Pyrazolo[4,3- d ]pyrimidine bioisostere of roscovitine: Evaluation of a novel selective inhibitor of cyclin-dependent kinases with antiproliferative activity

Article abstract of DOI:10.1021/jm200064p

Inhibition of cyclin-dependent kinases (CDKs) with small molecules has been suggested as a strategy for treatment of cancer, based on deregulation of CDKs commonly found in many types of human tumors. Here, a new potent CDK2 inhibitor with pyrazolo[4,3-d]

Full text of DOI:10.1021/jm200064p

ARTICLE
pubs.acs.org/jmc
Pyrazolo[4,3-d]pyrimidine Bioisostere of Roscovitine: Evaluation of a
Novel Selective Inhibitor of Cyclin-Dependent Kinases with
Antiproliferative Activity†
‡
‡,§
||
||
^
‡
ꢀ
Radek Jorda, Libor Havlíꢀcek, Iain W. McNae, Malcolm D. Walkinshaw, Jiꢀrí Voller, Antonín Sturc,
Jana Navrꢁatilovꢁa,‡ Marek Kuzma,# Martin Mistrík,¥ Jiꢀrí Bꢁartek,¥ Miroslav Strnad,‡ and Vladimír Kryꢀstof *,‡
ꢀ
‡
ꢀ
Laboratory of Growth Regulators, Faculty of Science, Palackꢁy University and Institute of Experimental Botany ASCR, Slechtitelu 11,
78371 Olomouc, Czech Republic
§Isotope Laboratory, Institute of Experimental Botany ASCR, Videnska 1083, 142 20 Prague, Czech Republic
Structural Biochemistry Group, University of Edinburgh, Michael Swann Building, King’s Buildings, Edinburgh, EH9 3JR, Scotland
^Centre of the Region Hanꢁa for Biotechnological and Agricultural Research, Faculty of Science, Department of Growth Regulators,
ꢀ
ꢀ
Palackꢁy University, Slechtitelu 11, Olomouc, CZ-783 71, Czech Republic
#Laboratory of Molecular Structure Characterization, Institute of Microbiology ASCR, Videnska 1083, 142 20 Prague, Czech Republic
ꢀ
¥
ꢀ
Laboratory of Genome Integrity and Institute of Molecular and Translational Medicine, Palackꢁy University, Slechtitelu 11,
78371 Olomouc, Czech Republic
S Supporting Information
b
ABSTRACT: Inhibition of cyclin-dependent kinases (CDKs)
with small molecules has been suggested as a strategy for
treatment of cancer, based on deregulation of CDKs commonly
found in many types of human tumors. Here, a new potent
CDK2 inhibitor with pyrazolo[4,3-d]pyrimidine scaffold has
been synthesized, characterized, and evaluated in cellular and
biochemical assays. 7-Benzylamino-5(R)-[2-(hydroxymethyl)-
propyl]amino-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine,
compound 7, was prepared as a bioisostere of the well-known
CDK inhibitor roscovitine. An X-ray crystal structure of com-
pound 7 bound to CDK2 has been determined, revealing a
binding mode similar to that of roscovitine. Protein kinase selectivity profile of compound 7 and its biological effects (cell cycle
arrest, dephosphorylation of the retinoblastoma protein, accumulation of the tumor suppressor protein p53, induction of apoptosis,
inhibition of homologous recombination) are consistent with CDK inhibition as a primary mode of action. Importantly, as the
anticancer activities of the pyrazolo[4,3-d]pyrimidine 7 exceed those of its bioisostere roscovitine, compound 7 reported here may
be preferable for cancer therapy.
’ INTRODUCTION
During the past decade many CDK inhibitors have been
developed and characterized. Some of the most efficient ones
entered clinical trials as candidate drugs against various types of
cancer5,6 and/or advanced to preclinical evaluation of potential
value in treatment of other diseases linked with CDK deregula-
tion, such as neurodegenerative and cardiac disorders, viral and
protozoan infections, glomerulonephritis or other types of
chronic inflammation.3,7ꢁ10 The purine heterocycle became
one of the first systematically investigated scaffolds of CDK
inhibitors (due to a possibility of variable substitutions mainly at
positions 2, 6, and 9), leading to the discoveries of olomoucine
and roscovitine.11ꢁ13 Roscovitine is a pan-selective CDK
A growing body of evidence has linked abnormal protein
phosphorylation patterns with pathogenesis of various human
diseases and encouraged the search for compounds capable of
specifically inhibiting protein kinases. Indeed, therapeutic success of
several kinase inhibitors that had been approved for the treatment of
particular cancer type(s) during recent years established protein
kinases as an important class of novel drug targets. Among the 518
human genes encoding protein kinases, cyclin-dependent protein
kinases (CDKs) have originally attracted attention because of their
frequent deregulation in cancer.1,2 Cyclin-dependent kinases, listing
at least 13 members in humans, are serine/threonine kinases that
participate mainly in processes of cell cycle control, transcription,
and postranscriptional modifications but also in cell differentiation
and cell death.3,4
Received: January 21, 2011
Published: March 21, 2011
r
2011 American Chemical Society
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Figure 1. Structure of roscovitine and its known bioisosteres.26,30,39,42 CAS numbers indicate Chemical Abstracts Service compound identifiers.
inhibitor with multiple effects on cell proliferation, cell cycle
progression, p53 expression, and p53-dependent transcription
and/or induction of apoptosis in cancer cells.14 Because of these
effects, (R)-roscovitine was among the first CDK inhibitors that
entered clinical trials.
evaluated in diverse biological assays in comparison with roscov-
itine as a reference molecule. Our results demonstrate that a
change in a position of a single nitrogen atom can alter CDK
inhibitory properties of this class of compounds, analogous to
some other purine bioisosteres (Figure 1).23,25,26,33,39,42
Inspired by the success of roscovitine, further exploration of
purine-derived CDK inhibitors has been mainly oriented toward
either modifications of the roscovitine molecule in its substitu-
table positions or, more recently, redistribution of nitrogen
atoms of the purine scaffold. Both these directions have led not
only to the large number of purine inhibitors derived from
roscovitine15ꢁ22 but also to libraries of compounds with alter-
native core heterocyclic skeleton structure with side chain types
of roscovitine: pyrazolo[3,4-d]pyrimidines,23,24 pyrazolo[1,5-a]-
1,3,5-triazines,25ꢁ27 imidazo[2,1-f]-1,2,4-triazines,25,26 pyrazolo-
[1,5-a]pyrimidines,28ꢁ34 imidazo[1,2-a]pyrazines,35,36 triazolo-
[1,5-a]pyrimidines,37,38 imidazo[4,5-d]pyridines39 and pyrolo-
[3,2-d]pyrimidines.40
In this study, we describe synthesis of a novel bioisostere of
roscovitine with the pyrazolo[4,3-d]pyrimidine core. The 3,7-
disubstituted pyrazolo[4,3-d]pyrimidines were previously de-
scribed as CDK inhibitors,41 and we have found that introduction
of the third substituent to position 5 led to development of a new
class of potent purine-related CDK inhibitors. The representa-
tive 3,5,7-trisubstituted pyrazolo[4,3-d]pyrimidine 7 has been
’ RESULTS AND DISCUSSION
Chemistry. We have previously described synthesis of 5,7-
di(4-methoxybenzyl)amino-3-isopropyl-1(2)H-pyrazolo[4,3-
d]pyrimidine that was prepared as an analogue of microtu-
bule-interfering drug myoseverin.43 A key assumed intermediate
of the synthetic route was 5,7-dichloro-3-isopropyl-1(2)H-
pyrazolo[4,3-d]pyrimidine. However the intermediate was not
isolated and characterized. An attempt to synthesize pyrazolo-
[4,3-d]pyrimidine analogue of roscovitine, i.e., the compound
with different 5,7-substituents in contrast to 5,7-disubstituted
myoseverin derivative, via the same synthetic approach was not
successful. Therefore, we developed a completely new synthetic
route outlined in Scheme 1. This synthetic approach is simple,
and all intermediates are easily detectable and isolatable, with the
exception of the final compound 7. The last reaction step gives
only poor yield of the desired compound even in a complex
reaction mixture, and therefore, compound 7 has to be isolated
by a column chromatography.
