Structure-based design, synthesis, and evaluation of the biological activity of novel phosphoroorganic small molecule IAP antagonists

Article abstract of DOI:10.1007/s10637-020-00923-4

One of the strategies employed by novel anticancer therapies is to put the process of apoptosis back on track by blocking the interaction between inhibitor of apoptosis proteins (IAPs) and caspases. The activity of caspases is modulated by the caspases themselves in a caspase/procaspase proteolytic cascade and by their interaction with IAPs. Caspases can be released from the inhibitory influence of IAPs by proapoptotic proteins such as secondary mitochondrial activator of caspases (Smac) that share an IAP binding motif (IBM). The main purpose of the present study was the design and synthesis of phosphorus-based peptidyl antagonists of IAPs that mimic the endogenous Smac protein, which blocks the interaction between IAPs and caspases. Based on the structure of the IAP antagonist and recently reported thiadiazole derivatives, we designed and evaluated the biochemical properties of a series of phosphonic peptides bearing the N-Me-Ala-Val/Chg-Pro-OH motif (Chg: cyclohexylglycine). The ability of the obtained compounds to interact with the binding groove of the X-linked inhibitor of apoptosis protein baculovirus inhibitor of apoptosis protein repeat (XIAP BIR3) domain was examined by a fluorescence polarization assay, while their potential to induce autoubiquitination followed by proteasomal degradation of cellular IAP1 was examined using the MDA-MB-231 breast cancer cell line. The highest potency against BIR3 was observed among peptides containing C-terminal phosphonic phenylalanine analogs, which displayed nanomolar K_i values. Their antiproliferative potential as well as their proapoptotic action, manifested by an increase in caspase-3 activity, was examined using various cell lines.

Full text of DOI:10.1007/s10637-020-00923-4

Investigational New Drugs
https://doi.org/10.1007/s10637-020-00923-4
PRECLINICAL STUDIES
Structure-based design, synthesis, and evaluation of the biological
activity of novel phosphoroorganic small molecule IAP antagonists
Agnieszka Łupicka-Słowik1 & Mateusz Psurski2 & Renata Grzywa1 & Monika Cuprych2 & Jarosław Ciekot2
&
Waldemar Goldeman1 & Elżbieta Wojaczyńska3 & Jacek Wojaczyński4 & Józef Oleksyszyn1 & Marcin Sieńczyk1
Received: 29 January 2020 /Accepted: 9 March 2020
#
The Author(s) 2020
Summary
One of the strategies employed by novel anticancer therapies is to put the process of apoptosis back on track by blocking the
interaction between inhibitor of apoptosis proteins (IAPs) and caspases. The activity of caspases is modulated by the caspases
themselves in a caspase/procaspase proteolytic cascade and by their interaction with IAPs. Caspases can be released from the
inhibitory influence of IAPs by proapoptotic proteins such as secondary mitochondrial activator of caspases (Smac) that share an
IAP binding motif (IBM). The main purpose of the present study was the design and synthesis of phosphorus-based peptidyl
antagonists of IAPs that mimic the endogenous Smac protein, which blocks the interaction between IAPs and caspases. Based on
the structure of the IAP antagonist and recently reported thiadiazole derivatives, we designed and evaluated the biochemical
properties of a series of phosphonic peptides bearing the N-Me-Ala-Val/Chg-Pro-OH motif (Chg: cyclohexylglycine). The ability
of the obtained compounds to interact with the binding groove of the X-linked inhibitor of apoptosis protein baculovirus inhibitor
of apoptosis protein repeat (XIAP BIR3) domain was examined by a fluorescence polarization assay, while their potential to
induce autoubiquitination followed by proteasomal degradation of cellular IAP1 was examined using the MDA-MB-231 breast
cancer cell line. The highest potency against BIR3 was observed among peptides containing C-terminal phosphonic phenylal-
anine analogs, which displayed nanomolar Ki values. Their antiproliferative potential as well as their proapoptotic action,
manifested by an increase in caspase-3 activity, was examined using various cell lines.
.
.
.
Keywords IAP antagonist Smac mimetic Apoptosis Phosphoroorganic
Introduction
Apoptosis, a process of programmed cell death, is a funda-
mental mechanism under precise homeostatic control. It as-
sures a delicate balance between cell survival and death,
which is crucial for embryogenesis as well as for sustaining
a constant number of mature cells while preventing the spread
of infectious diseases. Suppression of apoptosis is utilized by
cancer cells for their uncontrolled proliferation and is ob-
served during the development of autoimmune diseases [1,
2]. Conversely, excessive apoptosis may be linked to various
neurodegenerative disorders and diabetes [3, 4].
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10637-020-00923-4) contains supplementary
material, which is available to authorized users.
* Marcin Sieńczyk
marcin.sienczyk@pwr.edu.pl
1
Faculty of Chemistry, Department of Organic and Medicinal
Chemistry, Wrocław University of Science and Technology,
Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
2
Department of Experimental Oncology, Hirszfeld Institute of
There are two pathways that lead to the activation of apo-
ptosis. The extrinsic pathway is initiated through the activation
of death receptors that belong to the TNF receptor superfamily,
such as tumor necrosis factor (TNF)-related apoptosis-
inducing ligand (TRAIL) or Fas. The signal is then transmitted
via Fas-associated death domain protein (FADD)-dependent
activation of caspase-8 and caspase-10, which in turn
Immunology and Experimental Therapy, Polish Academy of
Sciences, Weigla 12, 53-114 Wrocław, Poland
3
Faculty of Chemistry, Department of Physical and Quantum
Chemistry, Wrocław University of Science and Technology,
Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
4
Department of Chemistry, University of Wrocław, F. Joliot-Curie 14,
50-383 Wrocław, Poland

