inhibitors, using the same excitation
wavelength (340 nm) as for the fluorimetric assay. None of the compounds
displayed a significant fluorescence signal around 490 nm, the emission
wavelength used in the enzymatic test system. Thus, potential inner-filter effects
did not have to be taken into account in our experiments.
The principle of the assays and more details have been described elsewhere (39,
41). Briefly, time-dependent progress curves were fitted to a first-order
exponential (equation 1) to obtain an observed first-order inhibition rate constant
(kobs) (26). F is the product fluorescence (measured in arbitrary units), v0 is the
initial velocity, t is time, and D is a displacement term accounting for the non-zero
emission at the start of data collection. Since in the case of fast inactivation, the
measurement of Ki and k3 tends to be difficult, kobs/[I] was used as an
approximation of the pseudo-second-order rate constant, in order to evaluate all
inhibitors for easy comparison.
( ) DkvF tk
obs
obs +−⋅⎟⎟⎠
⎞⎜⎜⎝
⎛= ⋅−exp10
(1) 265
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Reactions were performed with 0.2 μM protease and 20 μM FRET peptide in
buffer A (20 mM Tris (pH 7.3), 100 mM NaCl, 1 mM EDTA) by addition of the
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enzyme to the reaction buffer containing substrate and inhibitor. Three to five
different inhibitor concentrations were tested, varied over a range from 0.1 μM to
100 μM. Data from the continuous assays were analyzed with the non-linear
regression analysis program Origin (OriginLab) to obtain kobs for enzyme
inactivation at each inhibitor concentration. The slope of a graph of kobs vs [I] was
calculated using Origin and is reported as kobs/[I] in Table 2.
Cells and viruses. A derivative of human hepatocellular carcinoma cells,
constitutively expressing a T7 RNA polymerase (Huh-T7) (34), was grown in
Dulbecco’s modified minimal essential medium (DMEM) supplemented with 2
mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin sulphate, 400
μg/ml geneticin (G-418 sulphate), and fetal calf serum (10% in growth medium
and 2% in maintenance medium). Other cells used were Vero A cells, a
derivative of Vero cells (African green monkey kidney cells), BGM (buffalo green
monkey kidney cells), and RD (rhabdomyosarcoma) cells, which were grown in
MEM Rega3 (Invitrogen) supplemented with 10% FCS for regular subculturing
and 2% FCS for antiviral assays (Integro), 5 ml of 200 mM L-glutamine, and 5 ml
of 7.5% sodium bicarbonate. Hela Rh cells, a HeLa cell clone selected for
susceptibility to HRV replication, were grown in MEM (41965-039, 500 ml per
bottle, Invitrogen) supplemented with 10% FCS for regular subculturing and 2%
FCS for antiviral assays (Integro), 5 ml of 200 mM L-glutamine, and 10 ml of 1 M
HEPES (15630-056, Invitrogen).
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Echovirus 11 (ECHO11; strain Gregory) and rhinoviruses (HRV types 2, 14, 29,
70, and 95) were a kind gift of K. Andries (Johnson & Johnson). Enterovirus 71
(EV71; strain BrCr) was provided by F. van Kuppeveld (Nijmegen, The
Netherlands). Poliovirus 1 (Sabin) was obtained from the NIBSC, Hertfordshire
(UK) (J. Martin).
Viral replicons and transfection. Subgenomic replicon DNAs of PV (pRLuc31,
(2)) and HAV (p18f-Luc, (14)) encoded a full-length copy of poliovirus (strain
Mahoney) and Hepatitis A Virus (18f strain), respectively, in which the P1 capsid-
coding sequence was replaced by the firefly luciferase gene. The whole
sequence was cloned into the pGEM1 or pGEM2 vector (Promega) behind a T7
RNA polymerase promoter. The EV71 replicon DNA (pACYC-RLuc), kindly
provided by Bo Zhang, contained the EV71 sequence placed into the pACYC/177
vector (New England Biolabs), under control of the T7 promoter. In this construct,
the P1 region was replaced by the Renilla luciferase gene. Replication-deficient
replicons containing a deletion or mutation within the 3D polymerase gene were
also used as a control. To prepare replicon RNA transcripts, replicon DNAs of PV
(pRluc31), HAV (p18f-Luc), and EV71 (pACYC-EV71-RLuc) were completely
linearized by digestion with MluI, BshTI (MBI, Fermentas), and HindIII (New
England Biolabs), respectively. Copy RNA transcripts were synthesized in vitro
using linearized DNA templates, T7 RNA polymerase, and the T7 RiboMax™
Large-Scale RNA Production System (Promega) according to the manufacturer’s
recommendations. Huh-T7 cell monolayers grown in 12-well plates to a
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confluency of 80 – 90% were washed with 1 ml OptiMEM (Invitrogen) and
transfected with 0.5 μg of the replication-competent PV, EV71, and HAV replicon
DNA pRLuc31, pACYC-EV71-RLuc, and p18f-Luc, as well as 4.5 μl
Lipofectamin2000 (Invitrogen) in 300 μl OptiMEM (final volume). To transfect
replicon RNAs, RLuc31, EV71-RLuc, and 18f-Luc, the DMRIE-C reagent (3 μl/μg
of purified RNA) was used as recommended (Invitrogen). After 4 to 5 hours of
incubation at 37°C, the transfection mixtures were replaced with growth medium
containing different concentrations of the candidate inhibitor to be tested (40 μM
in preliminary screening experiments or increasing concentrations, from 0 μM to
40 μM, when studying the concentration-dependence). After 24 hours of
incubation, the cells were washed with 1 ml phosphate-buffered saline (PBS) and
lysed in 0.15 ml Passive lysis buffer (Promega) at room temperature (RT) for 10
min. After freezing (-80°C) and thawing (RT), the cell debris was removed by
centrifugation (13,000 rpm, 1 min) and the supernatant (10 μl or 20 μl) was
assayed for firefly or Renilla luciferase activity (Promega) using an Anthos Lucy-3
luminescence plate reader (Anthos Labtec instruments). All experiments were
carried out in triplicate or quadruplicate and the results are presented as mean
values with standard deviations (SD).
Cell toxicity. The CellTiter 96Aqueous One Solution Cell Proliferation Assay
(MTS test, Promega) was used to determine the cytotoxic effect of inhibitors. In
brief, the confluent monolayer of cells was incubated overnight under the
pressure of different concentrations of the tested inhibitor in a total volume of 100
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μl/well. 100% of cell lysis was achieved by incubation with 0.1% Triton-X100
(positive control). After incubation, 50 μl of medium was discarded and 10 μl of
freshly prepared single-assay reagent was added to the cells. The color reaction
was measured at 490 nm using the Anthos Lucy-3 luminescence plate reader
(see above) after 1 or 2 hrs of cell incubation at 37°C. In addition, to detect subtle
differences in cytotoxicity of the tested compounds towards Huh-T7 cells,
analysis of cell death was performed using the ToxiLight Non-destructive
Cytotoxicity BioAssay kit as recommended by the manufacturer (Lonza
Rockland).