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Scheme 1
Table 1. Inhibition of Proliferation in a Panel of Human
Cancer Cell Lines by Compound 7
respectively) are consistent with the accepted opinion that p53
status does not play a major role in resistance to CDK
inhibitors.45ꢁ47
human tumor cell lines
In order to identify compounds with similar effects on the
NCI60 cell lines, we calculated Pearson correlation coefficients
(r) of the activity patterns (GI50 values for the individual cell
lines) of compound 7 and 16 533 other compounds tested on the
NCI60 panel (see Experimental Section for the criteria of
compound selection). Calculations were carried out on a
logꢁlog scale. The analysis identified 2H-pyrazolo[3,4-d]-
pyrimidine CGP-5738048 (r = 0.66, rank 1, p = 1.0 ꢂ 10ꢁ8),
an inhibitor of MNK1. MNK1 is a positive regulator of the
eukaryotic initiation factor 4E (eIF4E), which besides its role in
translation also regulates distribution of cyclin D1 mRNA.49
Treatment with MNK1 inhibitor leads to a decrease of the
cellular content of cyclin D1.49,50 A strong correlation was
observed for the cells with both the wild type (N = 16, r =
0.71, p = 0.0022) and mutant p53 gene (N = 43, r = 0.63, p = 7.0
ꢂ 10ꢁ6). It is tempting to speculate that the observed similarity
in the activity patterns of the two compounds might stem from an
effect on CDK activity, directly in the case of compound 7 and
indirectly in the case of MNK1 inhibitor. On the other hand,
comparison with experimentally validated (IC50 < 100 μM)
inhibitors of cyclin-dependent kinases 1 and 2 and related kinases
(CDKs 4, 5, 7, 8, 9 and/or glycogen synthase kinase-3β) included
in BindingDB (33 compounds, 37 activity patterns)51,52 shows
that GI50 pattern of compound 7 is distinctly different. This
observation suggests that other factors beyond the known shared
molecular target(s) influence the resulting biological activity.
Figure 2 shows signed coefficients of determination r2 (measure
of variability explained by a regression line) calculated separately
for the cell lines with the wild-type and mutant p53 gene. The
cumulation of the data points along the x axis indicates various
degrees of similarity in the activity of compound 7 and the
individual CDK/GSK3B inhibitors against the cell lines expres-
sing wild-type p53. On the other hand, the activity pattern of
compound 7 on the p53 mutant cell lines was distinctly different
(low signed r2). A possible explanation of this difference might be
a generally high sensitivity of certain p53 wild type cell lines to
chemical insults. Similar results were obtained when rank sum
correlation was used instead of Pearson's correlation for calcula-
tion of signed coefficient of determination (not shown). We
conclude that growth inhibitory activity of compound 7 differs
origin
type
IC50 (μM),a 7
breast
colon
MCF-7
HBL-100
BT-474
HT-29
HCT-116
RPMI-8226
U266
7.5 ( 2.3
11.3 ( 2.4
8.5 ( 0.5
6.6 ( 1.9
11.0 ( 1.8
3.6 ( 0.3
4.9 ( 0.1
7.7 ( 0.8
3.8 ( 0.8
7.1 ( 0.5
7.5 ( 1.9
4.8 ( 1.6
6.9 ( 1.7
7.1 ( 2.1
7.7 ( 0.3
multiple myeloma
leukemia
K-562
CEM
HL-60
HOS
osteosarcoma
melanoma
cervix
G-361
HeLa
lung
A-549
epidermis
A-431
a Average ( SD values from three determinations.
Anticancer Cytotoxicity. For a comparison of pyrazolo[4,3-d]-
pyrimidines with purines, the antiproliferative activity of com-
pound 7 was tested on a panel of human cell lines representing a
range of tumor types (Table 1). Determination of proliferation
clearly showed stronger activity of compound 7 over roscovitine.
This result was further confirmed through testing on the NCI60
panel (Figure S2 in Supporting Information),44 where com-
pound 7 showed a higher activity than both racemic R,S-
roscovitine and R-roscovitine in all three assay end points
(GI50, TGI, LC50) (Table 2). All the observed differences in
the activity are statistically highly significant (p < 10ꢁ7, one-sided
Wilcoxon paired test). In contrast to roscovitine, compound 7
showed not only cytostatic (GI50, TGI) but also significant
cytotoxic (LC50) effect against the majority (74.6%) of the
NCI60 cell lines in the concentration range applied. The
observed comparable median activities of compound 7 toward
the cell lines with either the wild-type or mutant p53 (7.5 vs 10.4,
23.2 vs 26.4, and 55.8 vs 66.5 μM for GI50, TGI, and LC50,
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Table 2. Cytostatic and Cytotoxic Effects of Compound 7 and R,S- and R-Roscovitine against NCI60 Panela
GI50 (μM)
range
TGI (μM)
range
LC50 (μM)
range
median
median
median
7
10.2
17.9
19.3
3.0ꢁ23.8 (0/59)
4.6ꢁ57.8 (0/59)
4.9 to >100 (1/52)
25.9
58.1
>100
10.6ꢁ60.8 (0/59)
20.9 to >100 (14/59)
14 to >100 (36/53)
65.3
39.1 to >100 (15/59)
51.9 to >100 (39/59)
82.0 to >100 (50/53)
R,S-roscovitine
R-roscovitine
>100
>100
a Given are median and range of GI50, TGI, and LC50 together with proportion of the cell lines for which the end point was not reached at the highest
concentration tested (100 μM) (data in parentheses).
Table 3. Kinase Selectivity Profile for Compound 7 and Its
Purine Analogue Roscovitine
kinase inhibition (%)a
IC50 (μM)b
protein kinase
7
roscovitine
7
roscovitine
CDK1/cyclin B
CDK2/cyclin A
CDK2/cyclin E
CDK5/p35
CDK7/cyclin H/MAT1
CDK9/cyclin T1
CK1
84
97
98
95
96
90
7
70
94
95
85
87
87
1
nd
nd
nd
nd
0.04
0.20
0.16
1.00
nd
0.22
0.94
0.48
1.77
nd
CK2
19
32
54
58
30
39
10
13
25
38
28
29
nd
nd
GSK3A
nd
nd
GSK3B
nd
nd
MEK1
nd
nd
PRAK
nd
nd
MSK1
nd
nd
a In the presence of 100 μM ATP with 10 μM compound. b nd: not
Figure 2. Effect of p53 status on relation (expressed as signed coeffi-
cient of determination r2) between GI50 patterns of compound 7 and
individual inhibitors of CDKs and/or GSK3B. Black circle designates
MNK-1 inhibitor.48 Black triangle designates R-roscovitine, and black
square designates racemic roscovitine.
determined.
