Design, synthesis and evaluation of inhibitor of apoptosis protein (IAP) antagonists that are highly selective for the BIR2 domain of XIAP

Article abstract of DOI:10.1016/j.bmcl.2013.04.096

We recently reported the systematic ligand-based rational design and synthesis of monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Expanded structure-activity relationship (SAR) studies around these peptidomimetics led to compounds with significantly improved selectivity (>60-fold) for the BIR2 domain versus the BIR3 domain of XIAP. The potent and highly selective IAP antagonist 8q (ML183) sensitized TRAIL-resistant prostate cancer cells to apoptotic cell death, highlighting the merit of this probe compound as a valuable tool to investigate the biology of XIAP.

Full text of DOI:10.1016/j.bmcl.2013.04.096

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4253–4257
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of inhibitor of apoptosis protein
(IAP) antagonists that are highly selective for the BIR2 domain
of XIAP
Robert J. Ardecky a,b, , Kate Welsh a, , Darren Finlay a, Pooi San Lee a,b, Marcos González-López a,b
,
,
Santhi Reddy Ganji a,b, Palaniyandi Ravanan a, Peter D. Mace a, Stefan J. Riedl a, Kristiina Vuori a,b
John C. Reed a,b, Nicholas D. P. Cosford a,b,
⇑
a Program in Apoptosis and Cell Death, NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
b Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently reported the systematic ligand-based rational design and synthesis of monovalent Smac
mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Expanded struc-
ture–activity relationship (SAR) studies around these peptidomimetics led to compounds with signifi-
cantly improved selectivity (>60-fold) for the BIR2 domain versus the BIR3 domain of XIAP. The potent
and highly selective IAP antagonist 8q (ML183) sensitized TRAIL-resistant prostate cancer cells to apop-
totic cell death, highlighting the merit of this probe compound as a valuable tool to investigate the
biology of XIAP.
Received 25 January 2013
Revised 22 April 2013
Accepted 29 April 2013
Available online 14 May 2013
Keywords:
Apoptosis
XIAP
Ó 2013 Elsevier Ltd. All rights reserved.
Monovalent Smac mimetics
Probe compounds
With an estimated 12.1 million new cases of malignant disease
diagnosed every year resulting in approximately 7.6 million deaths
worldwide, cancer represents a significant public health concern
and a substantial burden on society.1 Despite progress in the last
several years toward the development of improved cancer medica-
tions, current therapeutic agents remain prone to intrinsic or ac-
is the most potent caspase inhibitor in the IAP family.6 XIAP con-
tains three ꢀ70 amino acid motifs termed baculovirus IAP repeat
(BIR) domains (designated 1, 2, and 3), with two of these BIRs
exhibiting specificity for different caspases. The BIR2 domain and
an adjacent linker peptide between BIR1 and BIR2 of XIAP combine
to mediate the two-site interaction with the apoptosis effector pro-
teases caspases-3 and -7, whereas the BIR3 domain of XIAP targets
the apoptosis initiator protease caspase-9.7
In the intrinsic cell death pathway, apoptotic signaling is regu-
lated by the mitochondrial protein Smac, an endogenous dimeric
proapoptotic antagonist of XIAP. The release of Smac from the in-
ter-membrane space of the mitochondria into the cytosol perpetu-
ates the apoptotic signal by competing with caspases for binding to
the BIR domains of XIAP.8 The design of peptidomimetics that bind
to the BIR3 domain of XIAP and in part mimic the activity of Smac
has been investigated by a number of research groups and compa-
nies that have developed antagonists of XIAP.4,9 For example, the
Genentech group recently reported the discovery and characteriza-
tion of the clinical candidate peptidomimetic GDC-0152 (1,
Fig. 1).10 While some success has been achieved using this ap-
proach, it has become clear that other strategies for alleviating
the repression of caspases by XIAP should also be viable therapeu-
tically. Since all apoptotic signaling, triggered via either the intrin-
sic or the extrinsic pathway, converges on caspases-3 or -7, it
would seem logical to develop XIAP inhibitors with high affinity
quired resistance, which is
a
major barrier to effective
treatment.2 The evasion of apoptosis (programmed cell death) by
tumorigenic cells is one of the defining hallmarks of cancer and
is an underlying cause of therapeutic resistance.3 The development
of therapeutic agents that correct defective apoptotic signaling and
allow cell death to proceed selectively in malignant cells is there-
fore an approach with great promise for the treatment of cancer.
