From the Department of Organic Chemistry, and
¶ Department of Pharmaceutical Sciences, University of Padova,
Padova 35131, Italy, and
Department of Biological Chemistry,
University of Padova, Padova 35121, Italy and § Venetian
Institute for Molecular Medicine, Padova 35131, Italy
Received for publication, September 12, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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Protein kinases play key roles in signal
transduction and therefore are among the most attractive targets for
drug design. The pharmacological aptitude of protein kinase inhibitors
is highlighted by the observation that various diseases with special
reference to cancer are because of the abnormal expression/activity of
individual kinases. The resolution of the three-dimensional structure
of the target kinase in complex with inhibitors is often the starting point for the rational design of this kind of drugs, some of which are
already in advanced clinical trial or even in clinical practice. Here
we present and discuss three new crystal structures of ATP site-directed inhibitors in complex with "casein kinase-2" (CK2), a
constitutively active protein kinase implicated in a variety of
cellular functions and misfunctions. With the help of theoretical calculations, we disclose some key features underlying the inhibitory efficiency of anthraquinone derivatives, outlining three different binding modes into the active site. In particular, we show that a nitro
group in a hydroxyanthraquinone scaffold decreases the inhibitory
constants Ki because of electron-withdrawing and
resonance effects that enhance the polarization of hydroxylic substituents in paraposition.
The crucial role of protein kinases in cell signaling, gene
expression, and metabolic regulation is highlighted by the fact that
nowadays this family of enzymes is the second most important drug
target (1). Actually, abnormal activity of individual protein kinases
is often associated with human diseases, especially tumors whose
treatment has been so far restricted to cytotoxic and hormonal agents
(2). Many kinase inhibitors are currently in clinical trials, mostly as
anti-tumor drugs (1, 3), and two of them, Gleevec (STI-571) and
rapamycin, are in clinical use for the treatment of a form of leukemia
and to prevent tissue rejection after organ transplantation,
respectively. One major problem with kinase inhibitors is that the
human genome encodes for >500 different protein kinases; therefore,
inhibitors designed to target specifically an individual kinase are
likely to bind to closely related kinases as well, thus interfering
with other cell functions. In addition, the most promising inhibitors
are directed to the highly conserved ATP binding site with the
consequence that their selectivity is hardly absolute, and they have to
compete against high intracellular concentrations of ATP. In this
respect, peptide inhibitors directed to the phosphoacceptor substrate
binding site may in principle display higher specificity. Their
pharmacological utilization, however, is hampered by a number of
practical drawbacks, primarily reduced bioavailability.
Many of the chemical scaffolds or building blocks studied as ATP
site-directed kinase inhibitors are based on more or less complex
heterocyclic molecules (mainly with nitrogen and oxygen as
heteroatoms). The most common scaffolds are derivatives of the
following: quinazolines; phenylamino-pyrimidines,
pyrido-pyrimidines, pyrrolo-pyrimidines, pyrimido-pyrimidines, or
pyrazolo-pyrimidines; pyrrolo-pyridines; indolin-2-ones; purines;
pyridinyl-imidazoles or pyrimidinyl-imidazoles; and
phthalazines. Other examples are natural products such as
balanol and alkaloids, flavopiridol (belonging to the flavonoid
family), and staurosporine and its derivatives (4).
Besides the traditional medicinal chemistry and the relatively new
combinatorial approaches (with the employment of high throughput screenings on molecules libraries), the solution of the crystal structure of complexes between an individual kinase and its inhibitors also represents a powerful tool for the discovery of new drugs by a
rational drug design approach. In fact, the structural bases for
selectivity and potency are now being clarified by means of crystallization of a number of such targets in complex with inhibitors (5). A telling example is that of Cdk2 whose crystal structures in complex with a number of ligands have been exploited to design more
potent and selective inhibitors (6-8). It is now a common exercise to
run a virtual screen of thousands low molecular weight compounds on the
crystal structure of a kinase in complex with an inhibitor with the aim
to identify the most promising chemical scaffolds to develop
(9). The progress made in the crystallization of protein kinases
has corroborated the concept that the ATP-binding domain is an
attractive target for drug design. Three successful examples of drug
design using a tyrosine kinase as a molecular target are the following:
1) PKI166, a pyrrolo[2,3,-d]pyrimidine derivative that
inhibits both epidermal growth factor receptor and ErbB2 kinases, 2)
the anilino-phthalazine derivative PTK787/ZK222584, a potent and
selective inhibitor of the kinase domain receptor and Flt-1
kinases, and 3) the aforementioned STI-571 (10).