The electron density for the inhibitor is excellent with all its
atoms being well-defined in density and allowing the unambig-
uous positioning of the inhibitor in the binding cleft (Figure 3A).
The binding mode of compound 7 in the active site of CDK2 is near
identical in the positioning of the pyrazolo[4,3-d]pyrimidine core
when compared with other homologous ligand structures (Protein
Data Bank codes 2A0C, 1G5S, 1W0X, 1CKP, 2A4L, 3DDP, 3NS9)
with the pyrazolo[4,3-d]pyrimidine being sandwiched between the
side chains of Leu134 and Ile10. Similar to other homologous ligand
structures, compound 7 forms a conserved hydrogen bond pattern
to the backbone carbonyl of Leu83 and to the backbone NH of this
same residue. An additional conserved hydrogen bond is found at
the backbone carbonyl of Glu81, yet this interaction is considerably
shorter when compared to other similar ligand structures (2.77 Å
compared to 3.18 Å for the roscovitine complex, PDB code 2A4L)
(Figure 3A). Some variation between homologous structures is
found in the positioning of the phenyl ring of the benzylamino
group. In general this group is sandwiched between the side chain of
Ile10 and the backbone of His83. This is also the case with
compound 7, although the interaction of the His84 backbone
carbonyl at the ortho position of the ring is longer in this structure
(Figure 3A). A further difference in the binding mode is apparent
when comparing the position of the hydroxymethylpropyl group.
This group takes up a similar orientation to that found in the
olomoucine II structure (PDB code 2A0C) and is rotated in the
opposite direction to that found in the structure containing
from those of other structurally diverse CDK inhibitors, includ-
ing its isomer roscovitine, and this difference is typically more
pronounced when only the cell lines harboring mutant p53 are
considered.
Kinase Selectivity of Compound 7. The kinase selectivity of
compound 7 was determined in enzyme assays with recombinant
CDK2/cyclin E, CDK5/p35, CDK7/cyclin H/MAT1, and
CDK9/cyclin T1. The IC50 values for compound 7 were
comparable to that of roscovitine (Table 3), but isomer 7 showed
generally better efficiency in CDK inhibition with IC50 values in
the submicromolar range (Figure S1). In the following experi-
ment the selectivity of compound 7 was tested against a panel of
purified recombinant human protein kinases at a single concen-
tration of 10 μM. The assays confirmed that compound 7 inhibits
preferentially CDKs (Table 3). In addition to potent inhibition
of CDKs, moderate inhibition was observed for GSK3B, stress-
activated protein kinases PRAK and MSK1, and mitogen-acti-
vated kinase MEK, and these activities may contribute to the
observed antiproliferative effects of compound 7.
Crystal Structure of Compound 7 with CDK2. The crystal
structure of compound 7 in complex with CDK2 has been
determined at 1.96 Å resolution. Compound 7 binds in the
narrow cleft between the N- and C-terminal domains of CDK2.
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Figure 3. (A) Refined electron density for ligand compound 7 within the active site of CDK2. 2|Fo| ꢁ |Fc| density is colored cyan and is contoured at
1σ shown around the ligand and an interacting water molecule. Significant proteinꢁligand interactions are indicated by dashed red lines. (B) Overlay of
compound 7 and roscovitine within the active site of CDK2.
Cellular Effects of Compound 7. When exponentially grow-
ing human MCF-7 breast cancer cells were treated with com-
pound 7 for 24 h, a dose-dependent inhibition of retinoblastoma
protein (Rb) phosphorylation at Ser249/Thr252, Ser807/811,
Ser780, and Ser795 became apparent on immunoblots of total
cellular proteins probed with phosphospecific antibodies.
These results demonstrate the ability of compound 7 to affect
the activities of CDK4 and CDK2 in proliferating cells. These
CDKs play critical positive roles at the G1/S transition by
phosphorylating the Rb protein. Inhibition of cellular CDK
activity and consequent Rb dephosphorylation causes cell
cycle arrest in the G1 phase. Similar observations have been
published not only with roscovitine53ꢁ56 but also with roscov-
itine isomers imidazo[2,1-f]-1,2,4-triazine 1325 and pyrazolo-
[1,5-a]-1,3,5-triazine 7a.25
Moreover, changes of protein abundance for some cell cycle
regulators upstream of the Rb protein were monitored in MCF-7
cells treated with compound 7, compared with mock-treated
controls. A significant reduction in CDK4 protein level was
observed, and the abundance of cyclin D1, a positive regulator of
CDK4, also diminished. In contrast, no changes in protein levels
of CDK1, CDK6, and cyclins E and A were seen (Figure 4). This
inhibitory pattern, consistent with the cell-cycle effects of com-
Figure 4. Immunoblot analysis of Rb phosphorylation and some cell
pound 7, was observed in MCF-7 cells also upon treatment with
roscovitine46 and pyrazolo[1,5-a]pyrimidine BS-181.28 On the
cycle regulators. Asynchronous MCF-7 cells were exposed for 24 h to the
indicated concentrations of compound 7. Tubulin levels are included as
other hand, human HT-29 colon cancer cells responded to
a control for equal protein loading.
roscovitine treatment by a decrease in cyclin A protein level,
while the abundance of CDK4 remained unchanged.54
roscovitine (PDB code 2A4L, Figure 3B). The hydroxyl atom of this
group makes a strong hydrogen bond to a water molecule (water
71), which in turn forms strong interactions to the side chain of
Asp145 (Figure 3). This bridging interaction is not observed in any
of the homologous ligand/CDK2 complex structures. Therefore,
although ligand compound 7 is reminiscent of other roscovitine-like
ligands, these are significant differences in their respective binding
modes that make compound 7 distinct.
Cell Cycle Analysis. The antiproliferative activity of com-
pound 7 was verified by flow cytometry analysis of asynchro-
nously growing MCF-7 cells and the multiple myeloma RPMI-
8226 cell line, through double staining with propidium iodide
and 5-bromo-20-deoxyuridine (BrdU). As shown in Figure 5,
treatment with both compound 7 and roscovitine arrested the
cell cycle progression in the G2/M phase and resulted in
decreased S-phase populations in the two cell lines. Upon
treatment with higher concentrations of roscovitine and
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Figure 5. Compound 7 arrests cells at various stages of the cell cycle: flow cytometric analysis of BrdU and propidium iodide incorporation in RPMI-
8226 and MCF-7 cells treated for 24 h with roscovitine and compound 7, respectively.
compound 7 the populations of cells actively replicating DNA
(i.e., BrdU-positive cells) also decreased markedly. In addition,
accumulation of RPMI-8226 cells in G1 was found, an outcome
not observed in MCF-7 cells. In summary, compound 7 arrested
the cell cycle of two human cancer cell lines more efficiently than
roscovitine, but both compounds displayed similar patterns of
the cell-cycle blockade.
Besides cell cycle changes, increases of subG1 population
(indicative of apoptosis) upon treatment with the two com-
pounds were also observed (Figure 5). Notably, compound 7
triggered apoptosis already after 24 h of treatment at compound
concentrations above 10 μM in MCF-7, U266, and RPMI-8226
cells (Figure S3). These results correlate well with data on
induction of apoptosis obtained by other apoptotic assays (see
below and Figures 6 and S4).