The inhibitor of apoptosis proteins (IAPs) have attracted atten-
tion recently as potential targets for the development of new can-
cer therapeutics.4 Distinct members of the IAP family of
antiapoptotic proteins inhibit caspases, the group of intracellular
cysteine proteases which act as executioner enzymes by perform-
ing apoptosis in cells.5 Thus, inhibition of specific IAPs de-represses
caspases, allowing apoptosis to occur in malignant cells but not
normal cells. Human X-linked inhibitor of apoptosis protein (XIAP)
⇑
Corresponding author.
E-mail address: ncosford@sanfordburnham.org (N.D.P. Cosford).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.096

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R. J. Ardecky et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4253–4257
valine and the optimized C-terminal capping groups while varying
O
the substituents at P3 (Fig. 2). Modifications were also made to the
P1 substituents in some of the optimized compounds. The general
synthesis of the tripeptide analogs 8 is illustrated in Scheme 1.
Condensation of Boc-amino acid derivative 3 with amino acid
derivative 4, followed by acidic cleavage of the N-terminal Boc pro-
tecting group afforded dipeptide 5. Condensation of dipeptide 5
with the Boc-protected derivative 6 followed by hydrogenolysis
of the benzyl ester afforded the tripeptide acid 7. Finally, conden-
sation of 7 with the appropriate amine followed by removal of the
Boc group provided the target IAP inhibitors 8.13
All compounds synthesized were evaluated for their ability to
bind the BIR2 or BIR3 domains of XIAP by employing fluores-
cence-polarization (FP) competition assays.14 The results are ex-
pressed as competitive inhibition constants (Ki), derived from the
corresponding IC50 values by application of a mathematical equa-
tion developed by Wang and co-workers.15
We first synthesized and tested a series of analogs where
P1 = NMeAla, P2 = Val and R4 = 1,2,3,4-tetrahydronaphthyl, while
varying the P3 residue. The results are shown in Table 1. The spe-
cific amino acid residues that were incorporated into the tripeptide
scaffold were selected to maximize diversity and to probe the size
of the binding pocket in the P3 region of the protein. Thus, several
of the amino acids chosen for P3 contained long chain substituents,
some of which were hydrophilic and others with hydrophobic
characteristics. Compared with the lead structure 2c (P3 = Pro)
none of the new analogs had improved affinity for the BIR3 domain
of XIAP. For example, replacing Pro with Ala at P3 as in 8a led to a
reduction in potency of two-fold for BIR2 and 14-fold for BIR3 com-
pared with 2c. However, it is noteworthy that some analogs with
long chain residues at P3 (e.g., 8b, 8d, and 8g) bound with submi-
cromolar affinity to the BIR3 domain, including the ArgNO2 analog
H
N
O
N
H
N
Me
N
H
N
R4
Me
N
H
O
Me
N
H
O
Me
O
NH
O
2a R4 = NHPh
2b R4 = 1-naphthyl
2c R4 = tetrahydronaphthyl
S
1 GDC-0152
N
N
Figure 1. Tripeptide IAP antagonists.
for the BIR2 domain of XIAP. However, somewhat surprisingly
there have been few reports on the design and synthesis of com-
pounds that effect inhibition by binding to the BIR2 domain of
XIAP.11
We recently described the systematic ligand-based rational de-
sign and synthesis of new monovalent IAP antagonists that bind
preferentially to the BIR2 domain of XIAP.12 Using the AVPI motif
found at the N-terminus of Smac as an initial template, we devel-
oped the tripeptide model shown in Figure 2. We then synthesized
a series of analogs and determined their binding affinity for the
BIR2 or BIR3 domains of XIAP. In this initial study, we held con-
stant the N-methyl alanine moiety at the P1 position and the pro-
line residue at P3, while investigating the effects of varying the P2
position (R2) and the C-terminal substituent (R4). This led to the
discovery of compounds such as 2a (R4 = NHPh) and 2b (R4 = 1-
naphthyl) with preferential affinity for XIAP BIR2 versus BIR3 and
promising cell killing activity. We also confirmed that a tetrahy-
dronaphthyl C-terminal capping group (R4) confers selectivity for
BIR3 and potent cell killing activity, as observed for peptidomimet-
ic 2c. Herein we report our further investigation of the SAR around
this series of tripeptides leading to the discovery of novel com-
pounds with unique selectivity for the BIR2 domain of XIAP.
