Download TP219 acts as a selective inhibitor of in vitro enterovirus replication

TP219 acts as a selective inhibitor of in vitro enterovirus replication by indirectly
targeting morphogenesis
Hendrik Jan Thibaut1, Bert Thys2, Lonneke van der Linden3, Maria-Dolores Canela4, Mathy Froeyen1, Leire Aguado4, Jan Paeshuyse1, Armando De
Palma1, Bart Rombaut2, María-Jesús Pérez-Pérez3, Frank van Kuppeveld2 and Johan Neyts1
Rega Institute for Medical Research, University of Leuven, Belgium, 2Vrije Universiteit Brussel, MICH, Dept. Pharmaceutical Biotechnology & Molecular Biology,
Brussel, Belgium, 3Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, 4Instituto de
Química Médica (CSIC), Madrid, Spain
1
ABSTRACT
We identified a class of 9-arylpurines as selective inhibitors of the replication of CVB3 and several CVAs. Analogue TP219 [9-(3-acetylphenyl)-6-chloropurine], was selected for further studies. First, we demonstrated that TP219 inhibits virus-induced CPE formation, without affecting (i) viral RNA synthesis, (ii)
polyprotein synthesis/processing or (iii) the formation of replication vesicles. In addition no infectious virus particles were detected in TP219-treated infected cells, suggesting that the drug interferes with the formation of novel infectious viral progeny. TP219-resistant (TP219res) CVB3 was selected by serial
passaging in increasing sub-optimal concentrations of TP219. TP219res CVB3 carries several mutations in VP1 (T77M, V150I and N212S) and VP3 (A180T). By means of reverse genetics it was shown that T77M is sufficient for the drug-resistant phenotype. Analysis of the structural model of the CVB3
capsid revealed that T77M (and the other mutations) are located at the interface of two protomers, suggesting that the molecule may prevent (by targeting directly or indirectly this region) viral morphogenesis. To determine at which step during this process the compound interferes, we determined the effect
by means of sucrose gradient ultracentrifugation and SDS-PAGE on the presence of different assembly intermediates. The data suggest that TP219 prevents protomer:protomer interactions. Interestingly, the T77M mutant proved cross-resistant with L-BSO (buthionine sulfoximine), a known inhibitor of
glutamate-cysteine ligase enzyme which is key in the glutathione (GSH) metabolism. Addition of GSH-reduced-ethyl-ester (GEE) to the culture supernatant reversed the antiviral effect of TP219 (as was evident from a rescue of the formation of infectious virus particles). Next, we demonstrated that TP219
reduces rapidly and efficiently intracellular levels of GSH. This was corroborated by the observation that TP219 and GSH, when incubated in cell free conditions, form an adduct via a covalent bond. The effect of an oxidizing environment (as a result of the depletion of intracellular GSH) on VP1, and more
specifically on T77, is currently being explored by means of MALDI-MS and nanoLC-MS. Oxidation of T77 may possibly be detrimental for protomer:protomer interaction. Taken together, TP219 provides a most exciting tool to obtain insights into a poorly understood part of enterovirus replication.
[Fig 7]
IDENTIFICATION OF 9-ARYL PURINES
[TABLE1]. ANTI-COXSACKIEVIRUS B3 EVALUATION OF A SERIES OF
RELATED ANALOGUES
R1
R2
R3
EC50 (µM) ±
CC50 (µM) ±
SD
SD
TP186
H
CH2OH
H
14.2 ± 3.9
158 ± 23
TP219
H
COCH3
H
7.3 ± 2.3
206 ± 14
TP276
CH3
COCH3
OH
6.7 ± 2.1
>250
TP279
271
573
104
[Fig 7] HPLC separation of TP219 and/or GEE directly coupled to ES-MS analysis was
performed to identify this new fraction. These data suggest a covalent non-enzymatic
coupling of TP219 with GEE resulting in the formation of an adduct and may provide
an explanation on the rapid depletion of intracellular GSH levels.