Induction of Apoptosis. Most CDK inhibitors, including
roscovitine, semisynthetic flavone flavopiridol,57ꢁ59 and 3-sub-
stituted indolinone compound SU9516,60,61 exert a strong
proapoptotic effect on multiple myeloma cells through down-
regulation of Mcl-1 protein.53,62ꢁ64 Therefore, we studied in-
duction of apoptosis in multiple myeloma cell lines in more
detail. Compound 7 induced apoptosis in the multiple myeloma
cell line RPMI-8226, as documented by detection of a cleaved
fragment of caspase-3 and its enhanced enzymatic activity, by
fragmentation of poly(ADP ribose)polymerase 1 (PARP) and by
down-regulation of antiapoptotic protein Mcl-1 (Figure 6C). As
shown in Figure 6A, treatment with 20 μM compound 7 induced
strong activation of caspases 3 and 7 as quantified by a biochem-
ical assay. This result correlates well not only with the immuno-
blotting analysis, where the cleavage of caspase-3 zymogene was
observed under the same experimental conditions, but also with
the flow cytometric detection of the caspase-3 fragment using
the anticleaved caspase-3 (Asp175) antibody (Figure 6B,C).
Monitoring of the cleavage of PARP, a nuclear target of
caspase-3, further confirmed the above results. Taken together,
our results clearly showed that compound 7 induces apoptosis
in the RPMI-8226 multiple myeloma cell line in a concentra-
tion-dependent manner. Furthermore, consistent results were
found also for another multiple myeloma cell line, U266
(Figure S5).
Induction of p53-Dependent Transcription. Treatment of
cells harboring wild-type p53 with CDK inhibitors leads to
accumulation of p53 and to an increase of p53-dependent
transcription, as shown with roscovitine.65,66 A strong nuclear
immunofluorescence signal of p53 was also evident in MCF-7
cells following treatment with compound 7 (Figure 7A). These
results were then confirmed and extended by immunoblotting
analyses of the levels of p53 and its targets, p21WAF1 and Mdm-2
(Figure 7C). We found that after 24 h incubations of proliferating
MCF-7 cells with 20 μM roscovitine and 10 μM compound 7 the
level of p53 was strongly increased. Moreover, the accumulated
p53 was transcriptionally active, as indicated by the enhanced
expression of the cell cycle inhibitory protein p21WAF1, a well-
established transcriptional target of p53.
Next, the effect of compound 7 on p53-dependent transcrip-
tional activity was determined by the β-galactosidase activity
measurement in the human melanoma cell line ARN8.66 Com-
pound 7 showed a dose-dependent activity effect on p53-
regulated transcription, with the maximum impact observed
between 10 and 20 μM (Figure 7B), while the maximal effect
of roscovitine was observed at an approximately 1.5-fold higher
concentration (data not shown).
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Figure 6. Compound 7 induces apoptosis in RPMI-8226 cell line after continuous 24 h treatment. (A) The activities of caspases 3 and 7 measured using
a fluorogenic substrate Ac-DEVD-AMC in lysates of cells treated with increasing doses of compound 7. (B) Active fragment of caspase-3 (gray line)
detected by flow cytometry using specific anticleaved caspase-3 (Asp175) antibody in cells treated with 20 μM compound 7. Black and gray lines indicate
untreated and treated cells, respectively. (C) Fragmentation of PARP and caspase-3 and down-regulation of Mcl-1 detected by immunoblot analysis.
Tubulin levels were monitored to verify equal protein loading.
Compound 7 Inhibits DNA Repair via Homologous Re-
combination Independently of RAD51 Protein Abundance.
Homologous recombination (HR) is important for DNA double
strand break (DSB) repair, and its proper function is required for
the maintenance of genomic stability and cell survival.67 HR
repair seems to be restricted only to S and G2 phases of the cell
cycle, where homologous sequences are available, consistent with
regulation of HR activity via CDK dependent mechanism(s).
Indeed, in yeast, Sae2 protein phosphorylation by CDK was
identified as an important HR regulator68 and a similar CDK-
dependent mechanism was proposed also for mammalian cells.
However, in mammalian cells the identification of CDK(s)
responsible for regulation of HR is rather problematic because
studies with roscovitine or flavopiridol also showed rapid down-
regulation of a core HR-pathway protein Rad51,69,70 the effect
which may mask any other potential regulatory impact of CDK
inhibition on HR. Moreover, Rad51 down-regulation seems
unlikely to represent a physiological mode of HR regulation
because Rad51 abundance is not markedly altered throughout
G1, S, and G2 phases of the cell cycle.71 Thus, we examined
whether compound 7 could be used as an alternative CDK
inhibitor, possibly capable of modulating the HR process without
affecting the Rad51 status. We selected the highest concentra-
tions of roscovitine, flavopiridol, and compound 7 which do not
yet affect the cell cycle progression (Figure S6) and assessed HR
using an assay in which HR efficiency to repair DSB within a
reporter plasmid in human cells is quantified through measure-
ment of the repair-generated GFP fluorescence signal by flow
cytometry.72 In parallel we monitored the RAD51 protein level
by immunoblotting. In cells treated with compound 7 and
flavopiridol, the HR efficiency was reduced to 66% and 62% of
control values, respectively (Figure 8A). Notably, at the drug
concentrations used for the HR assay only flavopiridol caused a
significant RAD51 decrease (Figure 8B). Treatment with ros-
covitine at a concentration not affecting the cell cycle did not
influence HR significantly nor did it affect the RAD51 level
(Figure 8). On the basis of these results, obtained particularly
because of compound 7, we conclude that CDK inhibition can
affect HR efficiency independently of effects on RAD51 protein
abundance.
’ CONCLUSION
Compound 7 was prepared and characterized as a representa-
tive of a new group of CDK inhibitors, trisubstituted pyrazolo-
[4,3-d]pyrimidines. In several biochemical and biological assays,
the effects of compound 7 were compared with those of its
bioisostere, roscovitine. These analyses showed similar kinase
selectivity profiles of roscovitine and compound 7, yet apparently
higher efficiency of the latter compound. The overall molecular
and cellular effects of compound 7 were consistent with its ability
to inhibit CDKs and, furthermore, revealed evidence for a role of
CDKs in regulation of DNA repair by HR. The data suggest that
blocking HR-mediated repair by compound 7 and perhaps also
by other CDK inhibitors could potentially be exploited in cancer
therapy in at least three scenarios: (i) to sensitize cancer cells to
therapeutically used clastogens including ionizing radiation; (ii)
as single agents to affect cancer cells preferentially, because of the
tumor-specific oncogene-evoked replication stress and the ensu-
ing constitutive DNA damage73ꢁ75 whose repair requires HR;
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Figure 7. Induction of p53 in cells by compound 7. (A) Double labeling of asynchronous MCF-7 cells, treated with 10 μM compound 7 for 24 h, with
anti-p53 antibody and DAPI. (B) Dose-dependent effect of 7 on p53-dependent transcription in ARN8 cells stably transfected with a p53-responsive
β-galactosidase reporter construct and treated with 7 for 24 h. Fluorescence of cleaved product of 4-methylumbelliferon-β-D-galactopyranoside (MUG)
was determined in lysed cells. Results represent the average ( SD for three independent experiments. (C) Immunoblotting analysis of p53 and its
targets, p21WAF1 and Mdm-2, in MCF-7 or ARN8 cells treated for 24 h with the indicated concentration of compound 7 and roscovitine, respectively.
