Fluorescent cyclin-dependent kinase inhibitors block the proliferation of human breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2011.02.052

Inhibitors of cyclin-dependent kinases (CDKs) are an emerging class of drugs for the treatment of cancers. CDK inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in combination with radiation therapy and chemotherapies. Drugs that target CDKs could have important inhibitory effects on cancer cell cycle progression, an extremely important mechanism in the control of cancer cell growth. Using rational drug design, we designed and synthesized fluorescent CDK inhibitors (VMY-1-101 and VMY-1-103) based on a purvalanol B scaffold. The new agents demonstrated more potent CDK inhibitory activity, enhanced induction of G2/M arrest and modest apoptosis as compared to purvalanol B. Intracellular imaging of the CDK inhibitor distribution was performed to reveal drug retention in the cytoplasm of treated breast cancer cells. In human breast cancer tissue, the compounds demonstrated increased binding as compared to the fluorophore. The new fluorescent CDK inhibitors showed undiminished activity in multidrug resistance (MDR) positive breast cancer cells, indicating that they are not a substrate for p-glycoprotein. Fluorescent CDK inhibitors offer potential as novel theranostic agents, combining therapeutic and diagnostic properties in the same molecule.

Full text of DOI:10.1016/j.bmc.2011.02.052

Bioorganic & Medicinal Chemistry 19 (2011) 2714–2725
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorescent cyclin-dependent kinase inhibitors block the proliferation of
human breast cancer cells
Venkata Mahidhar Yenugonda a,b, Tushar B. Deb b, Scott C. Grindrod a,b, Sivanesan Dakshanamurthy a,b
,
Yonghong Yang a,b, Mikell Paige a,b, Milton L. Brown a,b,
⇑
a Drug Discovery Program, Georgetown University Medical Center, 3970 Reservoir Rd, NW, Research Building, EP-07, Washington, DC 20057, USA
b Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Research Building, Washington, DC 20057, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of cyclin-dependent kinases (CDKs) are an emerging class of drugs for the treatment of cancers.
CDK inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in com-
bination with radiation therapy and chemotherapies. Drugs that target CDKs could have important inhib-
itory effects on cancer cell cycle progression, an extremely important mechanism in the control of cancer
cell growth. Using rational drug design, we designed and synthesized fluorescent CDK inhibitors (VMY-1-
101 and VMY-1-103) based on a purvalanol B scaffold. The new agents demonstrated more potent CDK
inhibitory activity, enhanced induction of G2/M arrest and modest apoptosis as compared to purvalanol
B. Intracellular imaging of the CDK inhibitor distribution was performed to reveal drug retention in the
cytoplasm of treated breast cancer cells. In human breast cancer tissue, the compounds demonstrated
increased binding as compared to the fluorophore. The new fluorescent CDK inhibitors showed undimin-
ished activity in multidrug resistance (MDR) positive breast cancer cells, indicating that they are not a
substrate for p-glycoprotein. Fluorescent CDK inhibitors offer potential as novel theranostic agents, com-
bining therapeutic and diagnostic properties in the same molecule.
Received 16 December 2010
Revised 24 February 2011
Accepted 28 February 2011
Available online 4 March 2011
Keywords:
Fluorescent
CDK inhibitors
Purvalanol B
Breast cancer
In vitro anti-cancer
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Mutations in either CDK4 or cyclin D1 are implicated in pri-
mary solid tumors including breast, lung, pancreatic, gastrointes-
Cyclin-dependent kinases (CDK) are classic Ser/Thr kinases with
molecular weights of 30–40 kDa. This family of enzymes plays an
important and well-defined role in cell cycle regulation and prolif-
eration. CDK enzymatic activation requires the binding of specific
regulatory subunits, termed cyclins, CDK-cyclin complexes are
necessary for phosphorylation of key proteins that regulate the or-
derly progression through the cell cycle.1–4 Abnormal activation of
various CDKs can ultimately lead to deregulated cell cycle progres-
sion, a common feature in many cancers.5,6 Given the pivotal role
that dysregulation of CDK activity plays in cancers, targeting the
CDKs is a viable strategy for blocking and/or interfering with tumor
cell proliferation.7–9
Thirteen CDK’s and at least 29 cyclins have been discovered
from the human genome10 and have been extensively character-
ized in regards to controlling cell cycle. Mutations in CDK proteins
can result in the overexpression and altered function of CDK’s and
specific cyclins. In human tumors, mutations have been reported
which result in the specific overexpression of cyclins D1 and E1
and CDK4 and CDK6.11,12
tinal, head and neck, liver, and prostate. CDK6 is mutated in a
smaller number of solid human cancers such as lymphomas,
sarcomas and gliomas. CDK2 is rarely mutated in cancer. Aber-
rant activation of CDK1 has also been observed in a number of
primary tumors including breast, colon, prostate, oral and
lung.10,13
Approximately 24 CDK inhibitors are currently in development
or clinical trials. These molecules can be classified in at least four
general categories including the purines, alkaloids, butyrolactones,
and flavonoids.14–20 Despite differences in chemical structures,
most CDK inhibitors competitively inhibit ATP binding at the cata-
lytic site.21,22 The specificity of the inhibitors is a major concern
due to a high degree of sequence similarity within the active site
of the CDKs and a large number of other protein kinases. In addi-
tion, the ability to monitor intracellular delivery and intratumoral
distribution remains difficult. Therefore, development of potent
CDK inhibitors that provide an imageable ‘readout’ may be advan-
tageous in the development of clinically effective therapeutic
drugs.
The 2,6,9-trisubstituted purine analogs (olomoucine and rosco-
vitine) and the purvalanols have been shown to selectively inhibit
a subset of CDK’s.15 These inhibitors act by selectively competing
with ATP at its binding site in targeted CDK’s, however purvalanol
⇑
Corresponding author. Tel.: +1 202 687 8603; fax: +1 202 687 7659.
E-mail address: mb544@georgetown.edu (M.L. Brown).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.052

V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
2715
B has been shown to target CDK2 and CDK1 complexes at submi-
2.2. Molecular modeling
cromolar concentrations.16 Importantly, the carboxylic acid of the
6-anilino group of the purvalanols (Fig. 1A) can be modified with-
out negatively affecting their CDK-inhibitory activity,23,24 provid-
ing a strategy for the development of modified purvalanol B
analogs.
To better understand the binding interactions between CDK2
and VMY-1-103, a structural model of the CDK2/VMY-1-103 com-
plex was developed (Fig. 2B). To provide consistent results, the
docked position was remodeled by step-by-step manual docking
with a restrained MD simulations followed by minimization. In
the restrained MD simulations, the optimum Van der Waals and
H-bond distance constraints were set between the ligand and the
interacting residues.
Dansyl fluorochromes have been used in tracking small mole-
cules intracellularly.25–28 By synthetically coupling the fluorescent
compound dansyl-ethylenediamine to purvalanol B, two fluores-
cent analogs of purvalanol B, termed VMY-1-103 and VMY-1-101
were developed (Fig. 1B). These compounds were evaluated for
their CDK inhibitory activity and for their in vitro effects in two hu-
man breast cancer cell lines (p53-mutated, estrogen-independent
MDA-MB-231 cells and p53 wild type, estrogen dependent MCF-
7 cells).
The model suggests that the N7 imidazole nitrogen of the pur-
ine ring and the N6 amino group make a H-bond network with the
backbone –NH and carbonyl of Leu83, respectively. The acidic C8
atom of the purine ring may form a weak H-bond with the car-
bonyl oxygen of Glu81. The C2 side chain of VMY-1-103 is bound
within the ATP ribose-binding pocket (Fig. 2A), with the isopropyl
group interacting hydrophobically with the glycine-rich loop and
Val18. The hydroxyl group of the R isopropyl group can make a
H-bond with the backbone carbonyl of Gln131. The purine N9 iso-
propyl group of VMY-1-103 shows a strong hydrophobic interac-
tion with the hydrophobic side chains of Val18, Ala31, Phe80,
Leu134, and Ala144. The chlorinated benzene ring of VMY-1-103
shows a stacking interaction with Phe82, and a strong polar inter-
action between the Cl atom and the side chain of Asp86. Networks
of H-bonds may be formed by the carbonyl and –NH group of the
amide of VMY-1-103, the carbonyl can form a H-bond with
Trp541 and the –NH can form a H-bond with Asp86. Furthermore,
the sulfonamide group of the dansyl moiety can make a H-bond
with Leu88, and the aryl component of dansyl group itself can
make a hydrophobic contact with Leu89 and Asp92.
