Controlling the biological effects of spermine using a synthetic receptor

Article abstract of DOI:10.1021/ja062536b

Polyamines play an important role in biology, yet their exact function in many processes is poorly understood. Artificial host molecules capable of sequestering polyamines could be useful tools for studying their cellular function. However, designing synthetic receptors with affinities sufficient to compete with biological polyamine receptors remains a huge challenge. Binding affinities of synthetic hosts are typically separated by a gap of several orders of magnitude from those of biomolecules. We now report that a dynamic combinatorial selection approach can deliver a synthetic receptor that bridges this gap. The selected receptor binds spermine with a dissociation constant of 22 nM, sufficient to remove it from its natural host DNA and reverse some of the biological effects of spermine on the nucleic acid. In low concentrations, spermine induces the formation of left-handed DNA, but upon addition of our receptor, the DNA reverts back to its right-handed form. NMR studies and computer simulations suggest that the spermine complex has the form of a pseudo-rotaxane. The spermine receptor is a promising lead for the development of therapeutics or molecular probes for elucidating spermine's role in cell biology.

Full text of DOI:10.1021/ja062536b

Published on Web 07/13/2006
Controlling the Biological Effects of Spermine Using a
Synthetic Receptor
Laurent Vial,† R. Frederick Ludlow, Julien Leclaire,‡ Ruth Pe´rez-Ferna´ndez, and
Sijbren Otto*
Contribution from the Department of Chemistry, UniVersity of Cambridge, Lensfield Road,
Cambridge CB2 1EW, United Kingdom
Received April 12, 2006; E-mail: so230@cam.ac.uk
Abstract: Polyamines play an important role in biology, yet their exact function in many processes is poorly
understood. Artificial host molecules capable of sequestering polyamines could be useful tools for studying
their cellular function. However, designing synthetic receptors with affinities sufficient to compete with
biological polyamine receptors remains a huge challenge. Binding affinities of synthetic hosts are typically
separated by a gap of several orders of magnitude from those of biomolecules. We now report that a
dynamic combinatorial selection approach can deliver a synthetic receptor that bridges this gap. The selected
receptor binds spermine with a dissociation constant of 22 nM, sufficient to remove it from its natural host
DNA and reverse some of the biological effects of spermine on the nucleic acid. In low concentrations,
spermine induces the formation of left-handed DNA, but upon addition of our receptor, the DNA reverts
back to its right-handed form. NMR studies and computer simulations suggest that the spermine complex
has the form of a pseudo-rotaxane. The spermine receptor is a promising lead for the development of
therapeutics or molecular probes for elucidating spermine’s role in cell biology.
Introduction
action of spermine in most of these processes is still under
debate. Thus, there is a considerable incentive to develop
Spermine (1, Figure 1) is a polycation that plays a key role
in many cellular processes1,2 forming strong interactions with
DNA and RNA.1,3 Spermine has been shown to condense
DNA3,4 and chromatin,5,6 to facilitate the formation of nucleo-
somes,7 to induce8-10 and to stabilize8,9 specific DNA confor-
mations, to modulate DNA-protein interactions,11 and to protect
DNA from external agents.12 These phenomena may underlie
spermine’s ability to regulate cell proliferation1 and influence
tumor growth13,14 and apoptosis.15 The exact mechanism of
synthetic molecules that can sequester spermine under physio-
logical conditions. While receptors have been reported,16,17
designing a water-soluble synthetic receptor with sufficient
affinity to interact with spermine in biological systems remains
a huge challenge. Binding affinities of synthetic hosts are
typically in the millimolar range, while biological systems
usually exhibit submicromolar affinities.18 We now report that
a dynamic combinatorial selection approach19-21 can deliver a
synthetic receptor that bridges this gap and binds spermine with
nanomolar affinity, sufficient to remove it from its biological
host DNA in vitro, causing the nucleic acid to change its
conformation from a left-handed to a right-handed helix. NMR
studies and computer simulations suggest the spermine complex
has the form of a pseudo-rotaxane.
† Current address: LEDSS, UMR 5616 CNRS - Universite´ Joseph
Fourier, 301 Rue de la Chimie, BP 53, 38041 Grenoble cedex 9, France.