Tubulin is included as controls for equal protein loading.
warrants further evaluation of these purine-derived bioisosteres
as potential new candidate anticancer drugs.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were deter-
mined on a Kofler block and are uncorrected. NMR spectra were
measured on a Bruker AVANCE III 400 MHz spectrometer (400.13
MHz for 1H and 100.61 MHz for 13C) and a Bruker AVANCE III 600
MHz spectrometer (600.23 MHz for 1H and 150.93 MHz for 13C) and a
Varian Geminy 300 (300.1 MHz for 1H and 75 MHz for 13C) in DMSO-
d6 or CDCl3 at 303 K. The residual solvent signal was used as an internal
standard (δH 2.500 and δC 39.60 for DMSO-d6 or δH 7.265 and δC
1
77.00 for CDCl3). H NMR, 13C NMR, COSY, HSQC, and HMBC
results were obtained using standard manufacturers’ software (Topspin
2.1, Bruker Biospin GmbH, Rheinstetten Germany). Chemical shifts are
given in δ scale [ppm] and coupling constants in Hz. Digital resolution
enabled us to report chemical shifts of protons to 3 and coupling constants
Figure 8. Compound 7 reduces homologous recombination. DR-
U2OS-GFP cells expressing I-SceI nuclease to produce DSBs within
the HR-reporter sequences were used to measure the GFP product as a
to 1 and carbon chemical shifts to 2 decimal places. ESI or APCI mass
spectra were determined using a Waters Micromass ZMD mass spectro-
meter (direct inlet, coin voltage 20 V). IR spectra were recorded on an FT-
IR Nicolet 200 instrument with KBr tablet. Compound purity was
determined by elemental analyses (0.4%) or LCꢁMS analysis and was
confirmed to be >95% for all compounds. Merck silica gel Kieselgel 60
(230ꢁ400 mesh) was used for column chromatography.
readout for effects of CDK inhibitors on HR efficiency. (A) GFP level
and its fluorescence intensity in DR-U2OS-GFP cells reflect the degree
of successful HR-mediated recombination that was subject to modula-
tion by roscovitine (10 μM), compound 7 (5 μM), or flavopiridol (0.1
μM) for 56 h. (B) Protein levels of RAD51 were analyzed by immuno-
blotting in DR-U2OS-GFP cells exposed to increasing concentrations of
the drugs, as indicated. Frames highlight scenarios with drug concentra-
tions used in the HR assay.
Prepared Compounds. 4-Amino-5-isopropyl-1(2)H-pyrazole-
3-carboxamide (1). 1was prepared according to the published synthesis.43
3-Isopropyl-5-sulfanyl-1(2)H-pyrazolo[4,3-d]pyrimidin-7-ol (2).
A mixture of amide 1 (1.5 g, 9 mmol) and thiourea (3.8 g, 46 mmol) was
fused (195 ꢀC) for 30 min under argon atmosphere. After cooling, the
reaction melt was suspended in water (20 mL) and the solution was
(iii) to selectively kill cancer cells that often harbor defects in
DNA damage response pathways, taking advantage of the
synthetic lethality principle.67 Altogether, our present character-
ization of this novel trisubstituted pyrazolo[4,3-d]pyrimidine
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alkalized at 0 ꢀC by 2 M NaOH solution to pH 12.3. The resulting dark
solution was decolorized by carboraffin. The filtered solution was
acidified by HCl to pH 5.7ꢁ6.0. After 1 h the product was filtered off,
washed with cooled water, and dried at 80 ꢀC/20 Torr. Yield 82%, mp
303.1 K, CDCl3) δ 1.340 (d, J = 7.0 Hz, 6H, (CH3)2), 2.541 (s, 3H,
CH3S), 3.300 (sept, J = 7.0 Hz, 1H, CH), 4.762 (br s, 2H, NHCH2),
6.658 (br s, 1H, NHCH2), 7.213ꢁ7.277 (5H, m, H-ortho, H-meta,
H-para), 11.615 (br s, H-7 or H-8). 13C NMR (100 MHz, 303.1 K,
CDCl3) δ 14.42 (q, CH3S), 21.51 (q, (CH3)2), 26.01 (d, CH), 44.65 (t,
NHCH2), 127.53 (d, C-para), 127.79 (d, C-ortho), 128.62 (d, C-meta),
137.96 (s, C-ipso), 138.77 (s), 151.24 (s, C-9), 163.34 (s, C-2). Anal.
(C16H19N5S) C, H, N.
1
>305 ꢀC (dec). MS ESIꢁ: [M ꢁ H]ꢁ = 209.3. H NMR (400 MHz,
303.1 K, DMSO-d6) δ 1.225 (d, J = 6.9 Hz, 6H, (CH3)2), 3.370 (sept, J =
6.9 Hz, 1H, CH), 12.116 (br s, 1H), 12.651 (br s, 1H), 13.842 (br s, 1H).
13C NMR (100 MHz, 303.1 K, DMSO-d6) δ 21.91 (q, (CH3)2), 24.08
(d, CH), 124.04 (s), 126.91 (s), 134.08 (s, C-9), 143.13 (s, C-9), 152.57
(s), 155.34 (s). Anal. (C8H10N4OS) C, H, N, S.
7-Benzylamino-3-isopropyl-5-methylsulfonyl-1(2)H-pyrazolo[4,3-
d]pyrimidin (6). Thioether 5 (liberated base, 8 g, 26 mmol) was
dissolved in 40 mL of ethanol, and solution of 20 g of Oxone in
50 mL of H2O was added at 45ꢁ50 ꢀC. Oxygenation was checked by
TLC (silica gel, MeOH/toluene, 1/9) and was completed in 30 min.
Water (130 mL) and ethyl acetate (50 mL) were added. Product was
extracted in the organic phase once more with 20 mL of ethyl acetate.
The combined organic phase was purified by carboraffin and dried with
magnesium sulfate and evaporated. Product was crystallized from
methanol. Yield 7.5 g, 83%, mp 96 ꢀC. MS ESIꢁ: [M ꢁ H]ꢁ = 344.3
(100%, M ꢁ Hþ). MS ESIþ: 346.3 (100%, M þ Hþ). IR (cmꢁ1): 1626,
3-Isopropyl-5-methylsulfanyl-1(2)H-pyrazolo[4,3-d]pyrimidin-7-ol
(3). Thiol 2 (16 g, 70 mmol) was added to a mixture of 50 mL of EtOH
and 170 mL of water at 35 ꢀC. The pH of the solution was adjusted to 9.3
by adding a water solution of 30% NaOH, and thus, thiol 2 was dissolved.
During vigorous agitation dimethylsulfate (6.6 mL, 70 mmol) was added
at 20 ꢀC to the reaction mixture. The crystallized product was filtered off,
washed (2ꢂ) with a mixture of EtOH/H2O (1/2.5, 20 mL), and water.
The product was dried at 80 ꢀC/20 Torr. Yield 90%, mp 213ꢁ216 ꢀC.
