identification and characterization of influenza virus entry inhibitors
TRANSCRIPT
JVI Accepted Manuscript Posted Online 1 June 2016J. Virol. doi:10.1128/JVI.00898-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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in silico
in silico
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in silico
in silico
in vivo
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μ μ
μ μ
o pp
pI rac
Sμ μ
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B101
100
10-1
10-2
10-3
10-4
robu
st Z
-sco
re
0.1 1 10 100 1000% inhibition by plate
2.2
3.4
inactive compoundshit candidates selected for follow-up testing
nano blocker
inactive at counter
confirmed anti-IAV-WSN hitsconfirmed broad spectrum hits
robu
st Z
-Sco
re
72 % inhibition by Plate
selected for developmentcytotoxic hits
142.495compounds
co-infectionIAV-WSN-nanoLRSV-fireSMASh
inactives
IAV Inhibitor
dualinhibitor
RSVinhibitor
inactives
BEAS-2Bdose-response
5-0.039 μ M384 well
primary screen direct counterscreen
IAV Inhibitor
dualinhibitor
RSVinhibitor
metabolicactivity
inactives
BEAS-2Bdose-response3-day exposure10-0.078 μ M
cytotoxicity screen
IAV Inhibitor
dualinhibitor
RSVinhibitor
luciferaseinterference
reporterinterfering
BEAS-2BRSV-renillaVSV-nano
5-0.039 μ M384 well
IAV Inhibitor
dualinhibitor
RSVinhibitor
reporter interference screen
IAV-WSNRSV-A2
yield
inactive
BEAS-2B10-0.04 μ M
12 well
orthogonal counterscreen
BEAS-2B5 μ M
384 well384 well
IAV Inhibitor
dualinhibitor
RSVinhibitor
A
ind. infectionIAV-WSN-nanoLRSV-fireSMASh
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GRP-71271
IAV-
WS
N ti
ter (
pfu/
ml) IAV-N
L-nanoLuc(%
RLU
of vehicle)107
106
103
105
104
0
80
120
40
-3 -2 -1 0 1 2log10 concentration [ M]
0
80
120
40
metabolic activity
(% of vehicle treated)
GRP-115249
0
80
120
40
metabolic activity
(% of vehicle treated)
107
106
103
105
104
0
80
120
40
-3 -2 -1 0 1 2log10 concentration [ M]
GRP-103594
107
106
103
105
104
0
80
120
40
-3 -2 -1 0 1 2log10 concentration [ M]
B
robust Z-score IAV% inhibition IAV
robust Z-score RSV% inhibition RSV
3.0488
0.420
primaryscreen
(384-well)
direct- andorthogonal
counterscreen
(384-well DR)
WSN-Nano (EC50) 0.28 M cytotoxicity (CC50) >10 M VSV-Nano (EC50) >5 M
GRP-71271 (M=395 g/mol)A
robust Z-score IAV% inhibition IAV
robust Z-score RSV% inhibition RSV
2.9892
>60
primaryscreen
(384-well)
direct- andorthogonal
counterscreen
(384-well DR)
WSN-Nano (EC50) 0.45 M cytotoxicity (CC50) >10 M VSV-Nano (EC50) >5 M
GRP-103594 (M=393 g/mol)
robust Z-score IAV% inhibition IAV
robust Z-score RSV% inhibition RSV
2.7488
>60
primaryscreen
(384-well)
direct- and orthogonal
counterscreen
(384-well DR)
WSN-Nano (EC50) 0.33 M cytotoxicity (CC50) >10 M VSV-Nano (EC50) >5 M
GRP-115249 (M=396 g/mol)
IAV-
WS
N ti
ter (
pfu/
ml)
IAV-
WS
N ti
ter (
pfu/
ml)
IAV-NL-nanoLuc
(%R
LU of vehicle)
IAV-NL-nanoLuc
(%R
LU of vehicle)
0
80
120
40
metabolic activity
(% of vehicle treated)
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virucidal
80
120
40
0
rel.
RLU
(%
of v
ehic
le)
ns
GRP-7127
1
GRP-1035
94
GRP-1152
49
vehic
le
nsns ns
80
120
40
0
ns
A B C
0
80
120
40rel.
RLU
(%
of v
ehic
le)
0 31 6 9-1time post-infection [hours]
time of addition E minigenome
0
80
120
40rel.