CK21 (an acronym derived from
the misnomer "casein kinase-2") is one of the most pleiotropic
protein kinases with hundreds of protein substrates involved in a
variety of cellular functions with special reference to signaling,
nuclear organization, and gene expression (11). An intriguing hallmark
of CK2 is the high constitutive activity, which is believed to underlie
its pathogenic potential (12). Although there are no known mutations of
CK2 associated with neoplasia, CK2 is abnormally elevated in a wide variety of tumors and there are several experimental models where the
unscheduled expression of the catalytic subunits of CK2 cooperates with
the altered expression of proto-oncogene or tumor suppressors to
promote cell transformation and neoplastic growth (13-16). Because of
its constitutive activity, CK2 is also exploited by many viruses to
phosphorylate proteins essential to their life cycle. This has
triggered an increasing interest for CK2 inhibitors that could act as
anti-neoplastic and anti-infectious drugs. Although CK2 is essential to
viability, it is conceivable that, as pointed out in the case of the
MAPK cascade (1), its essential roles in proliferation and
differentiation are required only at individual developmental stages, a
circumstance that would make applicable a transient pharmacological
treatment with CK2 inhibitors.
Our interest has been recently focused on CK2 inhibitors belonging to
the anthraquinone (17) and xanthenone families. Anthraquinones have
been used for the purification of proteins by affinity techniques taking advantage of their nucleotide-specific ligand capability (18).
This enables them to interact with ATP, ADP, and NAD binding sites of
enzymes such as dehydrogenases, kinases, and ATPases. Anthraquinone and
xanthenone derivatives often obtained from natural sources have several
potential therapeutic applications for instance as antiviral,
antimicrobial, or anti-cancer drugs (19). A potential drawback of these
compounds is that their cyclic planar structure confers them the
feature of DNA-intercalators with expectable cytotoxic effects. Even
with this limit, the optimization of highly specific and selective
inhibitors of this category could be exploited for the elucidation of
the still somewhat enigmatic cellular functions of CK2. Another
important benefit of the improvement of the inhibition potency and
selectivity of anthraquinone and xanthone derivatives is the
rationalization of the effect of different substituents on a common
scaffold in order to draw information regarding their effects on the
interaction energies involved in target binding. This sort of
information is useful for the optimization of the force fields used in
drug design and docking studies.
Here we present the crystal structure of three different complexes of
maize CK2 Crystals Preparation and Data Collection--
The catalytic
Crystallization trials were made by mixing a 2-µl drop of
preincubated stock solution with 4 µl of water and 2 µl of
precipitant solution (10-20% PEG 4000, sodium acetate 0.2 M, Tris 0.1 M, pH 8.0). The drop was
equilibrated against a 500-µl reservoir of the same precipitant
solution (20% PEG 4000). Crystals grew in few days at 293 K. Data were
collected at a temperature of 100 K. Before mounting, crystals were
cryoprotected by soaking in a 40% PEG 4000 solution of the
precipitation buffer.
For the MNA complex, a data set was measured at the x-ray diffraction
beamline of ELETTRA synchrotron facility on a Mar CCD detector at a
wavelength of 1.2 Å and at a crystal to detector distance of 115 mm.
The completeness of the data set is 90.7% at a maximum resolution of
2.0 Å. Data sets of MNX and DAA complexes were collected at the
beamline ID-29 of the ESRF on a Quantum4 CCD detector at a wavelength
of 0.918 Å and with a crystal to detector distance of 190 mm. The
completeness of the data sets for the two complexes is 87.6 and 89.7%
at a maximum resolution of 1.8 and 1.7 Å, respectively.
All the three crystals of the enzyme-inhibitor complexes belong to the
space group C2 with one molecule in the asymmetric unit in analogy with
the emodin complex and the apoenzyme structures published previously
(17, 20). It is worth noticing that the b-cell parameter and the Structure Determination and Refinement--
Data were indexed
with MOSFLM (22) and then scaled with SCALA from the CCP4 software
package (23). To solve the three new structures, a rigid body
transformation on the model of the apoenzyme was adequate using the CNS
software package (24). Some of the MNA reflections were affected by
systematic errors because of the presence of ice rings and therefore
were excluded from the set. The presence of the inhibitor in the active
site was clear since the beginning of the refinement in both
Fo
The definition files for the inhibitors were initially created by
Hic-Up (25) corrected with the adequate parameters and used in CNS in
the whole refinement procedure that was carried out alternating
automated cycles and manual inspection steps using the graphic program
QUANTA (26). During the final steps of the refinement, water molecules
were added and the stereochemistry was checked with the program
Procheck (27).