The present study is a continuation of work performed within
the Molecular Libraries Probe Production Centers Network
(MLPCN; http://mli.nih.gov/mli/mlpcn/) with the goal of develop-
ing novel XIAP BIR2-selective probes. In this phase of the study
we synthesized analogs that retained the P2 substituent (R2) as
8b, with a Ki value of 0.29
BIR3 versus BIR2. Interestingly, the BIR3 affinity for 8b (0.29
comparable with the clinical compound 1 (GDC-0152) (0.26
l
M at XIAP BIR3 and 12:1 selectivity for
M) is
M).
l
l
These results suggest that the BIR3 domain of XIAP tolerates rela-
tively bulky substituents in the P3 region.
We next investigated a set of analogs that retained P1 = NMe-
Ala, P2 = Val and introduced phenylhydrazine into the R4 position.
The results of varying the P3 residue among the analogs are shown
in Table 2. For compound 2a, where P3 = Pro, we had previously
found that a phenylhydrazine C-terminal capping group (R4) con-
fers selectivity for BIR2 of XIAP.12 In the new series, this trend
proved to hold true regardless of the P3 residue. In fact, the BIR2
versus BIR3 selectivity ratio of potencies for all the new com-
pounds was equal to or better than the 3:1 ratio exhibited by the
potent XIAP antagonist 2a. Furthermore, the BIR3 potency of the
P1
P2
P3
H
N
O
R2
R3
N
R
N
H
R4
O
R1
N
O
H
N-Terminus
C-Terminus
Figure 2. Tripeptide binding model.
R2
R2
O
O
H
N
a, b
H2N
BOC
OH
+
N
H
OBn
H2N
OBn
R3
O
R3
O
3
4
5
BOC O
N
a, c
R
OH
R1
6
R2
O
BOC O
N
R2
O
R4NH2
a, b
O
H
H
H
R4
N
N
R
N
R
N
H
OH
N
H
N
H
R1
R3
O
R1
R3
O
8
7
Scheme 1. General synthetic sequence for the synthesis of small molecule IAP antagonists. Reagents and conditions: (a) EDC, HOBt, N-methylmorpholine, DMF; (b) TFA,
CH2Cl2; (c) H2, Pd/C, MeOH.

R. J. Ardecky et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4253–4257
4255
Table 1
Binding affinities to BIR2 or BIR3 domains of XIAP for compound 1, and for analogs where R4 = 1,2,3,4-tetrahydronaphthyl (2c, 8a–g)
O
O
H
N
H
N
Me
N
H
N
H
O
Me
R3
8a-g
Compds
P3
BIR2 Ki,
l
Ma
BIR3 Ki,
l
Ma
BIR2 versus BIR3 selectivity
1
—
Pro
Ala
ArgNO2
Gln
LysCBZ
Orn
Ser
29.80 ( 4.60)
1.37 ( 0.11)
2.65 ( 0.15)
3.51 ( 0.40)
10.77 ( 1.70)
4.30 ( 0.18)
4.36 ( 0.18)
4.57 ( 0.38)
2.75 ( 0.07)
0.26 ( 0.015)
0.06 ( 0.011)
0.78 ( 0.11)
0.29 ( 0.03)
2.02 ( 0.01)
0.67 ( 0.04)
1.13 ( 0.06)
2.05 ( 0.11)
0.53 ( 0.01)
1:115
1:23
1:3.4
1:12
1:5.3
1:6.4
1:3.9
1:2.2
1:5.2
2c
8a
8b
8c
8d
8e
8f
8g
OrnCBZ
a
Values are determined in duplicate and reported standard deviation which is given in parentheses.