Invest New Drugs
proteolytically process executioner caspase-3 and caspase-7,
leading to apoptosis [5–8]. The intrinsic (mitochondrial) path-
way is activated by cell stress, such as DNA damage, cytoskel-
etal disruption, accumulation of unfolded proteins, hypoxia or
metabolic stress, which results in permeabilization of the outer
mitochondrial membrane [9–11]. As a consequence, mito-
chondrial intermembrane cytochrome c and secondary mito-
chondrial activator of caspases (Smac) proteins are released
into the cytosol. Cytochrome c is involved in the formation
of the apoptosome followed by the activation of caspase-9
and, finally, caspase-3 and caspase-7, whereas the Smac pro-
tein binds to the X-linked inhibitor of apoptosis protein
(XIAP), which liberates the caspases from inhibitor control [8].
XIAP is a member of the family of inhibitor of apoptosis
proteins (IAPs) that are considered to be negative regulators of
caspases and cell death [12]. Among the human IAPs, eight
different proteins have been distinguished: neuronal apoptosis
inhibitory protein (NAIP/BIRC1), cellular IAP1 (cIAP1/
BIRC2), cellular IAP2 (cIAP2/BIRC3), survivin (BIRC5),
BIR-containing ubiquitin-conjugating enzyme (BRUCE/
Apollon/BIRC6), melanoma IAP (ML-IAP/BIRC7), IAP-
like protein 2 (ILP2/BIRC8) and X-chromosome-linked IAP
(XIAP/BIRC4) [13]. The structures of IAPs are characterized
by the presence of at least one zinc-binding baculoviral do-
main (baculovirus inhibitor of apoptosis protein repeat, BIR),
which is essential for their antiapoptotic activity [14, 15].
Additionally, some IAPs contain a really interesting new gene
(RING) finger domain that promotes ubiquitination of IAPs
and other associated proteins, a ubiquitin (Ub)-associated do-
main (UBA) capable of binding the poly-Ub chains and a
conserved caspase recruitment domain (CARD) [16]. The
structural organization of human IAP proteins is the subject
of several excellent reviews [17–21].
Fig. 1 The interaction network between the N-terminal tetrapeptide (Ala-
Val-Pro-Ile) of Smac and the XIAP BIR3 domain (based on the NMR
data) [30] (a) and representation of the peptide-binding groove of the
BIR3 domain containing the bound N-terminal AVPI motif of Smac
(based on 1G73.pdb) (b) [31]
Increased expression of XIAP has been observed during
the neoplastic processes of prostate cancer [22], non-small-
cell lung carcinoma [23, 24], acute myeloid leukemia [25]
and acute mixed lineage leukemia [26]. In addition, the
XIAP expression level correlates with tissue resistance to che-
motherapeutic agents [27]. The XIAP protein contains three
different BIR domains (BIR1-BIR3), a C-terminal RING do-
main and a UBA domain. The interdomain fragments of BIR1
and BIR2 together with the BIR2 domain act as an inhibitor of
caspase-3 and caspase-7, whereas the BIR3 domain is able to
inhibit caspase-9, preventing the dimerization of caspase-9
[28, 29]. The inhibitory activity of the BIR3 domain can be
diminished by Smac released into the cytosol. The key role in
molecular recognition between the XIAP BIR3 domain and
Smac is played by the N-terminal fragment (AVPI) of the
Smac protein [30]. The fundamentals of these complex inter-
actions have been determined by crystal structure analysis
[31] and NMR spectroscopy [30] (Fig. 1).
currently being tested in clinical trials for cancer treatment [32,
33]. Monovalent compounds mimic the binding of a single
AVPI motif to BIR domains, whereas bivalent mimetics are
composed of two such binding units. Although bivalent an-
tagonists are more potent than monovalent compounds, their
pharmacological profile, including bioavailability, is less fa-
vorable [34].
Work in our laboratory has focused on the design and syn-
thesis of monovalent phosphoroorganic-based Smac analogs
as antagonists of the XIAP protein. These analogs mimic the
interaction of the endogenous AVPI binding motif of Smac
with the BIR3 domain. Our attempts to introduce
phosphoroorganic function into the IAP antagonist scaffold
were based on previously reported structures (Fig. 2) [35–41].
Herein, we report the design, synthesis and biological ac-
tivity of novel peptidyl derivatives of phosphine oxides and α-
aminoalkylphosphonic acid esters as antagonists of the XIAP
BIR3 domain. The ability of these compounds to interact with
the binding groove of the XIAP BIR3 domain was evaluated
via a fluorescence polarization assay. Based on the
Several potential therapeutics and therapy-supportive
agents acting as monovalent or bivalent Smac mimetics are

Invest New Drugs
Fig. 2 Chemical structures of
previously reported monovalent
Smac mimetics with therapeutic
potential: AT-406/SM-406 [35],
LCL-161 [36, 37], GDC-0152
[38, 39], and GDC-0917/CUDC-
427 [40, 41]
fluorescence polarization data, we selected the synthesized
phosphoroorganic derivative compounds that displayed the
most favorable kinetic parameters and further examined their
ability to induce cellular cIAP1 autoubiquitylation and
proteasomal degradation. Additionally, we ascertained their
antiproliferative activity as well as their proapoptotic potential
in a chemoresistant, highly aggressive breast cancer cell line.
compounds with endogenous Val or Chg derivatives, although
compounds with a larger side chain at this position might
exhibit drug-like properties because of the improved proteo-
lytic stability of the peptide bond between Chg and Pro [38].
Contained within the XIAP BIR3 domain is a hydrophobic
pocket between the aliphatic region of the Lys297 and Lys299
side chains and Leu292, Gly306 and Thr308 that enables in-
teractions with aromatic rings. This area is responsible for
interactions with the Ile side chain in the Ala-Val-Pro-Ile se-
quence of Smac and is the region in which we decided to
introduce a phosphoroorganic moiety. The data obtained by
Flygare et al. revealed that the presence of an aromatic ring at
this position rather than on the side chain of the endogenously
occurring isoleucine is preferred [38]. The presented data in-
dicated that a phosphoroorganic, bulky moiety introduced to
the N-Me-Ala-Val/Chg-Pro-OH scaffold can be an alternative
to the endogenous Ile residue. The designed and synthesized
structures of aliphatic and aromatic phosphine oxide
peptidomimetics, as well as phosphonate derivatives, are
schematically shown in Fig. 3.
The first step of the present study was the preparation of the
peptidyl scaffolds (N-Me-Ala-Val-Pro-OH (1) and N-Me-Ala-
Chg-Pro-OH (2)) that would then be used for further synthesis
of the antagonists; the tetrapeptide H2N-Ala-Val-Pro-Trp-OH
(3), which was used as a reference compound for the fluores-
cence polarization competition assay; and the fluorescent
probe H2N-Ala-Val-Pro-Dpm-Ala-Lys(5(6)-Fam)-Lys-NH2
(4). All peptides were synthesized manually on a solid sup-
port. The N-terminal methyl group was introduced into the
H2N-Ala-Val/Chg-Pro-OH peptides via the Mitsunobu reac-
tion [47] with o-nitrobenzenesulfonyl as the protecting group
and MeOH as the methyl donor yielding N-Me-Ala-Val-Pro-
OH (1) and N-Me-Ala-Chg-Pro-OH (2), which were further
Results and discussion
Based upon the structure of the endogenous IAP antagonist
Smac and the recently discovered thiadiazole derivatives
GDC-0152 [38] and LCL-161 [42], we introduced various
C-terminal phosphoroorganic functionalities into N-Me-Ala-
Val/Chg-Pro-OH scaffolds (Chg: cyclohexylglycine). As
shown in previous studies, the methyl group of the Ala residue
fits into the hydrophobic pocket formed by the Leu131,
Trp134 and Glu143 side chains of ML-IAP, and because of
steric limitations, it is difficult to replace with more complex
substituents [38]. The amino group of Ala interacts with the
carboxylates of Asp138 and Glu143 in ML-IAP, Glu314 in
XIAP and Asp320 and Glu325 in cIAP1 [38, 43, 44]. N-meth-
ylation of the N-terminal Ala leads to increased cellular stabil-
ity of these peptidyl compounds without reducing affinity for
the BIR domain [45] and is one of the strategies used for
improving pharmacokinetic parameters of potential
intercalators [46]. The proline residue assures the most favor-
able orientation of Ala; however, the Chg-Pro dipeptide toler-
ates some level of structural variation. The Chg/Val side chain
has a bulky character, and the rotation of this residue is re-
stricted by Pro. According to Flygare et al., there is little dif-
ference in the binding affinity between corresponding