Statistics on data collection and final models are reported in Table I.
The final model for the complex with the MNA inhibitor presents an
overall crystallographic R-factor of 22.2 (Rfree 24.3) with 177 water molecules and a good
stereochemistry with no residues in disallowed regions of the
Ramachandran plot. For the complexes with MNX and DAA, the
R-factors are 19.8 (Rfree 23.3) and
19.0 (Rfree 21.6), respectively, with 161 and
205 final water molecules and no residues with disallowed stereochemistry.
Computational Methodologies--
Calculations were performed on
a Silicon Graphics Octane R12000 work station. The ground state
geometry of charged and uncharged docked structures was fully optimized
without geometry constraints using Restricted
Hartree-Fock/3
Because not all x-ray crystallographic files contain hydrogen atoms,
they were added to the protein by using the MOE modeling suite (29)
before carrying out docking studies. To minimize contacts among
hydrogens, the structures were subjected to a Amber94 (30) energy
minimization protocol until the root mean square of conjugate gradient
was <0.15 kcal mol Coordinates--
Coordinates have been deposited in the Protein
Data Bank with the following accession codes: 1M2P (for MNA·CK2
complex); 1M2Q (for MNX·CK2 complex); and 1M2R (for DAA·CK2 complex).
The optimization of a co-crystallization protocol for inhibitors
MNA, MNX, and DAA allowed data collections with a maximal resolution
higher than that obtained by soaking methods used in the case of emodin
and TBB complexes (2.63 and 2.19 Å, respectively) (17, 20). The new
protocol consists of a preincubation for 1-2 h of the protein with the
inhibitor dissolved in Me2SO (final Me2SO
concentration
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(70% identical to its human homologue and almost 100%
conserved in the catalytic core) with two anthraquinone derivatives and
one related xanthenone. The three inhibitors studied whose chemical
formulae and Ki values with CK2 are shown in
Fig. 1 are: 1,8-dihydroxy-4-nitro-anthraquinone (MNA),
1,8-dihydroxy-4-nitro-xanthen-9-one (MNX), and
1,4-diamino-5,8-dihydroxy-anthraquinone (DAA). These compounds were
selected from a panel of many anthraquinones and xanthenones because of
their relatively low Ki values and ability to
originate diffracting quality crystals in co-crystallization trials. A
relevant issue we also wanted to address was the rationalization of the
higher inhibitory efficiency of these molecules as compared with
another inhibitor of the anthraquinone family, emodin (also shown in
Fig. 1), whose crystal structure in
complex with CK2 has previously been solved (17). Emodin
(3-methyl-1,6,8-trihydroxyantraquinone) is extracted from the medicinal
herb Rheum palmatum and has been used for a long time in the
Orient to cure inflammatory and neoplastic diseases. Our studies will
provide hints on how to improve the efficiency and selectivity of
emodin-related compounds toward CK2 and possibly other protein
kinases.
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Fig. 1.
Chemical structure of the anthraquinone and
xanthenone derivatives studied in this work with the respective values
of the inhibitory constants Ki on CK2.
Predicted pKa values are indicated on the
top right side of the hydroxyl functions. Emodin is also
reported for comparison.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunit of Zea mays CK2 was expressed in Escherichia
coli and purified according to a method described previously (20).
The crystals of the three inhibitor-enzyme complexes with MNA, MNX, and
DAA were obtained by co-crystallization with the sitting drop
vapor-diffusion technique. The synthesis and characterization of the
three inhibitors are described
elsewhere.2 The 8-mg/ml
protein stock solution was preincubated with 100 mM
inhibitor solution (100% Me2SO) in the proper
amount to have an inhibitor-protein molar ratio of 3 to 1 and not to
exceed a 5% Me2SO concentration in the final protein solution.
angle are different for MNA crystal with respect to MNX and DAA (see
below), which implies a different Matthews coefficient:
VM is 2.08 Å3 Da
1 for
MNX and DAA against a VM of 2.40 Å3
Da
1 for MNA. In addition, the solvent content is
different: 41% in MNX and DAA complexes and 49% in MNA.