Table 2
Binding affinities to BIR2 or BIR3 domains of XIAP for tripeptide analogs where R4 = NHPh (2a, 8h–p)
O
O
H
N
H
N
H
N
Me
N
H
N
H
O
Me
R3
8h-p
Compds
P3
BIR2 Ki,
l
Ma
BIR3 Ki,
l
Ma
BIR2 versus BIR3 selectivity
2a
8h
8i
8j
8k
8l
8m
8n
8o
8p
Pro
Ala
Val
ArgNO2
Arg
Thr
OrnCBZ
Orn
Ser
Lys
0.39 ( 0.17)
2.29 ( 0.37)
3.04 ( 0.28)
1.18 ( 0.11)
3.65 ( 0.23)
4.67 ( 0.37)
1.70 ( 0.13)
13.14 ( 1.19)
5.46 ( 0.56)
1.88 ( 0.13)
1.08 ( 0.06)
51.79 ( 2.16)
65.62 ( 6.22)
8.79 ( 1.11)
21.15 ( 1.77)
30.07 ( 0.55)
9.82 ( 1.17)
ꢀ39
>39
21.48 ( 1.52)
2.8:1
22.6:1
21.6:1
7.5:1
5.8:1
6.4:1
5.8:1
3:1
>7.1:1
11.4:1
a
Values are determined in duplicate and reported standard deviation which is given in parentheses.
compounds was poor, with none of them exhibiting Ki values bet-
ter than 8 lM. Although the BIR2 Ki value of 0.39 lM for 2a was
not improved upon, the selectivity of several compounds for BIR2
was significantly better. For example, the next most potent com-
60-fold selective for BIR2 over BIR3. Interestingly, the correspond-
ing analog in the R4 = N-phenylhydrazine series (8x, Table 3) did
not quite exhibit the same BIR2 potency enhancement compared
with the parent compound (8h, Table 2). Analogs of 8t in which
R1 = CH2OH (8u) or R1 = Et (8v) were also highly selective for
BIR2, although less potent. An analog of 8q in which R4 = naphtha-
len-1-ylmethanamine (8w) retained the BIR2 selectivity of the par-
pounds in this series were 8j (BIR2 Ki = 1.18
lM) and 8p (BIR2
Ki = 1.88 M) with ArgNO2 and Lys residues at P3, respectively.
l
The selectivity of these compounds was encouraging, with BIR2:-
BIR3 ratios of 7.5 and 11.4 to 1, respectively. Of particular note
were the data for 8h (P3 = Ala) and 8i (P3 = Val) with BIR2 Ki values
ent compound but also with reduced BIR2 potency (Ki = 3.00 lM).
Overall, the combination of P3 = Ala and R4 = 1-naphthyl provides
XIAP antagonists with excellent BIR2 selectivity and good in vitro
potency. Despite the excellent potency and selectivity of 8t, unfor-
tunately when tested in a cellular assay 8t did not show any activ-
ity, presumably due to a lack of cell permeability (see Fig. S1 in the
Supplementary data). We therefore proceeded with additional
characterization of 8q as the optimal probe for studies in cells.
Based on these encouraging results, we then sought to explore
whether the selectivity of compound 8q for the BIR2 domain of
XIAP might be explained by the relative structural properties of
the BIR2 and BIR3 domains. Thus we modeled the binding of 8q
onto the Smac binding groove of a BIR2 domain. We chose the
structure of XIAP BIR2 from our earlier structure of the caspase-
3–BIR2 complex.7 In addition to being the only available crystal
structure of a XIAP/cIAP BIR2 domain, this structure also depicts
‘Smac-like’ binding by the N-terminus of the caspase-3 small sub-
unit to XIAP BIR2 (Fig. 3A).7 Using this interaction, we overlaid the
structure of 8q into the Smac-binding groove, conserving the
of 2.29 lM and 3.04 lM, respectively, and an impressive BIR2:BIR3
selectivity ratio of more than 20:1. Taken together, these data sug-
gest that analogs in which P3 = Ala or Val exhibit favorable selec-
tivity for XIAP BIR2 versus BIR3.
The data for our final iteration of analogs are shown in Table 3.