Antiviral assays in virus-infected cells. Compound dilutions (dilution factor of
5, 4-point dose-response curve for initial screening; dilution factor of 2, 8-point
dose-response curve for further refinement) were prepared in the respective
assay medium (see above) and added to empty wells of a 96-well assay plate by
a liquid-handling robot (EVO200, Tecan). Subsequently, 50 µl of a 4x virus
dilution in assay medium (supplemented with 15 ml of 1.0 M MgCl2 (Sigma) in
case of HRV) was added, immediately followed by addition of 50 µl of the
respective cell suspension in assay medium, yielding a total assay volume of 200
µl. The assay plates were returned to the incubator (35°C for HRV, 37°C for the
other viruses) for 3 – 4 days, the time at which a maximal cytopathic effect is
observed.
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For the evaluation of the antiviral effect, the assay medium was aspirated by a
plate washer (Hydroflex, Tecan), replaced with 75 μl of a 5% MTS (Promega)
solution in phenolsulfonphthalein-free medium (EVO200, Tecan) and incubated
for 1.5 hours (37°C, 5% CO2, 95-99% relative humidity). Absorbance (OD cell
control condition varying between an OD of 0.6-0.8) was measured at a
wavelength of 498 nm (Safire², Tecan) and OD values were converted to
percentage of untreated controls and compiled into antiviral dose-response
curves.
Analysis of the raw data and calculation, if possible, of the EC50 and CC50 values
were performed employing a custom-made data processing software package
(Accelrys). The EC50 (value derived from the dose-response curve) represents
the concentration at which 50% inhibition of viral replication would be observed.
The CC50 (value derived from the dose-response curve) represents the
concentration at which the metabolic activity of the cells is reduced to 50% of the
metabolic activity of untreated cells. All assay wells yielding a percentage of
inhibition larger than 50 were inspected microscopically and scored for inhibition
of virus-induced cytopathic effects as well as evaluated for minor adverse effects
on the host cells.
RESULTS
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Substrate specificity and kinetics of EV68 3Cpro 383
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The substrate peptides used in this study corresponded to the presumable
processing sites of EV68 3Cpro within the viral polyprotein but carried N-terminal
and C-terminal tryptophan residues for easy UV-spectroscopic detection of
cleavage products at 280 nm. The individual peptides used were
WHAITQ↓GVPTYW (VP2↓VP3), WPDIAQ↓LDHLDW (VP3↓VP1),
WDAMEQ↓GITDYW (2A↓2B), WYVPRQ↓SESWLW (2B↓2C),
WEALFQ↓GPPQFW (2C↓3A), WFAGIQ↓GPYTGW (3A↓3B),
WTAKVQ↓GPGFDW (3B↓3C), and WFTDTQ↓GEIVSW (3C↓3D). All these
peptides, with the exception of VP3↓VP1, contain a Gln followed by a small
amino-acid residue (Gly or Ser) framing the cleavage site. Overall, Gly in the P1’
site leads to higher cleavage activities than Ser or Leu (Fig. 2). We observed the
highest cleavage rates for 3B↓3C and 2C↓3A; the latter was chosen for kinetic
studies. Consequently, the peptide substrate used for the FRET assay was
Dabcyl-KEALFQ↓GPPQFE-Edans amide. As Lee et al. (20) reported that
Coxsackievirus B3 (CVB3) 3Cpro exhibits higher activity with the SARS-CoV Mpro
substrate (SAVLQ↓SGFRK) than with the best CVB3 3Cpro substrate peptide, we
also tested this substrate against EV68 3Cpro, but could not detect any activity.
The kcat and Km values of the protease using the substrate corresponding to the
2C↓3A cleavage site were determined as 0.54 s-1 and 22.4 μM, respectively (not
shown).
Crystal structures of EV68 3Cpro and its complexes
18
EV68 3Cpro was cloned into the pOPINE vector, which codes for an extra Lys
residue and a (His)6-tag at the C-terminus. We decided to have these extra
residues at the C-terminus of the enzyme, as in case of the picornaviral 3C
proteases, the C-terminus is oriented away from the globular protein and
additional residues are unlikely to influence the structure or catalytic activity. This
is supported by Kuo et al. (18), who showed that C-terminally tagged EV71 3Cpro
has the same catalytic activity as the tag-free enzyme. The expression yield was
about 5.0 mg of soluble protein per liter of medium. The protein was concentrated
to 10.0 mg/ml for crystallization. Crystals grew from 0.07 M sodium acetate (pH
4.6), 5 – 15% PEG 4000, and 10 – 30% glycerol. For both the free enzyme and
its inhibitor complexes, these crystals belonged to space group P3121, with one
3Cpro monomer per asymmetric unit (Table 1). In solution, EV68 3Cpro is a
monomer as well, as indicated by gel filtration and Dynamic Light-Scattering (not
shown). The crystal structure of the free enzyme was determined by molecular
replacement using the structure of rhinovirus 3C protease (25) as a search
model, and refined to 2.4 Å resolution. All amino-acid residues were defined by
electron density, including four histidines of the C-terminal His6-tag. Alternative
conformations were detected in the electron density for the side-chains of active-
site His40 and for Cys60. The final R-factor for this crystal structure was 20.7%
(Rfree = 23.8%). The complex structures were determined based on the crystal
structure of the free enzyme, at resolutions between 1.80 and 2.65 Å. The
inhibitors could all be easily modeled according to the Fo-Fc difference density.
More details can be found in Table 1.
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
19
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
Structure of EV68 3Cpro
EV68 3Cpro adopts a chymotrypsin-like fold consisting of two β-barrel domains.
These two domains comprise residues 15 – 77 and 99 – 173, respectively, and
pack against each other to form a shallow substrate-binding cleft (Fig. 3A) that
harbors the catalytic triad of Cys147, His40, and Glu71. There are two
conformations for the side-chain of His40. One conformation, with side-chain
torsion angles χ1 = 69.0o ((+)-synclinal = +sc) and χ2 = -98.7o (anticlinal = ac),
corresponds to the catalytically competent form, in which the sulfur atom of
Cys147 is in plane with the imidazole of His40 and the carboxylate group of
Glu71. In this conformation, the distance between Cys147 Sγ and His40 Nε2 is
3.3 Å and the one between His40 Nδ1 and the carboxylate of Glu71 is 2.7 Å. In
the other conformation, with side-chain torsion angles χ1 = 179.7o (antiperiplanar
= ap) and χ2 = 86.6o (+sc), the imidazole of His40 is exposed to the solvent, with
a distance of 6.8 Å between Cys147 Sγ and His40 Nε2. A likely reason for the
occurrence of this second conformation is the low pH (4.6) of crystallization, at
which part of the His40 side-chains may be protonated, unable to interact with
the sulfhydryl of Cys147. Unfortunately, crystallization trials at higher pH
invariably led to precipitation of the protein and equilibrating low-pH crystals in
buffer of higher pH resulted in destruction of the crystals.
Substrate hydrolysis by cysteine and serine proteases occurs through a covalent
tetrahedral intermediate resulting from attack of the active-site nucleophile onto
20
the carbonyl carbon of the scissile bond. The developing oxyanion is stabilized by
hydrogen bonds donated by amide groups of the enzyme. In EV68 3Cpro, the
oxyanion hole is formed by the main-chain amides of Gly145 and the active-site
Cys147. In the structure of the free enzyme, the oxyanion hole is occupied by a
water molecule that interacts with the first of these amides.