2. Results
2.1. Chemistry
The CDK2–purvalanol B crystal structure (PDB ID: 1CKP) re-
vealed that the carboxylic acid of the 6-anilino substituent points
outside of the ATP-binding pocket, and does not interfere with
interior interactions of the kinase active site.13,27,28 Taking advan-
tage of this structural arrangement, we synthetically coupled a
fluorescent tag, dansyl ethylenediamine to the carboxylic acid of
the 6-anilino substituent of purvalanol B. The newly synthesized
fluorescent compounds VMY-1-101 and VMY-1-103 were pre-
pared following a four-step synthetic route outlined in Figure 2A.
Briefly, starting from commercially available 2-fluoro-6-chloropu-
rine (1), standard regioselective N-9 alkylation with 2-propanol
under Mitsunobu conditions gave 2-fluoro-6-chloro-9H-isopropyl-
purine. The 6-position was substituted with dansyl ethylenedia-
mine, followed by substitution at the 2-position with (R)-2-
amino-1-butanol to afford VMY-1-101, or (R)-valinol to give
VMY-1-103.
Upon overlay of purvalanol B with VMY-1-103 (Fig. 2B), we
noted that the both occupy a similar space in the binding pocket
and form similar interactions. The only difference noted was that
the –COOH group in purvalanol B, forms a H-bond with Trp541
and Leu89, whereas VMY-1-103 cannot because of the steric con-
straints imposed by the amide group substituent. The increased
affinity of VMY-1-103 over purvalanol B can be rationalized from
A
HO
O
NH2
Site for
Modification
OH
O
86
Asp
Hydrophobic
Pocket
HO
83
Cl
O
Leu
Cl
O
H2N
O
OH
OH
O
HN
131
HN
NH
Gln
H2N
HO
N
N
N
N
N
N
O
HO
HO
N
H
N
N
H
N
Ribose
Pocket
Hydrophobic
Pocket
Cl
O
B
H
N
N
N
H
S
O2
HN
OH
R=
R=
VMY-1-101
VMY-1-103
HN
N
N
N
R
N
OH
HN
Figure 1. (A) Design strategy for a new class of fluorescent CDK molecules and (B) fluorescent purvalanol B analogues.

2716
V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
Cl
COOH
Cl
N
Cl
HN
N
N
N
N
N
N
N
N
(b)
(a)
(c)
N
H
49%
100%
44%
F
F
N
F
N
1
2
3
Cl
O
H
N
N
N
H
S
O2
HN
Cl
O
H
N
N
N
N
N
VMY-1-101
N
H
S
O2
(d)
HO
N
H
N
63%
HN
N
N
Cl
O
N
H
N
S
N
N
H
F
N
O2
HN
4
N
N
VMY-1-103
(e)
N
HO
42%
N
H
N
Figure 2A. Synthetic scheme. Reagents and conditions: (a) iPrOH, DEAD, PPh3, THF, À10 °C–rt; (b) 4-amino-2-chloro-benzoic acid, DIEA, n-butanol, 80 °C; (c) Dansyl
ethylenediamine, DIEA, EDCI, HOBt, DMF–CH2Cl2–dioxane (1:1:1); (d) (R)-2-amino-1-butanol (excess), DIEA, 120 °C, 48 h; (e) (R)-2-amino-3-methyl-1-butanol, DIEA, n-
butanol, 120 °C, 72 h.
these binding interactions formed by the extended substituent at
the N6-position of the benzene ring outside of the ATP-binding
pocket.
by P-glycoprotein-dependent or P-glycoprotein-independent
mechanisms) that transports drugs out of the cells. In order to eval-
uate the MDR effect on VMY-1-103, we tested the effect of VMY-1-
103 on the proliferation in CL 10.3 (MDR-positive) cells, which over
express P-glycoprotein,29 and compare with MDR-negative MCF-7
cells. Paclitaxel, a known P-glycoprotein substrate30 was included
as a positive control. In Table 3, the IC50 of the compounds are rep-
resented along with the ratio of difference between MDR-positive
and MDR-negative cell lines. In contrast to paclitaxel, VMY-1-103
had comparable activity in both cell lines, and the activity did
not seem to be modulated by MDR.
The anti-proliferative effects of VMY-1-101 and VMY-1-103
further correlate with morphological changes associated with can-
cer cells. As shown in Figure 3, a marked decrease in the cell num-
ber and aberrant gross morphological changes was observed in
cells exposed to 10 lM of the agent for 48 h. These results further
confirm that the newly synthesized fluorescent compounds induce
3. Biological evaluation of compounds
3.1. Inhibition of Cdk isoforms activity by VMY-1-101 and VMY-
1-103
The inhibitory activities of newly synthesized compounds were
evaluated against several cell-free cyclin-dependent kinases. Inter-
estingly, VMY-1-101 and VMY-1-103 were found to be potent
competitive inhibitors of ATP as tested against various cell cycle ki-
nases compared to roscovitine. In fact VMY-1-101 and VMY-1-103
showed a greater percent inhibition of CDK2/cyclin E complex as
compared to other kinases (Table 1).
less density of cells by promoting induction of cell death.
3.2. VMY-1-101 and VMY-1-103 compounds inhibit the in vitro
growth of breast cancer cells or multidrug resistant cells
3.3. Compounds (VMY-1-101 and VMY-1-103) induce cell cycle
arrest at G2/M phase
Based on the potency in the in vitro cell-free enzyme kinase as-
say, we further evaluated the anti-proliferative effects of our ana-
logs on estrogen-independent (MDA-MB-231) and estrogen
dependent (MCF-7) human breast cancer cell by WST-1 assay.
The data (Table 2) indicate that the newly synthesized fluorescent
compounds possess potent in vitro anti-proliferative activity
To examine the relationship between growth inhibitory effect of
the newly synthesized compounds and the expression of cell cycle
proteins, we first analyzed the effects of the compounds on cell cy-
cle distribution. Asynchronous breast cancer cells were treated for
16, 24 and 40 h with DMSO alone (control), 5 lM, or 10 lM of
(IC50 = 4.86–4.06 lM or 10–19 lM for VMY-1-103 or VMY-1-101,
respectively) independent of cell type. No significant anti-prolifer-
ative effects were observed for purvalanol B or dansyl ethylenedi-
amine in either cell lines even at a concentration of 100 lM.
Advanced stages of cancer often display resistance to anticancer
drugs, this is considered one of the major obstacles to practical
chemotherapeutic interventions. One mechanism of multidrug
resistance (MDR) is the expression of a reflux pump (mediated
VMY-1-101 or VMY-1-103 or Roscovitine and purvalanol B. Fig-
ure 4A are representative cell cycle histograms for 24 h time inter-
vals. Results shows with the percentage of cells in each phase after
subtracted from untreated cells. After treatment with compounds,
a clear accumulation of cells in the G2-M phase was detected irre-
spective of origin of the cell lines. There was also a moderate in-
crease in cells containing sub-G1 amount of DNA indicating that
the present fluorescent compounds were inducing cell death. Sim-

V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
2717
Table 2
Effects of VMY-1-101 and VMY-1-103 on the survival of human breast cancer cell
lines
Cell survival (WST-1 reduction, IC50, lM, 48 h)
Compound
MDA-MB-231 (ERÀ)
MCF-7 (ER+)
VMY-1-101
VMY-1-103
R-Roscovitine
Purvalanol B
4.86 1.24
4.06 1.32
54.64 1.27
>100
19.05 1.14
10.03 1.25
55.01 1.1
>100
Dansyl ethylenediamine
>100
>100
Note: VMY-1-101 and VMY-1-103 and controls were tested at various concentra-
tions for effects on cell survival of breast cancer. Cell survival was estimated 48 h
after the addition of each compound using the WST-1 reduction assay. Results
shown are mean values of triplicate experiments. The IC50 value (the concentration
yielding 50% growth inhibition) was interpolated from the graph of the log of
compound concentration versus the fraction of surviving cells. The IC50 was cal-
culated using graph pad prism. Data are expressed as mean (SEM) of triplicate
experiments.