In this series we initially synthesized analogs of the potent XIAP
antagonist 2b, in which R4 = 1-naphthyl, by varying the P3 substi-
tuent. As noted above, we previously found that compound 2b
(with P3 = Pro) is a potent inhibitor of XIAP BIR2 but the BIR2:BIR3
selectivity ratio is only 3.4:1. Using the knowledge gained from the
series with R4 = NHPh (Table 2), we prepared analogs with P3 = Ala
(8q), Val (8r) or OrnCBZ (8s). Gratifyingly, we found that all three
analogs were selective for BIR2, and essentially inactive against
BIR3 with Ki values >39
ited very respectable potency against XIAP BIR2 with Ki = 1.74
Remarkably, the analog 8t in which P1 = Ala (R = H) rather than
NMeAla is highly potent against BIR2 (Ki = 0.64 M) and more than
l
M. In particular, compound 8q16 exhib-
M.
l
l

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R. J. Ardecky et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4253–4257
Table 3
Binding affinities to BIR2 or BIR3 domains of XIAP for tripeptide analogs 2b and 8q–8y
O
H
O
H
N
N
R4
R
N
H
N
H
O
R1
R3
8
Compds
R
R1
P3
R4
BIR2 Ki,
l
Ma
BIR3 Ki,
1.51 ( 0.13)
>39
>39
>39
40.15 ( 2.69)
>39
>39
l
Ma
BIR2 versus BIR3 selectivity
2b
8q
8r
8s
8t
8u
8v
8w
8x
8y
CH3
CH3
CH3
CH3
H
H
H
CH3
H
CH3
CH3
CH3
CH3
CH3
Pro
Ala
Val
OrnCBZ
Ala
Ala
Ala
Ala
Ala
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
CH2-1-naphthyl
NHPh
NH-3-FPh
0.44 ( 0.02)
1.74 ( 0.30)
4.54 ( 0.23)
1.94 ( 0.11)
0.64 ( 0.01)
2.43 ( 0.25)
3.78 ( 0.37)
3.00 ( 0.40)
1.83 ( 0.19)
2.38 ( 0.28)
3.4:1
>22.4:1
>8.6:1
>20.1:1
62.7:1
>16:1
>10.3:1
11.4:1
22.6:1
>16.4:1
CH3
CH2OH
CH2CH3
CH3
CH3
CH3
34.18 ( 2.15)
41.32 ( 0.86)
>39
Val
a
Values are determined in duplicate and reported standard deviation which is given in parentheses.
Figure 4. TRAIL-resistant PPC-1 cells challenged with TRAIL, or compounds 2a–c or
8q (5 M) alone or in the presence of TRAIL.
l
moiety of 8q allows a similar backbone conformation in P3 (Fig. 3B).
Secondly, the model (Fig. 3B) visualizes that the 1-naphthyl ring is
positioned between the aliphatic stems of Lys206 and Lys208 with
a plausible positive effect on the interaction of 8q with the BIR2 do-
main. These residues correspond to glycine and threonine residues,
respectively, in the BIR3 domain of XIAP, thus negating a potentially
favorable interaction.
We next determined the activity of the new inhibitors in a dis-
ease-relevant cellular assay. In particular, the ability to sensitize
TRAIL-resistant cells to apoptosis was investigated. Thus, PPC-1
Figure 3. Model of compound 8q binding to XIAP BIR2. (A) Smac-like binding to
BIR2 of XIAP (according to PMID 11257232; PDB 1i3o). Residues 310–312 from a
neighboring Caspase-3 molecule are presented in stick form (orange) binding to the
Smac-binding groove of BIR2. (B) An equivalent view of the model of 8q (green)
bound to XIAP BIR2. Residues that potentially contribute to the selectivity of 8q are
represented as spheres (C = gray, N = blue).
backbone geometry of residues 310–312 from the N-terminus of
the caspase-3 small subunit. Conformational freedom of the 1-
naphthyl ring permitted by the surrounding protein was explored
followed by energetic minimization of the compound, and the low-
est energy result is displayed in Figure 3B.