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
Comparison of EV68 3Cpro and related picornaviral proteases
A sequence alignment (Fig. 3B) shows that the identity between the EV68 3Cpro
and the corresponding enzymes of HRV2, EV71, CVB3, EV93, and PV is 49%,
53%, 67%, 67%, and 66%, respectively, whereas the 3C proteases of HAV and
FMDV are more distantly related (sequence identities of 25 - 26%). In terms of
three-dimensional structure, the closest relatives of the EV68 3Cpro are the
enzymes of HRV2 ((25); r.m.s.d. 0.58 Å for 170 superimposed Cα atoms) and PV
(27), a member of subgroup C of human enteroviruses (HEV-C; r.m.s.d. 0.64 Å
for 173 Cα atoms). In contrast to the amino-acid sequence comparisons (see
above), the superimpositions of the three-dimensional 3Cpro structures reflect the
classification into different subgroups of enteroviruses. Thus, the enzymes of
HEV-B members CVB3 (20) (Tan et al., in preparation) and EV93 (10) exhibit
r.m.s. distances from EV68 3Cpro of 0.78 Å (for 173 Cα atoms) and 0.81 Å (for
159 Cα atoms), respectively. The 3Cpro of EV71 (23), a member of the HEV-A
subgroup, exhibits an r.m.s.d. of 0.74 Å for 174 Cα atoms. The relatively large
distance between the enteroviruses on the one hand and Hepatitis A Virus (HAV)
as well as Foot-and-Mouth Disease Virus (FMDV) on the other is reflected in high
21
r.m.s. distances between the EV68 3Cpro and the corresponding proteases of
these viruses (3,5), with r.m.s.d. values of 2.04 Å for 166 Cα atoms, and 1.93 Å
for 170 Cα atoms, respectively. The intermediate position of the EV68 3Cpro
between the proteases of human rhinoviruses and those of other enteroviruses
suggested that compounds optimized for inhibition of the EV68 enzyme might
exhibit activities against a wide spectrum of picornaviruses.
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
Peptidic α,β-unsaturated esters: design, crystal structures of complexes
with the target enzyme, and inhibitory activities
General strategy
It was reported that several α,β-unsaturated esters acted as Michael acceptors
with excellent inhibitory activity against HRV 3C protease. The compounds
exhibited encouraging antiviral activity, stability in the presence of non-enzymatic
thiols, low cellular toxicity, and were relatively easy to synthesize (25). Based on
the substrate sequence (2C/3A site) and the three-dimensional structure of the
EV68 3Cpro (see above), we have designed and synthesized a series of α,β-
unsaturated ethyl ester compounds of increasing length directed at the enzyme
Fig. 1A and B). Immediately after synthesis, new inhibitor candidates were
cocrystallized with the target protease and had the X-ray structures of their
complexes determined, in order to visualize details of ligand binding as a basis
for the subsequent round of inhibitor design.
22
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Using the above-mentioned FRET-peptide substrate for the kinetic assay, the
inhibitory activities of these compounds have been determined. Being Michael
acceptors, these inhibitors initially form a reversible encounter complex with the
3Cpro, and then undergo a chemical step (nucleophilic attack by Cys147 leading
to irreversible covalent-bond formation). The observed second-order rate
constant for inactivation (kobs/[I]) depends on both the equilibrium binding
constant, Ki = k2/k1, and the chemical rate for covalent bond formation, k3. For fast
irreversible protease inhibitors, Ki tends to be very difficult to obtain; therefore,
usage of kobs/[I] is more suitable (25, 26). In this case, a higher value of kobs/[I]
indicates that the inhibitor is more active.
P1 position
The P1 residue was not altered in our series of inhibitor candidates. A five-
membered lactam was chosen as a surrogate for the glutamine side-chain amide
in this position, as the relatively rigid lactam side chain will likely lead to a
reduced loss of conformational entropy upon binding to the S1 pocket, compared
to the more flexible Gln, and therefore might lead to tighter binding of the inhibitor
to the 3C protease. This can cause an increase of kobs/[I] by a factor of 10,
compared to the Gln-containing compound (25). In fact, in all of our inhibitor
complex structures, the lactam occupies the S1 subsite (Fig. 4), which involves
residues Thr142, Arg143mc (“mc” stands for main chain), Ala144, Cys147,
His161, and Gly163. At the bottom of the site, the Nε2 atom of His161 donates a
hydrogen bond to the oxygen atom of the lactam; the length of this hydrogen
23
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521
522
523
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525
526
527
528
529
530
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533
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535
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537
538
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540
541
542
bond among all of the complexes is 2.8 Å, indicating that this is a very stable and
conserved interaction.
P2 position
The first compound to be synthesized, SG74 (Boc-GlnLactam-CH=CH-COOEt),
just spans positions P2 – P1'. Its P2-Boc group partly fills the S2 pocket. This
compound does not show any inhibition of the EV68 3Cpro (Table 2). However, it
still binds to the enzyme and features clear electron density in the crystal
structure (Fig. 4A), an interesting demonstration of the sensitivity of X-ray
crystallography.
On the basis of the structure with SG74, we decided that the S2 subsite should
be occupied by a larger hydrophobic residue, and kept Phe invariant in the P2
position in all subsequent inhibitor candidates. The S2 subsite is formed by the
catalytic His40…Glu71 pair on one side, and Leu127 and Gly128 on the other,
and closed off by Thr130 and Asn69 "at the top" (referring to the orientation
shown in Fig. 4H). The pocket is hydrophobic in nature, explaining the preference
of the EV68 3C protease for hydrophobic residues such as Phe, Leu, or Val in
position P2 of the substrates. Surprisingly, the side-chain of His40 has moved out
of the catalytic site in all of the complexes (Fig. 4). As mentioned previously, we
detected two alternative conformations for this side-chain in the free enzyme, one
(+sc, ap) in hydrogen-bonding contact to the catalytic Cys147, and the other (ap,
+sc) exposed to the solvent. However, with χ1 = -80.3 (-sc), χ2 = 59.0 (+sc), the
24
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conformation of His40 in the complexes corresponds to a third, totally different
orientation. This conformation is stabilized by an ion-pair interaction (but not a
hydrogen bond) between His40 and the carboxylate group of Glu71. However,
while Glu71 is held roughly in the same position as in the free enzyme through a
hydrogen-bond from Asn69, the mutual orientation of Glu71 and His40 is different
from the arrangement in the catalytically active triad and the distance between
the Sγ atom of Cys147 and the Nε2 atom of His40 is about 10.8 Å in all of the
complex structures (see Fig. 4). In other complex crystal structures of
picornavirus 3C proteases, such as that of rhinovirus (25), no such
conformational change was reported. In the EV68 3Cpro complexes with P2-Phe,
the unusual conformation of His40 appears to be stabilized by a C-H...π
hydrogen bond (7, 40) between the Cβ of His40 and the P2 phenyl ring of the
inhibitors. This interaction may also contribute to an unusual orientation of the
P2-Phe residue, which does not really fill the S2 pocket (see Fig. 4H), but moves
towards S1', in concert with the unusual orientation of His40. In order to fill the S2
pocket, the χ1 torsion angle of the phenylalanine side chain should be in the -sc
range, but in fact, it is ap in the structures of the complexes described here.
