Discovery of a novel small molecule binding site of human survivin

Article abstract of DOI:10.1016/j.bmcl.2007.03.042

Survivin is one of the most tumor-specific genes in the human genome and is an attractive target for cancer therapy. However, small-molecule ligands for survivin have not yet been described. Thus, an interrogation of survivin which could potentially both

Full text of DOI:10.1016/j.bmcl.2007.03.042

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3122–3129
Discovery of a novel small molecule binding site of human survivin
Michael D. Wendt,a,*,ꢀ Chaohong Sun,b,ꢀ Aaron Kunzer,a Daryl Sauer,b Kathy Sarris,b
Ethan Hoff,b Liping Yu,b David G. Nettesheim,b Jun Chen,a Sha Jin,a
Kenneth M. Comess,b Yihong Fan,b Steven N. Anderson,b Binumol Isaac,b
Edward T. Olejniczak,b Philip J. Hajduk,b Saul H. Rosenberga and Steven W. Elmorea
aCancer Research, Global Pharmaceutical R&D, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6101, USA
bAdvanced Technology, Global Pharmaceutical R&D, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6101, USA
Received 13 February 2007; revised 12 March 2007; accepted 12 March 2007
Available online 16 March 2007
Abstract—Survivin is one of the most tumor-specific genes in the human genome and is an attractive target for cancer therapy. How-
ever, small-molecule ligands for survivin have not yet been described. Thus, an interrogation of survivin which could potentially
both validate a small-molecule therapy approach, and determine the biochemical nature of any of survivin’s functions has not been
possible. Here we describe the discovery and characterization of a small molecule binding site on the survivin surface distinct from
the Smac peptide-binding site. The new site is located at the dimer interface and exhibits many of the features of highly druggable,
biologically relevant protein binding sites. A variety of small hydrophobic compounds were found that bind with moderate affinity
to this binding site, from which one lead was developed into a group of compounds with nanomolar affinity. Additionally, a subset
of these compounds are adequately water-soluble and cell-permeable. Thus, the structural studies and small molecules described
here provide tools that can be used to probe the biochemical role(s) of survivin, and may ultimately serve as a basis for the devel-
opment of small molecule therapeutics acting via direct or allosteric disruption of binding events related to this poorly understood
target.
Ó 2007 Elsevier Ltd. All rights reserved.
Survivin, a unique member of the inhibitors of apoptosis
(IAP) family of proteins,1 is highly overexpressed in
many common human cancers, but undetectable in most
terminally differentiated adult tissues.1–3 Clinically, the
level of expression of survivin in tumor cells is often
associated with poor prognosis and shorter patient sur-
vival rates.4,5 As with other IAP family members, survi-
vin has been implicated in protection from apoptosis. In
cell culture systems, survivin overexpression has been
strongly associated with inhibition of both the intrinsic
and extrinsic cell death pathways.3,6 Consistent with this
role, a number of binding partners have been proposed
for survivin to exert its anti-apoptotic activities, includ-
ing caspases-36 and -7,7 Smac/Diablo,8 hepatitis B
X-interacting protein (HBXIP),9 and XIAP.10 However,
interactions with caspases6,11,12 and Smac-derived pro-
teins and peptides13 either cannot be confirmed or are
so weak that their relevance in a cellular context can
be questioned.
Survivin also appears to play an important role in cell
division.4 Survivin displays highly cell-cycle-dependent
expression, with a pronounced accumulation at mitosis,3
and has also been shown to be a member of the group of
chromosomal passenger proteins, including INCENP,
Aurora B kinase, and Borealin.14,15 The chromosomal
passenger proteins associate with centromeres at meta-
phase, central spindle microtubules at anaphase, and
remain located at cytokinesis remnants at end of
telophase. In particular, survivin has been shown to be
important for localization of Aurora B kinase to the mi-
totic machinery,16 and this complex is important in com-
municating lack of tension to microtubules.17,18 In vitro
knock-down of survivin gene expression results in cas-
pase-dependent cell death as well as defects in mitotic
progression such as multinucleation and polyploidy.19–21
In vivo, mouse survivin knock-out studies show severe
defects in cytokinesis, such as disrupted microtubule
formation and polyploidy, leading to embryonic death.
