From the Departments of Virology,
§ Biochemistry, and ¶ Medicinal Chemistry, The
Metabolic and Viral Diseases Center of Excellence for Drug Discovery,
GlaxoSmithKline Pharmaceuticals and the
Department of Biology,
Indiana University, Bloomington, Indiana 47405
Received for publication, October 24, 2002, and in revised form, December 20, 2002
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ABSTRACT |
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The hepatitis C virus (HCV) NS5B protein
encodes an RNA-dependent RNA polymerase (RdRp), the primary
catalytic enzyme of the HCV replicase complex. Recently, two
benzo-1,2,4-thiadiazine compounds were shown to be potent, highly
specific inhibitors of the genotype 1b HCV RdRp containing a
carboxyl-terminal 21 residue truncation ( Hepatitis C virus
(HCV),1 a positive strand RNA
virus of the Flaviviridae family, is the major etiological agent of
post-transfusion and sporadic non-A, non-B hepatitis (2). HCV causes
significant liver disease, cirrhosis and can eventually lead to the
development of hepatocellular carcinoma. This disease is of significant
concern because more than 2% of the world population is chronically
infected with HCV. In infected cells, translation of the viral RNA
yields a polyprotein (3-5) that is subsequently cleaved to yield
structural proteins required for the assembly of new virus particles,
as well as nonstructural enzymes essential for viral replication (6-8).
Nonstructural protein 5B (NS5B) encodes RNA-dependent RNA
polymerase (RdRp) activity (9, 10); the catalytic activity associated
with this enzyme has been confirmed to be required for infectivity in
chimpanzees (11). RdRp initiates RNA synthesis preferentially from the
3' terminus of the template RNA (12-14), although in vitro
it has been shown to lack specificity for viral RNA because it readily
utilizes heterologous nonviral templates (9). Like poliovirus (15), the
HCV RdRp is capable of initiating viral RNA synthesis in
vitro by a primer-dependent mechanism (9, 10, 16).
However, Flaviviridae RdRps have also been shown to initiate RNA
synthesis by a de novo mechanism (12, 13, 17-19). De
novo initiation of virus replication is the likely preferred mechanism for HCV in infected cells (20, 21). Recently, our understanding of HCV replication has been significantly improved by
Hong et al. (22), whose work resulted in the
identification of a novel Cell-based HCV replicon systems, useful for studies of viral
replication and antiviral agents, were developed in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7
cells (23, 24). In combination with studies using recombinant RdRps,
such systems are proving to be invaluable in developing a better
insight into the mechanisms of Flaviviridae RNA synthesis. This report
describes the mechanism of action studies for the heterocyclic
inhibitor of the HCV RdRp, compound 4 (C21H21N3O4S, as
described in Ref. 1), based on biochemical and cellular studies.
Protein Purification--
HCV NS5B protein was purified as
described previously (25).
Heparin RdRp Assay--
The RdRp assay was performed as
described previously (25), with the following modifications. Briefly,
after a 15-min incubation, heparin was added to the reaction at a final
concentration of 10 ng/ml. At the same time that heparin was added, 15 µM 3'-ribodeoxy-GTP (3'-rdGTP), compound
1 (C20H19N3O4S) or compound
4 was also added. The reaction was continued for an
additional 60 min and then terminated, processed, and analyzed as
described previously (1).
Initiation Assays--
To specifically examine whether the
thiadiazines can abrogate dinucleotide polymerization, the initiation
step of replication, a direct initiation assay, was established. 100 ng
(1 µM) of a 17-mer RNA (5'-GCGCACAGCGCACUAAC-3') was
utilized as the template, and a 2-fold molar excess of the initiation
NTP, either GTP or the dinucleotide 5'-GpU3-', and the second NTP,
[
Additionally, primer-dependent and de novo
initiation was measured with a single-stranded RNA template, LE21, that
can direct both de novo initiation (which produces a 21-nt
product) and primer extension (which produces a 34-nt product).