P3 position
The first inhibitors to contain P2 = Phe, SG75 (Cbz-Phe-GlnLactam-CH=CH-
COOEt) and SG81 (Boc-Phe-GlnLactam-CH=CH-COOEt), span positions P3 to
P1'. Carrying a Cbz cap in the P3 position, SG75 exhibits significant inhibition
(kobs/[I] = 3246 M-1s-1), whereas SG81, which has a Boc cap in the same position,
25
is not an inhibitor (kobs/[I] = 42 M-1s-1) (Table 2). From the crystal structures of the
SG75 and SG81 complexes (Fig. 4B and 4C), it can be seen that both the Cbz
and Boc protecting groups follow the binding path of the main chain, rather than
binding in the orientation usually taken by a P3 side-chain, and the bulky Cbz
moiety even partially extends into the S4 subsite. In addition, the carbonyl
oxygen of the protecting group of SG75 accepts a 3.3-Å hydrogen bond (3.4 Å in
case of SG81) from the NH of Gly164. When we increased the lengths of the
compounds by one more amino-acid residue, i.e. having the protecting group in
P4 and an amino acid in P3, orientation of the side-chain of the latter towards the
solvent was observed. The EV68 3Cpro (like its homologues from other viruses)
has no S3 pocket, and hence, a large number of side-chains in P3 are tolerated
by the enzyme. However, there was an interesting correlation between the length
of an aliphatic side-chain in P3 and cell toxicity. Carrying a tert-butyl ester of
glutamate in P3, SG82 (Cbz-GluOtBu-Phe-GlnLactam-CH=CH-COOEt; kobs/[I] =
43225 M-1s-1; Table 2 and Fig. 4D) was slightly toxic to Huh-T7 cells and also
exhibited some adverse effects in BGM and Hela Rh cells (see Table 3).
However, when we shortened the P3 side-chain by one methylene group to give
the tert-butyl ester of aspartate in SG83 (Cbz-AspOtBu-Phe-GlnLactam-CH=CH-
COOEt, kobs/[I] = 174500 s-1M-1; Table 2 and Fig. 4E), the compound was non-
toxic (CC50 = 295 µM). Finally, the P3 side-chain is a tert-butyl ether of serine in
our most potent compound so far (SG85, Cbz-Ser(OtBu)-Phe-GlnLactam-
CH=CH-COOEt, kobs/[I] = 202200 s-1M-1; Table 2 and Fig. 4G). This compound is
also non-toxic (CC50 = 265 μM) for cells (see Supplemental Fig. S1).
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
26
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
P4 position
The S4 pocket can only tolerate small side chains, with alanine being the most
favored one by the 3Cpro of EV68. Since our inhibitors have much larger groups
at this position, such as Cbz in SG82, SG83, and SG85, Fmoc in SG84, and Boc
in SG98, these groups cannot penetrate into the S4 pocket; rather they follow the
path of the substrate main-chain and protrude partly into S5. There is no clear
electron density for Fmoc in SG84 (Fig. 4F), and the compound, which was an
intermediate in the synthesis of other inhibitors, is both toxic and has low in-vitro
activity (kobs/[I] = 9225 M-1s-1) when compared to SG83 or SG85. In contrast, the
Cbz and Boc caps of SG82, SG83, SG85, and SG98 can be easily modeled into
the Fo-Fc density. The P4-Cbz group makes strong hydrophobic interactions with
residues Leu125, Asn165, Phe170, and Tyr122 of the S4 site (Fig. 4D,E,G). A
comparison of SG85 (Cbz-Ser(OtBu)-Phe-GlnLactam-CH=CH-COOEt) with
SG98 (Boc-Thr(OtBu)-Phe-GlnLactam-CH=CH-COOEt; kobs/[I] = 11045 M-1s-1)
suggests that Cbz in P4 leads to stronger hydrophobic interactions (Fig. 4G,H)
and explains the much higher inhibitory activity of SG85.
Inhibitory effect on subgenomic replicons
Using enterovirus (PV and EV71) and hepatovirus (HAV) replicons, initial
evaluation of the inhibitory activity of our compounds was performed at 40 μM
concentration (Fig. 5). When examined microscopically, SG84 exhibited strong
27
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633
634
cellular toxicity and was therefore not studied further. However, at a
concentration of 100 µM, none of our compounds reduced by more than 10%
(Table 2 and Supplemental Fig. S1) the metabolic activity of Huh-T7 cells and
their viability according to cell toxicity assays (see Materials & Methods). SG81
did not result in inhibition of replicon replication and was therefore excluded as
well. SG75, SG83, SG85, and SG98 efficiently inhibited the replication of the PV
and EV71 replicons, but none inhibited the replication of the HAV replicon (Fig.
5). The HAV 3C protease is an outlier among the picornaviral 3C proteases, with
significant deviations in the specificity-determining subsites (overall r.m.s.d. from
EV68 3Cpro: 2.04 Å), probably explaining this lack of activity. The HAV replicon
was consequently excluded from further investigation. From the dose-response
curves presented in Fig. 6, SG85 and SG83 appear to be the most potent
inhibitors of PV replicon replication (EC50 = 0.5 µM and 1.0 µM, respectively).
SG98 and SG75 were markedly less effective (4.0 μM and 7.5 μM, respectively).
Comparable results were obtained with the EV71 replicon (Fig. 7), where SG85
and SG83 proved more potent than SG98 and SG75. Direct comparison of the
results of DNA and RNA replicon transfection in Huh-T7 cells excluded the
involvement of the cellular T7 RNA polymerase in inhibition, implying that mainly
replicon genome translation and replication steps were targeted by the inhibitors
(see Supplemental Material). Moreover, based on the observed predominant
inhibition of the replication-competent (up to 95%), as compared to the
replication-deficient (20 to 50%) replicon (Supplemental text and Fig. S2), the
picornavirus 3C protease, the proteolytic activity of which is a prerequisite for the
28
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657
formation of a functional replication complex (30, 32), was confirmed as the likely
in-vivo target of our compounds.
Antiviral activity of peptidic α,β-unsaturated esters against picornaviruses
The effect of most compounds was also evaluated on the replication of various
picornaviruses in live virus-cell-based assays employing ECHO virus 11, EV71,
multiple HRV types, and PV1. Both SG74 and SG81 proved to be inactive
against these viruses at the highest concentration tested or to produce high-µM
EC50 values. Interestingly and despite of the high EC50 values, both compounds
were shown to perfectly inhibit virus-induced cytopathic effects for at least one
concentration in the HRV29 assay (Table 3) without any adverse effects on the
host cell. Compound SG75, with Cbz in the P3 position (as opposed to SG81,
which has Boc in this position), exhibited potent and selective antiviral activity
against all viruses included in the test panel, except for PV1, for which it showed
a high EC50 value. Most potent and same order-of-magnitude activity (low-µM
EC50 values) was observed against the different rhinovirus types (order of
susceptibility HRV70 > 14 > 2 > 29 > 85). Upon comparison, SG82, SG83, and
SG85 showed a similar antiviral potency for each individual virus. Throughout the
entire data set and similar to SG75, SG82, SG83, and SG85 were shown to
completely protect host cells from virus-induced cytopathic effects except in the
PV1 assay and to some extent in the HRV2 assay. Compared to SG75, activity
was significantly more pronounced as evident from the lower EC50 values. For
most rhinoviruses, on average, even high nM values were reached. Activity
29
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663
664
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680
against ECHO11 and PV1 was markedly less pronounced. Most of the results of
the cell-based assays show the same trends as those from the enzymatic assay
and the replicons, except for SG82 that proved somewhat less active at the
enzymatic level as compared to SG83 and SG85.