Interestingly, the survivin knock-out phenotype is
Keywords: Survivin; Ligand.
*
Corresponding author. Tel.: +1 847 937 9305; fax: +1 847 938
1004; e-mail: mike.d.wendt@abbott.com
These authors contributed equally to this work.
ꢀ
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.042

M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3122–3129
3123
similar to that observed for INCENP, suggesting func-
tionally related roles between these two proteins.22 Sur-
vivin also has been shown to physically associate with
the cyclin-dependent kinase cdc2 on the mitotic appara-
tus, and is phosphorylated at Thr34 by cdc2/cyclin B1.23
This latter event is crucial to its activity as a cytokinesis
regulator, as overexpression of non-phosphorylatable
mutant survivin results in marked increases in apoptosis
and polyploidy.
has yet to be elucidated, while several reports bearing
on possible mechanisms of the apoptotic function of
survivin have been put forward. The nature of addi-
tional regulatory interactions such as those with Hsp90
or nuclear exporters is only now becoming
understood.25,26
Structurally, survivin has been thoroughly characterized
11,27,28
by both X-ray crystallography
and solution
NMR,13 where it forms a bow-tie shaped symmetrical
homodimer in solution. Each monomer contains a
zinc-binding fold similar to that found in other members
of the IAP family, but lacks the ring finger motif found
in XIAP.29,30 Instead, it contains a long amphipathic
a-helix C-terminal to its sole BIR domain. The dimer
interface is composed of a number of hydrophobic resi-
dues from its N-terminus as well as residues connecting
its BIR domain to the C-terminal helix. The BIR
domain contains a binding site analogous to those
possessed by other IAP family members, which are
known to bind to caspases and the Smac peptide. De-
tailed characteristics of this binding site and its interac-
tions with substrates have been described elsewhere.31,32
Survivin thus is an attractive therapeutic target in cancer
due to both its expression in tumors compared to nor-
mal tissues and its ability to promote tumor growth
and survival by multiple pathways involving multiple
mechanisms. Various strategies have been used to target
survivin, including antisense oligonucleotides and
siRNA,24 that have further validated survivin as a tar-
get, but small molecule inhibitors of survivin function
have yet to be described. A difficulty attendant to this
approach is the lack of understanding of the molecular
mechanisms responsible for affecting apoptosis and
mitosis. The nature of mitosis-related interactions of
survivin with other chromosomal passenger proteins
Table 1. Ligands for survivin identified through NMR and affinity-based screening
Compound
Structure
Source
Binding site
NMR KD (lM)
OH
H
N
O
Br
1
2
3
4
5
NMR/ ASMS
Dimer
Dimer
Dimer
Dimer
Dimer
5
CN
N
NMR
5
Cl
Cl
NMR
<10
<10
10
N
H
O
HN
N
OCF3
NMR
O
N
OH
O
Br
NMR
N
I
OH
I
6
7
ASMS
NMR
Dimer
8
O
O
H
N
NH
N
Peptide
130
O

3124
M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3122–3129
Here we demonstrate the existence of a newly discovered
small molecule binding site on survivin distinct from the
BIR site, and present details of an extension of this bind-
ing site through development of high-affinity ligands,
including NMR structural studies of the ligand–protein
interaction.
yielded 254 potential hits, from which two structurally
interesting compounds (Table 1, 1, 6) were confirmed
as ligands for human survivin by NMR. From an
1
inspection of H/13C-HSQC spectra, two distinct chem-
ical shift perturbation patterns were observed for the
leads (Fig. 1). Compound 7 shifted residue L64, similar
to what is observed for Hid/Smac peptide binding, sug-
gesting that this compound binds at or near the Hid/
Smac binding site on the protein.9 However, for the
remaining hits, no shifts were observed for L64. Instead,
for 1–6, most of whom share a roughly similar L-shaped
aromatic structural motif, a new set of residues includ-
ing L98, L6, and L14, was shifted, suggesting a different
binding site, with the affected residues being located near
the interface of the two survivin monomers (Fig. 2a).