MS ESIꢁ: [M ꢁ H]ꢁ = 223.3. 1H NMR (600 MHz, 303.1 K, DMSO-d6)
δ 1.349 (d, J = 7.0 Hz, 6H, (CH3)2), 2.531 (s, 3H, CH3S), 3.216 (sept,
J = 7.0 Hz, 1H, CH), 12.398 (s, 1H, NH or OH), 13.513 (s, 1H, NH or
OH). 13C NMR (150 MHz, 303.1 K, DMSO-d6) δ 12,94 (q, CH3S),
21.69 (q, (CH3)2), 26.32 (d, CH), 125.08 (s), 136.91 (s, C-4), 149.48 (s,
C-9), 151.94 (s, C-2), 153.51 (s). Tautomer: 1H NMR (600 MHz, 303.1
K, DMSO-d6) δ 1.369 (d, J = 7.0 Hz, 6H, (CH3)2), 2.50* (s, 3H, CH3S),
3.285 (sept, J = 7.0 Hz, 1H, CH), 11.959 (s, 1H, NH or OH), 13.770 (s,
1H, NH or OH). 13C NMR (150 MHz, 303.1 K, DMSO-d6) δ 12,86 (q,
CH3S), 21.69 (q, (CH3)2), 24.74 (d, CH), 133.60 (s), 134.69 (s),
140.34 (s, C-9), 151.21 (s, C-2), 157.35 (s). HSQC readout was done.
7-Chloro-3-isopropyl-5-methylsulfanyl-1(2)H-pyrazolo[4,3-d]pyri-
midine (4). Dimethylaniline (30 mL) was dropped under nitrogen
atmosphere into a mixture of thioether 3 (18 g, 80 mmol) and POCl3
(120 mL) during 30 min. Then the reaction mixture was refluxed for 5 h
(bath temperature of 108 ꢀC). After cooling to room temperature, the
reaction mixture was concentrated under vacuum (the excess of POCl3
was removed). Methyl tert-butyl ether (MTBE) (140 mL) was added,
and during agitation water (50 mL) was added. The organic phase was
separated off, and the water phase was extracted once more by MTBE.
Combined organic phases were diluted by 70 mL of water and alkalized
by solid NaHCO3 (2 g) to pH 7.5. The mixture was then agitated for 2 h.
The organic phase was separated by carboraffin trituration and dried by
MgSO4. Crystallization from MTBEꢁheptane afforded (after drying at
70 ꢀC/20 Torr) 16 g of a yellow product. Yield 80%, mp >170 ꢀC (dec).
MS ESIꢁ: [M ꢁ H]ꢁ = 241.3 (100%), 243 (30%). 1H NMR (400 MHz,
303.1 K, CDCl3) δ 1.500 (d, J = 7.0 Hz, 6H, (CH3)2), 2.644 (s, 3H,
CH3S), 3.488 (sept, J = 7.0 Hz, 1H, CH). 13C NMR (100 MHz, 303.1 K,
CDCl3) δ 14.59 (q, CH3S), 21.34 (q, (CH3)2), 27.12 (d, CH), 128.77
(s, C-6), 144.21 (s, C-5), 144.51 (s, C-4), 151.96 (s, C-9), 163.60 (s,
C-2). Anal. (C9H11ClN4S) C, H, N, Cl.
1
1534, 1453, 1359, 1297, 1128 (SO2), 1060, 926, 753. H NMR (400
MHz, 303.1 K, CDCl3) δ 1.325 (d, J = 5.0 Hz, 6H, (CH3)2), 3.222 (s,
3H, CH3SO2), 3.349 (br s, 1H, CH), 4.761 (br s, 2H, NHCH2),
7.259ꢁ7.185 (3H, m, H-meta, H-para), 7.310 (2H, m, H-ortho). 13C
NMR (100 MHz, 303.1 K, CDCl3) δ 21.56 (q, (CH3)2), 26.14 (d, CH),
39.13 (q, CH3SO2), 45.17 (t, NHCH2), 124.79 (s), 127.64 (d, C-para),
128.01 (d, C-ortho), 128.64 (d, C-meta), 137.27 (s, C-ipso), 150.68 (s),
151.39 (s), 157.79 (s, C-2). Anal. (C16H19N5O2S) C, H, N.
7-Benzylamino-5(R)-[2-(hydroxymethyl)propyl]amino-3-isopro-
pyl-1(2)H-pyrazolo[4,3-d]pyrimidine (7). Methylsulfone 6 (0.2 g, 0.58
mmol) and R-(ꢁ)-2-amino-1-butanol (2 mL, 23 mmol) were heated in
sealed ampule for 3 h to 160 ꢀC. Excess of the amine was evaporated at a
temperature below 70 ꢀC, and the residue was partitioned in CHCl3/
H2O. The combined organic phases were dried with magnesium sulfate
and evaporated. Product was purified by column flash chromatography
on silica gel stepwise with 1%, 2%, 4%, and 6% MeOH in CHCl3.
Product was obtained in noncrystallizable amorphous colorless glass
form. Yield 54 mg, 25%, [R]D þ53 (c1.35, CHCl3). MS ESIþ: [M þ H] þ
1
= 355.4 (100). MS ESIꢁ: [M ꢁ H]ꢁ = 353.3 (100). H NMR (300
MHz, DMSO-d6) 0.85 (t, J = 7,5 Hz, 3H, CH3CH2), 1.32 (d, J = 7.0 Hz,
6H, (CH3)2CH), 1.39ꢁ1.68 (m, 2H, CH2CH3), 3,16 (sept, J = 7,0 Hz,
1H, CH(CH3)2), 3.37 ꢁ3.51 (m, 2H, CH2OH), 3.78 (m, 1H, CHNH),
4.68 (br s, 2H, CH2Ph), 5.74 (br s, 1H, NH), 7.26 (m, 1H, H-para), 7.34
(m, 2H, H-meta), 7.39 (m, 2H, H-ortho), 11.79 bs (1/2 H, NH), 13,28
bs (1/2 H, NH). 13C NMR (75 MHz, DMSO-d6) δ 10.6, 21.5, 21.6, 23.8,
25.9, 43.0, 54.2, 63.4, 126.9, 127.5, 128.3, 139.3, 147.1, 157.6 . Anal.
(C19H26N6O) C, H, N.
Enzyme Inhibition Assay. CDK2/cyclin E kinase was produced
in Sf9 insect cells via baculoviral infection and purified on a NiNTA
column (Qiagen). CDK5/p35, CDK7cyclin H/MAT1, and CDK9/
cyclin T1 were purchased from ProQinase GmbH. The kinase reactions
were assayed with 1 mg/mL histone H1 (for CDK2 and CDK5) or
(YSPTSPS)2KK peptide (for CDK7 and CDK9) in the presence of 15/
0.15/1.5/1.5 μM ATP (for CDK2/CDK5/CDK7/CDK9), 0.05 μCi
[γ-33P]ATP, and the test compound in a final volume of 10 μL, all in a
reaction buffer (60 mM HEPESꢁNaOH, pH 7.5, 3 mM MgCl2, 3 mM
MnCl2, 3 μM sodium orthovanadate, 1.2 mM DTT, 2.5 μg/50 μL
PEG20.000). The reactions were stopped by adding 5 μL of 3% aqueous
H3PO4. Aliquots were spotted onto P-81 phosphocellulose (Whatman),
washed 3ꢂ with 0.5% aqueous H3PO4, and finally air-dried. Kinase
inhibition was quantified using digital image analyzer FLA-7000
(Fujifilm). The concentration of the test compounds required to
decrease the CDK by 50% was determined from dose-response curves
and designated IC50.