104
287
158
444
C(CH3)2OH
CH3
[Fig 6] HPLC separation of TP219 and/or GEE. Detection of TP219 or GEE at 254nm or
230nm respectively. Upon co-incubation peak intensities of TP219 and GEE decreases,
whereas a new fraction appears that has a retention time different than that of TP219
and GEE.
%
[FIG1]. IDENTIFICATION OF A LEAD COMPOUND WITH ANTICOXSACKIEVIRUS B3 ACTIVITY
271
100
H
8.0 ±
287
159
>250
3.4
574
444
378
575
0
[TABLE2]. ANTIVIRAL EVALUATION OF MOST POTENT
COMPOUNDS AGAINST A PANEL OF PICORNAVIRUSES
[Fig 1]. In a large screening effort, using an MTS-based cell protection assay 9-(3methoxyphenyl)-6-chloropurine [TP186] was discovered to exhibit antiCoxsackievirus B3 activity. Percentage CPE was calculated and antiviral activity
was expressed as EC50.
Virus
Strain
Cell line
IC50[µM]
EV-A
CVA16
G-10
MRC-5
2.7 0.76
239
EV71
BrCr
Hela
>100
114
14
12
CVA9
Bozek
Hela
>100
114
14
10
CVB3
Nancy
Vero
7.3 2.3
Hela
> 100
Echo9
EV-C
EV-D
Hill
3.3 1.2
RD
> 100
PV1
Sabin
Vero
>100
CVA21
Coe
MRC-5
4.5 1.6
RD
> 100
239
>250
239
4
2.7 0.87
239
4
EV-68
Clinical
MRC-5
ND
239
4
hRV-B
#5
Hela
/
Theilovirus
>100
Hela
[Table 1]. Using TP186 as a scaffold, 56 structurally related analogues were
synthesized and evaluated for anti-Coxsackievirus B3 activity and cellular toxicity.
TP219, TP276 and TP279 were identified as having the best potency.
4
MRC-5
/
114
>100
114
Mengovirus
BHK-21
>100
/
Saffold virus
Hela
>100
/
[Table 2]. TP219 and TP276 were subsequently evaluated for antiviral activity
against a panel of enteroviruses. Antiviral and cell dependent activity was
demonstrated for CVA16, CVA21, CVA24 and for echovirus 9. Cellular toxicity
was determined in three different cell lines, as assessed with the MTS-method
and luminescence (data not shown).
14
14
[Fig 2]
[Fig 3A]
Enviroxime
-
+
TP219
6
TP219
TP219
5
[Fig 8B]
+
-
[Fig 3B]
-
14S
6
2
2
1
0
0
3
5
7
9
11
13
15
17
19
21
23
25
75S
1
27
[Fig 8C]
18
[Fig 8]. To analyze the effect of BSO and TP219 on the
different assembly intermediates, metabolically labeled
viral proteins were separated on sucrose gradients.
Equal volumes of control and treated cell extracts were
fractionated over 5–20% (A and C) or 15–30% (B and D)
sucrose gradients. Following TCA precipitation, aliquots
were counted by liquid scintillation and expressed as
normalized CPM. Both TP219 and BSO prevented the
formation of 150S, 75S and 14S assembly
intermediates.
150S
3
4
1
Viruscontrol
Viruscontrol
TP219
TP219
4
Viruscontrol
16
Viruscontrol
L-BSO
14
TP219
12
3
5
7
9
11
13
15
17
19
21
23
[Fig 8D]
Viruscontrol
Viruscontrol
L-BSO
10
[Fig 9]. To analyze intensities of 5S and 14S
intermediates in the presence of absence of TP219 [Fig
9A] and BSO [Fig 9B]. Following immunoprecipitation
(using a polyclonal anti-enterovirus antibody), fractions
of 5-20% sucrose gradients were counted by liquid
scintillation and expressed as normalized CPM. In the
presence of TP219 or BSO, no 14S assembly
intermediates could be observed whereas 5S peak
intensities were only slightly reduced. Additional
assembly intermediates appeared between 5S and
14S.