DISCUSSION
It has been observed that enterovirus 68 shares properties with the rhinoviruses
and the enteroviruses (6, 17, 31). This is in line with our X-ray crystallographic
analysis of the EV68 3C protease. The three-dimensional structure of the
enzyme is closely related to those of its homologues from rhinovirus 2 and
poliovirus, whereas the structures of the 3Cpro of enterovirus 71, enterovirus 93,
and Coxsackievirus B3 are somewhat more distantly related and those of
Hepatitis A Virus and Foot-and-Mouth Disease Virus are markedly different.
We have shown that the EV68 3C protease is a target for anti-EV68 drug
discovery and, beyond that, is very useful for the design of broad-spectrum anti-
picornavirus inhibitors. A series of peptidic α,β-unsaturated ethyl esters of
growing lengths has been designed on the basis of the crystal structure of the
EV68 3Cpro and synthesized. In agreement with earlier reports on short inhibitors
of the EV71 3Cpro (18), SG74 (Boc-GlnLactam-CH=CH-COOEt), a compound
containing only residues P2 to P1', had no inhibitory activity against the enzyme
of EV68. In spite of that, we could identify clear electron density for the
compound in its complex with the enzyme. SG81, with Boc as the P3 cap, was
30
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686
687
688
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691
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693
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695
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703
an intermediate compound and did not show good inhibition. Again, however, it
did bind to the substrate-binding pocket in the crystal structure. With introduction
of the Cbz group at the P3 position (as in SG75), the inhibitory activity increased.
This can be explained by the hydrophobic contacts that the Cbz group makes by
occupying the S3 binding site for the substrate's main chain and, beyond that, by
partly extending into S4.
A comparison of SG82, SG83, SG84, SG85, and SG98, which comprise residues
P4 to P1’, reveals variable inhibitory potency and cytotoxicity. SG82 has a larger
group (GluOtBu) in P3 compared to SG83 and SG85; it showed lower inhibition
and some cytotoxicity in Huh-T7 cells. Upon shortening the side chain by one
methylene group to yield AspOtBu as in SG83, the cytotoxicity disappeared
completely. In contrast, SG84 has a fluorescein group in P4 and was cytotoxic.
After replacing this group by Boc, as in SG98, cytotoxicity was no longer
observed. However, with the Boc group in P4, the inhibitory activity of SG98 was
by far not as good as that of SG85, which has SerOtBu in P3 and Cbz in P4. So
far, SG85 proved to be the best inhibitor against the EV68 3C protease. We have
tested all these compounds as inhibitors of picornaviral replicons and also
against infection by EV71, poliovirus, ECHO virus 11, and various rhinovirus
types at the cellular level. Again, SG85 showed the best inhibitory activity with
(sub)micromolar EC50 values in the PV and EV71 replicons. SG83 and SG85
proved to be the most potent inhibitors of the series in terms of inhibition of
rhinovirus replication and, together with SG82, are about equipotent as inhibitors
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of EV71 replication. Our next aim is to optimize our lead compound, SG85,
further, to yield antivirals with improved stability, bioavailability, and activity
against a broad spectrum of picornaviruses.
Acknowledgements
We are grateful to Raul Andino and to Bo Zhang for providing PV and EV71
replicon DNAs, resp., to Jiajie Zhang for discussion, and to Doris Mutschall,
Walter Verheyen, Maria Michailow, Ekaterina Shimanovskaya, and Violaien
Lantez for expert technical assistance. We also would like to acknowledge Stijn
Delmotte, Mieke Flament, Tom Bellon, Annelies De Ceulaer, and Kim Donckers
for their help in collecting the live virus-cell-based data. The work presented here
was supported by the European Commission, initially through its VIZIER project
(contract no. LSHG-CT-2004-511960) and subsequently by its SILVER project
(contract no HEALTH-F3-2010-260644). We also acknowledge support from the
Schleswig-Holstein Innovation Fund. RH thanks the DFG Cluster of Excellence
"Inflammation at Interfaces" (EXC 306) and the Fonds der Chemischen Industrie
for continuous support. He is also supported by a Chinese Academy of Sciences
Visiting professorship for Senior International Scientists, grant no. 2010T1S6.
Céline Lacroix is supported by a Ph.D. grant of the Agency for Innovation by
Science and Technology (IWT). We acknowledge access to beamline BL14.1
operated by the Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron
storage ring (Berlin-Adlershof, Germany).
32
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References
1. Anand K, Ziebuhr J, Wadhwani P, Mesters JR, Hilgenfeld R. 2003.
Coronavirus main proteinase (3CLpro) structure: basis for design of anti-
SARS drugs. Science 300:1763-1767.
2. Andino R, Rieckhof GE, Achacoso PL, Baltimore D. 1993. Poliovirus
RNA synthesis utilizes an RNP complex formed around the 5`- end of viral
RNA. EMBO J. 12:3587-3598.
3. Bergmann EM, Mosimann SC, Chernaia MM, Malcolm BA, James MN.
1997. The refined crystal structure of the 3C gene product from hepatitis A
virus: specific proteinase activity and RNA recognition. J. Virol. 71:2436-
2448.
4. Berrow NS, Alderton D, Sainsbury S, Nettleship J, Assenberg R,
Rahman N, Stuart DI, Owens RJ. 2007. A versatile ligation-independent
cloning method for high-throughput expression screening applications.
Nucleic Acids Res. 35:e45
5. Birtley JR, Knox SR, Jaulent AM, Brick P, Leatherbarrow RJ, Curry
S. 2005. Crystal structure of Foot- and Mouth-disease virus 3C protease.
J. Biol. Chem. 280:11520-11527.
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755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
6. Blomqvist S, Savolainen C, Råman L, Roivainen M, Hovi T. 2002.
Human rhinovirus 87 and enterovirus 68 represent a unique serotype
with rhinovirus and enterovirus properties. J. Clin. Microbiol. 40:4218-
4223.
7. Brandl M, Weiss MS, Sühnel J, Hilgenfeld R. 2001. C-H...π
interactions in proteins. J. Mol. Biol. 307:357-377.
8. Collaborative Computational Project No. 4. 1994. The CCP4 suite:
programs for protein crystallography. Acta Crystallogr., Sect. D: Biol.
Crystallogr. 50:760-763.
9. Collett MS, Neyts J, Modlin JF. 2008. A case for developing antiviral
drugs against polio. Antiviral Res. 79:179-187.
10. Costenaro L, Kaczmarska Z, Arnan C, Janowski R, Coutard B, Sola
M, Gorbalenya AE, Norder H, Canard B, Coll M. 2011. Structural basis
of antiviral inhibition of the main protease, 3C, from enterovirus 93. J.
Virol. 85:10764-10773.