Standard RdRp assays consisted of 2.5 pmol of template LE21 with
50-100 ng of NS5B in 20 µl containing final concentrations of 20 mM sodium glutamate at pH 8.2, 12 mM
dithiothreitol, 4 mM MgCl2, 2 mM
MnCl2, 0.5% (v/v) Triton X-100, 200 µM GTP, 100 µM ATP, 100 µM
UTP, 250 nM [ Filter Binding Assay--
In vitro binding reactions
were carried out using 5 nM purified NS5B Surface Plasmon Resonance--
Surface plasmon resonance (SPR)
analyses were performed using a BIAcore 3000 equipped with a
streptavidin sensor chip in a running buffer of 0.01 M
HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%
(v/v) surfactant P20, and 0.1% Me2SO. A biotinylated
36-mer RNA (5'-AUAUUAUCCUAUAUACCGGCUUGCAUAGCAAGCCGG-3') was immobilized to a surface density of 30 response units (RU) by injecting 9 µl of a
2 nM solution at a flow rate of 10 µl/min. The following injection solutions were prepared in running buffer: 10 nM
GTP, 10 nM phosphonoformic acid (PFA), 10 nM
compound 4, 10 nM HCV Single Cycle Synthesis
The benzo-1,2,4-thiadiazines (compounds 1 and
4) were shown to be potent inhibitors of the HCV RdRp with
biochemical IC50 values between 80 and 100 nM and cell-based IC50 values around 500 nM (1). Calorimetry studies confirmed a direct interaction of these compounds with the viral polymerase and a window of
selectivity for nucleic acid interaction. The standard RdRp assay
described previously (25) represents a continuous polymerization assay rather than single cycle synthesis. The RdRp assay was modified by
adding heparin 15 min before initiation of RNA synthesis in an attempt
to reduce the number of newly initiated rounds of RNA synthesis.
Heparin functions as a nucleic acid mimic to titrate free enzyme,
including polymerase molecules, which dissociate from the RNA during
elongation. Therefore, in the presence of heparin, new reinitiation
events should be impeded, and only RNA synthesized from already
initiated complexes or complexes in the elongation phase should be measured.
Using this assay format, compound 4 (1) and a 3'-rdGTP chain
terminator were tested. The 3'-rdGTP is incorporated into the newly
synthesized RNA product. However, unlike GTP, this analog cannot be
extended further because a 3'-hydroxyl group is absent. This chain
terminator would be expected to have a similar potency profile
regardless of whether it is tested in the presence or absence of
heparin, because heparin should not interfere with elongative
synthesis. As shown in Fig. 1,
A and B, the IC50 for 3'-rdGTP
remained at ~100 nM, both in the presence and absence of
heparin. As expected, the total amount of radiolabel incorporated into
the newly synthesized RNA template decreased around 7-fold in the
presence of heparin, an observation consistent with single cycle
synthesis. Interestingly, although compound 4 is a highly
potent inhibitor in the standard assay (Fig. 1C), it failed
to impair the amount of label accumulated in the presence of heparin
(Fig. 1D). These data suggest that the benzothiadiazine may
interfere with initiation, rather than elongative synthesis. Furthermore, similar results were obtained when using a 3-fold molar
excess of poly(A) RNA to trap free polymerase in the
poly(rC)-oligo(rG) RdRp assay (data not shown). In these assays,
compound 1 activity was similar to that of compound
4 (data not shown).
21 HCV RdRp)
(Dhanak, D., Duffy, K., Johnston, V. K., Lin-Goerke, J.,
Darcy, M., Shaw, A. N. G. B., Silverman, C., Gates,
A. T., Earnshaw, D. L., Casper, D. J., Kaura, A.,
Baker, A., Greenwood, C., Gutshall, L. L., Maley, D.,
DelVecchio, A., Macarron, R., Hofmann, G. A., Alnoah,
Z., Cheng, H.-Y., Chan, G., Khandekar, S., Keenan, R. M., and Sarisky, R. T. (2002) J. Biol. Chem.
277, 38322-38327). Compound 4 (C21H21N3O4S) reduces
viral replication by virtue of its direct interaction with the viral
polymerase rather than by nonspecific titration of nucleic acid
template. In this study, we present several lines of evidence to
demonstrate that this inhibitor interferes with the initiation step of
RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether
replication was initiated by primer-dependent or de
novo mechanisms. Filter binding studies using increasing
concentrations of compound 4 did not interfere with the ability of
21 HCV RdRp to interact with nucleic acid. Furthermore, varying the
order of reagent addition in the primer extension assay showed no
distinct differences in inhibition profile. Finally, surface plasmon
resonance analyses provided evidence that a ternary complex is capable
of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent
interacts with the apoenzyme, as well as with the RNA-bound form of
21 HCV RdRp, and therefore does not directly interfere with the
RdRp-RNA interaction to mediate inhibition.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-loop near the catalytic site of the RdRp.