Competition experiments between 7 and 1 indicated that
Since no biochemical mechanism of survivin is known,
no robust in vitro functional assays have been estab-
lished to enable a conventional high-throughput screen
for lead identification. Thus, two types of affinity-based
screening methods, HTS-NMR33 and AS/MS,34,35 were
employed to identify leads for this unique target. A
number of different classes of ligands for survivin were
identified from the NMR screen (Table 1, 1–5, 7). Affin-
ity selection/mass spectrometry (AS/MS) screening
Figure 1. [13C, 1H]-HSQC spectra of selectively 13CH3-labeled (Leu, Val, and Ile-d1) survivin in the absence (black) and presence (red) of (a)
acylamino pyridine hit 7 (100 mM) binding to BIR3 site, and (b) benzofuran hit 6 (100 mM) binding to dimer site. Selected chemical shift changes are
indicated.
a
b
d
OH
Dimer site
H
C
N
B
O
CN
A
Peptide binding site
L96
F13
c
F101*
L98*
L96
F93
V89
F93
L104
L87
F101
L14
L102*
W10
L98*
F101
L104
F86
F86
L102*
I74
K15
R18
L6
Figure 2. Details of survivin binding sites. (a) ribbon diagram of human survivin (8 in green), with Connolly surfaces of dimer binding site (gold) and
peptide binding site (blue). (b) structure of 8 with A-B-C-ring nomenclature. (c) expanded view of dimer site. Residues making important contacts
with ligand are rendered. Residues belonging to adjacent survivin monomer are starred and in blue with surface in yellow. (d) NMR-derived structure
of 23b bound to survivin, with Connolly surface of binding site.

M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3122–3129
3125
these two compounds can bind simultaneously to the
protein.
Alternatively, the intrinsic tryptophan fluorescence of
survivin was monitored, but compound interference
was a significant problem. Fortunately, most of the
pyridinone analogs exhibit relatively high fluorescence
(with excitation and emission wavelengths of 450 and
530 nm, respectively) increasing in intensity upon com-
plex formation. Compound concentrations as low as
100 nM could be utilized on standard instrumentation,
allowing for the quantitation of binding affinities down
to the 20 nM range (see Supplementary Material). This
assay was utilized for the lead optimization described
below.42 As this assay is non-competitive, we also veri-
fied the site of binding by NMR for all compounds.
Compounds 1–6 are relatively insoluble and were not
suitable for structural studies. However, a soluble, des-
bromo analog of 1, compound 8 (Fig. 2b), exhibiting a
KD of 75 lM for survivin, was amenable for NMR
study. Structural study of the survivin/8 complex
revealed details of a binding site that is located at the
interface of the two survivin monomers (Fig. 2a
and c). A total of 24 intermolecular NOEs were ob-
served between 8 and survivin, which defined extensive
hydrophobic contacts. In particular, the bottom or A-
ring of 8 packs deeply into a pocket formed by the side
chain of residues F101 and L6 from one monomer and
residue L102* and L98* from the adjacent monomer.