11. Dragovich PS, Prins TJ, Zhou R, Webber SE, Marakovits JT,
Fuhrman SA, Patick AK, Matthews DA, Lee CA, Ford CE, Burke BJ,
34
Rejto PA, Hendrickson TF, Tuntland T, Brown EL, Meador JW, Ferre
RA, Harr JEV, Kosa MB, Worland ST. 1999. Structure-based design,
synthesis, and biological evaluation of irreversible human rhinovirus 3C
protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine
replacements. J. Med. Chem. 42:1213-1224.
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
12. Emsley P, Cowtan K. 2004. Coot: Model-building tools for molecular
graphics. Acta Crystallogr. D Biol. Crystallogr. D60:2126-2132.
13. Evans P. 2006. Scaling and assessment of data quality. Acta Crystallogr.
D Biol. Crystallogr. 62:72-82.
14. Gauss-Müller V, Kusov Y. 2002. Replication of a hepatitis A virus
replicon detected by genetic recombination in vivo. J. Gen. Virol.
83:2183-2192.
15. Hayden FG, Turner RB, Gwaltney JM, Chi-Burris K, Gersten M, Hsyu
P, Patick AK, Smith III GJ, Zalman LS. 2003. Phase II, randomized,
double-blind, placebo-controlled studies of rupintrivir nasal spray 2-
percent suspension for prevention and treatment of experimentally
induced rhinovirus colds in healthy volunteers. Antimicrob. Agents
Chemother. 47:3907–3916.
35
16. Hilgenfeld R. 2010. Structure-based antivirals for emerging and
neglected RNA viruses: an emerging field for medicinal chemistry in
academia. Future Med. Chem. 2:1061-1067.
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
17. Ishiko H, Miura R, Shimada Y, Hayashi A, Nakajima H, Yamazaki S,
Takeda N. 2002. Human rhinovirus 87 identified as human enterovirus
68 by VP4-based molecular diagosis. Intervirology 45:136-141.
18. Kuo CJ, Shie JJ, Fang JM, Yen GR, Hsu JTA, Liu HG, Tseng SN,
Chang SC, Lee CY, Shih SR, Liang PH. 2008. Design, synthesis, and
evaluation of 3C protease inhibitors as anti-enterovirus 71 agents. Bioorg.
Med. Chem.16:7388-7398.
19. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. 1993.
PROCHECK: a program to check the stereochemical quality of protein
structures. J. Appl. Crystallogr. 26:283-291.
20. Lee CC, Kuo CJ, Ko TP, Hsu MF, Tsui YC, Chang SC, Yang S, Chen
SJ, Chen HC, Hsu MC, Shih SR, Liang PH, Wang AH. 2009. Structural
basis of inhibition specificities of 3C and 3C-like proteases by zinc-
coordinating and peptidomimetic compounds. J. Biol. Chem. 284:7646-
7655.
36
21. Lee ES, Lee WG, Yun SH, Rho SH, Im I, Yang ST, Sellamuthu S, Lee
YJ, Kwon SJ, Park OK, Jeon ES, Park WJ, Kim YC. 2007.
Development of potent inhibitors of the coxsackievirus 3C protease.
Biochem. Biophys. Res. Commun. 358:7-11.
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
22. Leslie AGW, Powell HR. 2007. Evolving Methods for Macromolecular
Crystallography, eds Read RJ, Sussmann JL (Springer, Berlin), vol 245,
pp 41–51.
23. Lu GW, Qi JX, Chen ZJ, Xu X, Gao F, Lin DZ, Qian WK, Liu H, Jiang
HL, Yan JH, Gao GF. 2011. Enterovirus 71 and Coxsackievirus A16 3C
proteases: Binding to rupintrivir and their substrates and anti-Hand, Foot,
and Mouth Disease Virus drug design. J. Virol. 85:10319-10331.
24. Matthews BW. 1968. Solvent content of protein crystals. J. Mol. Biol.
33:491-497.
25. Matthews DA, Dragovich PS, Webber SE, Fuhrman SA, Patick AK,
Zalman LS, Hendrickson TF, Love RA, Prins TJ, Marakovits JT,
Zhou R, Tikhe J, Ford CE, Meador JW, Ferre RA, Brown EL, Binford
SL, Brothers MA, DeLisle DM, Worland ST. 1999. Structure-assisted
design of mechanism-based irreversible inhibitors of human rhinovirus
37
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
3C protease with potent antiviral activity against multiple rhinovirus
serotypes. Proc. Natl. Acad. Sci. USA 96:11000-11007.
26. Meara JP, Rich DH. 1995. Measurement of individual rate constants of
irreversible inhibition of a cysteine proteinase by an epoxysuccinyl
inhibitor. Bioorg. Med. Chem. Lett. 5:2277-2282.
27. Mosimann SC, Cherney MM, Sia S, Plotch S, James MN. 1997.
Refined X-ray crystallographic structure of the poliovirus 3C gene
product. J. Mol. Biol. 273:1032-1047.
28. Mou K, Xu B, Ma C, Yang X, Zou X, Lü Y, Xu P. 2008. Novel CADD-
based peptidyl vinyl ester derivatives as potential proteasome inhibitors.
Bioorg. Med. Chem. Lett. 18:2198–2202.
29. Murshudov GN, Vagin AA, Dodson EJ. 1997. Refinement of
macromolecular structures by the maximum-likelihood method. Acta
Crystallogr. D Biol. Crystallogr. 53:240-255.
30. Norder H, De Palma AM, Selisko B, Costenaro L, Papageorgiou N,
Arnand C, Coutard B, Lantez V, De Lamballerie X, Baronti C, Solà M,
Tan J, Neyts J, Canard B, Coll M, Gorbalenya AE, Hilgenfeld R. 2011.
Picornavirus non-structural proteins as targets for new anti-virals with
38
broad activity. Antiviral Res. 89:204-218. 863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
31. Oberste MS, Maher K, Schnurr D, Flemister MR, Lovchik JC, Peters
H, Sessions W, Kirk C, Chatterjee N, Fuller S, Hanauer JM,
Pallansch MA. 2004. Enterovirus 68 is associated with respiratory illness
and shares biological features with both the enteroviruses and the
rhinoviruses. J. Gen. Virol. 85:2577-2584.
32. Pallansch M, Roos R. 2007. Enteroviruses: Polioviruses,
coxsackieviruses, echoviruses, and newer enteroviruses. In: Knipe, D.
M., and P. M. Howley, eds., Fields Virology, 5th Edition. Lippincott
Williams & Wilkins. pp. 840-893.
33. Rossmann MG. 1990. The molecular replacement method. Acta
Crystallogr. A Foundations Crystallogr. 46:73-82.
34. Schultz DE, Honda M, Whetter LE, McKnight KL, Lemon SM. 1996.
Mutations within the 5` nontranslated RNA of cell culture-adapted
hepatitis A virus which enhance cap-independent translation in cultured
African green monkey cells. J. Virol. 70:1041-1049.
39
35. Steuber H, Hilgenfeld R. 2010. Recent advances in targeting viral
proteases for the discovery of novel antivirals. Curr. Top. Med. Chem.
23:323-345.
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
36. Tan J, Verschueren KH, Anand K, Shen J, Yang M, Xu Y, Rao Z,
Bigalke J, Heisen B, Mesters JR, Chen K, Shen X, Jiang H,
Hilgenfeld R. 2005. pH-dependent conformational flexibility of the
SARS-CoV main proteinase (Mpro) dimer: molecular dynamics
simulations and multiple X-ray structure analyses. J. Mol. Biol. 354:25-
40.