24 h. Further exposure to drug free media for additional 48 h. As
shown in Figure 4B, VMY-1-103 induced cell cycle arrest at G2/M
phase for 24 h and remains at G2/M phase after 48 h with drug free
medium. In contrast, the control roscovitine induces the G2/M
phase arrest at 24 h and returns to normal stage after 48 h with
drug free medium. These results further indicate that the anti-pro-
liferative effect of VMY-1-103 is irreversible.
3.5. Anti-proliferative mechanism of VMY-1-103
We evaluated purvalanol B and VMY-1-103 for their effects on
G2/M cell cycle proteins. MDA-MB-231 cells were treated with
purvalanol B and VMY-1-103 at 10 lM and a comparison of cell cy-
cle proteins expression at different time points were evaluated by
western blotting. We specifically assessed the expression levels of
principal component proteins for G2/M checkpoint, such as phos-
pho-cdc2, cdc2, and cyclin B. As shown in Figure 5, after 6 h treat-
ment, the VMY-1-103 treated cells resulted in a time-dependent
decrease in expression of phospho-cdc2 at both the catalytic
(T161) and regulatory (Y15) sites compared to control. Purvalanol
B had no affect on the phospho-cdc2 expression throughout the
time period tested. The inhibition of the expression level of phos-
pho-cdc2 in MDA-MB-231 cells further supports that VMY-1-103
decreases the cdc2 (CDK1) kinase activity, which halts cell division
at the G2/M check point. Quantitative analysis of these bands
showed a statistically significant difference between VMY-1-103
and purvalanol B in terms of phospho expression levels of cdc2.
Figure 2B. VMY-1-103 modeled in the ATP-binding pocket of CDK2 (A): H-bond
interactions are indicating by dotted lines (red) connecting the respective residues.
CDK2 is represented by ribbon model and residues interacting with VMY-1-103
shown by ball and stick model. VMY-1-103 is shown in a stick representation with
its carbon atoms colored in green. (B) Overlay of VMY-1-103 with purvalanol B
(yellow).
ilar patterns of G2/M phase arrest were observed at 16- and 40-h
time intervals (data not shown).
3.4. Reversible/irreversible effect of VMY-1-103 on breast cancer
cell proliferation
3.6. Intracellular localization of VMY-1-103
Activation of CDK1/cyclin B kinase activation is responsible for
driving the G2/M phase of the cell cycle in the cytoplasm as the
cells progress into metaphase.31 Taking advantage of the inherent
fluorescent property of VMY-1-103, we examined the intracellular
In order to examine the reversible/irreversible mechanism of
the anti-proliferative effect of the fluorescent compounds, we ex-
posed MDA-MB-231 cells to 5 lM or 10 lM of VMY-1-103 for
Table 1
Inhibitory effects of VMY-1-101 and VMY-1-103 on CDK’s activity
Protein kinase
VMY-1-101 (0.1
lM)
VMY-1-103 (0.1
lM)
R-Roscovitine (0.1
l
M)
Purvalanol B (0.1 lM)
CDK1/cyclin B
CDK2/cyclin A
CDK2/cyclin E
CDK3/cyclin E
CDK5/p25
CDK5/p35
CDK7/cyclin H/MATI
CDK9/cyclin T1
16
62
87
40
76
67
57
44
3
0
0
3
1
1
0
1
35
72
89
65
75
66
69
67
1
1
1
3
5
5
2
3
10
41
57
20
40
41
26
19
5
4
4
7
1
1
5
1
78
96
98
89
97
97
66
22
6
0
0
0
0
0
1
0
Note: CDK, cyclin-dependant kinase. The inhibitor concentrations are shown in parenthesis. Kinase inhibition represented as a percentage of control (N = 2) with SD.
Compounds were tested at 100 nM concentrations on 8 purified kinases as described in Section 5.1.

2718
V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
Table 3
Effects of VMY-1-103 and paclitaxel on the survival of multidrug resistant breast cancer cell lines
Cell survival (WST-1 reduction, IC50, lM, 72 h)
Cell line
Origin
VMY-1-103
Paclitaxel
MCF-7
CL 10.3
MDR IC50/non-MDR IC50
Breast
Breast (MDR)
4.5 1.19
44.1 1.18
9.8
0.002 1.12
>50
>25,000
Note: VMY-1-103 was tested at various concentrations for effects on cell survival of MDR cell line. Cell survival was estimated 72 h after the addition of each compound using
the WST-1 reduction assay. Results shown are mean values of triplicate experiments. The IC50 value (the concentration yielding 50% growth inhibition) was interpolated from
the graph of the log of compound concentration versus the fraction of surviving cells. The IC50 was calculated using graph pad prism. Data are expressed as mean (SEM) of
triplicate experiments.
dide staining (red) was used to visualize the nucleus (Fig. 6B), and
differential interference contrast (DIC) was used to reveal the mor-
phology of the cells. In both cell lines (MDA-MB-231 and MCF-7),
VMY-1-103 localized into the cytoplasm. Similar results were also
found at different time intervals such as 3, 6, 12, and 24 h (data not
shown). The data supports our hypothesis that VMY-1-103 binds
to the inactive form of cdc2, which resides in the cytoplasm.
3.7. Binding of VMY-1-103 to human breast cancer tissue
We evaluated the binding ability of VMY-1-103 as compared to
dansyl ethylenediamine (fluorophore alone) on human breast can-
cer tissue (Georgetown University tissue bank). Tissues were trea-
ted with
a
10
lM
solution of VMY-1-103 or dansyl
ethylenediamine and imaged by two-photon confocal microscopy.
As shown in Figure 6C, VMY-1-103 has strong binding affinity to
binding to breast tissues as compared to the fluorophore alone.
These results support binding of VMY-1-103 and not chromophore
fragment to the tissue, which is tethered on the solvent-exposed
region of the VMY-1-103.
3.8. Apoptotic signaling
In order to understand whether the fluorescent compounds
were able to induce apoptotic cell death, we evaluated the expres-
sion of anti and pro-apoptotic proteins in MDA-MB-231 cells after
treatment with a 10 lM solution of each compound for 48 h. Wes-
tern blot analysis of regulatory anti-apoptotic Bcl2 and pro-apop-
totic Bax proteins for intrinsic apoptosis are shown in Figure 7A.
These data indicate that the decreased level of anti-apoptotic pro-
teins and increased level of pro-apoptotic protein for cells treated
with VMY-1-101 and VMY-1-103 could upset the balance of pro-
and anti-apoptotic proteins towards the expression of proteins
favoring apoptosis. This was confirmed with cleaved of pro-apop-
totic 116 kDa poly (ADP-ribose) polymerase protein (PARP) into
its 89-kDa fragments. As shown in Figure 7B VMY-1-101 and
VMY-1-103 promote moderate cleavage of the 116-kDa full length
PARP into an 89-kDa fragment, a mechanism known to impair
genomic integrity before apoptosis. We also observed decreased
protein level of full length caspase 3 and 7 in cells treated with
VMY-1-101 and VMY-1-103 (data not shown). Interestingly, there
was no change in protein levels in roscovitine treated cells even at
40 lM. This further supports the hypothesis that, in contrast to
roscovitine the present fluorescent compounds induce apoptosis
mediated cell death in MDA-MB-231.
