Design, synthesis and biological studies of Survivin Dimerization Modulators that prolong mitotic cycle

Article abstract of DOI:10.1016/j.bmcl.2013.07.034

Survivin, a member of the inhibitor of apoptosis protein (IAP) family proteins, has essential roles in cell division and inhibition of apoptosis. Several clinical studies in cancer patients have shown that the elevated levels of survivin correlate with aggressiveness of the disease and resistance to radiation and chemotherapeutic treatments. Survivin is an integral component of chromosomal passenger complex (CPC) where it binds to borealin and INCENP through its dimerization interface. Thus, disruption of functional survivin along its dimer interface with a small molecule is hypothesized to inhibit the proliferation of cancer cells and sensitize them to therapeutic agents and radiation. Recently, a small molecule (Abbott8) was reported to bind at the dimerization interface of survivin. Further development of this compound was accomplished by computational modeling of the molecular interactions along the dimerization interface, which has led to the design of promising survivin dimerization modulators. Two of the most potent survivin modulators, LLP3 and LLP9 at concentrations between 50 and 100 nM, caused delay in mitotic progression and major mitotic defects in proliferating human umbilical vein endothelial cells (HUVEC) and prostate cancer cells (PC3).

Full text of DOI:10.1016/j.bmcl.2013.07.034

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5429–5433
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological studies of Survivin Dimerization
Modulators that prolong mitotic cycle
Somsundaram N. Chettiar a, James V. Cooley b, In-Hee Park a, Deepak Bhasin a, Arnab Chakravarti b,
Pui-Kai Li a, Chenglong Li a, , Naduparambil Korah Jacob b,
⇑
⇑
a Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Rm 612 Riffe Building, 496 West 12th Avenue, Columbus, OH
43210-1291, United States
b Department of Radiation Oncology, College of Medicine, The Ohio State University, 366 Wiseman Hall, 410 West 12th Avenue, Columbus, OH 43210, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Survivin, a member of the inhibitor of apoptosis protein (IAP) family proteins, has essential roles in cell
division and inhibition of apoptosis. Several clinical studies in cancer patients have shown that the ele-
vated levels of survivin correlate with aggressiveness of the disease and resistance to radiation and che-
motherapeutic treatments. Survivin is an integral component of chromosomal passenger complex (CPC)
where it binds to borealin and INCENP through its dimerization interface. Thus, disruption of functional
survivin along its dimer interface with a small molecule is hypothesized to inhibit the proliferation of
cancer cells and sensitize them to therapeutic agents and radiation. Recently, a small molecule (Abbott8)
was reported to bind at the dimerization interface of survivin. Further development of this compound
was accomplished by computational modeling of the molecular interactions along the dimerization inter-
face, which has led to the design of promising survivin dimerization modulators. Two of the most potent
survivin modulators, LLP3 and LLP9 at concentrations between 50 and 100 nM, caused delay in mitotic
progression and major mitotic defects in proliferating human umbilical vein endothelial cells (HUVEC)
and prostate cancer cells (PC3).
Received 30 April 2013
Revised 28 June 2013
Accepted 16 July 2013
Available online 24 July 2013
Keywords:
Survivin
LLP3
LLP9
Ó 2013 Elsevier Ltd. All rights reserved.
Survivin is a bi-functional protein with critical roles in mitosis
and inhibition of apoptosis. Survivin is an integral component of
chromosomal passenger complex (CPC) and plays an essential role
in kinetochore attachment, bipolar spindle assembly and cytokine-
sis.1 Survivin inhibits apoptosis by binding to caspases through its
baculovirus IAP repeat (BIR) domain, which is potentially contrib-
uting to therapeutic resistance in cancer cells.2–6
Survivin is important for multiple processes associated with cell
division and is a critical regulator of mitotic progression.5 In prolif-
erating cells, survivin levels are low in interphase and starts
I
OH
OH
OH
OH
H
H
H
H
N
O
N
O
N
O
N
O
Cl
Cl
Cl
CN
CN
CN
CN
O
O
O
LLP9
LLP6
LLP3
Abbott8
Figure 1. Survivin dimerization modulators.
⇑
Corresponding authors. Tel.: +1 (614) 247 8786; fax: +1 (614) 292 2435(C.L.); tel: +1 (614) 247 6484; fax: +1 (614) 688 4882(N.K.J.).
E-mail addresses: li.728@osu.edu (C. Li), Naduparambil.Jacob@osumc.edu (N.K. Jacob).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.034