‡ Current address: UMR 6180, Case A62, EGIM - Sud, Faculte´ de
Saint-Je´roˆme, Av. Escadrille Normandie-Nie´men, 13397 Marseille cedex
20, France.
(1) Tabor, C. W.; Tabor, H. Annu. ReV. Biochem. 1984, 53, 749-790.
(2) Igarashi, K.; Kashiwagi, K. Biochem. Biophys. Res. Commun. 2000, 271,
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Results and Discussion
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(4) Flock, S.; Labarbe, R.; Houssier, C. Biophys. J. 1996, 70, 1456-1465.
(5) Hougaard, D. M.; Bolund, L.; Fujiwara, K.; Larsson, L. I. Eur. J. Cell
Biol. 1987, 44, 151-155.
As spermine lacks a well-defined geometry in solution,
designing a receptor for it is inherently difficult. We therefore
took a selection approach, generating mixtures of potential
receptors by linking building blocks together using a reversible
reaction. The resulting dynamic combinatorial libraries (DCLs)
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1988, 5, 1149-1161.
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Biochem. 1997, 243, 247-258.
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32, 1067-1071.
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Chem., Int. Ed. 2000, 39, 4257-4260.
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1991, 30, 2543-2549.
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Ed. 2003, 42, 4872-4897.
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Res. Commun. 1998, 244, 298-303.
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(21) Rowan, S. J.; Cantrill, S. J.; Cousins, G. R. L.; Sanders, J. K. M.; Stoddart,
J. F. Angew. Chem., Int. Ed. 2002, 41, 899-952.
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A R T I C L E S
Vial et al.
Figure 1. Oxidation of thiol building blocks 2 and 3 produces a DCL of linear and macrocyclic disulfides that contains potential receptors for protonated
spermine (1).
are equilibrium mixtures that are responsive to influences that
result in the selective stabilization of specific library members.
Thus, introducing a guest into a DCL of potential hosts will
shift the equilibrium, amplifying strong binders at the expense
of their inferior counterparts.20,21 Amplification allows new
synthetic receptors to be identified and, in small DCLs, even
provides a method for their preparation.22-24
We have set up a DCL from building blocks 225 and 3
equipped with carboxylate groups (Figure 1) tailored to recog-
nize the protonated amine groups of spermine through hydrogen-
bonding and cation-anion interactions. We deliberately intro-
duced a large number of carboxylate groups as each amine group
of spermine is able to interact with several of these units. An
excess of carboxylate groups also ensures the solubility of the
library members in water by preventing neutralization of all
negative charges by the cationic spermine. The thiol functional-
ity allows the building blocks to be oxidatively linked together
to form disulfide bonds, which can subsequently undergo
Figure 2. HPLC analyses of the libraries made from thiols 2 and 3 (10
mM each) in the absence of any template (a), and in the presence of spermine
(2.5 mM) (b). HPLC analyses of the libraries made from thiol 2 only (10
mM) in the absence of any template (c),27 and in the presence of spermine
(2.5 mM) (d).
disulfide exchange to generate the DCL.26
As it was not clear whether linear or cyclic architectures
would be more suitable for spermine recognition, we designed
our libraries to contain both architectures (Figure 1). Thus, while
the oxidation of dithiol 2 will give macrocyclic species of
various ring sizes, the presence of monothiol 3 will enable the
additional formation of linear species.
The structure of the complex between 6 and spermine was
investigated using proton NMR spectroscopy and computer
modeling.
Equimolar amounts of the building blocks 2 and 3 were
allowed to oxidize and equilibrate in aqueous solution at pH
8.25 in the presence of air following standard protocols.26 The
composition of the ensuing DCL was analyzed by HPLC and
mass spectrometry (Figure 2a). Linear tetramer 4 was the major
compound in the absence of spermine. When spermine was
introduced into the DCL, the cyclic tetramer 6 was amplified,
alongside linear dimer 5 built up from two units of monothiol
building block 3. The strong affinity of spermine for 6 (vide
infra) drives the conversion of dithiol 2 into this receptor and
leaves monothiol 3 with no alternative but to dimerize.
A biased DCL made from only building block 2 and spermine
gives macrocycle 6 in more than 90% yield allowing straight-
forward isolation by preparative HPLC (Figure 2c and d).