The top phenol or C-ring packs against the side chains
of residues L96, F93, V89, L104, and F86. Compared
to the apo-protein, a large side-chain rearrangement of
F93 occurs, and results in exposure of a hydrophobic
pocket on the protein which is blocked by F93 in the
apo-protein. There is no rearrangement of the protein
backbone in this region as a result of small-molecule
binding. In addition, the N-terminus of the protein,
which is disordered in the apo form, becomes more or-
dered in the complex and exhibits NOEs to ring A of
the ligand. A hydrogen bonding network of the B- and
C-ring functional groups probably serves to lock the
two rings into a coplanar arrangement. Also of note is
that the stoichiometry of binding is one ligand per survi-
vin monomer, thus the complex exists as a doubly occu-
pied dimer. Subsequent compound complexes showed
similar features.
Initial work focused on separate studies of modifications
to each of the three rings of 1. Synthesis was normally
carried out by a one-pot procedure.43 We found that
the central pyridone ring did not tolerate modification
of any kind, and the top, C-ring phenol likewise was re-
quired for good binding. Our hypothesis, later verified
by NMR structure, was that the two groups form a
hydrogen bonding network, via the 2-hydroxypyridine
central ring tautomer, which serves to lock the two rings
into a near-coplanar arrangement. Surprisingly, we also
found the cyano group to be necessary for binding, in
spite of NMR structures providing no indication of a
specific interaction with the protein. Thus, replacement
with carboxamide, methyl, halogen, or hydrogen all
greatly diminished binding (data not shown). Substitu-
tion elsewhere on the C-ring tolerated substitution at
positions ortho and para to the hydroxy group, in keep-
ing with structure-based prediction. Para substituents
are completely buried by protein; we found various alkyl
groups and halogens to be roughly equal in affinity to
that of the bromo group of 1. NMR structures suggested
that larger substituents such as phenyl resulted in com-
pounds being pushed out toward solvent to some de-
gree, with still larger groups resulting in a loss of
affinity (9–12).
The functional role of this dimer interface site is cur-
rently unknown. However, several pieces of evidence
suggest that the dimer interface site is probably involved
in binding to other proteins that regulate survivin func-
tion in vivo. First, the number and variety of chemical
structures that bind to this pocket are indicative of a
biologically relevant protein binding site that can be tar-
geted with small molecule drugs. The NMR hit rate for
this binding site was 0.35%, which is comparable to
other druggable protein targets, and much higher than
the rate of 0.01% observed for the BIR site.36 Also,
the hit rate for the XIAP BIR3 site was 0.09%, again
much higher than for the corresponding survivin do-
main. Finally, the large conformational rearrangements
that are observed upon ligand binding are similar to
those observed for other protein–protein interaction
sites37 including Bcl-xL,38 IL-2,39 MDM-2,40 and IgG
domains.41
Likewise, A-ring substitution found only hydrophobic
groups to be preferred. Both 2,4- and particularly 2,5-
disubstitution were favored, with Cl and Me preferred
at the 2 position, and Cl, Me, and CF3 at the 4 and 5
positions, resulting in a roughly tenfold improvement
in affinity. Larger groups at the bottom of the pocket
were not tolerated, in keeping with the complexed
NMR structure with 8. The cumulative result of these
investigations resulted in a number of compounds in
the 300–550 nM binding range (Table 2).
We next sought to access the well-defined, though rela-
tively shallow cleft adjacent to the C-ring, formed lar-
gely by residues E40, I74, F86, L87, and K91. The
substituents of 17 were chosen for their unreactivity to-
ward the reaction conditions necessary for further elab-
oration. Experimentation with various substituents
ortho to the C-ring phenol led to use of an aminomethyl
group as a substrate for high-throughput organic syn-
thesis (HTOS). Fully elaborated compounds 22–26 were
synthesized from 17 as in Scheme 1. Iodination and car-
bonylation gave 20,44 which could easily be condensed
with a variety of amines or primary amides.45 Subse-
As there is, again, no clearly defined biochemical func-
tion, for survivin, and no known endogenous ligand
for the dimer site, a variety of biophysical techniques
were investigated in order to quantitatively measure
ligand binding. NMR can monitor ligand binding over
a wide affinity range, but cannot quantitate KD values
of less than ꢀ10 lM. Initially a variety of fluorophores
were attached to analogs of 8 to serve as probes in
fluorescence polarization competition assays, but high
non-specific binding precluded their routine use.