TP219
12
8
10
6
8
6
4
4
2
0
0
1
3
5
7
9
11
13
15
17
19
21
23
+
25
5
7
9
11
13
15
17
19
21
23
25
Fraction Number
VP2
17
19
181.8
115.5
P2-P3
23
92.2
25
64.2
27
48.8
P2
2BC3AB
37.1
VP0/2C
VP1
29
VP3
31
-1
0
1
2
3
4
5
6
7
25.9
VC
19.4
19.0
Fig 2. To define the stage in the viral replication cycle at which TP219
2A
exerts its antiviral activity, time of drug addition studies were performed.
8 hours post infection intracellular viral RNA was quantified by means of
RT-qPCR (expressed as Ct-values). No inhibition of viral RNA replication was observed after a single replication cycle (8 h) when added at
any given time point. Enviroxime was used as a control and acts at a stage in the replication cycle that coincides with viral RNA synthesis.
5
10000
4
1000
3
100
2
10
1
1
0
VC TCA precipitation
TP219 TCA precipitation
18
16
18
14
16
14
16
14
12
12
10
10
8
8
6
6
11
13
15
17
19
21
23
25
8
8
6
6
2
2
0
0
0
0
9
10
2
2
7
12
10
4
4
5
14
12
4
4
3
20
1
27
3
5
7
9
11
13
15
17
19
21
23
25
27
Fraction Number
Renilla
Luciferase
VPg
VP4 VP2 VP3
VP1
2A 2B
2C
3A
VPg
100000
TP219 immunoprecipitation
16
IRES
6
Virus titers [Log TCID50/ml]
1000000
BSO TCA precipitation
VC immunoprecipitation
18
1
[Fig 4]
VC TCA precipitation
[Fig 9B]
20
18
The effect of TP219 on protein
synthesis
and
processing
was
monitored
using
Western
Blot
(targeting VP1) [Fig 3B] and pulselabeling experiments [Fig 3A]. TP219
has no effect on polyprotein
processing.
BSO immunoprecipitation
Normalized c.p.m. after TCA
P2-3ABC/P1
P3
P2-3AB/3CD
VC immunoprecipitation
Normalized c.p.m. after
immunoprecipitation
[Fig 9A]
Normalized c.p.m. after
immunoprecipitation
21
Time of Addition [hours p.i.]
Luciferase activity [8h post infection]
Viruscontrol
Viruscontrol
2
Normalized c.p.m. after TCA
Reduction in intracellular viral RNA at 7
hours p.i. [Ct values]
TP219 WORKS LATE IN THE REPLICATION CYCLE
15
5S
8
Clinical
# 10
[Fig 8A]
>250
CVA24
hRV-A
Cardiovirus
MRC-5
4
Normalized CPM after TCA precipitation
EV-B
CC50[µM]
TP219 AFFECTS MORPHOGENESIS
Species
3C
3D
AAAAAAA
5’ UTR
3’ UTR
[Fig 4]. The effect of TP219 (50µM) on RNA replication was monitored using a CVB3
expressing a Renilla luciferase (RLuc-CVB3). Virus yields were determined by endpoint titration according to the method of Reed and Muench and expressed as 50%
tissue culture infective doses (TCID50). GuaHCl (inhibits viral RNA replication) and
Geldanamycin (inhibits morphogenesis by targeting Hsp90) are used as controls.
TP219 has no effect on RNA replication, but inhibits the production of mature
virions
VIRAL RESISTANCE TO TP219
Three independent TP219-resistant CVB3-clones were generated by serially passaging wildtype CVB3 in the presence of increasing concentrations
of the compound [Fig 10].