This model suggests several factors that could contribute to the
BIR2 selectivity of 8q. Firstly, as already visualized in the contact
from the caspase-3 BIR2 structure, a small nonproline entity is
highly compatible with P3-binding adjacent to His223 of BIR2
(Fig. 3). In the template caspase-3/BIR2 contact structure (Fig. 3A)
this residue is valine, and recent publications suggest an alanine
in this position to be favorable for BIR2 selectivity.17,18 The alanine
prostate cancer cells were exposed to each compound (5 lM) for
4 h, followed by the addition of various concentrations of TRAIL,
and incubated for 20 h.19 Cell viability was then assessed using
ATP content as a readout (ATPlite). As shown in Figure 4, the cell
penetrant IAP antagonists 2a–c or 8q all act synergistically with
TRAIL to reduce the number of viable cells to much lower levels
than those observed with TRAIL alone. The LD50 values for TRAIL
in the presence of the compounds 2b, 2c, or 8q (5
lM) are in the
0.073–0.089 nM range. Compound 2b (5 M) also acts synergisti-
l
cally with various concentrations of TNF to induce cell death in
MDA-MB-231 cells (see Fig. S2 in the Supplementary data). To
evaluate the effects in normal cells, compounds 2b and 8q were

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Figure 5. Normal human foreskin fibroblasts (HFF) challenged with TRAIL, or
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l
tested against human foreskin fibroblasts (HFF) (Fig. 5). No toxicity
was observed, even in the presence of TRAIL (100 ng/mL). Further-
more, 8q showed no toxicity against Fa2N-4 immortalized human
12. González-Lopez, M.; Welsh, K.; Finlay, D.; Ardecky, R. J.; Ganji, S. R.; Su, Y.;
Yuan, H.; Teriete, P.; Mace, P. D.; Riedl, S. J.; Vuori, K.; Reed, J. C.; Cosford, N. D.
P. Bioorg. Med. Chem. Lett. 2011, 21, 4332.
hepatocytes (LC50 > 50 l
M).20 Taken together, these data suggest
13. All synthesized compounds were purified by either medium pressure silica gel
chromatography or preparative HPLC using
a XBridge Prep C18 column
that our new compounds are cell penetrant and exhibit effects
on prostate cancer cells consistent with their observed in vitro
activity as IAP antagonists.
(19 Â 100 mm) eluted with 0.05% formic acid in a methanol-water gradient
of 10:90 to 100:0 over 10 to 15 min. with a flow rate of 25 mL/min. Compound
purity was determined using analytical HPLC/MS and NMR (1H and 13C). The
yields for the first two steps ranged from 70–90% while the yields for the last
step ranged from 50–90%. The purity of each final product was greater than
95% as determined by HPLC/MS analysis.
In conclusion, by using a ligand-based rational design approach,
we have systematically optimized small molecule monovalent IAP
antagonists that inhibit XIAP by binding selectively to the BIR2 do-
main without interacting with the BIR3 domain at detectable lev-
els. The in vitro probe compound 8t binds to the BIR2 domain
with submicromolar affinity and >60-fold selectivity for BIR2 over
BIR3. In addition, the XIAP inhibitory potency observed in vitro
translates into cell killing activity in prostate cancer cells for the
cell penetrant probe 8q. Consistent with our previous observations
with IAP antagonists,21 the highly BIR2 selective, cell penetrant
analog 8q showed no toxicity as a single agent (Figs. 4 and 5 and
S1) but rather sensitized the cells to TRAIL-induced apoptosis.
Our results suggest that these compounds are potentially useful
tools for probing apoptosis signaling pathways in cells. Further
optimization of these compounds towards the discovery of potent
and selective drug-like analogs is currently in progress.
14. Fluorescence polarization assays were run in 20 lL volume in 384 well format
with black plates. The assay solution was 25 mM Hepes at pH 7.5/1 mM tris(2-
carboxyethyl)phosphine/0.005% Tween 20 and 20 nM AVPIAQK-rhodamine.