Fc and
2Fo
Fc maps for all of the three compounds. The electron density for the position of the nitro
group was clear for MNX, whereas it was ambiguous for MNA, suggesting a
double orientation for this inhibitor in the active site. Because of
the symmetry of DAA, the assignment of the hydroxyl or amino groups of
the inhibitor H-bonded to the protein was made on the basis of the
H-bonds length (longer for an NH acceptor than for an OH one)
and on the results of DAA binding simulations (see below).
21G ab initio calculations. Vibrational frequency analysis was used to characterize the minimal stationary points (zero imaginary frequencies). The software package Spartan O2 was utilized for all quantum mechanical calculations
(28).
1 Å
1, keeping the heavy
atoms fixed at their crystallographic positions. All anthraquinone
derivatives were docked into both intercalation sites using flexible
MOE-Dock methodology. The purpose of the MOE-Dock procedure is to
search for favorable binding configurations between a small flexible
ligand and a rigid macromolecular target. The search is carried out
within a user-specified three-dimensional docking box using the
Tabù Search protocol (31) and the MMFF94 force field (32-38).
MOE-Dock performs a user-specified number of independent docking runs
(50 in our case) and writes the resulting conformations and their
energies to a molecular data base file. The resulting docked complexes
were subjected to a MMFF94 energy minimization protocol until the root
mean square of the conjugate gradient was <0.1 kcal mol
1
Å
1. The charges for the ligands were imported from the
Spartan output files. To model the solvent effects more directly,
corrections for the electrostatic interactions were used. The MOE suite
utilized includes an implemented version of generalized born/surface
area contact function (39) that models the electrostatic
contribution to the free energy of solvation in a continuum solvent
model. The interaction energy values were calculated as the energy of the complex minus the energy of the ligand minus the energy of protein:
Einter = E(complex)
(E(L) + E(protein)). Apparent pKa values of all anthraquinone and xanthenone
derivatives were theoretically calculated by using ACD/pKa DB (version
6.0) software (40).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
5%) and then the set up of the crystal trials as
described under "Experimental Procedures." With this new procedure, we could collect data at 2.0-Å resolution for MNA, 1.79-Å resolution for MNX, and 1.70-Å resolution for DAA. It may be interesting to note
that the CK2 complexes crystallized so far are not perfectly isomorphous. As outlined in Table
I, although the differences in the
a and c axis lengths can be considered within the
experimental errors, axis b varies from 59.5 ± 1 Å (with ATP or MNA bound and in the apo-form) to 52.2 ± 0.5 Å (in
the case of emodin, MNX, and DAA). This change is coupled with an
adjustment in the
angle from 103.0 ± 0.5 to 90.5 ± 0.2° and a decrease of the solvent content from 49 to 41%. From the
analysis of the final three-dimensional structures, we have noted that
these variations reflect two different conformations of the protein
loop between
strand 4 and 5 (residues 102-108) (Fig.
2). This segment has a bent conformation
with the short b axis and an extended one with the stretched
b axis. This is the only evident structural difference
between the two diverse groups of cell parameters, and it involves a
shrink in the crystal packing roughly along the b axis. One
possible explanation is that high PEG concentrations (
30%) as
precipitating agent or glycerol as cryoprotectant are responsible of
the cell shrinkage. A direct implication of the different inhibitors
located ~20 Å far away from the 102-108 loop is hardly
conceivable.
Data collection and final model statisticsa
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Fig. 2.
Overview of CK2 three-dimensional
folding. The ribbon diagram of CK2 in complex with MNA (in
ball-and-stick) bound in the ATP binding site between the N-
and the C-terminal lobes is shown (gray). The location of
the inhibitor DAA is also shown (ball-and-stick in
black). On the top right are shown the two
different positions of the loop 102-108 in the case of the MNA complex
(gray) and DAA (or MNX) complex (black) that
correspond to a long b-cell axis (around 59.5 Å) and a short one
(around 52.2 Å), respectively.
As expected by analogy with emodin, the three inhibitors bind in the
co-substrate binding cavity of CK2 between the N- and C-terminal lobes
in the proximity of the Gly-rich loop (Fig. 2). As already noted (20),
the C-terminal domain of the kinase is quite rigid, whereas the
N-terminal one is more inclined to alterations induced by the presence
of different ligands. A noteworthy exception is helix C that is
conformationally very well conserved in all structures solved until
now. Among the three complexes described here, the one with MNX shows
the greatest variation in the N-terminal domain with special reference
to the positions of the backbone between residues 72 and 75 and of the
Gly-rich loop (residues 45-51). The latter collapses into the
co-substrate binding cavity, but this movement is not accompanied by
the rotation of His-160 and Asn-118 side chains as in the case of the
emodin complex (17).