37. Vagin A, Teplyakov A. 1997. MOLREP: an automated program for
molecular replacement. J. Appl. Cryst. 30:1022–1025.
38. Vaguine AA, Richelle J, Wodak SJ. 1999. SFCHECK: a unified set of
procedures for evaluating the quality of macromolecular structure-factor
data and their agreement with the atomic model. Acta Crystallogr. D Biol.
Crystallogr. 55:191-205.
39. Verschueren KHG, Pumpor K, Anemüller S, Chen S, Mesters JR,
Hilgenfeld R. 2008. A structural view of the inactivation of the SARS-
coronavirus main proteinase by benzotriazole esters. Chem. Biol.
15:597-606.
40
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
40. Weiss MS, Brandl M, Sühnel J, Pal D, Hilgenfeld R. 2001. More
hydrogen bonds for the (structural) biologist. Trends Biochem. Sci.
26:521-523.
41. Wilkinson GN. 1961. Statistical estimations in enzyme kinetics,
Biochem. J. 80:324-332.
42. Yang F, Ren L, Xiong Z, Li J, Xiao Y, Zhao R, He Y, Bu G, Zhou S,
Wang J, Qi J. 2009. Enterovirus 71 outbreak in the People’s Republic of
China in 2008. J. Clin. Microbiol. 47:2351-2352.
43. Yang H, Xie W, Xue X, Yang K, Ma J, Liang W, Zhao Q, Zhou Z, Pei
D, Ziebuhr J, Hilgenfeld R, Yuen KY, Wong L, Gao G, Chen S, Chen
Z, Ma D, Bartlam M, Rao Z. 2005. Design of wide-spectrum inhibitors
targeting coronavirus main proteases. PLoS Biol. 3:e324.
41
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
Figure legends
Fig. 1. (A) SG74 was synthesized using a published procedure (28). Reagents
and conditions: (a) (i) Me3SiCl, MeOH, 0-25°C, 18 h; (ii) Boc2O, Et3N, THF, 0-
25°C, 12 h, 96%; (b) LiN(SiMe3)2, THF, -78°C, 1 h; then BrCH2CN, 4 h; (c)
NaBH4, CoCl2.6H2O, MeOH, 0-25°C, 12-48 h, 73%; (d) NaBH4, LiCl, EtOH, THF
(1:1), 0-25°C, 12 h, 50%; (e) (i) SO3.Py, CH2Cl2, 0-25°C, 2 h; (ii) PPh3CHCO2Et,
25°C, 2 h, 96%; (f) (i) TFA:CH2Cl2 (1:2), 2 h; (ii) EDCI, HOBt, Z-Phe-OH, DMF, 0-
25°C, 18 h, 62%; (g) (i) TFA:CH2Cl2 (1:2), 2 h; (ii) EDCI, HOBt, Boc-Phe-OH,
DMF, 0-25°C, 18 h, 74%. (B) Reagents and conditions: (a) (i) TFA:CH2Cl2 (1:2),
2 h; (ii) EDCI, HOBt, protected amino acid, DMF, 0-25°C, 18 h, 46-70%.
Fig. 2. Substrate specificity of EV68 3Cpro. Among the peptides corresponding to
its polypeptide cleavage sites, the EV68 3Cpro displayed high activities towards
EALFQ↓GPPQF (2C↓3A) and TAKVQ↓GPGFD (3B↓3C); the former was
selected for further characterization of the enzyme's activity (not shown).
Fig. 3. (A) The three-dimensional structure of EV68 3Cpro. The catalytic triad
consisting of Cys147, His40, and Glu71 is shown in stick mode. Hydrogen bonds
between these residues are indicated by dashed lines, with the corresponding
distances given in Å. (B) 2Fo-Fc electron density (contoured at 1σ above the
mean) for the catalytic center. Note the two alternative conformations for His40;
42
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
only one of these interacts with Cys147. (C) Structure-based sequence alignment
of 3C proteases of various picornaviruses. The secondary structure (as found in
the crystal structure of the EV68 enzyme) is shown at the top, invariant residues
are highlighted with red background, and conserved residues are shown in red.
Fig. 4. Binding-mode analysis by X-ray crystallography of α,β-unsaturated ethyl
ester inhibitors. (A) to (H) show the binding modes of SG74, SG75, SG81, SG82,
SG83, SG84, SG85, and SG98, respectively. The catalytic triad is shown in
yellow, while other residues involved in the binding site are shown in pink.
Inhibitors are shown in green along with an omit 2Fo-Fc map (blue) contoured at
1.0 σ above the mean. A stereo view of this figure can be found in the on-line
Supplemental Material.
Fig. 5. Initial evaluation of selected Michael acceptor compounds (40 µM) for
inhibition of PV, EV71, and HAV replicons in Huh-T7 cells. “-” – no inhibitor
added to the cell-growth medium after PV, EV71, or HAV replicon transfection. *
– compound SG82 (yellow bar) was not tested in the EV71 replicon. RLU –
relative light units of firefly (PV and HAV replicons) or Renilla (EV71 replicon)
luciferase. The results are means of quadruplicate +/- SD.
Fig. 6. The 50% effective concentration (EC50) of SG75 (filled circles), SG83
(empty circles), SG85 (filled triangles), and SG98 (empty triangles) on the
43
replication of the PV replicon in Huh-T7 cells. The values represent the means of
quadruplicate wells +/- SD.
969
970
971
972
973
974
975
976
977
978
979
980
981
Fig. 7. Concentration-dependent inhibition of EV71 replication-competent
replicon DNA by SG75 (filled circles), SG83 (empty circles), SG85 (filled
triangles), and SG98 (empty triangles).