Figure 3. Morphological changes in human breast cancer cell lines after exposure
to different compounds. MDA-MB-231 (A–D) and MCF-7 (E–H) were treated with
To investigate the programmed cell death (apoptosis) in MCF-7
cells (caspase-3 negative), we performed a biparametric cytofluori-
metric analysis, using propidium iodide (PI) and annexin V-fluores-
cein isothiocyanate (FITC) which stain DNA and phosphatidylserine
(PS) residues. Because externalization of PS occurs in the first
stages of apoptosis, annexin-V staining identifies apoptosis at an
earlier stage than sub-G1 appearance. After 24 h treatment of the
compounds, MCF-7 cells were labeled with the two dyes and mon-
10 lM of purvalanol B, VMY-1-101 and VMY-1-103 for 48 h. After treatment, media
was removed and washed with PBS. The images were captured with an olympus
X71 inverted digital microscope system with a 20Â bright field.
localization of VMY-1-103 in breast cancer cells. Cells were treated
with 10
lM of VMY-1-103 for 1 h followed by fixation and visual-
ization of cells by two-photon confocal microscopy. Propidium io-

V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
2719
A
B
15
10
5
15
GOG1
S
G2M
SUBG1
GOG1
S
G2M
SUBG1
10
5
0
-5
0
-10
-15
-20
-5
MDA-MB-231, 10 μM
MDA-MB-231, 5 μM
-10
C
D
15
15
10
5
GOG1
S
G2M
SUBG1
GOG1
S
G2M
SUBG1
10
5
0
0
-5
-5
-10
-15
MCF-7, 5 μM
MCF-7, 10 μM
-10
Figure 4A. Effect of compounds on cell cycle distribution. Asynchronised MDA-MB-231 cells were treated with 5
lM (A) and 10
lM (B) for 24 h and similarly asynchronized
MCF-7 cells were treated with 5 M (C) and 10 lM (D) for 24 h. Cell cycle distributions were determined by flow cytometry using propidium iodide staining of fixed cells
l
(details as described in Section 5). The percentage of cells in each phase of the cell cycle is represented in the graph as subtracted values from DMSO treated cells (control).
Results are represented as a mean of triplicate experiment.
itored by flow cytometry. As shown in Figure 7C, the newly synthe-
sized fluorescent compounds provoked a significant induction of
apoptotic cells in both early and late apoptotic cell population after
24 h treatment at 10 lM. These findings further support the
hypothesis that the fluorescent compounds induced apoptotic cell
The strategy described herein involved the use of the fluores-
cent property of a dansyl moiety in our compound design. We
chemically modified purvalanol B (a potent CDK inhibitor) at the
solvent exposed carboxylic acid region. Evaluation of the fluores-
cent compounds for inhibition of CDKs resulted in the discovery
of two novel potent fluorescent CDK inhibitors (Fig. 1). VMY-1-
101 and VMY-1-103 fluoresced with an excitation of 410 nm and
emission of 512 nm (Fig. 6A). As shown in Table 1, VMY-1-101
and VMY-1-103 showed the greatest activity against CDK2/cyclin
E (87% and 89% at 100 nM, respectively). The compounds were also
found be effective in inhibiting other CDK’s, such as CDK1/cyclinB,
and to have strong anti-proliferative activity against two human
adenocarcinoma cell lines (p53-mutated, estrogen-independent
MDA-MB-231 cells, and wild type p53, estrogen dependent MCF-
7 cells) in vitro. The potent anti-proliferative activity of fluorescent
compounds (VMY-1-103 and VMY-1-101) were observed in two
death in MCF-7 cells.
4. Discussion
Aberrant expression and activation of cell cycle regulatory pro-
teins is a common feature of many cancers. Components of the
cell cycle machinery, such as the CDKs, may therefore represent
excellent molecular targets for the design of anticancer agents.32
Several classes of CDK inhibitors, including both natural33 and
chemically synthesized agents11,12 have been reported and sev-
eral are in clinical trials.32 However as because the target specific-
ity of CDK inhibitors is challenging, and in the absence of an
efficient method of determining real time pharmacodynamics,
there is a need to develop trackable, selective and potent CDK
inhibitors.
breast cancer cell lines (IC50 4.0–4.8
lM in MDA-MB-231 cells
and 10.0–19.0 M in MCF-7 cells), and was 13-fold more potent
l
in inhibiting MDA-MB-231 cell growth and 2.8–5.5-fold more po-
tent in inhibiting MCF-7 cell growth as compared to roscovitine.

2720
V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
103 (Table 1). This suggests that purvalanol B has poor cell penetra-
40
30
tion. Confocal imaging of treated cells confirmed the intracellular
delivery of VMY-1-103, which localized in the cytoplasm of human
breast cancer cells (Fig. 6B). The lipophilic dansyl ethylenediamine
group may provide increased cell membrane permeability, enhanc-
ing intracellular delivery and concentration of VMY-1-103.42
Treatment of cancer cells with the fluorescent CDK inhibitors
resulted in the modulation of several key apoptotic and cell sur-
vival proteins, mediating apoptosis by down-regulating the anti-
apoptotic protein Bcl-2, and inducing the pro-apoptotic protein,
Bax (Fig. 7A). Induction of apoptosis was further confirmed by in-
creased PARP cleavage in MDA-MB-231 cells (Fig. 7B) and in-
creased annexin-V positivity in MCF-7 cells (Fig. 7C).
In summary, we have designed and synthesized a new class of
fluorescent CDK inhibitors that are irreversible agents with potent
anti-proliferative and pro-apoptotic capabilities in human breast
cancer cell lines beyond that seen with the parent compound.
These new CDK analogs also allowed for the visualization of their
delivery to the cytoplasm of human breast cancer cells and tissues,
and therefore provide a platform for future preclinical studies.
MDA-MB-231, 5 μM
G1
S
G2M
SUBG1
20
10
0
-10
-20
-30
-40
24h
24h/MC/48h
5. Experimental section
5.1. Materials
40
MDA-MB-231, 10 μM
G1
S
G2M
SUBG1
30
20
All reagents and solvents were commercially available and used
without further purification. Chromatography was performed for
purification of final compounds using a Biotage SP-1 system with
silica gel cartridges. NMR spectra were recorded on a Varian 400
MR spectrometer at 400 MHz for H-1 and 100 MHz for C-13. Chem-
ical shifts (d) are given in ppm downfield from tetramethylsilane,
and coupling constants (J-values) are provided in hertz (Hz). The
purity of final compounds was evaluated by reverse-phase HPLC
analysis (Shimadzu Model LC 2010) using two conditions as fol-
lows A = water, B = acetonitrile, method 1: 2–60% B in A over
20 min, method 2: 50–60% B in A over 20 min (Restek Ultra IBD
10
0
-10
-20
-30
-40
5
lm column (4.6 mm  50 mm) at a flow rate of 1 mL/min).
5.2. Chemical synthesis
24h
24h/MC/48h
5.2.1. 6-Chloro-2-fluoro-9-isopropyl-9H-purine (2)
Figure 4B. Anti-proliferative effect of VMY-1-103 is irreversible/reversible. Expo-
nentially growing MDA-MB-231 cells were treated with VMY-1-103 at 5 M (A)
and 10 M (B). Cells were treated for 24 h and additionally, after post-incubation
for 48 h in a compound free media (24 h/MC/48 h). Cell cycle distributions were
determined by flow cytometry using propidium iodide staining of fixed cells
(details as described in Section 5). The percentage of cells in each phase of cell cycle
represented in the graph as subtracted values from untreated cells (control). Results
are represented as a mean of triplicate experiments.