Invest New Drugs
Fig. 4 Determination of the binding affinity of fluorescent probe 4 and
the XIAP BIR3 domain. The assay was performed at a constant
fluorescent probe concentration (2 nM) and serially diluted XIAP BIR3
domain (ranging from 2.5 µM to 0.0763 nM)
Fig. 3 General structure of phosphoroorganic peptide derivatives
designed as potential IAP antagonists
of IAP family proteins [50], and control tripeptides 1 and 2,
which did not interact with the XIAP BIR3 domain at a sig-
nificant level. The selection of H2N-Ala-Val-Pro-Trp-OH as a
scaffold peptide instead of H2N-Ala-Val-Pro-Ile-OH (which
endogenously binds to the BIR domain as the Smac N-termi-
nal part) was dictated by its higher binding affinity to the BIR
domain despite its low biological activity in cell-based assays
[50, 51]. The kinetic parameters (EC50 and Ki) of the obtained
phosphoroorganic derivatives were determined by a fluores-
cence polarization assay performed at constant concentrations
of the XIAP BIR3 domain (50 nM) and fluorescent probe 4 (2
nM), while the concentrations of potential antagonists ranged
from 50 µM to 0.640 nM (for screening purposes) or from 50
µM to 0.0537 nM (for detailed analysis). The fluorescence
polarization results were used for the determination of the Z´
factor, which provides information regarding the dynamic
range and data quality (Z´ = 0.737; Supplementary
Figure S1, Supplementary Materials). Most of the
phosphoroorganic compounds exhibited the ability to interact
with a binding groove on the surface of the XIAP BIR3 do-
main at a concentration of 50 µM, as manifested by the inhi-
bition of the protein-probe interaction. Although the obtained
data make it difficult to distinguish which R1 substituent struc-
ture (isopropyl or cyclohexyl, Fig. 3) has the greatest impact
on the organophosphorus derivative affinity toward the target
protein, slightly higher activity was observed for compounds
containing a cyclic R1 ring (Tables 1 and 2). Exceptions may
include compounds 41 and 42, which were found to be the
most active derivatives obtained in this study. One possible
explanation for the increased activity of these two compounds
could be that the phosphonic phenylalanine can influence the
ability of the N-terminal residue of the peptide to interact with
the binding groove of the BIR domain.
modified with various phosphoroorganic moieties.
Modification of the peptide probe with fluorescein was
achieved through selective removal of the 4-methyltrityl
protecting group (Mtt) [48] followed by fluorescein conjuga-
tion to the ε-amino group of Lys, yielding the peptide H2N-
Ala-Val-Pro-Dpm-Ala-Lys(5(6)-Fam)-Lys-NH2 (4) required
for fluorescence polarization assays.
Different phosphoroorganic derivatives were designed and
synthesized for introduction as the C-terminal functionality of
the N- m e t h y l a t e d pe p t i d e s c a ff o l d s . Th e α -
aminoalkylphosphine oxides were obtained from tritylimines
and phosphine oxides followed by a trityl group deprotection,
while the diaryl esters of α-aminoalkylphosphonic acids were
synthesized via the α-amidoalkylation reaction described by
Oleksyszyn [49].
A fluorescence polarization assay was used to examine the
ability of the obtained compounds to interact with the binding
groove located on the surface of the XIAP BIR3 domain. First,
the conditions of the assay were optimized, and the Kd value
for the protein-probe interaction was determined. The binding
assay was performed with serial dilutions of the XIAP BIR3
domain (2.5 µM to 0.0763 nM) and fixed concentrations of
fluorescent probe 4 (2 nM). We determined the affinity of the
fluorescent probe and the XIAP BIR3 domain (Kd = 49.85
5.37 nM) with a dynamic range of ΔmP = 196.2 4.5 (Fig. 4).
We also analyzed the interaction of the synthesized
phosphoroorganic peptide derivatives with the binding groove
of the BIR3 domain. As reference compounds, commercially
available XIAP antagonists GDC-0152 and LCL-161 were
used together with tetrapeptide H2N-Ala-Val-Pro-Trp-OH
(3), which is characterized by its high affinity to BIR domains