Upon binding of spermine to 6 a strong ring-current-induced
upfield shift was observed for the central protons d and e of
spermine, while protons a, b, and c near the termini of the
molecule are hardly shifted (Figure 3). These observations
suggest that the central fragment of spermine is located within
the cavity of the receptor and imply the formation of a pseudo-
rotaxane in which spermine is threaded through the cyclic
receptor.16,17
The presence of multiple signals for the host-guest complex
is most likely a result of stereoisomers resulting from different
conformations of the disulfide linkages. The preferred 90°
torsional angle of the disulfide bond makes this linkage
inherently chiral as it can exist in a P or M configuration. In
most disulfides, rapid rotation around the S-S bond leads to
fast exchange between stereoisomers. However, when spermine
is threaded through macrocycle 6, this rotation is likely to be
hindered, thus freezing out up to four different diastereomers
that arise from the fact that the four disulfide bonds can have
different configurations. The NMR spectrum of the complex is
dominated by a singlet for the host protons, indicating that the
(22) Otto, S.; Furlan, R. L. E.; Sanders, J. K. M. Science 2002, 297, 590-593.
(23) Lam, R. T. S.; Belenguer, A.; Roberts, S. L.; Naumann, C.; Jarrosson, T.;
Otto, S.; Sanders, J. K. M. Science 2005, 308, 667-669.
(24) Corbett, P. T.; Tong, L. H.; Sanders, J. K. M.; Otto, S. J. Am. Chem. Soc.
2005, 127, 8902-8903.
(25) Field, L.; Engelhardt, P. R. J. Org. Chem. 1970, 35, 3647-3655.
(26) Otto, S.; Furlan, R. L. E.; Sanders, J. K. M. J. Am. Chem. Soc. 2000, 122,
12063-12064.
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Controlling the Biological Effects of Spermine
A R T I C L E S
Figure 3. 1H NMR (500 MHz, cryoprobe) analyses of receptor 6 (a),
spermine (b), and receptor 6 (1.3 mM) in the presence of 1 equiv of spermine
(c). Spectra were recorded at 300 K in 89 mM phosphate buffer (pH 7.4,
11% D2O) with water presaturation.
Figure 5. (a) UV spectra of calf thymus DNA (33 µM in 3.0 mM TRIS
buffer; pH 7.4; 298 K) without additive (blue O); in the presence of spermine
(2.2 equiv with respect to the number of base pairs) (red ]); and in the
presence of spermine and 6 (2.2 equiv each) (green 4). (B) CD spectra of
poly(dA-dC)‚poly(dG-dT) (97 µM in 3.0 mM TRIS/EDTA buffer; pH 7.4;
1.0 mM NaCl; 298 K) without additive (blue O); after addition of spermine
(0.5 equiv with respect to the number of base pairs) (red ]); and after
addition of 6 (0.5 equiv) (green 4). The UV and CD traces correspond
exclusively to DNA; contributions from the buffer, spermine, and 6 have
been subtracted.
Figure 4. Side and top views of the energy minimized complex of receptor
6 and spermine obtained by computer modeling.32 Some hydrogen atoms
have been omitted for clarity.
Binding experiments were performed in the presence of DNA
to assess the ability of the receptor to (i) inhibit the formation
of DNA-spermine complexes, (ii) unbind spermine from DNA,
and (iii) control the conformation of DNA.
major stereoisomer is highly symmetric with all four disulfide
bonds having the same configuration.
Computer modeling studies on the free receptor 6 and on its
complex with spermine provide further support for these
conclusions. In the absence of spermine, the four diastereomers
of the receptor were found to be similar in energy. Modeling
of the four different spermine-receptor complexes showed that
the diastereomer with the highest symmetry was significantly
lower in energy than the three others. Furthermore, the energy
barrier for the interconversion of the diastereomers was found
to be substantially higher in the spermine complex than in the
free receptor (see Supporting Information).
When an excess of spermine (2.2 equiv with respect to the
number of DNA base pairs) was added to a solution of calf
thymus DNA, essentially complete precipitation of the biopoly-
mer was observed as was apparent from the disappearance of
the UV absorbance of the DNA (Figure 5a, red and blue traces,
respectively). However, in the presence of 6 (1.0 equiv with
respect to spermine), the addition of spermine does not give
any precipitation of DNA and the UV absorbance is unchanged
(green trace).