[Fig 10]
[Fig 11A]
Phenotype
Virus Strain
EC50 [µM] (fold change)
wildtype
7.3
2.3 (1)
TP219r
>100 (>13.7)
TP219 DEPLETES INTRACELLULAR GSH
[Fig 5A]
[Fig 5B]
10
TP219
Untreated Control
Nucleotide
L-BSO
LOG10 TCID50/ml
GSH per well [µM]
8
Data are mean values ± SD for three independent experiments as determined
by a MTS-based CPE-reduction assay
6
4
2
8
6
4
2
0
0
0h
1h
3h
6h
24h
48h
Env
TP219
GEE
GEE
GEE
GEE
GEE
GEE
(5µM) (50µM) (0,5mM) (1mM) (2mM) (5mM) (10mM) (10mM)
72h
Time point [hours post incubation]
protein
AA Substitution
Substitution
Position
Pool
VP3
K115R
AAG AGG
1337
2
VP3
A180T
GCT  ACT
1531
1
VP1
T77M
ACG  ATG
1937
1,2,3
VP1
V150I
GTA  ATA
2155
3
VP1
N212S
AAC  AGC
2342
2
2A
E54K
GAG  AAG
2797
1,2
2A
E59V
GAA  GTA
2813
1,2
3A
I22V
ATT  GTT
4348
1,2
3A
K39R
AAA  AGA
4400
1,2
WT
-
+ TP219 50µM
[Fig 6]
Cl
TP219
N
N
TP219 254nm
N
New
254nm
TP219
N
[Fig 11B]
[Table 3]. Amino acid mutations identified in 3
independently generated TP219-resistant variants
VC
The effect of TP219 and BSO on glutathione (GSH) depletion was quantified in a luminescence-based system in which GSH-dependent conversion
of a GSH probe (Luciferin-NT) to luciferin by GST is coupled to a firefly luciferase reaction. TP219 reduces efficiently and rapidly intracellular
GSH levels, in a way different than that of BSO [Fig 5A]. Addition of GSH-reduced-ethyl-ester (GEE) reversed the antiviral effect of TP219
(as was evident from a rescue of the formation of infectious virions) [Fig 5B].
[Fig 11A] PDB1-based structure as determined with X-ray diffraction (1COV) of a single
pentamer of CVB3, which consists of one copy of VP1 (yellow), VP2 (green), VP3
(blue). Identified amino acids are labeled in red and are located on VP1:VP3 interace
+
α-Tubulin
T77M
-
+
On membrane trypsinisation
Analysis with nanoLC-MS
α -VP1
[Fig 12]
[Fig 12] Mass spectometry analysis to determine the
effect of TP219-induced oxidative stress on wild type
and mutant VP1
[Fig 11B] Ligplot analysis of possible interaction partners of Thr-77 based on 1COV.
Thr-77, a solvent exposed residue, interacts with Tyr-75 (van der Waals). Tyr-75 might
be implicated in stabilization of the antiparallel β-sheets through aromatic stacking with
Trp-90. Theoretically, oxidation of Thr-77 could be detrimental for this process.
H3C
CONCLUSIONS
O
GEE (2 mM) + TP219 (10 µM)
pH 7.4 , 37 ºC
GEE
GEE 230nm






230nm
GEE
Retention Time [min]
Retention Time [min]
TP219 does not affect entry, RNA replication or polyprotein processing, but targets morphogenesis
TP219 depletes very rapidly intracellular glutathione levels in way different than that of L-BSO
TP219 may form covalent bonds with glutathione
TP219 may interfere with protomer > pentamer assembly intermediates
TP219-resistant CVB3-clones were generated.
T77M and A180T/N212S/V150I mutations in VP1 and VP3 were identified in the drug-resistant phenotype.
1. Identified mutations are located on the VP1-VP3 interface
2. NanoLC-MS experiments are optimized to determine possible oxidation of VP1
3. Oxidation of Thr-77 might be affecting the stability of pentamers
Wallace A C, Laskowski R A and Thornton J M (1995). LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions. Prot.Eng 8,127-34.