RLU
(%
of v
ehic
le)
100 42 6 8concentration [ M]
GRP-71271GRP-103594
JMN3-003DMSO
D F
G
DMSO JMN-3-003 GRP-71271 GRP-103594 GRP-115249
hemolysis inhibition assay
cell priming
80
120
40
0
rel.
RLU
(%
of v
ehic
le)
nsns ns
GRP-7127
1
GRP-1035
94
GRP-1152
49
vehic
le
receptor binding
rel.
RLU
(%
of v
ehic
le)
ns
GRP-7127
1
GRP-1035
94
GRP-1152
49
vehic
le
GRP-115249
log10 concentration [ M]-2 -1 0 1
abso
rban
ce
0.0
0.8
0.4
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200 nM400 nM3200 nM6400nM
GRP-103594
0
0.01
0.02
nm
0 50 100 250200150time [sec]
400 nM800 nM6400 nM1.28x104 nM5.12x104 nM
KD=5.40x10-7 MR2=0.8460
inactive GRP-71271 analog
0
0.01
0.02
nm
0 50 100 250200150
200 nM400 nM800 nM3200 nM2.56x104 nM
time [sec]
mock
0
40
80
120
% o
f veh
icle
trea
ted ns
ns
ns
***
-IAV-HA-TfR
75
A
GRP-7127
1
GRP-1035
94
GRP-1152
49
vehic
le
mock
GRP-7127
1
GRP-1035
94
GRP-1152
49
vehic
le
C GRP-71271
0
0.01
0.03
nm
0 50 100 250200150time [sec]
400 nM800 nM1600 nM3200 nM6400 nM
KD=9.10x10-7 MR2=0.8569
0.02
2.56x104 nM
0
0.01
0.03
nm
0 50 100 250200150time [sec]
0.02
GRP-115249
KD=1.8x10-6 MR2=0.9714
onpu
t
elutio
n
flow-th
rough
onpu
t
elutio
n
flow-th
rough
Ag stain
elutio
n
WB
-streptavidin-HRP
75
WB -IAV HA
B
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A I257T
V323ID85N
K58Q
N154S
T156A
F110L
D
IAV-
WS
N-n
anoL
uc(%
RLU
of v
ehic
le)
80
120
40
0-1.5 -1.0 -0.5 0 0.5 1.0
log10 concentration [ M]
K58Q
B
A: triple mutation in HA; adapted to GRP-71271B: double mutation in HA; adapted to GRP-115249C: single mutation in HA; adapted to GRP-103594D: single mutation in HA; adapted to GRP-71271
C
recIAV-WSN-nanoLuc-HA-
recIAV-WSN-nanoLuc-HA-
GRP-71271
GRP-71271 recIAV-WSN-nanoLuc
GRP-103594GRP-115249
*
K58
Q
FP TM
HA2HA1
FP
S-S
*
F110
L
*
N15
4S
*
T156
A
*
D85
N
*
M59
I/M
59L
*
I257
T
*
V323
I
340 2221
LH
A A A
C DB
B
B-loop
SH
IAV-
WS
N-n
anoL
uc(%
RLU
of v
ehic
le)
80
120
40
0-1.5 -1.0 -0.5 0 0.5 1.0
log10 concentration [ M]
V323I
80
120
40
0-1.5 -1.0 -0.5 0 0.5 1.0
log10 concentration [ M]
T156A
80
120
40
0-1.5 -1.0 -0.5 0 0.5 1.0
log10 concentration [ M]
V323I/T156A
80
120
40
0-1.5 -1.0 -0.5 0 0.5 1.0
log10 concentration [ M]
I257T
recIAV-WSN-nanoLuc-HA-
IAV-
WS
N-n
anoL
uc(%
RLU
of v
ehic
le)
IAV-
WS
N-n
anoL
uc(%
RLU
of v
ehic
le)
IAV-
WS
N-n
anoL
uc(%
RLU
of v
ehic
le)
IAV-
WS
N-n
anoL
uc(%
RLU
of v
ehic
le)
80
120
40
0-1.5 -1.0 -0.5 0 0.5 1.0
log10 concentration [ M]
N154S
80
120
40
0-1.5 -1.0 -0.5 0 0.5 1.0
log10 concentration [ M]
D85N/N154S
E
80
120
40