7-Benzylamino-3-isopropyl-5-methylsulfanyl-1(2)H-pyrazolo[4,3-
d]pyrimidine Hydrochloride (5). 7-Chloro derivative (8.5 g, 35 mmol),
benzylamine (9 mL, 85 mmol), and triethylamine (23 mL, 230 mmol) in
50 mL of 1-butanol were heated with stirring at 100 ꢀC for 3 h. The
solution was evaporated to dryness in vacuum, and the residue was
partitioned between H2O and EtOAc. The combined organic phase was
purified by carboraffin and dried with magnesium sulfate and evapo-
rated. Product was dissolved in a mixture of 30 mL of MTBE, 10 mL of
H2O, and 4 mL of isopropanol. After acidification by 5 N HCl to pH 0.5,
hydrochloride 5 crystallized. Product was filtered off, washed twice with
a mixture of MTBE/2-propanol (7/3, 10 mL), and dried at 70 ꢀC/20
Torr. Yield 11 g, 88%, mp 197ꢁ204 ꢀC (after recrystallization from
boiling EtOH, mp 205ꢁ210 ꢀC). MS ESIꢁ: [M ꢁ H]ꢁ = 312.3 (100%),
348 (50%, M þ Clꢁ). MS ESIþ: 314.3 (100%, M þ Hþ). IR (cmꢁ1):
1618,1581,1532, 1353, 1246, 1181, 1062, 924, 698. 1H NMR (400 MHz,
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Kinase Selectivity. All kinase assays were carried out by the
SelectScreen Kinase Profiling Service in the presence of 100 μM ATP
and 10 μM compound and performed according to the standard
protocol of Invitrogen.
Dr. B. Vojtꢀeꢀsek (anti-p53, clone DO-1, anti-p21WAF1, clone 118, anti-
CDK4, anti-cyclin D1, anti-CDK6, anti-cyclin A).
BrdU Incorporation and Cell Cycle Analysis. Subconfluent
MCF-7 cells were treated with compound 7 or roscovitine at different
concentrations for 24 h. The cultures were fed and pulse-labeled with
10 μM 5-bromo-20-deoxyuridine (BrdU) for 30 min at 37 ꢀC before
harvesting. The cells were trypsinized, washed by PBS containing 1%
BSA (PBS/BSA), fixed with ice-cold 70% ethanol, incubated on ice for
30 min, washed with PBS/BSA again, and resuspended in 2 M HCl for
30 min at room temperature in order to denature their DNA. Following
neutralization with 0.1 M Na2B4O7, the cells were harvested by
centrifugation and washed with PBS/BSA containing 0.5% Tween-20.
They were then stained with anti-BrdU fluorescein-labeled antibody
(1:50) for 30 min at room temperature in the dark. The cells were then
washed with PBS, incubated with propidium iodide (0.1 mg/mL) and
RNase A (0.5 mg/mL) for 1 h at room temperature in the dark and
finally analyzed by flow cytometry using a 488 nm laser (Cell Lab Quanta
SC, Beckman Coulter).
p53-Dependent Transcriptional Activity. To measure p53-
dependent transcriptional activity, β-galactosidase activity was deter-
mined in the human melanoma cell line ARN-8, stably transfected with a
p53-responsive reporter construct pRGCΔfoslacZ.66 After 24 h of
incubation with the inhibitors the cells were permeabilized with 0.3%
Triton X-100 for 15 min, and then 4-methylumbelliferon-β-D-galacto-
pyranoside was added as a substrate to a final concentration of 80 μM.
After 1 h the fluorescence was measured at 355 nm/460 nm (excitation/
emission) with a Fluoroskan Ascent microplate reader (Labsystems).
Immunofluorescence. MCF-7 cells grown on coverslips were
treated with increasing concentrations of compounds for 24 h. Slips were
then rinsed in PBS, and cells were fixed in methanol/acetone (1:1)
at ꢁ20 ꢀC for at least 1 h. The coverslips were then rehydrated in PBS for
10ꢁ20 min, rinsed with 10% fetal bovine serum, and incubated with the
mouse monoclonal anti-p53 antibody (DO-1) for 1 h at room tempera-
ture. The samples were then washed three times with PBS before being
incubated for 1 h with a secondary fluorescein isothiocyanate-conju-
gated anti-mouse IgG antibody. After incubation the coverslips were
rinsed three times in PBS and then the nuclei were stained by DAPI (MP
Biomedicals). After the final wash by water the coverslips were mounted
on microscope slides using Mowiol mounting medium (Calbiochem)
and observed using a fluorescence microscope (Olympus BX50)
coupled with a digital camera (Olympus DP71).
Caspase-3/7 Assay. The cells were harvested by centrifugation
and homogenized in an extraction buffer (10 mM KCl, 5 mM HEPES,
1 mM EDTA, 1 mM EGTA, 0.2% CHAPS, inhibitors of proteases, pH
7.4) on ice for 20 min. The homogenates were clarified by centrifugation
at 10000g for 30 min at 4 ꢀC. The proteins were quantified by the
Bradford method and diluted to equal concentrations. Lysates were then
incubated for 1 h with 100 μM Ac-DEVD-AMC as substrate in the assay
buffer (25 mM PIPES, 2 mM EGTA, 2 mM MgCl2, 5 mM DTT, pH
7.3). For negative controls, the lysates were supplemented with 100 μM
Ac-DEVD-CHO as a caspase-3/7 inhibitor. The fluorescence of the
product was measured using a Fluoroskan Ascent microplate reader
(Labsystems) at 355 nm /460 nm (excitation/emission).
Flow Cytometry Analysis of Cleaved Caspase-3. RPMI-8226
cells were collected by centrifugation, and the pellets were resuspended
in 4% formaldehyde solution for 10 min at 37 ꢀC. The cells were then
permeabilized by adding ice-cold methanol to a final concentration of
90% and incubated for 30 min on ice. Subsequently the cells were rinsed
in BSA/PBS, pelleted, resuspended in a few drops of BSA/PBS, and
incubated for 10 min at room temperature. Then anticleaved caspase-3
antibody conjugated with Alexa Fluor 488 (Beckman Coulter) was
added, and the cells were incubated for 1 h in the dark at room
temperature. Finally the cells were washed in BSA/PBS, resuspended
Crystallization and Structure Determination. Human recom-
binant CDK2 was purchased from ProQinase GmbH, and crystals were
grown following the protocol of the supplier. The compound 7/CDK2
complex was prepared by transferring a coverslip containing a drop of
native CDK2 crystals over a well solution of 35% PEG 6000 and
equilibrating at 17 ꢀC for 24 h. A single crystal of CDK2 was transferred
from this drop into a 1 μL drop of 35% PEG 6000, 100 mM Na HEPES
buffer (pH 7.5), 1 mM compound 7, and 5 mM DMSO and placed over
a well of the same solution. Crystals were left to soak for 2 days. The
crystal of about 0.1 mm in length was mounted in a 0.1ꢁ0.2 mm
cryoloop (Hampton Research) and was flash-frozen in liquid nitrogen.
The soaking solution acted as a cryoprotectant. All diffraction data were
collected at 100 K (Cryostream) using a Rigaku Micro7 rotating anode
generator and a Mar345 detector (MarResearch). Data processing was
carried out using the programs MOSFLM and SCALA.76 Initial struc-
ture solution was performed using the program PHASER77 using an
available CDK2 structure (PDB code 2A0C). The programs
REFMAC78 and PHENIX79 were used for refinement, with manual
refinement and waterfitting being performed by the program COOT.80
Crystallographic processing and refinement statistics are summarized in
Supporting Information (Table S1). Atomic coordinates have been
deposited in the Brookhaven Protein Data Bank under the accession
code 3PJ8.