The orientation and precise location of MNA and MNX differ from that of
emodin, which enters the active site with hydroxyl groups 1 and 8 on
the side of the hinge region. In contrast, both MNA and MNX penetrate
with the nitro group oriented toward the hinge region, and
consequently, the hydroxyl groups make contacts with Lys-68. The
structures of MNA and MNX in the active site are fully superimposable
(Fig. 3). Emodin and TBB bind to the protein mainly through hydrophobic and van der Waals interactions. In
the case of MNX and MNA, additional polar interactions between the
inhibitors and the active site of the enzyme contribute both to
increase the affinity as indicated by the lower Ki values and to orient the molecules in a different way.
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In MNA, the nitro group is not co-planar with the aromatic
rings because of the steric hindrance of the adjacent carbonyl (ortho effect). In fact, in our structure, the nitro
group is roughly perpendicular to the ring plane. The two hydroxyl
groups and the carbonyl of the same side are involved in four hydrogen bonds with the side chains of Asp-175 and Lys-68 and a water molecule (Fig. 4). The latter is also bound to
Glu-81 and Trp-176 as in the other CK2 complexes presented here. The
nitro group is preferentially oriented toward the opening of the
binding pocket (0.67 occupancy), but it is also present in the depth of
the cavity (0.33 occupancy); therefore, MNA was refined with a double
conformation with final B-factors of 32.3 and 33.8, respectively.
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A similar network of interaction is present also in the case of MNX (Fig. 4) with a notable difference that the nitro group is found only in the inner position. In this case, the nitro group is co-planar with the aromatic plane because there is no steric hindrance by carbonyls.
From the analysis of the crystal structures of MNA and MNX complexes,
the nitro group does not seem to play a direct role in the recognition
process between the ligand and the protein. In fact, no essential
direct chemical interactions between the nitro group and the protein
binding cavity are detectable. Nevertheless, in the absence of the
nitro group as in the cases of 1,8-dihydroxy-anthraquinone and
1,8-dihydroxy-xanthen-9-one, the inhibitory constants
Ki rise to values higher than 40 µM.
These results outline the relevance of the nitro group in enhancing the
potency of anthraquinone derivative inhibitory activity as also
confirmed by the higher IC50 of the chrysophanic acid (>40
µM) compared with the nitro derivative (0.30 µM) (41). A probable explanation is that the nitro group in paraposition considerably increases the dissociation constant of the
phenolic hydroxyl group because of an electron-withdrawing effect and
thereby stabilizes the negative charge on the oxygen. The formation of
the monoanionic state can drastically modify several chemical and
biochemical properties of both compounds including solubility, chemical
reactivity, and molecular recognition. To better investigate this point
using the additive Hammett-type equation method implemented by ACD/pKa
DB software, we theoretically calculated the acid ionization constants
of both 1,8-dihydroxy-anthraquinone and 1,8-dihydroxy-xanthen-9-one and
of the corresponding 4-nitro-substituted derivatives (Fig. 1). As
experimentally demonstrated (42), the first dissociation constant of
both 1,8-dihydroxy-anthraquinone and 1,8-dihydroxy-xanthen-9-one is at
least three orders of magnitude lower than that of phenol itself
(pKa = 10.0). The presence of the nitro group
further reduces the first pKa value. In the case of
the anthraquinone derivative, the pKa1 value is 5.3, whereas for the xanthenone derivative, the value is 4.8 because of the resonance effect that is not possible in anthraquinones.