This has provided a direct structural basis for de novo
initiation and suggests that the HCV RdRp can select against primer
extension (22).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
32-P]UTP, were used to initiate RNA synthesis in 20 mM Tris-HCl, pH 7.5, 25 mM KCl, 3 mM dithiothreitol, 2.5 mM
MgCl2, 1 mM MnCl2, and 0.5% (v/v)
Triton X-100. After 1 µM polymerase was added, the
reaction was allowed to proceed for 20 min, and then total RNA products
were analyzed on a 25% polyacrylamide gel containing 7.5 M
urea. Reaction products (3-mer) were analyzed by autoradiography.
-32P]CTP (400 Ci/mmol, 10 mCi/ml, Amersham Biosciences), and ~0.2 µl of 10 mCi/ml
[
-32P]CTP. Reactions were performed at 25 °C.
Compound 4 was used at a final concentration between 2.5 and
25 nM. The reaction products were separated on 20%
polyacrylamide 7.5 M urea gel, exposed to a phosphorimaging
screen, and quantified. Quatifications were normalized to the control
reaction lacking compound 4 but containing an equivalent
amount of Me2SO. Therefore, the percent inhibition
of 21-nt product was expressed as percentage relative to that of the
control reaction (taken as 0% inhibition). The amount of 34-nt product
was also normalized in the same manner.
21 in a 50-µl
reaction volume containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 25 mM KCl, 3 mM dithiothreitol, and 0.05% bovine serum albumin.
Reaction mixtures containing increasing amounts of 33P
end-labeled SLD8 RNA
(5'-GGCUUGCAUAGCAAGUCUGAUAUGCGUCCAGAGACCA-3') were incubated for 15 min
at 30 °C and then filtered immediately onto an 0.45 µM
MF-membrane filter (Whatman), washed with 1.0 ml of ice-cold binding
buffer, and counted in a scintillation counter. For studies using
compound 4, two distinct preincubation protocols were
followed. For one study, compound 4 was preincubated with
polymerase and buffer for 15 min prior to the addition of RNA.
Alternatively, the polymerase, RNA, and buffer were preincubated for 15 min prior to the addition of compound 4.
21NS5B, 10 nM GTP with 10 nM HCV NS5b
21, 10 nM PFA with 10 nM HCV
21NS5B, and 10 nM compound 4 with 10 nM HCV
21NS5B. All solutions were preincubated at 25 °C for 15 min prior to injection. Samples were injected for 60 s at a flow rate of 75 µl/min and allowed to dissociate for 60 s. A regeneration
injection of 2 M NaCl at 20 µl/min for 1 min followed
each sample injection. The binding curves were analyzed in BIAevaluation.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Single cycle RdRp assay in the presence of
heparin. RdRp reactions were initiated and incubated for 15 min
prior to the addition of heparin and compound. Panels A and
C represent RdRp in the absence of heparin, and B
and D represent RdRp in the presence of heparin. The
3'-rdGTP chain terminator activity is shown in A and
B, and compound 4 in C and
D. The x axis displays 3-fold dilutions of
compound (in nM) and the y axis total cpm of
newly synthesized [33P]rGTP-labeled RNA product.
Initiation of RNA Synthesis
To extend the above described study, a more direct assay was
developed to examine initiation of RNA synthesis. Recently, Bressanelli et al. (21) identified three NTP-binding sites in the HCV
RdRp: a low affinity site specific for GTP and the two
nucleotide-binding sites in the active site of the RdRp (NTP1 and
NTP2). To discern the mode of action for compound 4, RNA
synthesis on a 17-mer RNA (5'-GCGCACAGCGCACUAAC-3') was primed using a
dinucleotide 5'-GpU-3' as the initiation nucleotide (NTP1). NTP1
provides the 3'-hydroxyl for nucleotidyl transfer to a radiolabeled NTP
(NTP2; [-32P]UTP) as shown in Fig.
2A. In the presence of both
NTP1 and NTP2, a radiolabeled 3-mer RNA product is present (Fig.
2B, lanes 4 and 5). This 3-mer RNA is
a functional substrate for additional nucleotide incorporation, with
elongative synthesis products forming after the addition of ATP
(Fig. 2B, lane 3), ATP and GTP (lane 2) or all four nucleotides (lane 1). The addition of
compound 4 was shown to abrogate the initial phosphodiester
bond formation between 5'-GpU3-' and radiolabeled UTP (Fig.
2C, compare lane 4 with lanes 5 and
6), regardless of whether ATP and CTP were present (Fig. 2C,
lane 1). Furthermore, compound 4 also inhibits
the addition of a second nucleotide (ATP) onto the GUU-primed RNA (Fig.
2D, lanes 1-4). Taken together, these data suggest that the thiadiazines interfere directly with the process of
RNA initiation and prevent initial phosphodiester bond formation prior
to the commitment to elongative synthesis.