3126
M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3122–3129
1
Table 2. SAR of survivin compound A- and C-rings
ized by H NMR and MS. Both 21 itself and tertiary
amines derived from it exhibited reduced affinity, but
many N-methyl amides bound at least as well as the core
structure 9 (Table 3). In addition, some analogous sec-
ondary amides (22a,b) were also synthesized; these were
roughly equal in affinity and more water-soluble than
their N-methyl counterparts, but were highly crystalline,
and insoluble in organic solvents. Placement of an amine
(23c,d) two or three carbon atoms away from the amide
carbonyl improved affinity by another order of magni-
tude. Other compounds such as 23e, which situate het-
eroatoms in a similar location, were somewhat less
potent. The secondary amine 24 bound no better than
9, or the cyclohexyl amide 23a. However, we employed
24 in a second round of HTOS, and many resulting ter-
tiary amines such as 26a were an order of magnitude
more potent. Also, a number of amides, such as 25a,b,
exhibited similar affinity gains. Small alkyl or hetero-
alkyl groups were tolerated in both of these series, and
small alkyl branching or cycloalkyl groups, such as in
25c, were only slightly less potent, but much larger, or
longer alkyl groups such as in 26b had a deleterious
effect on binding.
OH
H
N
O
R2
CN
R1
Compound
R1
R2
KD (lM)
1
8
—
—
Br
—
5.7
75
9
2,5-Cl
2,5-Cl
Ph
0.70
0.51
7.56
2.98
0.70
1.17
0.81
0.49
0.52
>4.3
10
11
12
13
14
15
16
17
18
c-C5H9
i-Bu
c-C6H11
Br
2,5-Cl
2,5-Cl
2,4-Me
2,5-Me
2,5-Cl
Br
Br
2-Cl, 5-CF3
2-Cl, 5-CF3
2-Cl, 5-Ph
Br
Cl
Br
We obtained an NMR-derived structure for 23b
bound to the extended cleft (Fig. 2d). NOE data
indicate that the pyridinone core of 23b binds in a
similar location to compound 8 with minor changes
to accommodate the amide linker. The hydrogen
bonding network of the B- and C-rings is again
quent coupling of 21 gave a series of amides 23; the Boc-
protected amide from this group was deprotected and
amides 25 and amines 26 were again made largely via
parallel synthesis. All compounds 22–26 were character-
NH
O
R
O
H
N
N
N
X
OH
OH
OH
OH
H
H
H
H
N
O
N
O
N
O
N
O
f
c
d
Cl
Cl
Cl
Cl
NBOC
R =
CN
CN
CN
CN
Cl
Cl
Cl
Cl
CF3
CF3
CF3
CF3
17 X = H
19 X = I
21
23
24
a
b
d
g
20 X = CHO
O
e
R
N
R
N
O
O
O
R
N
N
NH
OH
OH
OH
H
H
H
N
O
N
O
N
O
Cl
Cl
Cl
CN
CN
CN
Cl
Cl
Cl
CF3
CF3
CF3
22
25
26
Scheme 1. Reagents and conditions: (a) NIS, CH3CN, D; (b) Pd(OAc)2, dppf, Et3N, Et3SiH, DMF, CO, 80 °C; (c) MeNH2ÆHCl, NaBH(OAc)3,
(CH2Cl)2; (d) RCO2H, polymer-supported-DCC, HOBtÆH2O, DIPEA, DMA; (e) RCONH2, TFA, Et3SiH, toluene, 100 °C; (f) aq HCl, dioxane; (g)
RCHO, NaBH(OAc)3 (CH2Cl)2.