At lower concentrations, spermine binding can switch the
conformation of DNA from a right-handed (B-form) to a left-
handed (Z-form) helix, a change that can be monitored by
circular dichroism (CD).8,10 Thus, the addition of one-half an
equivalent of spermine (with respect to the number of DNA
base pairs) to a buffered solution of poly(dA-dC)‚poly(dG-dT)
induced the appearance of a negative CD-band at 294 nm and
a shift in the positive band from 280 to 263 nm (Figure 5b).
In the lowest-energy conformation of the major pseudoro-
taxane complex, 8 of the 10 amine protons of the guest are
involved in hydrogen bonds with the host (Figure 4). In solution,
rapid exchange between different hydrogen-bonding arrange-
ments must occur as any long-lived hydrogen bonds would
desymmetrize the receptor and no such effect is observed in
the proton NMR analysis.
The dissociation constant for binding of spermine to receptor
6 was found to be 22 ((1) nM, as determined by isothermal
titration calorimetry (ITC) (see Supporting Information). This
affinity is higher than that reported for the binding of spermine
to DNA,8,28-30 suggesting that 6 should be able to inhibit DNA-
spermine interactions.
(27) The complexity of the trace can be explained by the presence of peaks
corresponding to linear species in which the terminal thiols have oxidized
to sulfinic or sulfonic acids.
(28) Morgan, J. E.; Blankenship, J. W.; Matthews, H. R. Arch. Biochem. Biophys.
1986, 246, 225-232.
(29) Ouameur, A. A.; Tajmir-Riahi, H. A. J. Biol. Chem. 2004, 279, 42041-
42054.
(30) Patel, M. M.; Anchordoquy, T. J. Biophys. J. 2005, 88, 2089-2103.
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Vial et al.
Scheme 1. Synthesis of Building Block 2
bath. Dimethylthiocarbamoyl chloride (19 g, 157 mmol) dissolved in
dry DMA (50 mL) was added dropwise under nitrogen. The mixture
was stirred for 16 h at room temperature. The off-white precipitate
was filtered, washed extensively with water (300 mL), and dried under
vacuum, yielding compound 8 (16 g, 97%). 1H NMR (400 MHz,
CDCl3): δ 7.73 (s, 2H, CHAr), 4.30 (q, J ) 7.1 Hz, 4H, CH2), 3.46
(s, 6H, CH3), 3.40 (s, 6H, CH3), 1.33 (t, J ) 7.1 Hz, 6H, CH3).
2,5-Bis(dimethylthiocarbamoylsulfanyl)terephthalic Acid Diethyl
Ester (9).25 Compound 8 (650 mg, 1.51 mmol) was heated under
nitrogen at 215 °C for 1 h. The brownish mixture was cooled to 70 °C,
and EtOH (20 mL) was added. Pale brown crystals appeared as the
sample was slowly cooled to room temperature. The crystals were
filtered off, yielding compound 9 (626 mg, 97%). 1H NMR (400 MHz,
CDCl3): δ 8.11 (s, 2H, CHAr), 4.34 (q, J ) 7.1 Hz, 4H, CH2), 3.12
(s, 6H, CH3), 3.02 (s, 6H, CH3), 1.37 (t, J ) 7.1 Hz, 6H, CH3).
2,5-Dimercaptoterephthalic Acid (2). A solution of compound 9
(1.3 g, 3.03 mmol) in degassed 1.3 M KOH in EtOH/H2O (1:1, 40
mL) was refluxed under an inert atmosphere for 3 h. The reaction
mixture was cooled in ice, and concentrated HCl (15 mL) was added.
A bright yellow precipitate was formed, filtered, and washed extensively
with water, yielding compound 2 as a yellow solid (663 mg, 95%). 1H
NMR (400 MHz, CD3OD): δ 8.02 (s, 2H, CHAr).
Subsequent addition of one-half an equivalent of 6 to the
resulting Z-DNA solution reversed the transition, regenerating
the original B-DNA, indicating that the receptor is indeed able
to unbind spermine from DNA and thereby control the helicity
of DNA.31
Dynamic Combinatorial Libraries. In a typical experiment, the
thiols (10 mM overall) were suspended in water, and the pH was
adjusted to 8.25 by addition of a 1 M solution of NaOH. Where
appropriate, spermine (2.5 mM) was added. The mixtures were allowed
to oxidize and equilibrate by stirring in an open vial at room
temperature. Evaporated water was replenished every day.