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De Novo and Primer-dependent RNA Synthesis
Additional studies were performed using RNA LE21 (Fig.
3A), to allow direct and
quantitative measurement of both de novo and primer-dependent RNA synthesis within a single reaction.
GTP is required for de novo initiation of LE21 RNA, and the
resultant product is 21 nt (Fig. 3B, lane 1). Two
molecules of LE21 can form a heteroduplex (Fig. 3A)
to allow primer extension, which can occur in the absence of
GTP, to generate a 34-nt product (Fig. 3B, lanes
1 and 2) as reported by Ranjith-Kumar et al.
(26). Multimers of 21-nt product (i.e. 42, 63, etc.) are
usually observed; this is because of the end-to-end template switch
(recombination) (27). Reactions without compound 4 received
a mock aliquot of Me2SO to normalize for any effects of
Me2SO on RNA synthesis (Fig. 3B, lanes
1 and 2). Upon the addition of 25 nM
compound 4 to the reaction (Fig. 3B, lanes
3-6), both de novo and primer-dependent
RNA synthesis were inhibited by ~39% (Fig. 3C). Significantly less inhibition was observed at a lower concentration of
the heterocyclic inhibitors.
|
Nucleic Acid-Polymerase-Compound Interaction
As shown above, the benzothiadiazines disrupted the initiation step of RNA synthesis. It is unclear from these studies whether the heterocyclic inhibitors form a ternary complex with polymerase-bound RNA or interact preferentially with the RdRp apoenzyme prior to template binding. To address this question, (a) an RdRp assay varying the order of reagent addition, (b) filter binding studies, and (c) surface plasmon resonance studies were performed.
Order of Addition--
The order of reagent addition was varied
for the standard RdRp assay. Specifically, free polymerase was
preincubated with RNA prior to the addition of compound 4,
was preincubated with compound 4 prior to the addition of
template RNA, or was used to initiate reaction for a mixture containing
preincubated RNA and compound. The order of addition variation did not
affect compound potency; IC50 values were 101, 102, and 110 nM, respectively (Fig.
4). Hence, compound 4 did not
demonstrate preferential inhibition for the apoenzyme or appear to
disrupt the polymerase-RNA complex.
|
Filter Binding--
Titration of polymerase with SLD8 RNA
in the RdRp buffer showed an apparent Kd of 62 nM (Fig. 5A).
Titration of compound 4 with the polymerase-RNA complex did
not result in a measurable IC50, suggesting that under
these conditions the heterocyclic inhibitor did not interfere with the
polymerase-RNA interaction (Fig. 5B). This result was
consistent with the order of addition experiment shown in Fig. 4.
Compound 4 preincubated with the polymerase prior to
addition of RNA or added to the polymerase-RNA mixture resulted in
similar amounts of radioactivity on the membrane (data not shown).
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Surface Plasmon Resonance--
Finally, SPR studies were performed
to measure more directly the potential of ternary complex formation
between compound 4, RNA, and the HCV RdRp. The response
difference shown in Fig. 6 corresponds to
a direct measure of the change in molecular weight of the RNA tethered
to the streptavidin chip surface. The differential for compound
4, GTP, or the herpesvirus DNA polymerase inhibitor PFA was
0 RU, confirming that these agents alone do not bind the RNA directly
under these experimental conditions. The response differential
for the HCV RdRp alone or the HCV RdRp with PFA was ~5 RU, thereby
indicating that the viral polymerase is capable of binding the
chip-tethered RNA and that the addition of PFA does not modulate that
interaction. Thus, under these conditions PFA most likely does not
interact with the RNA-bound polymerase, which is consistent with the
inability of PFA to inhibit the viral RdRp in primer extension assays
(data not shown). Conversely, the response differential for the
RNA-bound HCV RdRp upon adding compound 4 or GTP increased
to around 27 RU. This change in RU corresponds to an increase in mass
on the chip, potentially indicating an increase in affinity for RNA by
HCV RdRp bound to compound 4 or GTP. Taken together, these
studies confirm that both GTP and compound 4 are capable of
interacting directly with the RNA-bound viral RdRp.
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Terminal Transferase Activity--
Recombinant HCV RdRp was
reported to possess the ability to add nontemplated nucleotides to the
3' end of viral RNA, referred to as terminal transferase
(TNTase) activity (9, 25). Further, this RdRp-associated activity has
been implicated in maintaining the integrity of the termini of the
viral RNA genome (25). Because compound 4 was shown not to
interfere with the formation of an enzyme-RNA complex necessary for
initiation of RNA synthesis, it was evaluated for inhibition of other
RdRp functions such as TNTase activity. Compound 4 was found
to inhibit TNTase activity of the HCV RdRp with a potency profile
equivalent to inhibition of RdRp activity (data not shown).