6-Chloro-2-fluoro-9H-purine (1) (1.8 g, 10.4 mmol) and tri-
phenylphosphine (6 g, 20.8 mmol) in 60 mL of anhydrous tetrahy-
drofuran was added to a flame-dried flask under N2. Anhydrous 2-
propanol (1.6 ml, 20.8 mmol) was added and the mixture was
cooled to À10 °C. After dropwise addition of 40% (w/v) diethyl azo-
dicarboxylate in toluene (9 ml, 20.8 mmol), the mixture was
warmed gradually to room temperature. After 18 h, the reaction
mixture was quenched with 1 mL of water and the solvent was re-
moved under reduced pressure. The resulting yellow oil was puri-
fied by column chromatography and the solid was triturated with
methanol (to remove the diethyl hydrazine-N,N’-dicarboxylate es-
ter by-product) to yield 1.1 g (49%) of a white solid.
l
l
Interestingly, no anti-proliferative activity was found in these cell
lines even at 100 lM (Table 2). Morphological changes (Fig. 3)
were induced by compounds VMY-1-101 and VMY-1-103 in both
cell lines. Treatment of breast cancer cells with our compounds in-
duced G2/M cell cycle arrest, independent of the estrogen status of
the cells (Fig. 4A). Interestingly, in contrast to roscovitine, VMY-1-
103 showed irreversible anti-proliferative effects (Fig. 4B). VMY-1-
103 was also moderately active at inhibiting a multidrug resistance
(MDR) positive cell line and we show that our analogs are not sub-
strates for p-glycoprotein (Table 3).
Inhibition of the levels of phospho-cdc2 in MDA-MB-231 (Fig. 5),
further support the hypothesis that VMY-1-103 decreases the cdc2
kinase activity, and may play a central role in halting cell division in
G2/M. Purvalanol B does not show any effect on levels of phospho-
cdc2 in cellular assays, although it demonstrated more potent
CDK1-inhibitory activity in the in vitro kinase assay than VMY-1-
1H NMR (400 MHz, CDCl3): d 1.65 (d, 6H), 4.85 (m, 1H), 8.16 (s,
1H).
5.2.2. 2-Chloro-4-(2-fluoro-9-isopropyl-9H-purin-6-
ylamino)benzoic acid (3)
4-Amino-2-chlorobenzoic acid (0.32 g, 1.9 mmol), 2-fluoro-6-
chloro-9H-isopropylpurine (2) (0.4 g, 1.9 mmol), and diisopropyl-
ethylamine (0.8 mL, 2.5 mmol) were combined with n-butanol in
a sealed tube and heated for 12 h at 80 °C. After removal of the sol-
vent under reduced pressure, the crude product was triturated
with CH2Cl2, the precipitate was collected by filtration, and washed
with 0.05 M cold HCl (10 mL), CH2Cl2 (20 mL), and ether (20 mL) to

V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
2721
1h
3h
6h
12h
24h
A
P-cdc2 (Y15)
P-cdc2 (T161)
cdc2
cyclin-B1
β-Actin
P-Cdc2 (T161)
P-Cdc2 ( Y15)
B
C
Purvalanol B
VMY-1-103
Purvalanol B
VMY-1-103
1
0
1
0
1
3
1
12
Time ( hours)
12
Time ( hours)
6
3
6
24
24
Figure 5. Mechanism of in vitro action of VMY-1-103. MDA-MB-231 cells treated with 10 lM of purvalanol B and VMY-1-103 at different time intervals (1, 3, 6, 12 and 24 h)
and was subjected to western blot analysis to analyze the phosphorylation status of cdc2 substrates. Actin in each sample was employed as a standard. Below (B and C) are
shown the quantification of bands of phospho-CDC2 (Y15), phospho-CDC2 (Y15), which were performed using NIH image analysis normalized to actin. Data are expressed as
mean SD for three values.
yield 3 as a white solid (0.68 g, 100%). The crude product was used
without purification.
13C NMR (100 MHz, CDCl3) d 166.9, 157.1, 152.8, 152.6, 151.9,
150.9, 150.8, 140.9, 139.4, 134.5, 131.2, 130.5, 129.783, 129.5,
129.4, 128.6, 128.3, 123.1, 120.9, 118.9, 118, 115.1, 47.7, 45.3,
42.9, 39.8, 22.4.
1H NMR (400 MHz, DMSO-d6): d 1.51 (d, 6H, J = 6.8 Hz), 4.69 (m,
1H), 7.83 (d, 1H, J = 8.7 Hz), 7.93 (dd, 1H, J = 2.1 Hz, J = 8.7 Hz), 8.16
(d, 1H, J = 2.0 Hz), 8.46 (s, 1H), 10.74 (s, 1H).
5.2.4. (R)-2-Chloro-N-(2-(5-(dimethylamino)naphthalene-1-
sulfonamido)ethyl)-4-(2-(1-hydroxybutan-2-ylamino)-9-
isopropyl-9H-purin-6-ylamino)benzamide (VMY-1-101)
A mixture of compound (4) (0.1 g, 0.16 mmol), (R)-(À)-2-ami-
no-1-butanol (5 mL, 53 mmol), and diisopropylethylamine
(0.2 mL, 1.21 mmol) was placed in a sealed tube and heated at
120 °C for 48 h. After cooling to room temperature, the reaction
mixture was diluted with chloroform (100 mL), and washed with
water and brine. The organic phase was separated and dried over
anhydrous sodium sulfate, filtered, and concentrated under re-
duced pressure to give the crude product. The product was purified
by flash chromatography to yield a pure amorphous sticky solid of
VMY-1-101 (0.070 g, 63%). HRMS ([M+H]) calcd, 694.2691; found,
694.2437.
5.2.3. 2-Chloro-N-(2-(5-(dimethylamino)naphthalene-1-
sulfonamido)ethyl)-4-(2-fluoro-9-isopropyl-9H-purin-6-
ylamino)benzamide (4)
Compound (3) (0.67 g, 1.9 mmol) and dansyl ethylenediamine34
(0.74 g, 2.5 mmol) were dissolved in 30 mL of CH2Cl2–dioxane–
DMF (1:1:1 solution) and the resulting solution was added to a
mixture of diisopropylethylamine (0.70 mL, 4.1 mmol) and HOBt
hydrate (0.33 g, 2.5 mmol) followed by addition of EDCI (0.48 g,
2.5 mmol). The mixture was stirred for 24 h and then concentrated
under reduced pressure. The residue was dissolved in chloroform
(100 mL) and washed with 10% citric acid (30 mL), saturated LiCl
solution and brine. The organic phase was separated, dried over
anhydrous sodium sulfate, filtered, and concentrated under re-
duced pressure to give the crude product. The crude product was
purified by column chromatography yielding 0.52 g (44%) of com-
pound 4 as a yellow amorphous sticky solid. HRMS ([M+H]) calcd,
625.1912; found, 625.1724.
1H NMR (400 MHz): 0.98 (t, 3H, J = 7.4 Hz), 1.44 (dd, 6H,
J = 5.7 Hz, J = 20.9 Hz), 1.62 (m, 2H), 2.84 (s, 6H), 3.18 (brs, 2H),
3.49 (tt, 2H, J = 7.1 Hz, J = 19.7 Hz), 3.65 (m, 1H), 3.91–4.03 (m,
2H), 4.47 (s, 1H), 5.43 (d, 2H), 7.10 (dd, 2H, J = 2.7 Hz, J = 7.9 Hz),
7.45 (m, 4H), 7.57 (s, 1H), 7.86 (s, 1H), 8.23 (dd, 1H, J = 1.1 Hz,
J = 7.3 Hz), 8.34 (d, 1H, J = 8.6 Hz), 8.50–8.55 (m, 1H).
1H NMR (400 MHz, CDCl3): d 1.58 (d, 6H, J = 6.7 Hz), 2.83 (s, 6H),
3.18 (dd, 2H, J = 5.7 Hz, J = 10.8 Hz), 3.49 (m, 2H), 4.75 (m, 1H), 6.95
(d, 2H, J = 5.6 Hz), 7.08 (d, 1H, J = 7.6 Hz), 7.34 (m, 2H), 7.47 (t, 1H,
J=7.9 Hz), 7.56 (dd, 1H, J = 1.5 Hz, J = 8.6 Hz), 7.87 (s, 1H), 7.94 (s,
1H), 8.25 (m, 2H), 8.49 (d, 1H, J = 8.6 Hz), 8.65 (s, 1H).