Library Analysis. Analyses were performed using an Agilent 1100
series HPLC and Agilent XCT ion-trap mass spectrometer. Solvents
and formic acid were acquired from Romil. Analyses were performed
using a reversed phase HPLC column (Agilent C8 Zorbax Eclipse XBD,
4.6 × 150 mm, 5 µm), using an injection volume of 10 µL, a flow rate
of 1 mL/min, and a gradient (5-95% in 40 min) of acetonitrile in water
(both containing 0.1% formic acid) at 303 K. Negative ion mass spectra
were acquired in ultrascan mode using electrospray ionization (drying
temperature, 350 °C; nebulizer pressure, 55 psi; drying gas flow, 12
L/min; HV capillary, 4000 V; ICC target, 200 000).
Conclusions
Our results demonstrate that, after the design of only
rudimentary molecular recognition elements, dynamic combi-
natorial chemistry can be used to guide their assembly into a
high-affinity receptor. Thus, starting from a dynamic combi-
natorial library made from two building blocks that contain
carboxylate groups as recognition units, a new macrocyclic
receptor for the oligoamine spermine was obtained that binds
this guest with a dissociation constant of 22 ((1) nM. Water-
soluble synthetic receptors for biologically relevant guests with
such high affinities are extremely rare. Analysis of the host-
guest complex by NMR and computer simulations indicates a
pseudo-rotaxane structure in which spermine is threaded through
the macrocyclic host and bound through a series of hydrogen
bonds and anion-cation interactions. The host was shown to
interfere efficiently with spermine-DNA interactions, being able
to prevent and reverse the binding of spermine to DNA and
thereby indirectly control the conformation of DNA; while
spermine can induce a change in the conformation of DNA from
a right-handed (B-form) to a left-handed (Z-form) helix, the
addition of the synthetic spermine receptor reverts the DNA
back to its original B-form. These results suggest that our
spermine receptor may be a promising lead for the development
of novel therapeutics or molecular probes that may help unravel
the still poorly understood role of oligoamines in cell biology.
Purification of Receptor 6. Receptor 6 was isolated from a dynamic
“library” made from thiol 2 (10 mM) and spermine (2.5 mM) in water
at pH 8.25. Aliquots of 700 µL of this solution were injected onto a
reversed phase preparative HPLC column (Agilent C8 Zorbax Eclipse
XBD, 21.2 × 150 mm, 5 µm) at 303 K. Using a flow rate of 5 mL/
min and a gradient (5-95% over 30 min) of acetonitrile in water (both
containing 0.1% formic acid), the receptor was collected from 14 runs.
The solvents were removed under reduced pressure keeping the
temperature below 40 °C. The last traces of solvent were removed under
high vacuum for 2-3 h. The crude host was dissolved in 12 mL of
borate buffer (10 mM pH 9.0) and sonicated for 2 min. The resulting
solution was centrifuged and decanted. Hydrochloric acid (3 mL of a
2.0 M solution) was added to the supernatant. The resulting suspension
was centrifuged and the pellet resuspended in dilute hydrochloric acid
(30 mL of a 40 mM solution) and centrifuged. The solid (6 mg) was
dried under vacuum overnight. The LC-MS analysis of 6 is shown in
Experimental Section
Materials and Methods. Chemicals were purchased from Aldrich
or Fluka. Building block 2 was synthesized on the basis of a method
described by Field and Engelhardt (Scheme 1).25 NMR analyses were
performed using an Advance 500 Bruker instrument equipped with a
standard TCI Cryoprobe. Spectra were recorded at 300 K with water
presaturation during relaxation delay and mixing time.
2,5-Bis(dimethylthiocarbamoyloxy)terephthalic Acid Diethyl Es-
ter (8).25 2,5-Dihydroxyterephthalic acid diethyl ester 7 (10 g, 39.00
mmol) and DABCO (18 g, 157 mmol) were dissolved in dry DMA
(100 mL) under a nitrogen atmosphere and cooled to 0 °C in an ice
1
the Supporting Information. H NMR (500 MHz, DMSO-d6): δ 8.34
(s, 8H). Anal. Calcd for 6‚5H2O: C, 38.32; H, 2.61. Found: C, 38.06;
H, 2.76.