Cell Maintenance and Cytotoxicity Assay. The cytotoxicity of
the studied compounds was determined with cell lines of different
histological origin. The cells, cultured in DMEM (supplemented with
10% fetal calf serum, 4 mM glutamine, 100 IU/mL penicillin, 100 μg/mL
streptomycin) in a humidified CO2 incubator at 37 ꢀC, were redis-
tributed into 96-well microtiter plates at appropriate densities for their
respective cell sizes and growth rates. After preincubation, test com-
pounds in 3-fold dilutions were added in triplicates. Treatment lasted for
72 h and then calcein AM solution was added. The fluorescence of the
live cells was measured at 485 nm/538 nm (excitation/emission) with a
Fluoroskan Ascent microplate reader (Labsystems). IC50 values, the
drug concentrations reducing number of viable cells to 50%, were
determined from the dose-response curves.
Immunoblotting. For direct immunoblotting, total cellular pro-
tein lysates were prepared by harvesting treated cells in Laemmli sample
buffer. Proteins were separated on SDS-polyacrylamide gels and elec-
troblotted onto nitrocellulose membrane. The blotted membranes were
stained with 0.2% Ponceau-S in 1% aqueous acetic acid to verify equal
protein loading, destained, and blocked in PBS and 0.1% Tween 20
(PBS-T) with 5% low fat milk or 3% bovine serum albumin (BSA). The
membranes were then incubated with specific antibodies overnight at
4 ꢀC. After being washed three times in PBS-T, the membranes were
incubated with a 1:2000 dilution of peroxidase-conjugated secondary
antibodies. After another three washes in PBS-T, peroxidase activity was
detected using ECLþ reagents (AP Biotech) according to the manu-
facturer’s instructions.
Antibodies. Specific antibodies were purchased from Cell Signaling
Technology (antitotal pRb, clone 4H1, and anti-pRb antibodies phos-
phorylated at S780, S795, and S807/811), Sigma-Aldrich (anti-pRb
phosphorylated at Ser249/Thr252, anti-R-tubulin, clone DM1A, perox-
idase-labeledsecondary antibodies), Santa Cruz Biotechnology (anti-Mcl-
1, clone S-19, anti-PARP, clone F-2, anti-Mdm-2, clone SMP14, anti-
RAD51, clone H-92, anti CDK1, clone B-6; anti-cyclin E, clone HE12),
DAKO Cytomation (anti-caspase-3), Roche Applied Science (anti-5-
bromo-20-deoxyuridine-fluorescein, clone BMC 9318), Jackson Immu-
noResearch Laboratory (fluorescein-conjugated Goat Anti-Mouse IgG),
Beckman Coulter (anti-cleaved caspase-3) or were a generous gift from
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Journal of Medicinal Chemistry
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again, and analyzed by flow cytometry using a 488 nm laser (Cell Lab
Quanta SC, Beckman Coulter).
2010, the European Commission (Projects Infla-Care and
CZ.1.05/2.1.00/01.0030), and Grant MSM6198959216 and Grant
ED0007/01/01 of Centre of the Region Hanꢁa for Biotechnological
and Agricultural Research. Cytotoxicity testing on NCI60 panel was
done at the Developmental Therapeutics Program (DTP) of the
National Cancer Institute (Bethesda, MD, U.S.).
Homologous Recombination Assay. A U2OS clone containing
a single complete copy of the integrated HR reporter hprt-DR-GFP was
obtained from Pierce et al.81 DR-U2OS-GFP cells (3.3 ꢂ 105 per well)
were seeded in 100 mm dishes and transfected with 1 μg of pCKA-I-SceI
plasmid using Fugene 6 reagent. Sixteen hours later, the medium was
replaced with fresh medium containing tested compounds (5 μM for
compound 7, 10 μM for roscovitine, 0.1 μM for flavopiridol). Cells were
harvested after 2 days for flow cytometric analysis on a Cell Lab Quanta
SC cytometer (Beckman Coulter).
NCI60 Cytotoxicity Assay. Tests of toxicity on NCI60, a set of 59
human cancer cell lines derived from nine tissue types, were performed
at the Developmental Therapeutics Program of the National Cancer
Institute (Bethesda, MD, U.S.). The cytotoxicity of compound 7 was
evaluated by measuring total cell protein using the sulforhodamine B
method according to the standard protocol at time 0 and after 48 h. The
highest concentration tested was 100 μM. GI50, TGI, and LC50
(concentration of a drug inducing 50% reduction of growth, total
growth inhibition, and 50% reduction of initial cell population, re-
spectively) were estimated from the dose-response curves.
Correlation Analysis of NCI60 Activity. The activity pattern
(GI50 values for individual NCI60 cell lines) of compound 7 was
correlated with the drug activity patterns in the DTP cancer screening
data set, May 2009 release (http://dtp.nci.nih.gov/docs/cancer/
cancer_data.html). Pearson correlation coefficients (r) were calculated
on a logꢁlog scale. Signed version of coefficient of determination is
defined as [r/abs(r)]r2. Only the activity patterns fulfilling following
criteria were analyzed: (1) GI50 values for at least 50 cell lines, (2) GI50
reached against more than 50% of the cell lines tested, and (3) GI50 for
the most sensitive cell line at least 5 times lower than GI50 for the most
resistant cell line. Experimentally validated low molecular inhibitors of
CDKs 1, 2, 4, 5, 7, 8, 9 and glycogen synthase kinase-3β with IC50 < 100
μM were extracted from BindingDB.51,52 SDfile with the data was
downloaded on November 17, 2009. Pubchem ID was used for
conversion of BindingDB and NCI60 identifiers. Data manipulation
and analysis was done in R 2.8.1.
’ ABBREVIATIONS USED
BrdU, 5-bromo-20-deoxyuridine;CDK, cyclin-dependentkinase; CK,
casein kinase; DSB, double strand breaks; SAR, structureꢁactiv-
ity relationship; GI50, growth inhibition 50%; GSK, glycogen
synthase kinase; HR, homologous recombination; LC50, reduc-
tion of initial cell population 50%; MEK1, MAPK/ERK
kinase 1; MNK-1, MAPK-interacting kinase 1; MSK, mito-
gen- and stress-activated protein kinase; MTBE, methyl tert-
butyl ether; MUG, 4-methylumbelliferon-β-D-galactopyra-
noside; NCI, National Cancer Institute; PARP, poly(ADP
ribose)polymerase 1; PCNA, proliferating cell nuclear anti-
gen; PDB, Protein Data Bank; PRAK, p38-regulated-acti-
vated protein kinase; Rb, retinoblastoma protein; TGI, total
growth inhibition
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’ ASSOCIATED CONTENT
S
Supporting Information. Crystallographic data collec-
b
tion and refinement statistics, kinase inhibition curves, NCI60
screening of compound 7, cell cycle analysis of K562, U266, and
DR-U2OS-GFP cells upon treatment with roscovitine, com-
pound 7, and flavopiridol, and the influence of compound 7 on
subG1 cell population in different cell lines. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
†PDB code 3PJ8.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ420585634854. Fax: þ420585634870. E-mail: vladimir.
krystof@upol.cz.
’ ACKNOWLEDGMENT
The authors thank J. Hudcovꢁa and D. Parobkovꢁa for their
technical assistance. Dr. B. Vojtꢀeꢀsek is acknowledged for the gift
of antibodies. This work was supported by GACR Grants 204/
08/511, 301/11/P554, and 301/02/0475, GAAV Grant IAA50-
1370902, IGAMZ Grants NS10282-3/2009 and NT/11065-5/
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