13C NMR (100 MHz, CDCl3): d 167.1, 159.2, 151.8, 151.3, 151.1,
142.0, 135.4, 134.9, 130.8, 130.3, 129.8, 129.5, 129.3, 128.2, 127.2,

2722
V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
Figure 6. Panel A: Fluorescence spectrum of dansyl ethylenediamine (left, excitation/emission, 410/512 nm) and VMY-1-103 (right, excitation/emission, 410/512 nm). Both
compounds were dissolved in methanol and fluorescence spectrums were measured by fluorescent spectrophotometer. Panel B: Confocal images of MDA-MB-231 (A–D) and
MCF-7 (E–H) of human breast cancer cell lines treated with VMY-1-103. Cells (5 Â 105) were treated with the 10
lM VMY-1-103 for 1 h. After treatment, cells were washed
with PBS, fixed with 4% formaldehyde in PBS, stained with PI, and mounted on glass slides. The Zeiss LSM510/META/NLO live imaging multiphoton microscope was used to
visualize the fluorescence with 63Â magnification. Excitation was at 720 nm and absorbance was read at bandpath filter of 480–520 nm. Panel C: Binding of VMY-1-103 in
human breast tissues. After epitope retrieval of human breast cancer tissue as described in Section 5.1, exposed to 10 lM VMY-1-103 and dansyl ethylenediamine (control)
for 1 h. After treatment, tissues were washed with water, mounted with aqua mount. Zeiss LSM510/META/NLO live imaging multiphoton microscope was used to visualize
the fluoroscence with magnification Â63. Excitation was at 720 nm and absorbance was read at bandpath filter of 480–520 nm.
123.1, 120.0, 118.8, 117.2, 115.1, 114.5, 65.3, 55.5, 53.4, 46.5, 45.3,
42.7, 40.2, 24.8, 22.5, 22.3, 10.7.
1.2 mmol) were dissolved in n-butanol in a sealed tube and
heated to 120 °C for 24 h. After 24 h, (R)-(À)-2-amino-3-
methyl-1-butanol (0.04 g, 0.38 mmol) and 0.2 ml (1.2 mmol) of
diisopropylethylamine were added and the reaction was allowed
to stir for 48 h. After removal of the solvent under reduced pres-
sure, the crude product was dissolved in CH2Cl2, and washed
with water (50 mL) followed by brine (50 mL). The organic phase
was separated and dried over anhydrous sodium sulfate, filtered,
and concentrated under reduced pressure to give 0.1 g of crude
5.2.5. (R)-2-Chloro-N-(2-(5-(dimethylamino)naphthalene-1-
sulfonamido)ethyl)-4-(2-(1-hydroxy-3-methylbutan-2-
ylamino)-9-isopropyl-9H-purin-6-ylamino)benzamide (VMY-1-
103)
Compound (4) (0.15 g, 0.24 mmol), (R)-(À)-2-amino-3-methyl-
1-butanol (0.12 g, 1.2 mmol), and diisopropylethylamine (0.2 mL,

V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
2723
5.3. Molecular modeling
Docking simulations were carried out using the program Sur-
flexDock within Sybyl 8.2 (Tripos Inc., St. Louis, USA) with the
parameters set to default, except the number of ligand conforma-
tions generated which was set to 90. After consistent manual inter-
vention of the best selected pose, a final model was obtained. The
CDK2/VMY-1-103 complex structure was then refined by molecu-
lar dynamics simulation using the AMBER 9 program suite35 with the
PARM98 force-field parameter. The charge and force-field parame-
ters of VMY-1-103 were obtained using the most recent Ante-
chamber module in the AMBER 9 program, where VMY-1-103 was
minimized at the MP2/6-31G⁄ level. The SHAKE algorithm36 was
used to keep all bonds involving hydrogen atoms rigid. Weak cou-
pling temperature and pressure coupling algorithms37 were used
to maintain constant temperature and pressure, respectively. Elec-
trostatic interactions were calculated with the Ewald particle mesh
method38 with a dielectric constant at 1Rij and a nonbonded cutoff
of 12 Å for the real part of electrostatic interactions and for van der
Waals interactions. The system was then solvated in a 14 Å cubic
box of water where the TIP3P model8 was used. A 3000 step min-
imization of the system was performed in which the CDK2 com-
plex was constrained by a force constant of 100 kcal/mol/Å.2
After minimization, a 5 ps simulation was used to gradually raise
the temperature of the system to 298 K while the complex was
constrained by a force constant of 20 kcal/mol/Å2. Another 10 ps
equilibration run was used where only the backbone atoms of
the complex were constrained by a force constant of 5 kcal/mol/
Å2. Final production run of 50 ps was performed with no con-
straints. When applying constraints, the initial complex structure
was used as a reference structure. The PME method was used with
a time step of 5 fs. The neighboring pairs list was updated every 30
steps.
5.4. Biological assays
5.4.1. Cell lines and culture
The human breast cell lines MDA-MB-231 (HTB-26) and MCF-7
(HTB-22) were provided by the tissue culture core facility at Lom-
bardi Comprehensive Cancer Center, Georgetown University Med-
ical Center. MCF-7MDR (CL 10.3) cells were gifted by Dr. Robert from
the Georgetown University Medical Center. MDA-MB-231, MCF-7,
and CL 10.3 cells were maintained at 37 °C in a humidified incuba-
tor in DMEM supplemented with heat inactivated fetal bovine ser-
Figure 7. Effects of compounds on the expression of apoptosis related proteins
(Panel and B): MDA-MB-231 cells were treated with vehicle or indicated
concentrations of compounds for 48 and 72 h. Cells were harvested and lysed for
western blot analysis as described in Section 5.1. Blots were probed against
apoptosis related proteins (anti-Bax, anti-Bcl-2, anti PARP). Panel C: Annexin
A
um (10%), 2 mM L-glutamine, and 50 lg/mL each of antibiotics,
binding assay: MCF-7 cells treated 10 lM of corresponding compounds for 24 h and
trypsinized and resuspended in 1 ml of 1 X annexin binding buffer as described in
Section 5.1 and subjected to double staining with annexin V-FITC and propidium
iodide followed by analysis by flow cytometry.
namely penicillin, streptomycin, and neomycin.
5.4.2. Cell viability assay (WST-1)
Human breast cancer cells were seeded into a 96-well plate at
3000 cells per well in DMEM containing 10% FBS and allowed to
incubate for 24 h. Compounds were dissolved in DMSO, serially
product. The crude product was purified by column
chromatography to yield
a pure amorphous sticky solid of
diluted in tissue culture media (1–100 lM), added to cells in trip-
VMY-1–103 (0.070 g, 42%). HRMS ([M+H]) calcd, 708.2847;
found, 708.2659.
licate, and incubated for 48 h at 37 °C. Control cells were treated
with an equal amount of DMSO. After 48 h of incubation, cell via-
bility was measured by a WST-1 assay according to the manufac-
1H NMR (400 MHz, CDCl3): d 1.00 (dd, 6H, J = 1.3 Hz, J = 6.7 Hz)
1.44 (dd, 6H) 1.96 (m, 1H) 2.84 (s, 6H), 3.19 (d, 2H, J = 4.3 Hz),
3.50 (m, 2H), 3.75 (t, 1H, J = 9.1 Hz), 3.99 (m, 2H) 4.43 (bs, 1H),
5.34 (brs, 1H), 5.56 (brs, 1H), 7.10 (t, 2H, J = 8.0 Hz), 7.46 (m,
2H), 7.55 (s, 1H), 7.87 (s, 1H), 8.23 (dd, 1H, J = 1.0 Hz,
J = 7.3 Hz), 8.34 (d, 1H, J = 8.6 Hz), 8.50 (d, 1H, J = 8.5 Hz), 8.57
(s, 1H).
turer’s instructions (Roche). Briefly, 20 lL of WST-1 solution was
added in each well and incubated for 2–3 h. The water soluble
tetrazolium salt of WST-1 is converted into orange formazan by
dehydrogenase in the mitochondria of living cells. The formazan
absorbance, which correlates to the number of living cells, was
measured at 450 nm and 630 nm as reference filter using a
microplate reader (Ultramark, Microplate Imaging System, Bio-
Rad). The IC50 was calculated from the graph of the log of the
compound concentration versus the fraction of the surviving
cells.