Invest New Drugs
Table 1 Kinetic parameters of α-aminoalkylphosphine oxide peptidyl derivatives
No.
Structure
log EC50 [µM]
EC50
[µM]
Ki
[µM]
% Inh
(50 µM)
R 1
R 2
R 3
5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-H
-C(CH3)3
1.471 0.043
1.299 0.044
-
29.58
19.90
-
14.68
9.87
-
63.2
72.0
32.0
41.3
25.9
24.2
63.6
67.1
100.0
100.0
61.6
57.0
74.0
62.0
66.2
50.0
74.4
59.2
6
-H
-C(CH3)3
7
-H
-CH(CH3)2
-CH(CH3)2
-CH(CH2)5
-CH(CH2)5
-C6H5
8
-H
-
-
-
9
-H
-
-
-
10
11
12
13
14
15
16
17
18
19
20
21
22
-H
-
-
-
-H
1.468 0.055
1.420 0.051
0.186 0.080
0.118 0.059
1.489 0.045
1.606 0.045
1.285 0.041
1.466 0.042
1.448 0.044
1.681 0.042
1.353 0.032
1.511 0.051
29.33
26.29
1.53
1.31
30.83
40.32
19.25
29.27
28.03
47.96
22.54
32.46
14.55
13.04
0.75
0.64
15.30
20.01
9.55
14.52
13.91
23.80
11.18
16.11
-H
-C6H5
-CH2(C6H5)
-C6H5
-CH2(C6H5)
-C6H5
-H
-C6H4-4-CH3
-C6H4-4-CH3
-C6H4-4-CH3
-C6H4-2-CH3
-C6H4-2-CH3
-C6H3-3,5-(CH3)2
-C6H3-3,5-(CH3)2
-C6H3-3,5-(CH3)2
-H
-CH3
-H
-H
-H
-H
-CH3
Among the obtained peptidyl derivatives of α-
aminoalkylphosphine oxides with an aliphatic R3 substituent,
the ability to interact with the BIR3 domain was observed only
at high antagonist concentrations (5–8; Table 1), enabling the
determination of EC50 values only for the tert-butyl derivatives
(5, 6). Within the group of derivatives bearing the cyclohexyl
ring at R3 (9, 10), an interaction with the BIR3 domain was
demonstrated by approximately 25% inhibition at the highest
antagonist concentration (Table 1). Among compounds that
vary at the R2 positions and have a fixed R3 phenyl ring (11–
14), we observed that introducing a bulky, aromatic R2 substit-
uent resulted in an improved interaction with the BIR3 domain.
For the R2 phosphonic analogs of Gly, the EC50 values were
29.33 µM (11) and 26.29 µM (12), whereas for the Phe analogs,
the EC50 values decreased significantly, to 1.53 µM and 1.31
µM for R1 Val (13) and Chg (14), respectively. The last group
of peptidyl α-aminoalkylphosphine oxides includes derivatives
containing small R2 (-H or -CH3) and aryl R3 (-C6H4-2-CH3, -
C6H4-4-CH3 or -C6H3-3,5-(CH3)2) substituents (15–22,
Table 1). Within this group, the lowest EC50 value was associ-
ated with compound 17, containing R2 = -CH3 and R3 = -C6H4-
4-CH3 (EC50 value of 19.25 µM).
ester ring substituent (R3 = -CH2CH3, -CH(CH3)2 and -
C(CH3)3) did not significantly change the activity, and EC50
values ranged between 19.83 µM (26) and 34.25 µM (23).
The corresponding diphenyl derivatives demonstrated EC50
values of 31.86 µM (29) and 30.24 µM (30). Investigating the
effect of R2 structure on the ability to interact with the BIR3
domain revealed that among derivatives with an aliphatic R2
substituent (31–40), the Chg-Leu analog had the highest poten-
cy (38, EC50 = 10.61 µM). Similar activity was found for R2 Ile
derivatives (35 and 36, EC50 values of 17.84 and 16.24 µM,
respectively). Similar to the α-aminoalkylphosphine oxide IAP
antagonists, phosphonic Phe analogs were found to be the most
potent compounds not only of this group but also of this study,
displaying EC50 values of 0.13 µM (41) and 0.74 µM (42).
Interestingly, replacing Phe with Hph (43 and 44) led to a dra-
matic decrease in activity compared to parent compounds 41
and 42, thus highlighting the strong preference toward phenyl-
alanine at this position (Table 2).
In summary, the obtained phosphonic peptides represent a
novel class of BIR3 antagonists. Among all of the obtained
compounds, the highest activity toward the XIAP BIR3 do-
main was observed for phosphonic Phe analogs, either deriv-
atives of α-aminoalkylphosphine oxides (13, 14) or α-
aminoalkylphosphonate diaryl esters (41, 42; Fig. 5). The
Among peptidyl derivatives of α-aminoalkylphosphonate
diaryl esters (23–28, Table 2), increasing the size of the phenyl

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Table 2 Kinetic parameters of α-aminoalkylphosphonate diaryl ester peptidyl derivatives
No.
Structure
log EC50
[µM]
EC50 [µM]
Ki
[µM]
% Inh
(50 µM)
R 1
R 2
R 3
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH(CH2)5
-CH(CH3)2
-CH3
-CH2CH3
1.535 0.031
1.313 0.053
1.342 0.026
1.297 0.033
1.328 0.039
1.427 0.041
1.503 0.042
1.481 0.060
1.580 0.056
1.380 0.074
1.543 0.052
1.378 0.034
1.251 0.037
1.211 0.036
1.449 0.062
1.026 0.051
1.418 0.041
1.420 0.028
-0.884 0.036
-0.132 0.034
1.235 0.048
1.184 0.046
34.25
20.54
21.98
19.83
21.26
26.76
31.86
30.24
37.97
23.97
34.96
23.85
17.84
16.24
28.13
10.61
26.20
26.31
0.13
17.00
10.19
10.90
9.84
64.1
74.1
81.8
85.8
83.3
77.3
62.1
69.0
57.0
71.0
58.5
70.5
77.4
81.4
67.4
84.2
65.8
73.8
100.0
100.0
85.4
86.5
-CH3
-CH2CH3
-CH3
-CH(CH3)2
-CH3
-CH(CH3)2
-CH3
-C(CH3)3
-C(CH3)3
-H
10.54
13.27
15.81
15.00
18.84
11.89
17.35
11.83
8.84
-CH3
-CH3
-CH3
-H
-CH2CH3
-H
-CH2CH3
-H
-CH(CH3)2
-CH(CH3)2
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
-CH2CH(CH3)2
-CH2CH(CH3)2
-CH2CH2SCH3
-CH2CH2SCH3
-CH2(C6H5)
-CH2(C6H5)
-CH2CH2(C6H5)
-CH2CH2(C6H5)
-H
-H
-H
-H
8.05
-H
13.96
5.25
-H
-H
13.00
13.05
0.05
-H
-H
-CH(CH 2
-CH(CH3)2
-CH(CH2)5
)
-H
0.74
0.35
5
-H
17.17
15.27
13.00
7.57
-H
EC50 value for the most potent obtained phosphoroorganic
compound (41, EC50 = 0.13 µM) was more than ten times
higher than that of the GDC-0152 reference compound
(EC50 = 0.01 µM) and almost three and a half times higher
than that of LCL-0161 (EC50 = 0.04 µM). Nevertheless, in
relation to the H2N-Ala-Val-Pro-Trp-OH reference
tetrapeptide (EC50 = 0.34 µM), the EC50 value of 41 was over
two and a half times lower, indicating the efficacy of 41 when
interacting with the XIAP BIR3 domain. Interestingly, replac-
ing Phe with Hph led to decreased BIR domain binding po-
tency. This finding highlights the high preference for the BIR3
domain toward Phe mimetics at this position of the antagonist.
It is worth mentioning that the presence of Val or Chg residues
in the scaffold seems to have a secondary/less significant role
in activity.
methods of molecular docking using the Ala-Val-Pro-Ile
tetrapeptide as a template. The obtained data showed that the
group present at the R2 position of the phosphoroorganic com-
pound interacts with the hydrophobic pocket of the BIR3 do-
main (formed by aliphatic regions of Lys297 and Lys299, Fig.
1) in a similar fashion to the side chain of Ile of the Smac
protein when it interacts with the BIR3 domain. The
phosphoroorganic moiety together with its substituents is ex-
posed to the solvent (Fig. 6). On the basis of the obtained
fluorescence polarization data and molecular docking studies,
we showed that phosphoroorganic derivatives of N-Me-Ala-
Val/Chg-Pro represent a new class of potent IAP antagonists.
Further research should focus on detailed structural optimiza-
tion, as altering the structure of the phosphorus atom substit-
uents of the phenylalanine mimetic might result in the gener-
ation of compounds displaying improved potency.
In order to examine whether compounds exhibiting activity
in the fluorescence polarization assay interact with the binding
groove of the XIAP BIR3 domain in a manner analogous to
the N-terminal sequence of Smac, we applied in silico
Smac mimetics, in addition to their direct interaction with
the BIR domains of the XIAP protein that leads to the lib-
eration of caspases from inhibitor binding, are able to induce