5430
S. N. Chettiar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5429–5433
I
OH
OH
O
OH
Cl
O
H
N
O
CN
R
I
Cl
ii
i
Cl
ii
LLP9 (R = 3-benzyloxy)
OH
H
N
O
Cl
CN
R
LLP3 (R = 3,5-dibenzyloxy)
LLP6 (R = H)
Figure 2. Lowest docking energy poses of LLP3 (cyan), LLP6 (magenta) and LLP9
(green) in the binding pocket of survivin monomer at the dimerization interface
(hydrophobic regions are colored by orange and sky blue regions are hydrophilic).
Scheme 1. Reagents and conditions: (i) N-iodosuccinimide, p-toluenesulfonic acid,
acetonitrile, rt, 36 h; (ii) benzaldehyde or 3,5-dibenzyloxybenzaldehyde or 3-
benzyloxybenzaldehyde, ethyl cyanoacetate, ammonium acetate, ethanol, reflux,
overnight.
increasing during G2-M transition phase of cell division. During
mitosis, survivin is associated with borealin, inner centromere pro-
tein (INCENP) and aurora B kinase to form the CPC, which plays an
essential role in proper chromosomal segregation and cytokine-
sis.7–9 Aurora B kinase is the enzymatic core of the complex and
CPC ensures its proper localization for timely phosphorylation of
critical targets for mitotic progression. In CPC, survivin binds to
borealin and INCENP through its dimerization interface. The inter-
action between survivin and borealin in CPC are similiar to those
present in the homodimer survivin structure.9 Delay in mitotic
progression and polyploidy is observed in cells lacking functional
survivin.10 Survivin is over-expressed in many cancers including
breast, colon, brain, and pancreatic cancer.11–16 Increased survivin
expression is correlated with decrease in overall survival, increased
recurrence, metastasis and angiogenesis in cancer patients.17–20
Thus, inhibition of survivin expression or its functional association
Figure 3. Lowest docking energy conformation of (a) LLP3, (b) LLP6 and (c) LLP9 and its 4 Å proximity residues of survivin monomer represented by translucent surface.

S. N. Chettiar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5429–5433
5431
1
2
a
b
Actin
GFP-Survivin
Endogenous
Survivin
1- Cells with GFP-Survivin
2- Control
c
d
300
*
250
200
150
100
50
*
0
Figure 4. The effect of LLP3 and LLP9 on chromosomal passenger complex organization and mitotic phase completion were compared by real-time video microscopy. (a)
Western blot showing physiological level of GFP-survivin expression. (b) Images of GFP-survivin associated with CPC in HUVEC cells were captured at different time points
beginning chromosome condensation and various stages in mitosis. Untreated control cells show a timely movement of GFP-survivin during mitosis, and the overall length of
mitosis was 51.2 2.3 min. Cells treated with LLP6 at 100 nM showed little or no difference in their ability to mobilize and coordinate each phase of mitosis, and length of
mitosis value similar to control cells 62.45 11.2 min. Cells treated with LLP3 at 100 nM showed an elongated mitotic phase of 154.23 44 min and difficulties in resolving
cleavage. Cells treated with LLP9 had the greatest effect, where mitotic stage was greatly prolonged. (c) Quantification of the length of mitosis with various compounds at
100 nM concentration in HUVEC cells. The average length of mitosis was calculated from 50 cells except for the LLP9, where only 14 cells were counted. Asterisks (⁄) indicate
statistical significance (P <0.005). (d) The images of the GFP-survivin in PC3 cells with various treatment showing similar effects in proliferating cancer cells. Cells treated with
LLP3 and LLP9 showed a significantly elongated mitotic phase in comparison to that of LLP6. Those cells treated with LLP9 at 100 nM exhibited more distorted structure and
difficulty in assembling GFP-signal due to increased cytotoxicity and hence were not quantifiable.
at the dimerization interface in CPC can serve as an attractive tar-
get for cancer therapeutics. Different strategies have been em-
ployed to inhibit survivin expression or its function including
antisense oligonucleotides, ribozymes, and small molecule antago-
nists.21,22 Recently, Abbott Laboratories identified a potential
small-molecule binding site near the dimerization interface of sur-
vivin, distinct from the Smac-peptide binding site, and also re-
ported a new set of small molecules, including Abbott8 (Fig. 1),
which binds at the dimerization interface of survivin.23
Herein, we report the design, synthesis and functional evalua-
tion of small molecules that bind to the dimerization interface of
survivin and interfere with survivin function in proliferating cells.
Two of the newly designed survivin dimerization modulators, LLP3
and LLP9, caused delay in mitotic progression and caused mitotic
defects at low nanomolar concentration in proliferating prostate
cancer (PC3) cells as well as normal human umbilical vein endo-
thelial cells (HUVEC) that expresses survivin.
The optimal survivin dimer coordinates, mimicking the NMR
experimental binding mode of Abbott8 compound, from our previ-
ous computational study on survivin dimer was used to design sur-
vivin dimerization modulators.24 Quantitative structure–activity
relationship (QSAR) analysis was performed with the extended Ab-
bott8 derivative compound set and residue-by-residue decomposi-
tion analysis of the dimerization energy of survivin for the rational
modulator design approach (Supplementary data section 4). The
strategy to disrupt survivin dimerization via a small molecule is