0-1.5 -1.0 -0.5 0 0.5 1.0
log10 concentration [ M]
D85N
recIAV-WSN-nanoLuc-HA-
IAV-
WS
N-n
anoL
uc(%
RLU
of v
ehic
le)
IAV-
WS
N-n
anoL
uc(%
RLU
of v
ehic
le)
IAV-
WS
N-n
anoL
uc(%
RLU
of v
ehic
le)
80
120
40
0-1.5 -1.0 -0.5 0 0.5 1.0
log10 concentration [ M]
F110L
on April 4, 2018 by guest
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B
stand
ard H
A
V323I/
T156A
K58Q
mock
D85N/N
154S
V323I/
N154S
D85N/T15
6AF11
0L
75
PNGase F
80
120
40
01.5 0.5 0 0.5 1.0
log10 concentration [ M]
V323I/N154S
C
80
120
40
01.5 0.5 0 0.5 1.0
log10 concentration [ M]
D85N/T156A
stand
ard H
A
V323I/
T156A
D85N/N
154S moc
k
V323I/
N154S
D85N/T15
6AK58
QF11
0L
120
80
40
0
ns nsns
ns ns ns
***
A
75
stand
ard H
A
V323I/
T156A
D85N/N
154S
mock
V323I/
N154S
D85N/T15
6A
K58Q
F110L
D
max
]
1
0.01
0 126 18 24
standard HAV323I/T156AD85N/N154SV323I/N154SD85N/T156AK58QF110L
100
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pH 5.2 pH 5.3 pH 5.4pH 5.2 pH 5.3 pH 5.4
V323
I/T15
6AD
85N
/N15
4SV3
23I/N
154S
D85
N/T
156A
stan
dard
HA
F110
LK5
8Q
pH 5.7pH 5.5 pH 5.6pH 5.2 pH 5.3pH 5.2pH 5.2pH 5.2 pH 5.3pH 5.3pH 5.3 pH 5.4pH 5.4
V323
I/T15
6AD
85N
/N15
4SV3
23I/N
154S
D85
N/T
156A
stan
dard
HA
F110
LK5
8Q
pH 5.7pH 5.5 pH 5.6
IAV-
WSN
HA
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GRP-71271
PheHA2 88
(B)
TrpHA2 92
(B) LysHA1 308
(B)
ProHA1 300
(B)
AspHA2 90
(A)
ValHA1 301
(B)
LeuHA2 89
(B)
GlnHA2 62
(B)
AlaHA2 65
(B)Tyr
HA1 309(B)
LysHA2 83
(A)
GlyHA2 87
(A)
AspHA2 86
(A)
AspHA2 85
(B)
AsnHA2 79
(A)
GlyHA2 67
(B)
ThrHA2 64
(B)
ValHA2 66
(B)
AspHA2 85
(A)
LysHA1 308
(A)LysHA2 83
(B)
ProHA1 300
(A)
LeuHA2 89
(A)
AlaHA2 65
(A)Trp
HA2 92(A)
ValHA2 66
(A)
TyrHA1 309
(A)
GlnHA2 62
(A)
PheHA2 88
(A)
A 400 nM800 nM1600 nM3200 nM6400 nM
GRP-71271 (HA2 M59I)0.02
0 50 100 250200150 time [sec]
( )
0
0.01
nm
KD=1.00x10-6 MR2=0.9603
0
0.01
0.02
0 40 80 140120 time [sec]
nm
50 nM800 nM1600 nM3200 nM6400 nM
KD=5.40x10-5 MR2=0.8407
GRP-115249 (HA2 M59I)
GRP-71271 (HA2 D85N/N154S)
0
0.01
0.02
0 50 100 250200150 time [sec]
400 nM800 nM1600 nM3200 nM6400 nM
KD=6.7x10-7 MR2=0.9638
0.03
nm
GRP-115249 (HA2 D85N/N154S)
0
0.01
0.02
0 20 60 16012080 time [sec]
nm
400 nM800 nM1600 nM12800 nM25600 nM
KD=5.8x10-6 MR2=0.8684
B
AspHA2 85
LysHA1 308
(A)
AspHA2 85
(A)
AspHA2 90
(B)
AspHA2 86
(B) LysHA2 83
(B)
GlyHA2 87
(B)
TyrHA1 309
(A)
GlnHA2 62
(A)
ProHA1 300
(A)
LeuHA2 89
(A)
AlaHA2 65
(A)
TrpHA2 92
(A)
PheHA2 88
(A)
GRP-103594 GRP-115249
AsnHA2 82
(A)
PheHA2 63
(B)
C
D
polaracidicbasicgreasy
backbone acceptorbackbone donor