Invest New Drugs
Fig. 5 Competitive binding curves obtained for BIR incubated with 2 (negative control, (a)), 3 (positive control, (b)), reference compounds (c,
d), and the most potent derivatives obtained in this study (e-h)
autodegradation of the proteins cIAP1 and cIAP2.
Autoubiquitination occurs through the activation of E3 ubiq-
uitin ligase activity, which results in proteasome-mediated
cIAP degradation [52]. Therefore, we determined whether
the obtained phosphoroorganic Smac mimetics were able
to induce autoubiquitination followed by cIAP1 degradation.
For this purpose, MDA-MB-231 breast cancer cells were
incubated either with a phosphoroorganic peptide derivative
(13, 14, 41 or 42) or a reference compound (GDC-0152 or
LCL-161). Cell lysates were subjected to Western blot
Fig. 6 Molecular docking best-scoring models obtained using the
Protein-Ligand ANT System (PLANTS v. 1.2) studies of compounds
13 (a), 14 (b), 41 (c) and 42 (d). Oxygen, nitrogen, and phosphorus atoms
are colored in red, blue and orange, respectively. The solvent accessible
surface area of the binding groove of the XIAP protein (1G73.pdb) was
made transparent in order to visualize the main residues involved in the
interaction with the Ala-Val-Pro-Ile tetrapeptide (stick representation)

Invest New Drugs
Fig. 7 The ability of obtained phosphoroorganic peptide mimetics to
induce autoubiquitination and proteasomal degradation of cIAP1.
Western blot analysis of MDA-MB-231 cell lysates subjected to incuba-
tion with reference compounds: G – GDC-0152, L – LCL-161, selected
phosphoroorganic peptide derivatives (13, 14, 41 and 42) and C –
negative control treated only with DMSO; A and B – samples taken after
15 and 120 min incubation with antagonists (a). The dose response for
compound 41 (1, 2.5, 5 and 10 µM) on the degradation of cIAP1 protein
after 15 min incubation with MDA-MB-231 cells; C – negative control
treated only with DMSO (b)
analysis using anti-cIAP1 IgG antibodies (Fig. 7a). The re-
sults clearly indicated the ability of the developed deriva-
tives to induce autoubiquitination and degradation of
cIAP1 in MDA-MB-231 breast cancer cells. The reference
compounds showed slightly higher induction of cIAP1 pro-
tein degradation than the developed compounds. Phosphonic
Phe analogs (41 and 42) showed the greatest ability to in-
duce cIAP1 degradation in the MDA-MB-231 cell line.
Because of its high potency of action in the fluorescence
polarization assay, compound 41 was subjected to a more
detailed, concentration-dependent analysis of its ability to
induce cIAP degradation (Fig. 7b and Supplementary
Figure S2).
Additionally, we examined the activity of N-Me-Ala-Val/
Chg-Pro-PheP(OPh)(OH) derivatives (details concerning their
structure, synthesis, and kinetic parameters are included in the
Supplementary Materials, Supplementary Figure S3). Despite
their relatively low EC50 values (EC50 = 0.43 µM (41 m) and
0.52 µM (42 m), Supplementary Figure S4) when compared
aminoalkylphosphonic acids (EC50 = 0.13 µM (41), EC50 =
0.74 µM (42)) that demonstrated an effective interaction with
the BIR3 domain via the fluorescence polarization assay, they
were devoid of the autoubiquitination activity and thus did not
lead to the degradation of cIAP1 (Supplementary Materials,
Supplementary Figure S5). This lack of effectiveness might
due to the inability of compounds 41 and 42 m to cross the cell
membrane. This result provides important guidance for further
modification of the phosphoroorganic structure for the design
of BIR3 domain antagonists.
The in vitro antiproliferative activity of the most promising
phosphoroorganic derivatives was evaluated in a set of five
diverse human cancer cell lines: two breast cancer cell lines
(MDAMB231 and MCF-7), a representative of transitional
cell carcinoma (urinary bladder; UMUC3), nonsmall-cell lung
carcinoma (A549) and colon adenocarcinoma (LoVo). MCF-
10A cells (a nontumorigenic mammary gland epithelial cell
line) was used as a reference cell line, whereas GDC0152,
LCL161 and cisplatin (CDDP) were used as reference anti-
proliferative agents. In the case of all XIAP antagonists, the
MDAMB231 cell line was significantly more sensitive than
to the corresponding α-aminoalkylphosphine oxides (EC50
=
1.53 µM (13), EC50 = 1.31 µM (14)) or diaryl esters of α-
Table 3 Antiproliferative activity
of selected compounds
Compd.
IC50 SD (n = 3) [µM]
MDA-MB-231
MCF-7
MCF-10A
UMUC-3
LoVo
A549
GDC-0152
0.88 0.21
0.76 0.23
3.39 0.42
2.72 0.69
2.58 0.32
2.46 0.38
22.14 5.26
[18]*
[19]*
[22]*
[20]*
[20]*
[16]*
4
2
6
3
7
2
[1]*>
[2]*
[5]*
[1]*
[2]*
[2]*
1
[13]*
[16]*
[6]*
3
5
[9]*
4
[13]*
[11]*
[2]*
4
3
LCL-161
13
1
2
1
1
1
[26]*
[6]*
7
2
3
2
3
2
3
1
14
[8]*
[7]*
[5]* 2
41
[5]*
[11]*
[12]*
6
4
[10]*
[23]*
3
6
42
[6]*
CDDP
5.36 1.8
6.76 1.2
3.73 0.63
3.9 1.3
1.6 0.4
* - mean proliferation inhibition [%] at highest concentration used (10 µM)