44
Table 1. Data collection and refinement statistics for crystals of EV68 3Cpro and
its complexes
982
983
EV68 3Cpro EV68 3Cpro/SG74
EV68 3Cpro/SG75
EV68 3Cpro/SG81
EV68 3Cpro/SG82
Data collection statistics
Space group P3121 P3121 P3121 P3121 P3121 Unit cell parameters (Å)
56.34, 56.34, 171.00
55.99, 55.99, 170.55
56.10, 56.10, 170.41
56.32, 56.32, 170.34
56.49, 56.49, 170.36
Estimated solvent contenta (%)
67.05 66.55 66.65 66.90 67.10
Wavelength (Å) 0.9184 0.9184 0.9184 0.9184 0.9184 Resolution range (Å) 42.75-2.40
(2.53-2.40) 34.11-2.05 (2.16-2.05)
36.92-2.20 (2.32-2.20)
48.77-2.65 (2.79-2.65)
34.07-2.00 (2.11-2.00)
Number of reflections measured
100289 (14756)
183050 (26814)
118096 (17334)
172402 (25467)
200417 (29073)
Unique reflections 13045 (1878) 20341 (2929) 16608 (2389) 9730 (1394) 22202 (3176) Completeness (%) 100 (100) 100 (100) 100 (100) 100 (100) 100 (100) Rmerge (%)b,c 0.099 (0.569) 0.092 (0.480) 0.089 (0.455) 0.137 (0.480) 0.086 (0.454) Redundancy 7.7 (7.9) 9.0 (9.2) 7.1 (7.3) 17.7 (18.3) 9.0 (9.2) I/σ(I) 14.1 (3.9) 15.7 (4.6) 16.0 (5.4) 18.7 (6.8) 17.2 (5.4) Mosaicity (o) 0.48 0.47 0.45 0.33 0.34 Refinement statistics Rcryst/Rfree (%) 20.7 (23.8) 18.9 (21.8) 19.3 (22.8) 19.1 (25.9) 19.3 (22.2) r.m.s deviation from ideal geometry
Bonds (Å) 0.019 0.019 0.018 0.019 0.019 Angles (°) 1.953 1.600 1.624 1.936 1.662 Ramanchandran plot Favored (%) 95.2 96.2 95.2 93.0 97.3 Allowed (%) 4.8 3.2 3.8 7.0 2.7 Outlier (%) 0.0 0.5 1.0 0.0 0.0 PDB code 3zv8 3zv9 3zva 3zvb 3zvc
984
45
985
986
987
988
Table 1 (continued). Data collection and refinement statistics for crystals of EV68
3Cpro and its complexes
EV68
3Cpro/SG83 EV68 3Cpro/SG84
EV68 3Cpro/SG85
EV68 3Cpro/SG98
Data collection statistics
Space group P3121 P3121 P3121 P3121 Unit cell parameters (Å)
56.31, 56.31, 170.35
56.30, 56.30, 170.35
55.87, 55.87, 170.15
56.20, 56.20, 170.23
Estimated solvent contenta (%)
66.89 66.88 66.33 66.74
Wavelength (Å) 0.9184 0.9184 0.9184 0.9184 Resolution range (Å) 36.99-2.25
(2.37-2.25) 34.07-1.80 (1.90-1.80)
36.81-2.50 (2.64-2.50)
32.04-2.10 (2.21-2.10)
Number of reflections measured
140050 (20152)
255740 (36767)
95365 (13852)
131896 (19127)
Unique reflections 15635 (2205) 29927 (4268) 11333 (1606) 19052 (2724) Completeness (%) 100 (100) 100 (100) 100 (100) 100 (100) Rmerge(%)b,c 0.110 (0.468) 0.064 (0.445) 0.128 (0.554) 0.094 (0.445) Redundancy 9.0 (9.1) 8.5 (8.6) 8.4 (8.6) 6.9 (7.0) I/σ(I) 15.4 (5.4) 17.3 (4.2) 11.9 (4.0) 13.2 (4.6) Mosaicity (o) 0.39 0.46 1.31 0.29 Refinement statistics Rcryst/Rfree (%) 19.3 (24.7) 20.4 (23.1) 19.5 (24.5) 19.9 (23.8) r.m.s deviation from ideal geometry
Bonds (Å) 0.019 0.016 0.020 0.019 Angles (°) 1.660 1.622 1.969 1.680 Ramanchandran plot Core (%) 96.2 96.8 94.6 95.2 Allowed (%) 3.2 3.2 5.4 4.8 General (%) 0.5 0.0 0.0 0.0 PDB code 3zvd 3zve 3zvf 3zvg
989 990 991
992
Values in parentheses are for the highest resolution shell.
a Solvent content estimated according to Matthews (24).
b ( ) ( )| | ( )∑ ∑∑ ∑ −= iihkliihklmerge hklIhklIhklIR , where ( )hklI is the intensity of
reflection hkl and
993
( )hklI is the average intensity over all equivalent reflections. 994
46
c ( ) ( )| | ( )∑∑ −= hklFhklFhklFR ohklcohklcryst . freeR was calculated for a test set of
reflections (5%) omitted from the refinement.
995
996
997
998
999
47
Table 2. Enzyme inhibition and inhibition of replicons by α,β-unsaturated esters 1000 1001
EV68 3Cpro
inhibition inhibition of replicons
(EC50, μM)
Toxicity (CC50, μM)
Name Molecular structure kobs/[I] (M-1s-1) PV EV71 HAV Huh-T7
SG74 O N
H
OO
O
NHO
ND > 40 ND* ND* >40
SG75 NH
OO
O
NHO
HNO
O
3246±107 7.5±1.5 7.0±1.1 30±12 320±30
SG81 NH
OO
O
NHO
HNO
O
42±3 > 40 > 40 ≥40 ND*
SG82 NH
OO
O
NHO
HN
O
O O
O
NH
O
43225±1850 ND* 1.2±0.2 >20 >100
SG83 NH
NHO
O
O
OHN
ONH
O
O
O
O
174500±9245 1.0±0.3 1.3±0.5 12±2.5 295±15
SG84 NH
OO
O
NHO
OHN
O
O
NH
O
9225±423 ND* 2.5±0.4 ND* >100
SG85 NH
OO
O
NHO
OO H
N
ONH
O
202200±8103 0.5±0.1 1.0±0.2 15±5 265±15
48
SG98 NH
OO
O
NHO
HN
ONH
O
OO
11045±661 4.0±1.4 2.5±0.5 >40 250±10
1002 1003
* ND – not determined
49
50
1004 1005 1006 1007
Table 3. Antiviral activity of SG compounds on the replication of a panel of entero- and rhinoviruses in a live virus-cell-based assay
Code Echo11 (Gregory; BGM)
EV71 (BRCR; RD)
HRV02 (Hela Rh)
HRV14 (Hela Rh)
HRV29 (Hela Rh)
HRV70 (Hela Rh)
HRV85 (Hela Rh)
PV1 (Sabin; BGM)
SG74 97 ± 18 >307 57 ± 28 >307 55 ± 21 >307 242 >307
SG75 63 ± 10 9.3 ± 1.0 2.0 ± 0.5 1.5 ± 1.0 2.2 ± 1.2 1.5 ± 0.7 5 ± 2 132 ± 1
SG81 98 ± 28 >211 59 ± 33 53 ± 28 31 ± 16 >211 >211 >211
SG82 19 ± 5 0.26 ± 0.26 1.4 ± 1.4 0.11 ± 0.07 0.19 ± 0.14 0.4 ± 0.1 0.4 ± 0.1 41 ± 18
SG83 29 ± 3 0.15 ± 0.03 1.9 ± 1.9 0.05 ± 0.02 0.27 ± 0.18 0.8 ± 0.3 0.6 ± 0.1 43 ± 20
SG85 26 ± 2 0.18 ± 0.19 1.7 ± 2.3 0.06 ± 0.01 0.05 ± 0.03 0.5 ± 0.2 0.14 ± 0.04 47 ± 23
Enviroxime 0.8 ± 0.6 0.8 ± 0.6 0.04 ± 0.02 0.3 ± 0.1 0.15 ± 0.8 0.4 ± 0.3 0.2 ± 0.1 0.5 ± 0.2
1008 1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
Data represent the 50% effective concentration (EC50) expressed in µM and are
averages ± SD for at least 2 independent experiments. Note that relatively high
standard deviations are common in such experiments and may arise because of
minor instabilities of compounds when stored in DMSO, varying dilution factors,
or slight differences between assays. Data shown in grey shade indicate that,
following microscopic inspection, no compound-induced adverse effects were
observed on the host cell and the compound, for at least one concentration,
completely prevented the virus from inducing any cytopathic effects. For
comparison, data for enviroxime, an inhibitor of the 2C NTPase of picornaviruses,
are also shown.