13C NMR (100 MHz, CDCl3): d 167.2, 159.4, 151.8, 151.1, 142.1,
135.1, 134.9, 130.8, 130.3, 129.8, 129.7, 129.5, 129.3, 128.2, 127.0,
123.1, 119.9, 118.8, 117.0, 115.1, 114.4, 64.0, 59.3, 53.4, 46.3, 45.3,
42.8, 40.2, 30.2, 22.6, 22.1, 19.3, 19.1.

2724
V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
5.4.3. Cell cycle analysis
with 10 lM of VMY-1-103 for 1 h. Cells were washed twice with
The effect of CDK inhibitors on cell cycle progression was ana-
PBS, fixed with 4% formaldehyde in PBS for 15 min at room temper-
ature and washed again with PBS. For nuclear staining, cells were
incubated with propidium iodide for 5 min and washed with PBS
and mounted on glass slides using Antifade solution. Images were
taken with a multiphoton confocal microscope X63 oil immersion
(Zeiss510LSM/META/NLO live imaging) at excitation 720 nm and
imaged with a band path filter of 480–520 nm.
lyzed by flow cytometry. Cells were treated with 5
lM and
10 M of VMY-1-101 or VMY-1-103 compounds, purvalanol B or
l
roscovitine for 24 h. Cells were trypsinized, centrifuged
(2000 rpm), and the cell pellets were collected. Pellets were
washed once time with PBS, permeabilized with 70% (v/v) ethanol,
resuspended in 1 mL of PBS containing 1 mg/mL RNase and 50 mg/
mL propidium iodide, incubated in the dark for 30 min at room
temperature, and analysed using a FACSort Flow Cytometer (Bec-
ton Dickinson, San Jose, CA). The cell cycle distribution was evalu-
ated on DNA plots using the Modfit software (Verity
softwarehouse, Topsham, ME).
5.4.8. Kinase assay
VMY-1-101 and VMY-1-103 were screened for kinase activity
at Millipore in Dundee, UK. Briefly, assays were performed with a
Biomek 2000 Laboratory Automation Workstation in a 96-well for-
mat (Beckman instruments, Palo Alto, CA, USA) for 40 min at ambi-
5.4.4. Western blot analysis
ent temperature in 25
reaction volume of 25
10 mU) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA,
0.1 mg/mL histone H1, 10 mM Mg(OAc)2, and [
33P-ATP] (specific
lL incubations using [
c-
33P]-ATP. In a final
Western blotting was performed according to literature proto-
lL, corresponding CDK/cyclin (h) (5–
col.39 In brief, cell pellets were collected at the indicated times
after treatment with each compound, suspended in 100
l
L of lysis
c-
buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA,
20 mM NaF, 100 mM Na3VO4, 0.5% NP-40, 1% Triton X-100, 1 mM
PMSF, 5 lg/mL aprotinin, 5 lg/mL leupeptin), vortexed twice and
activity approx. 500 cpm/pmol, concentration as required). The
reaction was initiated by the addition of the MgATP mix. After
incubation for 40 min at room temperature, the reaction was
incubated in an ice bath for 30 min. Lysates were cleared by centri-
fugation at 12,000 rpm for 15 min at 4 °C and protein content was
estimated by detergent compatible BCA protein assay kit (Pierce).
Equivalent amounts of total protein were resolved by SDS–PAGE
(10%) and transferred to PVDF membrane. Membranes were
blocked by 5% non-fat powdered milk in TBST overnight. Mem-
branes were incubated with indicated primary antibodies for 2 h
followed by HRP-conjugated secondary antibodies for 1 h and
developed using enhanced chemiluminescence kit (Perkin Elmer).
stopped by the addition of 5
lL of a 3% phosphoric acid solution.
From the reaction, 10 L was spotted onto a P30 filtermat and
l
washed three times for 5 min in 75 mM phosphoric acid and once
in methanol prior to dying and scintillation counting.
5.5. Test compounds
Roscovitine and purvalanol B were purchased from Sigma (St.
Louis, MO), VMY-1-101 and VMY-1-103 were prepared as de-
scribed below. Drugs were dissolved in DMSO, stored at À20 °C,
and diluted in serum free medium immediately before use. All
experiments were performed in 5% media.
5.4.5. Annexin V binding assay
The loss of phospholipid asymmetry of the plasma membrane is
an early event of apoptosis.40,41 The annexin V binding assay was
performed according to protocol (BD Pharmingen™) to detect this
early event of apoptosis. Cells were washed twice with cold PBS
and then resuspended in 1Â binding buffer at a concentration of
5.6. Statistical analysis
All experiments were repeated three times, and the data ex-
pressed as mean standard deviation (SD). Two-tailed student t
test was used for statistical analysis of the data. A P <0.05 was ta-
ken as the level of significance.
1 Â 106 cells/mL. Solution (100
lL) was transferred to a 5 lL cul-
ture tube and 5 lL of annexin V-FITC were added followed by
5
lL of PI. Cells were vortexed gently and incubated for 15 min
at rt in the dark. Immediately after adding 400
l
L of 1Â binding
buffer to each tube, cells were analyzed using a FCSort Flow
Cytometer (Becton Dickinson, San jose, CA) and data were analyzed
using FCSExpress Denovo software (Los Angeles, CA). Viable cells
were FITC-/PIÀ, apoptotic cells were FITC+/PI-, and necrotic cells
were FITC+/PI+.
6. Disclosure of potential conflicts of interest
A patent application has been filed by Georgetown University
on behalf of the inventors that are listed as authors in this article.
Acknowledgements
5.4.6. Staining procedure for human breast tissue
Human breast tissues (Georgetown University Tissue Bank)
were subjected to the autoshaker program 7 for de-waxing and
re-hydration, followed by epitope retrieval for 20 min at 100 °C
in a steamer, and cooled to rt for 24 min. Tissue were blocked in
10% goat-serum, 10 min at rt, exposed to 5% goat-serum for 2 h
at rt and washed twice with distilled water. After epitope retrieval
We thank Dr. Karen Creswell for flow cytometry and Dr. Susette
Mueller for microscopy imaging expertise, the core facilities of the
Lombardi Comprehensive Cancer Center and the Drug Discovery
Program at Georgetown University Medical Center for financial
support. We thank Deborah L. Berry for histological staining of
the human breast tissue. We thank Dr. Chris Albanese for his valu-
able scientific discussions.
was performed, the breast tissues were exposed to 10 lM of VMY-
1-103 or dansyl ethylenediamine for 1 h at room temperature. The
tissues were washed twice with distilled water for 5 min and
mounted in aquamount. Images were taken with multiphoton con-
focal microscopy X63 oil immersion (Zeiss 510LSM/META/NLO live
imaging) at excitation 720 nm and imaged with a band path filter
of 480–520 nm.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.02.052.
References and notes
5.4.7. Confocal microscopy
Breast cancer cells were seeded at a density of 5 Â 105 on ster-
1. Nigg, E. A. Curr. Opin. Cell Biol. 1993, 5, 187.
2. Nigg, E. A. Bioessays 1995, 17, 471.
3. Senderowicz, A. M. Oncogene 2003, 22, 6609.
ilized microscope slides coated with poly-D-lysine. After 24 h incu-
bation, slides were washed with serum free medium and treated

V. M. Yenugonda et al. / Bioorg. Med. Chem. 19 (2011) 2714–2725
2725
4. Morgan, D. O. Annu. Rev. Cell Dev. Biol. 1997, 13, 261.
5. Sherr, C. J. Science 1996, 274, 1672.
25. Stearns, M. E.; Jenkins, D. P.; Tew, K. D. Proc. Natl. Acad. Sci. U.S.A. 1985, 82,
8483.