Invest New Drugs
Fig. 8 Potential of selected compounds as apoptosis inducers measured
via caspase-3 enzymatic activity assay after 24 h of exposure. For GDC-
0152 and LCL-161, the left bar corresponds to the sample treated with a
concentration of 0.1 µM, and the right bar corresponds to the sample
treated with a concentration of 1 µM. For selected phosphoroorganic
peptide derivatives (13, 14, 41 and 42), the left bar corresponds to 1
µM, and the right bar corresponds to 10 µM. CDDP and CPT were
applied at a concentration of 10 µM
any other cell line used. Although the IC50 values determined
for the commercial antagonists GDC-0152 and LCL-161 were
3–4 times lower (0.88 and 0.76 µM, respectively) than those
determined for the selected phosphoroorganic compounds
(3.39 µM (13), 2.72 µM (14), 2.58 µM (41), 2.46 µM (42)),
the antiproliferative potential of our newly described antago-
nists promotes motivation for further in vitro investigations.
It is notable that no significant differences in IC50 values
were observed between the tested phosphoroorganic antago-
nists. The activity of all tested compounds on the MCF7 cell
line was limited even at a concentration of 10 µM and was
negligible in the remaining cell lines, including the
nontumorigenic epithelial cell line MCF10A (Table 3). Such
selectivity strongly contrasts with the sensitivity of the tested
cell lines for cisplatin – MDAMB231 was the least sensitive
cell line with an IC50 value several times higher than those for
the other cell lines (IC50 = 22.14 µM). This distinctive feature
of the MDA-MB-231 cell line, which might contribute to the
observed selectivity, includes a lack of estrogenic and proges-
terone receptor expression and amplification of HER2, which
are reasons for the wide application of the MDA-MB-231 cell
line as a model of poorly differentiated triple negative breast
cancer. However, the observed selectivity might also arise
from other attributes. The high antiproliferative activity of
our novel phosphoroorganic compounds on a cell line that is
recognized as a model for chemoresistant, highly aggressive
breast cancer combined with a lack of toxicity in a
nontumorigenic cell line is an important observation.
newly synthesized derivatives were indicated to be signifi-
cantly more potent than cisplatin when used at 10 µM (1
µM for GDC-0152 and LCL-161) and at least equally active
when used at 1 µM (0.1 µM for GDC-0152 and LCL-161;
cisplatin was used at a fixed concentration of 10 µM) (Fig. 8).
In conclusion, the designed and synthesized derivatives
represent the first example of phosphorus-containing antago-
nists of the XIAP BIR3 domain. The observed selective anti-
proliferative activity associated with strong apoptosis induc-
tion might open an avenue for further development of novel,
low molecular weight IAP antagonists that might be useful as
potential anticancer agents.
Materials and methods
General
All chemical reagents and solvents were obtained from com-
mercial sources and used without purification. High-
resolution mass spectra (HRMS) were acquired on Waters
Acquity Ultra Performance LC, LCT Premier/XE (Waters,
Warszawa, Poland) or Bruker micrOTOF-QII (Bruker,
Poznań, Poland) spectrometers. The nuclear magnetic reso-
nance spectra (1H and 31P) were recorded on either a
400 MHz NMR Jeol PCZ 400S (Jeol, Warszawa, Poland) or
600 MHz NMR Bruker Avance spectrometer (Bruker). High-
performance liquid chromatography analysis and purification
were carried out on a Varian ProStar system (Varian,
Australia) or a Waters Binary Module System (Waters,) with
a dual λ absorbance detector system using a Discovery BIO
Wide Pore C8 HPLC column (250 mm × 21.2 mm, 10 µm)
with a 15 mL/min flow rate using a linear gradient from 0 to
100% B within 20 min or a Discovery BIO Wide Pore C8
Further studies on the proapoptotic potential of the devel-
oped compounds showed that they could induce strong cas-
pase3 activity in MDA-MB-231 cells, with an activity com-
parable to that induced by camptothecin (CPT) when used at a
concentration of 10 µM (1 µM for GDC-0152 and LCL-161).
Both the reference XIAP BIR3 domain antagonists and the