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S. N. Chettiar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5429–5433
Control
LLP3
LLP9
a
b
LLP6
8
7
6
5
4
3
2
1
0
50 nM
LLP3
LLP9
Control
LLP6
*
*
100 nM
Figure 5. The effect of LLP compounds on cell proliferation in HUVEC cells. Cells were treated with LLP compounds and the effect of these compounds on mitosis were
quantified by enumerating the cells at the t = 0 and 48 h. (a) Cells in the entire imaging field show visual disparity between the changes in mitosis. (b) The proportional
differences in the rates of growth between treatments are shown graphically by counting at least 100 cells. Asterisks (⁄) indicate statistical significance (P <0.05). LLP3 and
LLP9 showed a dose and time dependant effect.
6
a
b
Control
LLP6
LLP3
LLP9
5
4
3
2
1
0
*
*
50 nM
Figure 6. The effect of LLP compounds on cell proliferation in PC3 cells. (a) Cells in the entire imaging field are shown at the beginning of the experiment and at a time point
3-4 rounds of cell division that is 72 h. (b) The proportional differences in the rates of growth between treatments are shown graphically by counting at least 100 cells.
Asterisks (⁄) indicate statistical significance (P <0.05).
to replace the most contributing residue of the protein (Leu98 and
Leu102 on monomer A) with a fragment on the modulator, while
leaving the Phe93 site aromatic ring p-stacking intact on monomer
B. The most representative survivin dimer conformation was used
as a template structure for chemical library screenings and lead
modifications. The Abbott8 structure with added halogen atoms
(chlorine and iodine) to improve the activity was used as the lead
compound to design the LLP compounds (Fig. 1), by growing a frag-
ment pointing toward the Leu98 to reduce survivin’s dimer
interaction.
interactions between different aromatic rings of LLP3 and Phe86/
93/101 of survivin. The lowest docking energy conformation
showed that the core scaffold (3-cyano-2-pyridone) was aligned
parallel to Phe93. One of the benzyloxy ring of LLP3 penetrated to-
wards the back side of the pocket forming
p-p stacking with
Phe86, while the other benzyloxy ring was aligned parallel to
Phe101 at the front of the pocket. The docking energies of LLP6 var-
ied from À9.9 to À8.6 kcal/mol by various combination of
p-p
stacking between the two end rings of LLP6 and Phe93/101 while
the pyridone ring of LLP6 centered at Leu96 (Fig. 3b). LLP9 docking
energies were from À10.6 to À9.3 kcal/mol. As seen in Figure 3c,
the extended benzyloxy ring of LLP9 touched N-terminus region
of survivin besides similar interactions as those of LLP6. The ex-
tended benzyloxy moiety of LLP9 is pointed toward N-terminus
(Thr5 and Leu6 in Fig. 3c), whereas the extended benzyloxy moiety
of LLP3 penetrate towards the backside of the pocket (Phe86 site in
Fig. 3a).
The overall protein–ligand interaction was dominated by
p-
stacking interactions between the aromatic rings of the ligand
and Phe93/Phe101 of survivin monomer, as shown by the hydro-
phobic patch colored in orange (Fig. 2). There were no specific
hydrogen bond interactions observed between the ligand and pro-
tein. As seen in Figure 3a, the docking energies of LLP3 varied from
À11.3 to À10.2 kcal/mol by various combination of
p-stacking