M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3122–3129
Table 3. Survivin affinities for analogs of 22–26
3127
apparent. NOEs were observed between the amide
N-methyl and residues Q92, V89, K90, F93, and L96,
and between the piperidine acetyl methyl and residues
A41, I74, and L87, positioning this group in the
extended cleft adjacent to the C-ring. Also of note is
that the HSQC spectrum for 23b shows perturbation
of L64, which is part of the BIR binding site. This
result was reproduced by other compounds in Table
3. It is possible that compounds with sufficient bulk
may be able to allosterically disrupt the BIR site, most
likely through an effect on the intervening zinc finger
motif.
O
R2
N
R1
OH
H
N
O
Cl
Compound
KD (lM)
CN
Cl
CF3
R1
R2
A few other structural features are of note. The exo-
cyclic amide bond of 23b potentially forms a hydro-
gen bond to the terminal guanidine of R18, with an
23a
22a
23b
Me
0.62
0.88
0.086
˚
approximate distance of 3.0 A. The amide N-methyl
group fits snugly into a small pocket formed by the
residues linking the BIR-domain C-terminus to the
long terminal a-helix. We did find that larger amide
N-alkyl groups depressed binding (data not shown).
It is also apparent that larger terminal N-alkyl groups
such as those of 26b will extend beyond the end of
the cleft and into solvent, accounting for the reduced
affinity of long-chain compounds in the binding
assay.
H
O
Me
N
N
O
22b
H
0.048
In order to evaluate the potential of these compounds as
probes of intracellular processes, several of their physi-
cal properties were evaluated. Amines were determined
to have very low aqueous solubilities. However, both
23b and other amides among the group containing 25
possessed solubilities of >32 lM by nephelometry, and
23b and 25a were soluble to 300 and 138 lM, respec-
tively, at pH 7.4 by the shake-flask method. In addition,
several representative compounds in Tables 2 and 3 were
shown to readily pass through cell membranes, and were
highly associated with the cytosol in a red blood cell up-
take assay. Compound 23b had a [Cell]free/[Extracellu-
lar] ratio of 1.80, while the ratio for 23c was 2.61.
Thus, compounds such as 23b, 25a, and 25b should be
amenable to use as probes in experiments designed to
uncover the various mechanisms whereby survivin exerts
its biological effects.
23c
23d
Me
Me
0.044
0.076
N
N
O
O
23e
Me
0.20
NBOC
NH
23f
24
Me
Me
Me
0.26
0.53
0.064
O
N
Though survivin has been implicated in both apoptosis
and cell cycle regulation, neither function has been associ-
ated with a specific binding event; while the BIR binding
site was initially assumed to play some role, that remains
to be confirmed. A related but separate question concerns
whether small molecules are likely to be found that can
bind with biologically meaningful affinity. We have iden-
tified a second potential binding site on the survivin sur-
face that appears to possess a far greater propensity for
small-molecule binding than the BIR site. In preliminary
experiments, these ligands do not exert the broad pheno-
typic consequences associated with survivin knock-down
through antisense or siRNA approaches (e.g., apoptosis,
polyploidy), though global ablation of survivin should be
expected to exert a greater effect than inhibition of
selected pathways. Nonetheless, further study of this
binding site using these or other ligands should lead to
better understanding of the biochemical roles of survivin.
O
25a
O
N
25b
25c
Me
Me
0.060
0.10
O
N
N
N
26a
26b
Me
Me
0.037
0.22

3128
M. D. Wendt et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3122–3129
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In addition, the structural studies of these protein–ligand
complexes provide a framework for interpreting bio-
chemical studies with putative binding partners. We hope
that the work presented here stimulates renewed interest
in determining the biochemical behavior of this difficult
but important target.
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Acknowledgments
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We thank Jennifer Garton for nephelometry measure-
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Supplementary data
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Experimental details of protein preparation, NMR and
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structure calculations; and representative curves for
the fluorescence assay. Supplementary data associated
with this article can be found, in the online version, at
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