sidechain acceptorsidechain donor proximity contour
ligand exposurereceptor exposure
arene-H
on April 4, 2018 by guest
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nloaded from
GRP-71271 GRP-103594
LysHA2 71
(A)
AspHA2 89
(A)Glu
HA2 88(A)
GluHA2 72
(A)
LysHA2 85
(A)
GluHA1 87
(A)
LysHA1 269
(A)
AsnHA2 84
(A)
GluHA2 81
(A)
PheHA2 73
(A)
LeuHA1 300
(A)
LysHA1 307
(A)
LysHA2 86
(A)
LeuHA1 300
(A)
ProHA1 299
(A)
ValHA2 69
(A)
TrpHA2 86
(A)
LeuHA2 92
(A)
PheHA2 91
(A)
AsnHA2 63
(A)
GlnHA2 65
(A) GlyHA2 90
(A)
TyrHA1 308
(A)
HisHA1 298
(A)
ThrHA2 301
(A)
AspHA2 89
(A) GluHA2 67
(A)
AspHA2 93
(A)
AspHA2 89
(A)
AspHA2 93
(A)GluHA2 88
(A)GluHA2 67
(A)
LysHA2 86
(A)
LysHA1 307
(A)
TrpHA2 86
(A)
LeuHA2 92
(A)Pro
HA1 299(A)
PheHA2 91
(A)
ValHA2 69
(A)
TyrHA1 308
(A)
ThrHA1 301
(A)
GlyHA1 90
(A)
GRP-115249
polaracidicbasicgreasy
backbone acceptorbackbone donor
sidechain acceptorsidechain donor proximity contour
ligand exposurereceptor exposure
arene-H
on April 4, 2018 by guest
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Dow
nloaded from
PheHA2 88
(B)
TrpHA2 92
(B) LysHA1 308
(B)
ProHA1 300
(B)
AspHA2 90
(A)
ValHA1 301
(B)
LeuHA2 89
(B)
GlnHA2 62
(B)
AlaHA2 65
(B)Tyr
HA1 309(B)
LysHA2 83
(A)
GlyHA2 87
(A)
AspHA2 86
(A)
AspHA2 85
(B)
AsnHA2 79
(A)
GlyHA2 67
(B)
ThrHA2 64
(B)
ValHA2 66
(B)
AsnHA2 82
(A)
PheHA2 63
(B)
GRP-71271 (EC50 0.05 μ M)
GRP-71271-2 (EC50 3.6 μ M)
PheHA2 88
(A)
ProHA1 300
(A)
ValHA2 66
(A)
AlaHA2 65
(A)PheHA2 63
(A)
TrpHA2 92
(A)
LeuHA2 89
(A)
ValHA1 301
(A)
AspHA2 85
(A)
AspHA2 86
(B)
AspHA2 90
(B)
LysHA1 308
(A)
LysHA1 83
(B)
GlyHA2 67
(A)
TyrHA1 309
(A)Asn
HA2 82(B)
GlyHA2 87
(B)
ThrHA2 64
(A)
GlnHA2 62
(A)
AlaHA2 65
(A)
AsnHA2 82
(B)
GlyHA2 67
(A)
AlaHA2 65
(A)
AspHA2 85
(A)
AspHA2 90
(B)
AspHA2 86
(B)
PheHA2 88
(A)GlyHA2 87
(B)Thr
HA2 64(A)
GlnHA2 62
(A)
ProHA1 300
(A)
ValHA2 66
(A)
TrpHA2 92
(A)
ValHA1 301
(A)
TyrHA1 309
(A)
LeuHA2 89
(A)
LysHA1 83
(B)
LysHA1 308
(A)
LysHA1 308
(A)
AsnHA2 79
(B)
GRP-71271-8 (EC50 8.7 μ M)
GRP-71271-1 (EC50 10 μ M)
AspHA2 90
(B)
AspHA2 86
(B)
AspHA2 85
(A)
LysHA1 308
(A)
GlnHA2 62
(A)
TyrHA1 309
(A)
ValHA2 66
(A)
AlaHA2 65
(A)PheHA2 63
(A)
LysHA1 83
(B)
GlyHA2 67
(A)
AsnHA2 82
(B)
GlyHA2 87
(B)
ThrHA2 64
(A)
TrpHA2 92
(A)
ValHA1 301
(A)
LeuHA2 89
(A)
PheHA2 88
(A)
ProHA1 300
(A)
polaracidicbasicgreasy
backbone acceptorbackbone donor
sidechain acceptorsidechain donor proximity contour
ligand exposurereceptor exposure
arene-H
on April 4, 2018 by guest
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