S. N. Chettiar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5429–5433
5433
LLP3, LLP6 and LLP9 were synthesized according to the syn-
thetic route shown in Scheme 1. LLP3 and LLP6 were synthesized
using one pot reaction of 5-chloro-2-hydroxyacetophenone with
appropriate aldehyde, ethyl cyanoacetate and ammonium acetate
in ethanol.25 LLP9 was synthesized in 2 steps. 5-Chloro-2-hydroxy-
acetophenone was first iodinated using N-iodosuccinimide in pres-
ence of p-toluenesulfonic acid;26 followed by condensation
reaction using 3-benzyloxybenzaldehyde, ethyl cyanoacetate and
ammonium acetate (Supplementary data section 1).
Cell lines expressing physiological levels of GFP-tagged survivin
relative to endogenous levels of survivin were developed in order
to test the effect of compounds on the CPC and mitosis. Prostate
cancer (PC3) cells as well as normal proliferating primary cells hu-
man umbilical vein endothelial cells (HUVEC) were selected based
on their relatively higher levels of endogenous survivin. Cells with
physiological levels of GFP-tagged survivin expression were se-
lected by single cell cloning and Fluorescence Activated Cell Sort-
ing (FACS). The expression of GFP-survivin in these cells was
confirmed by Western blot analysis (Fig. 4a). Immunofluorescence
studies confirmed that the N-terminal GFP tagged survivin assem-
ble functionally to the chromosomal passenger complex, a multi-
subunit complex with characteristic localization pattern at distinct
stages in mitosis. Time and dose dependent effects of each LLP
compounds on mitotic progression and assembly of CPC were com-
pared by real-time tracking of green fluorescence signal. The effect
of compounds on cell division was compared by counting the time
elapsed by a cell during mitosis. For each compound at its respec-
tive concentration, evaluations were made in regard to changes in
proliferation rate, cell morphology, alterations in chromatin orga-
nization, delay in initiation and progression of mitosis, and in-
crease in mitotic cell death.
Cells were synchronized at G1/S phase of cell cycle by adding
2 mM thymidine in the culture medium for 15–18 h. Following re-
lease from the thymidine block in the untreated HUVEC and PC3
cells, mitosis began respectively at about 6 and 8 h, and was com-
pleted in 1 h. During the mitotic stages in the control cells, CPC
associated GFP-survivin was found moving as a sharp signal in a
concerted manner (Fig. 4). GFP signal was observed to be localized
to the chromosome arms in cells at prometaphase, condensed
chromatin in cells progressing to metaphase and anaphase, and
midzone in cells at telophase. Variations to this pattern were used
as a parameter to evaluate the effect of compounds that bind to the
dimerization interface of survivin, leading to defects in CPC assem-
bly and function.
In this Letter, we report the design, synthesis and testing of LLP
compounds that modulate cell cycle progression. Earlier studies
have shown that survivin is essential for mitotic progression, and
higher levels of expression of survivin in cancer cells provide better
leverage to the mitotic machinery.27 The effect of LLP3 and LLP9 on
mitotic progression was predicted as they bind to the dimerization
interface of survivin, which is essential for structural and func-
tional organization of chromosomal passenger complex. These
compounds were effective in prolonging mitosis at nanomolar con-
centration. Ongoing studies include, understanding other potential
mechanisms and factors such as photosensitizing effects contribut-
ing to the cytotoxicity of some LLP compounds. The current study
also reports a novel method to screen mitotic inhibitors directly by
the real-time video microscopy based assay.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2013.07.034.
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Delay in mitosis and increased cell death was observed in cells
treated with LLP3 and LLP9 even at low nanomolar concentrations;
however LLP6 showed no significant effect. In both HUVEC and PC3
cells, LLP9 and LLP3 caused defects in CPC organization and mitotic
progression at 50 nM and 100 nM respectively, but had little or no
effect at 10 nM. The defects noticed in LLP3 or LLP9 treated cells
include distorted, diffused, or decondensed CPC in comparison to
the sharp signals observed in control cells. Cells treated with
LLP3 exhibited a two to three fold delay in completion of mitosis,
primarily due to extended metaphase and anaphase. Cells that
managed to complete the first division were found to be arrested
or defective for subsequent divisions. This effect was more pro-
nounced in case of LLP9, with 50–70% reduction in cell number de-
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PC3 cells, respectively), by which the control untreated cells
completed 3–4 rounds of cell division (Figs. 5 and 6).
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