6. Harper, J. W.; Elledge, S. J. Curr. Opin. Genet. Dev. 1996, 6, 56.
7. Shapiro, G. I. J. Clin. Oncol. 2006, 24, 1770.
26. Summerer, D.; Chen, S.; Wu, N.; Deiters, A.; Chin, J. W.; Schultz, P. G. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 9785.
8. Vermeulen, K.; Van Bockstaele, D. R.; Berneman, Z. N. Cell Prolif. 2003, 36, 131.
9. Collins, I.; Garrett, M. D. Curr. Opin. Pharmacol. 2005, 5, 366.
10. Malumbres, M.; Barbacid, M. Nat. Rev. Cancer 2009, 9, 153.
11. Wölfel, T.; Hauer, M.; Schneider, J.; Serrano, M.; Wölfel, C.; Klehmann-Hieb, E.;
De Plaen, E.; Hankeln, T.; Meyer zum Büschenfelde, K. H.; Beach, D. Science
1995, 26, 1281.
12. Easton, J.; Wei, T.; Lathi, J. M.; Kidd, V. J. Cancer Res. 1998, 58, 2624.
13. Malumbres, M.; Barbacid, M. Nat. Rev. Cancer 2001, 1, 222.
14. Chang, Y. T.; Gray, N. S.; Rosania, G. R.; Sutherlin, D. P.; Kwon, S.; Norman, T. C.;
Sarohia, R.; Leost, M.; Meijer, L.; Schultz, P. G. Chem. Biol. 1999, 6, 361.
15. Gray, N. S.; Wodicka, L.; Thunnissen, A. M.; Norman, T. C.; Kwon, S.; Espinoza, F.
H.; Morgan, D. O.; Barnes, G.; LeClerc, S.; Meijer, L.; Kim, S. H.; Lockhart, D. J.;
Schultz, P. G. Science 1998, 281, 533.
27. Damianovich, M.; Ziv, I.; Heyman, S. N.; Rosen, S.; Shina, A.; Kidron, D.; Aloya,
T.; Grimberg, H.; Levin, G.; Reshef, A.; Bentolila, A.; Cohen, A.; Shirvan, A. Eur. J.
Nucl. Med. Mol. Imaging 2006, 33, 281.
28. Cohen, A.; Ziv, I.; Aloya, T.; Levin, G.; Kidron, D.; Grimberg, H.; Reshef, A.;
Shirvan, A. Technol. Cancer Res. Treat. 2007, 6, 221.
29. Clarke, R.; Currier, S.; Kaplan, O.; Lovelace, E.; Boulay, V.; Gottesman, M. M.;
Dickson, R. B. J. Natl. Cancer Inst. 1992, 84, 1506.
30. Beck, W. T.; Qian, X. D. Biochem. Pharmacol. 1992, 43, 89.
31. Meijer, L.; Raymond, E. Acc. Chem. Res. 2003, 36, 417.
32. Lapenna, S.; Giordano, A. Nat. Rev. Drug Disc. 2009, 8, 547.
33. Besson, A.; Dowdy, S. F.; Roberts, J. M. Dev. Cell 2008, 14, 159.
34. Doyle, E. L.; Hunter, C. A.; Phillips, H. C.; Webb, S. J.; Williams, N. H. J. Am. Chem.
Soc. 2003, 125, 4593.
16. Senderowicz, A. M.; Sausville, E. A. J. Natl. Cancer Inst. 2000, 92, 376.
17. Brasca, M. G.; Amboldi, N.; Ballinari, D.; Cameron, A.; Casale, E.; Cervi, G.;
Colombo, M.; Colotta, F.; Croci, V.; D’Alessio, R.; Fiorentini, F.; Isacchi, A.;
Mercurio, C.; Moretti, W.; Panzeri, A.; Pastori, W.; Pevarello, P.; Quartieri, F.;
Roletto, F.; Traquandi, G.; Vianello, P.; Vulpetti, A.; Ciomei, M. J. Med. Chem.
2009, 52, 5152.
35. Case, D. A.; Darden, T. A.; Cheatham, T. E.; Simmerling, C. L.; Wang, J.; Duke, R.
E.; Luo, R.; Merz, K. M.; Pearlman, D. A.; Crowley, M.; Walker, R. C.; Zhang, W.;
Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Wong, K. F.; Paesani, F.; Wu, X.;
Brozell, S.; Tsui, V.; Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V.; Cui, G.;
Beroza, P.; Mathews, D. H.; Schafmeister, C.; Ross, W. S.; Kollman, P. A. AMBER 9;
University of California: San Francisco, 2006.
18. Popowycz, F.; Fournet, G.; Schneider, C.; Bettayeb, K.; Ferandin, Y.; Lamigeon,
C.; Tirado, O. M.; Mateo-Lozano, S.; Notario, V.; Colas, P.; Bernard, P.; Meijer, L.;
Joseph, B. J. Med. Chem. 2009, 52, 655.
19. Rizzolio, F.; Tuccinardi, T.; Caligiuri, I.; Lucchetti, C.; Giordano, A. Curr. Drug
Targets 2010, 11, 279.
20. Krystof, V.; Uldrijan, S. Curr. Drug Targets 2010, 11, 291.
21. Davies, T. G.; Pratt, D. J.; Endicott, J. A.; Johnson, L. N.; Noble, M. E. Pharmacol.
Ther. 2002, 93, 125.
36. Hanson, R. N.; Lee, C. Y.; Friel, C. J.; Dilis, R.; Hughes, A.; DeSombre, E. R. J. Med.
Chem. 2003, 46, 2865.
37. Berendsen, H. J. C.; Postma, J. P. M.; Vangunsteren, W. F.; Dinola, A.; Haak, J. R. J.
Chem. Phys. 1984, 81, 3684.
38. Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, 10089.
39. Deb, T. B.; Coticchia, C. M.; Dickson, R. B. J. Biol. Chem. 2004, 279, 38903.
40. Fadok, V. A.; Voelker, D. R.; Campbell, P. A.; Cohen, J. J.; Bratton, D. L.; Henson, P.
M. J. Immunol. 1992, 148, 2207.
22. Knockaert, M.; Greengard, P.; Meijer, L. Trends Pharmacol. Sci. 2002, 23, 417.
23. Knockaert, M.; Gray, N.; Damiens, E.; Chang, Y. T.; Grellier, P.; Grant, K.;
Fergusson, D.; Mottram, J.; Soete, M.; Dubremetz, J. F.; Le, R. K.; Doerig, C.;
Schultz, P.; Meijer, L. Chem. Biol. 2000, 7, 411.
24. Becker, F.; Murthi, K.; Smith, C.; Come, J.; Costa-Roldan, N.; Kaufmann, C.;
Hanke, U.; Degenhart, C.; Baumann, S.; Wallner, W.; Huber, A.; Dedier, S.; Dill,
S.; Kinsman, D.; Hediger, M.; Bockovich, N.; Meier-Ewert, S.; Kluge, A. F.; Kley,
N. Chem. Biol. 2004, 11, 211.
41. Koopman, G.; Reutelingsperger, C. P.; Kuijten, G. A.; Keehnen, R. M.; Pals, S. T.;
van Oers, M. H. Blood 1994, 84, 1415.
42. Ringer, L.; Sirajuddin, P.; Yenugonda, V. M.; Ghosh, A.; Divito, K.; Trabosh, V.;
Patel, Y.; Brophy, A.; Grindrod, S.; Lisanti, M. P.; Rosenthal, D.; Brown, M. L.;
Avantaggiati, M. L.; Rodriguez, O.; Albanese, C. Cancer Biol. Ther. 2010, 10, 326.