XIAP BIR2 was present at 1
within a range of 100 M–6.1 nM using two fold dilutions starting at 100
generating a 16 point curve. Plates were read on an Analyst HT in fluorescence
polarization mode with excitation at 530 nm, emission at 580 nm and
lM or XIAP BIR3 at 0.2 lM. Compound was present
l
lM
a
dichroic mirror at 565 nm. Resulting data in mP was fit in GraphPad Prism 5
with a nonlinear regression using a sigmodial curve with variable slope.
15. Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Roller, P.
P.; Krajewski, K.; Saito, N.; Stuckey, J.; Wang, S. Anal. Biochem. 2004, 332, 261.
16. Spectroscopic data for compound 8q: 1H NMR (400 MHz, DMSO-d6) d 9.99 (1H,
s), 8.80 (1H, b), 8.60 (1H, d, J = 8.5 Hz), 8.38 (1H, d, J = 8.5 Hz), 7.75 (1H, m), 7.59
(1H, m), 7.52 (1H, d, J = 5.9 Hz), 7.51 (1H, m), 7.50 (4H, br m), 4.71 (1H, m), 4.34
(1H, m) 3.74 (1H, m), 3.64 (1H, b), 2.05 (1H, m), 1.40 (3H, d, J = 5.9 Hz), 1.35
(3H, d, J = 5.9 Hz), 0.88 (6H, m), 13C NMR (100 MHz, DMSO-d6) d 171.8, 170.2,
168.5, 133.7, 133.3, 128.1, 127.9, 126.0, 125.8, 122.7, 121.6, 57.5, 56.0, 48.9,
30.7, 19.1, 17.9, 15.8. Exact mass for chemical formula: C22H30N4O3 398.23 [M],
HR-MS (ESI) 399.30 [M+H].
17. Speer, K. F.; Cosimini, C. L.; Splan, K. E. Biopolymers 2012, 98, 122.
18. Eckelman, B. P.; Drag, M.; Snipas, S. J.; Salvesen, G. S. Cell Death Differ. 2008, 15,
920.
Acknowledgments
19. Cell viability was assessed using the ATPlite 1step Luminescence Assay System
(PerkinElmer Inc., Waltham, MA). Briefly, cells were seeded at 2000 cells/well
This work was supported by NIH grants HG005033 and
R01CA163743 to J.C.R., and R01AA017238 to S.J.R. The authors
thank Andrey Bobkov for expert technical assistance. M.G.L.
in 38
(1 L) was added to a final concentration of 5
at 37 °C for 4 h. TRAIL (1 L) was then added to the desired final concentration
and the cells again incubated at 37 °C for 20 h. The plates were removed to
room temperature for 30 min. before addition of one half volume (20 L) of
l
L medium with 5% FBS and allowed to attach overnight. Test compound
l
lM and the cells were incubated
l
acknowledges Fundación Ramón Areces for
fellowship.
a postdoctoral
l
ATPlite reagent. The plates were shaken at 1100 rpm for 2 min to ensure
proper mixing before luminescence measurement on a BMG POLARstar Omega
plate reader. PPC-1 cells were maintained in RPMI 1640 (Cellgro, Manassas,
Supplementary data
VA) with 10% (v/v) FBS (HyClone, Logan, UT), and penicillin/streptomycin/
glutamine (Life Technologies, Carlsbad, CA).
L-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
096.
20. Lopez, M.; Welsh, K.; Yuan, H.; Stonich, D.; Su, Y.; Garcia, X.; Cuddy, M.;
Houghten, R.; Sergienko, E.; Reed, J. C.; Ardecky, R.; Reddy, S.; Finlay, D.; Vuori,
K.; Dad, S.; Chung, T. D. Y.; Cosford, N. D. P. Antagonists of IAP-family anti-
apoptotic proteins—Probe 2. In Probe Reports from the NIH Molecular Libraries
Program [Internet]; National Center for Biotechnology Information (US);
Bethesda, MD, 2010–2009.
References and notes
21. Vamos, M.; Welsh, K.; Finlay, D.; San Lee, P.; Mace, P. D.; Snipas, S. J.; Gonzalez,
M. L.; Reddy Ganji, S.; Ardecky, R. J.; Riedl, S. J.; Salvesen, G. S.; Vuori, K.; Reed, J.
C.; Cosford, N. D. P ACS Chem. Biol. 2013, 8, 725.
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