Invest New Drugs
HPLC column (250 mm × 4.6 mm, 10 µm) with a 1 mL/min
flow rate using a linear gradient from 0 to 100% B over 15 min
(solvent A: H2O with 0.05% trifluoroacetic acid, solvent B:
MeCN with 0.05% trifluoroacetic acid).
TFA:TIPS:H2O (95:2.5:2.5, v/v/v; 2 h, r.t.). The obtained pep-
tides were purified with HPLC and analyzed by HRMS and
NMR.
The tetrapeptide H2N-Ala-Val-Pro-Trp-OH (3) was synthe-
sized on a solid support (2-Cl-Trt resin; 1.6 mmol/g) using the
Fmoc strategy and HBTU as a coupling agent described
above, starting with Fmoc-Trp(tBoc)-OH. The peptide cleav-
age from the resin was performed with TFA:TIPS:H2O solu-
tion (95:2.5:2.5, v/v/v; 2 h, r.t.), followed by HPLC purifica-
tion and HRMS and NMR analysis.
Docking studies
Molecular docking studies of the designed IAP protein
phosphoroorganic antagonists were performed using the crys-
tal structure of the XIAP BIR3 domain as a receptor
(1G73.pdb) and the tetrapeptide H2N-Ala-Val-Pro-Ile-OH as
a template whose structure was a pattern of constraints used
for the docked antagonists. The geometry of
phosphoroorganic compounds 13, 14, 41 and 42 was opti-
mized with the MM2 force field (as implemented in
ChemBio3D Ultra 11.0). Atom types and structural proton-
ation were set with SPORES [53]. Docking studies were car-
ried out by means of the Protein-Ligand ANT System
(PLANTS ver. 1.2) [54] with an IAP peptide-binding groove
defined in the binding site with a 20 Å radius and a center
corresponding to the Cα of Leu307.
Synthesis of the α-aminoalkylphosphine oxides
Tritylamine was synthesized from trityl chloride and ammo-
nium chloride in a 25% ammonium hydroxide solution.
Briefly, to an ice bath-cooled solution of ammonium chloride
(0.2 mol) in ammonium hydroxide (200 mL), trityl chloride
(0.2 mol) dissolved in toluene (200 mL) was added dropwise
over 2 h. The reaction continued overnight at r.t. with vigorous
stirring. The organic layer was washed with water until a neu-
tral pH was reached, dried over sodium sulfate and evaporated
under vacuum to dryness, yielding the final product as a white
solid (94%) [55].
Chemistry
Tritylimines were obtained by reacting tritylamine (1 eq.)
with different aldehydes (4 eq.): formaldehyde, acetaldehyde,
phenylacetaldehyde or 2-methylbutyraldehyde in anhydrous
toluene (for 24 h at r.t.). The progress of the reaction was
monitored by TLC. The reaction mixture was washed with
water, dried over anhydrous sodium sulfate and evaporated
to dryness, yielding the target tritylimines that were used in
the next step without purification.
The synthesis of α-aminoalkylphosphine oxides started
with the preparation of diaryl or dialkyl phosphine oxides
obtained via the oxidation of diaryl or dialkyl
chlorophosphines with 1 M HClaq (0 °C → r.t., 24 h).
The resulting crude phosphine oxide (1 eq.) was dissolved
in toluene, and a tritylimine (1 eq.) was added. The reac-
tion was performed under reflux for 8 h. The volatile
components were removed under vacuum, and the trityl
protecting group was removed with TFA (2 h, r.t.) for the
aryl derivatives or HCl (10 eq.) in MeOH (30 min, reflux)
for the alkyl derivatives. The crude products were used in
the next steps of peptidyl derivative preparation. The syn-
thesis of the α-aminoalkylphosphine oxides is outlined in
Fig. 9a.
Synthesis of the peptides
Peptides N-Me-Ala-Val-Pro-OH and N-Me-Ala-Chg-Pro-OH
were synthesized manually on solid support using the Fmoc
strategy. For this purpose, 2-Cl-Trt resin (1.6 mmol/g) was
preloaded with Fmoc-Pro-OH (1.2 eq.). The resulting level
of substitution (0.88 mmol/g) was determined spectrophoto-
metrically at 300 nm after deprotection with 30% piperidine in
dimethylformamide (DMF). During peptide synthesis, the
Fmoc group was removed by means of 20% piperidine in
DMF, while Fmoc-protected amino acids (3 eq.) were subse-
quently introduced with 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU) as a cou-
pling agent (3 eq. ) in the presence of N, N-
diisopropylethylamine (DIPEA, 5 eq.). The N-terminal methyl
group was introduced into the peptide on the resin via the
Mitsunobu reaction [47]. Briefly, the N-terminal Fmoc
protecting group was removed with 20% piperidine in DMF,
followed by a 15 min incubation with 4-nitrobenzenesulfonyl
chloride (o-NBS-Cl, 4 eq.) and collidine (10 eq.) in NMP.
Next, the resin was washed with NMP and THF followed by
the addition of MeOH (10 eq.), Ph3P (5 eq.) and DIAD (5 eq.)
in THF. The reaction was performed at r.t. for 30 min, and the
resin was washed with THF and NMP. Removal of the o-NBS
group was performed using HSCH2CH2OH (10 eq.) and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU, 5 eq.) in THF (2 × 10
min). Cleavage of the obtained N-methylated tripeptides (N-
Me-Ala-Val-Pro-OH (1) and N-Me-Ala-Chg-Pro-OH (2))
from the resin was achieved by means of a solution of
Synthesis of the α-aminoalkylphosphonate diaryl esters
The synthesis started with the preparation of triaryl phos-
phites, which were obtained from the appropriate phenol (3
eq.) and PCl3 (1 eq.) in refluxing acetonitrile [56]. The α-
amidoalkylation reaction of the obtained triaryl phosphite with
benzyl carbamate (1.1 eq.) and an aldehyde (1.1 eq.) was

Invest New Drugs
Fig. 9 Synthesis of phosphoroorganic derivatives. The synthesis of α-
aminoalkylphosphine oxides: (i) R1-CHO, toluene, r.t.; (ii) 1 M HCl, r.t.;
(iii) Trt-N = CHR1, toluene, 8 h, reflux; (iv) TFA, r.t. or HCl/MeOH,
reflux (a). The synthesis of α-aminoalkylphosphonate diaryl esters: (i)
PCl3, acetonitrile, 4 h, reflux; (ii) Cbz-NH2, R2-CHO, AcOH, 4 h, reflux;
(iii) 33% HBr/AcOH, 2 h, r.t. (b)
performed in glacial acetic acid (80–90 °C, 2 h) [57]. After
evaporation under reduced pressure, the resulting oil was dis-
solved in methanol, and the product crystallized at -20 °C. The
Cbz protecting group was removed with 33% HBr/AcOH (2
h, r.t.) and the product crystallized in diethyl ether as an HBr
salt. The synthesis pathway of α-aminoalkylphosphonate
diaryl esters is shown in Fig. 9b.
bound peptide was labeled with 5(6)-carboxyfluorescein
(5(6)-Fam, 2.2 eq.) using HATU as a coupling agent (3 eq.)
and HOBt as an additive (3 eq.) in the presence of DIPEA (3.5
eq.). The obtained fluorescent probe was cleaved off of the
resin with a TFA:TIPS:H2O solution (95:2.5:2.5, v/v/v, 2 h,
r.t.) yielding H-Ala-Val-Pro-Dpm-Ala-Lys(5(6)-Fam)-Lys-
NH2 (4), which was purified by HPLC and analyzed
spectroscopically.
Peptide scaffold modification with phosphoroorganic
functionality
Fluorescence polarization assay
Peptide scaffolds (1 and 2) were coupled with α-
aminoalkylphosphine oxides or α-aminoalkylphosphonate
diaryl esters (1.2 eq.), using HBTU as a coupling agent (1.2
eq.) in the presence of DIPEA (5 eq.) in MeCN or DMSO. The
reaction was performed overnight at r.t. The solvent was re-
moved in vacuo, and the target compounds were isolated di-
rectly using HPLC, followed by spectroscopic analysis.
The fluorescence polarization assay was performed at r.t. in
Corning 384-well black, polystyrene flat bottom plates
(Corning, NY, USA) on a Synergy H4 Hybrid Reader
(BioTek, Bad Friedrichshall, Germany). For fluorescence po-
larization measurements, (milipolarization units, mP) 484/20
nm excitation and 528/20 nm emission filters were selected.
Phosphate buffer (pH 7.5) supplemented with γ-globulin (100
µg/mL) and NaN3 (0.02% w/v), fluorescent probe 4 and the
XIAP BIR3 domain (Sino Biological, Beijing, China) were
used for all measurements. All experiments were performed in
duplicate. The obtained fluorescence polarization assay data
were analyzed with Prism 5.0 Software (GraphPad Software,
San Diego, ca.).
Synthesis of the fluorescent probe
Synthesis of the fluorescein-labeled peptide was performed on
solid support applying the Fmoc strategy (Rink Amide resin;
0.6 mmol/g) using HBTU (3 eq.) and DIPEA (3 eq.) for amino
acid coupling in DMF. Fmoc deprotection was performed
with 20% piperidine in DMF. In the resulting resin-bound
peptide, Boc-Ala-Val-Pro-Dpm-Ala-Lys(Mtt)-Lys(Boc)-res-
in, the Mtt group was removed with 1% TFA solution in
DCM. The progress of Mtt removal was monitored spectro-
photometrically at 460 nm [50]. Subsequently, the resin-
Binding assay
In order to determine the dissociation constant (Kd) between
the XIAP BIR3 domain and fluorescent probe 4, serial dilu-
tions of the XIAP BIR3 domain (two-fold dilutions, ranging