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DISCUSSION |
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The HCV RNA-dependent RNA polymerase, the central catalytic enzyme of replication, represents a viable target for identification of antiviral agents to treat chronic HCV infections. In this study, we report the mechanism of action for the benzothiadiazine compound 4. We describe an assay using a short RNA template to investigate the early steps of HCV RdRp-catalyzed RNA synthesis. Specifically, we have demonstrated that this HCV-selective inhibitor interferes with the initiation process of viral replication rather than with elongative RNA synthesis. The inhibition of initial phosphodiester bond formation occurs regardless of whether replication initiates via a primer-dependent or a de novo mechanism.
The distribution of products suggests that the RdRp initiation complex readily accumulates dinucleotides but may be inefficient in forming a productive elongative complex. This abundance of di- or trinucleotides in the gel-based assays reported herein as well as by another group (28) suggest that the RdRp may dissociate rapidly from the RNA template rather than transition efficiently into elongative synthesis. This process of abortive initiation is not uncommon and has been described recently for T7 RNA polymerase (29-34), such that initiation complexes release short RNA templates prior to transition toward forming a stable elongative complex. This suggests that a conformational change may be inherent in this transition and that HCV may operate in a manner similar to T7 RNA polymerase.
As a first attempt at understanding the dynamic process of RNA
synthesis and utilizing compound 4 as a tool, several key
experiments were performed. The goal was to assess whether compound
4 inhibits the apoenzyme form of the viral RdRp or is
capable of forming a ternary complex to mediate inhibition of RNA
synthesis. The order of reagent addition studies showed no difference
in the inhibition profile when preincubating the RdRp with RNA or with
compound prior to initiating the reaction. These data suggest that a
ternary complex may indeed form, although alternate possibilities for
the mode of action cannot be directly ruled out on the basis of this
data. Similar results were found using the filter binding study,
suggesting that an increasing concentration of compound 4 did not disrupt the ability of the RdRp to interact with RNA template.
Finally, to evaluate formation of a ternary complex more directly,
surface plasmon resonance was utilized. This study provided evidence
that a ternary complex is capable of forming between RNA template,
21 HCV RdRp, and compound 4. Together, these data suggest
that this heterocyclic agent interacts with both the apoenzyme form and the RNA-bound form of
21 HCV RdRp and does not interfere directly with RdRp-RNA interaction. The ability to interact with
21 HCV RdRp
that is tethered to an RNA template is consistent with its inhibition
in the replicon system, whereby the HCV RdRp is presumed to be
membrane-bound and continually in contact with the viral RNA. Further
studies using the full-length form of the viral polymerase will help
provide additional insight into this inhibition profile.
The TNTase activity of HCV RdRp, which is responsible for adding nucleotides to the 3' terminus of template RNA, may directly modulate the initiation of RNA synthesis. This activity potentially could be used by RNA viruses to restore the 3'-initiation site, which may be damaged by exonucleases present in the intracellular compartments (35). Herein, we report that compound 4 disrupts both RdRp and TNTase activity of the HCV RdRp. To date, all evidence indicates that TNTase activity is a property of the viral polymerase that requires the RdRp catalytic site, and inhibition of both enzymatic activities by compound 4 is in agreement with this model.
This report represents the first demonstration of an initiation
inhibitor for HCV RNA synthesis. This novel mode of action would be
expected to demonstrate synergy with nucleoside analogs capable of
inhibiting the HCV RdRp, with the latter class of agents representing
elongation inhibitors. Further investigations are ongoing to assess the
potential utility of this agent in the treatment of chronic HCV disease.
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ACKNOWLEDGEMENTS |
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We thank C. Rice for the BB7 HCV replicon and C. Silverman and S. Khandekar for HCV RdRp preparation.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Infectious Diseases Dept., Centocor, Inc., R-4-1, 200 Great Valley Parkway, Malvern, PA 19355-1307. Tel.: 610-240-8207; Fax: 610-240-4064; E-mail: rsarisky@cntus.jnj.com.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210891200
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ABBREVIATIONS |
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The abbreviations used are: HCV, hepatitis C virus; NS5B, nonstructural protein 5B; RdRp, RNA-dependent RNA polymerase; nt, nucleotide(s); SPR, surface plasmon resonance; RU, relative units; PFA, phosphonoformic acid; TNTase, terminal transferase; 3'-rdGTP, 3'-ribodeoxy-GTP.
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