Isothermal Titration Calorimetry (ITC). The binding studies
between 1 and 6 were conducted at three different concentrations using
isothermal titration microcalorimetry (MCS-ITC, Microcal LLC,
Northampton, MA). Solutions of guest 1 (in 3 mM TRIS buffer pH
7.4) were titrated into solutions of host 6 (in 3 mM TRIS buffer pH
7.4). Binding constants and enthalpies of binding were obtained by
curve fitting of the titration data using the one-site binding model
available in the Origin 2.9 software as shown in the Supporting
Information. The thermodynamic parameters were averaged over three
(31) The amplitude of the CD signals after addition of spermine and after addition
of 6 is somewhat lower than the original value as a result of partial
precipitation of the DNA-spermine complex. This does not affect the
conclusions.
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Controlling the Biological Effects of Spermine
A R T I C L E S
experiments (the enthalpy value for run C [see Supporting Information]
was discarded due to an inferior signal-to-noise ratio).
AMBER* force field with all atom treatment, were run for 20 ns at a
simulation temperature of 300 K, with a 1.5 fs time step and 1 ps initial
equilibration time. The variation in the average enthalpy (adjusted to
the simulation temperature) during the last 5 ns of each experiment
DNA Competition Experiments. Analyses were performed using
a Varian Cary 100 Bio UV-visible spectrophotometer and a Jasco J-810
spectropolarimeter with 1 cm path-length cells at 298 K. Calf thymus
DNA (highly polymerized), poly(dA-dC)‚poly(dG-dT), spermine‚4HCl,
and buffers were purchased from Sigma and used without further
purification. The solutions were prepared by dilution of concentrated
stock solutions of the different species (DNA, spermine‚4HCl, or 6) in
the desired buffer (3.0 mM TRIS buffer, pH 7.4, or 3.0 mM TRIS-
EDTA buffer, 1.0 mM NaCl, pH 7.4). The final concentration in DNA
was determined by measuring the absorbance at 260 nm (A260) and
assuming that 1 absorbance unit corresponds to 0.15 mM in base pairs.
Samples were incubated for at least 30 min at 298 K prior to the
spectroscopic measurements. The reported UV and CD traces cor-
respond exclusively to DNA (contributions from the buffers, spermine,
or 6 have been subtracted). Where appropriate, the traces have been
corrected to compensate dilution.
was determined, and in all cases was found to be less than 2.2 kJ mol-1
,
implying the simulation length was sufficient. The energies of the
spermine, free receptors, and complexes as determined by molecular
dynamics are given in the Supporting Information. The interconversion
barriers between P and M forms of the disulfide bonds were estimated
using the dihedral driving function of Macromodel 8.0. Starting from
the lowest energy conformer found in the conformational search, the
dihedral angle of one disulfide bond was driven, in 1° steps, through
360°, in each direction. The lowest energy barrier for the conversion
of the empty (P,P,P,P) host into the (P,P,P,M) conformer was found to
be 74 kJ mol-1. In the complex, the energy of interconversion was
determined for each disulfide bond, and the lowest barrier was found
to be 83 kJ/mol.
Acknowledgment. We thank J. M. Goodman for advice on
the simulations and J. K. M. Sanders for support. This work
was supported by EPSRC, the European Union (Marie Curie
Intra-European Fellowships), and the Royal Society.
Theoretical Calculations. Calculations were performed using Mac-
romodel 8.0 and the AMBER* all atoms force field32 using the
continuum water solvent treatment. Conformational searches were
carried out using the Monte Carlo Multiple Minimum method (MCMM).
For the unbound tetramers, 1000 steps were sufficient to find the 10
lowest energy conformations at least twice each. Between 20 000 and
40 000 steps were calculated for the complexes; this was sufficient for
at least 5 of the lowest 10 conformations to be found at least twice.
Subsequent molecular dynamics (MD) simulations, also using the
Supporting Information Available: COSY spectrum of the
complex between spermine and 6; LC-MS analysis of 6; ITC
data for binding of spermine to 6; computer simulation results.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(32) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.;
Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem.
1990, 11, 440-467.
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