At physiological pH, both nitro derivatives should be present, at least
partially, in their monoanionic forms. To better understand the role of
the monoanionic species in the CK2 recognition process, a molecular
docking study has been performed starting from our CK2 crystallographic
coordinates (see "Experimental Procedures"). In the case of MNX-
and MNA-monoanionic derivatives, the energetically most stable and
statistically most representative docked conformation is extremely
close to the MNX crystallographic structures with the nitro group
located only in the inner position and with the negatively charged
oxygen at position 1 near to the positive side chain of Lys-68 (see
Fig. 5). This result can support the
hypothesis that if the monoanionic form is present, it can bind CK2
with high efficiency. In this case, the strong electrostatic interaction between Lys-68 and the monoanionic form seems to play a
crucial role in the recognition process. However, considering the
neutral form of both MNX and MNA compounds, the modeling procedure sampled two different families of docked conformations: the first one
as observed for the monoanionic form with the nitro group located in
the inner position and the protonated phenolic oxygen at position 1 close to the positive side chain of Lys-68; and the second one exactly
in the opposite configuration with the nitro group located in the
external position and the hydroxyl group at position 1 close to the
negative side chain of Asp-175. The second one appears to be
thermodynamically more stable (~9 kcal mol1) because of
the formation of a strong hydrogen bond between the hydroxy group at
position 1 of the anthraquinone structure and the negatively charged
carboxyl group of Asp-175. This prediction is in good agreement with
the crystal structure of the MNA·CK2 complex (see Fig. 5). These data
indicate that the neutral and the monoanionic forms bind to the CK2
recognition cavity using a different network of stabilizing
interactions, depending on the ionization capabilities of inhibitors.
This can generate at least two different configurations inside the
binding pocket. Considering the increase of the first
pKa of MNA due to the ortho
effect, we can speculate that MNA is present in solution both as
a neutral and monoanionic form and that both can fit the CK2 binding
cavity whose local pH can be different from that of the external
solvent. In addition, the equilibrium between the two conformations of
MNA inside the cavity can also be affected by steric contributions
because of non-favorable contacts between the nitro group in the inner
location and Ile-66 and Val-116.
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In the case of DAA where the dissociation constant of hydroxyl groups is not affected by the presence of any nitro function, the binding mode is different (Figs. 3 and 4). DAA binds into the enzyme co-substrate pocket on the side of the hinge region, making two specific H-bonds with backbone carbonyls of Glu-114 and Val-116, which are also responsible for interactions with the adenine moiety of bound ATP (21). This specific binding mode confers to DAA the lowest Ki value (0.35 µM) among anthraquinone inhibitors tested so far.
In summary, the four anthraquinone derivatives analyzed so far display,
despite their common scaffold, three significantly different modes of
binding into the active site of CK2: (a) anchoring to the
hinge region (DAA); (b) anchoring to Lys-68 and Asp-175 albeit with different orientations (MNA and MNX); and (c)
sitting in the middle of the cavity with no strong polar interactions (emodin). These three binding modalities correlate with a gradual increase in the Ki value from (a) (0.35 µM) to (c) (1.85 µM). Based on
this information, a strategy to improve the affinity to CK2 could be to
design new molecules able to fill the entire cavity and therefore to
interact on both sides, with the hinge region on one hand and
Lys-68/Asp-175 on the other. However, a complication arises from the
fact that DAA lies on the same plane of the ATP adenosine moiety,
i.e. slightly tilted with respect of the other three
anthraquinone inhibitors (as shown in Fig. 3). This difficulty could be
overcome by replacing the hydroxyl group of the DAA scaffold with more
extended flexible chains bearing hydroxylated and/or negatively charged functions.
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ACKNOWLEDGEMENTS |
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We thank the staff of the x-ray diffraction beamline of ELETTRA (Trieste, Italy) and of beamline ID-29 of the ESRF (Grenoble, France) for technical assistance during data measurements.
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FOOTNOTES |
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* This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, the Italian Ministries of University and Research (COFIN 2000 and 2001) and of Health (Project AIDS) and CNR (T. P. Biotechnology).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.
The atomic coordinates and the structure factors (code 1M2P, 1M2Q, and 1M2R) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
** To whom correspondence may be addressed: Dept. of Organic Chemistry, via Marzolo 1, Padova 35131, Italy. Tel.: 39-049-8275265; Fax: 39-049-8275239; E-mail: roberto.battistutta@unipd.it. (R. B.) or Dept. of Biological Chemistry, viale G. Colombo 3, 35121 Padova, Italy. Tel.: 39-049-8276108; Fax: 39-049-8073310; E-mail: pinna@civ.bio.unipd.it (L. A. P.).
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209367200
2 S. Moro, S. Bosio, D. Dal Ben, E. De Moliner, R. Battistutta, G. Zanotti, F. Meggio, L. A. Pinna, and G. Zagotto, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: CK2, casein kinase-2; MNA, 1,8-dihydroxy-4-nitro-anthraquinone; MNX, 1,8-dihydroxy-4-nitro-xanthen-9-one; DAA, 1,4-diamino-5,8-dihydroxy-anthraquinone; MAPK, mitogen-activated protein kinase; PEG, polyethylene glycol; TBB, tetra-bromo-2-benzo-triazole.
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