Invest New Drugs
from 2.5 µM to 0.0763 nM) were incubated with the probe (2
nM) for 30 min. Fluorescence polarization values were plotted
as a function of the protein concentration, and the Kd value
was determined using GraphPad software (one site - total
binding model).
insulin, 0.05 µg/mL cholera toxin, 0.5 µg/mL hydrocorti-
sone, and 20 ng/mL hEGF (all from Sigma-Aldrich).
A549 and LoVo cells were cultured in a 1:1 (v/v) mixture
of RPMI1640 and Opti-MEM (both from HIIET) supple-
mented with 5% (v/v) FBS and 2 mM L-glutamine. The
UMUC-3 cell line was cultured in high glucose DMEM
(Thermo Fisher Scientific) supplemented with 10% FBS
and 2 mM Lglutamine. The MCF-7 line was cultured in
Eagle’s minimal essential medium (EMEM; Thermo
Fisher Scientific) supplemented with 10% (v/v) FBS,
2 mM L-glutamine, 1% (v/v) nonessential amino acids,
and 8 µg/mL insulin (all from Sigma-Aldrich). All culture
media contained 100 µg/mL streptomycin (Sigma-
Aldrich) and 100 U/mL penicillin (Polfa Tarchomin SA,
Warszawa, Poland). The cells were grown at 37 °C in a
humid atmosphere saturated with 5% CO2.
Inhibition assay
For all phosphoroorganic peptide derivatives, the inhibition
constant values were determined by the addition of the
XIAP BIR3 protein (50 nM; established experimentally based
on the Kd value) into the phosphoroorganic compound solu-
tion (5-fold dilutions, ranging from 50 µM to 0.640 nM for
screening purposes and 2.5-fold dilutions ranging from 50 µM
to 0.0537 nM for detailed analysis). After a 15 min incubation
at r.t., fluorescent probe 4 (2 nM) was added. The values were
measured 30 min after the moment the probe was added. The
assay was performed in phosphate buffer with a final DMSO
concentration of 1.4% (v/v). As a reference, inhibitors,
tetrapeptide H-Ala-Val-Pro-Trp-OH (3) and commercially
available IAP antagonists (GDC-0152 and LCL-161) were
used. In order to evaluate the quality and precision of the
performed screening assay, we determined the Z´ factor as
described by Zhang et al. [58]. The obtained fluorescence
polarization values were used to determine the percent inhibi-
tion, which was plotted as a function of the protein concentra-
tion. The EC50 and Ki values were calculated with GraphPad
software (log(agonist) vs. response (variable slope) model) in
combination with the web tool developed by Nikolovska-
Coleska et al. [59].
Western blot analysis
Prior to Western blot sample collection, cells were harvested
by trypsinization, counted and seeded on 50 mm Petri dishes.
After overnight incubation, the culture medium was replaced
with fresh medium containing the tested compounds at the
target concentration ranges with 0.1% (v/v) DMSO as a con-
trol. Next, the cells were washed with PBS and lysed with ice-
cold RIPA buffer containing protease and phosphatase inhib-
itors (Sigma-Aldrich, Poznań, Poland), and after debris re-
moval (4 °C, 15 min, 16,000 × g), the lysates were used in
further experiments.
MDA-MB-231 cell lysates (25 µg of total protein/lane)
were resolved by SDS-PAGE (4–12%, Tris-glycine, re-
ducing conditions) and transferred onto a nitrocellulose
membrane (0.45 µm pore size; Thermo Scientific,
Gdańsk, Poland) using the semi-dry blotting system
(Cleaver Scientific, Rugby, UK). The membrane was
washed with 10 mM PBS (pH 7.4; 5 min, r.t.) and
blocked with 5% skim milk in PBS supplemented with
0.05% Tween-20 (PBST) for 1 h at r.t. Subsequently, the
membrane was washed with PBST (3 times for 5 min, r.t.)
and incubated with rabbit anti-cIAP1 monoclonal IgG
(1:1,000 in 0.5% skim milk in PBST; 4 °C, overnight;
Cell Signaling Technology, Warszawa, Poland, Cat#
7065, D5G9). Next, the membrane was washed with
PBST (3 times for 5 min, r.t.) and incubated with goat
anti-rabbit IgG-HRP (1:1,000 in 0.5% skim milk in
PBST; Sigma-Aldrich, Cat# A6154). After incubation (1
h, r.t.), the membrane was washed with PBST (3 times for
5 min, r.t.), and chemiluminescent peroxidase substrate
was added (Super Signal WestPico, Thermo Scientific).
The bands were visualized by means of the blot imaging
system (GelLogic 1500, Carestream, Rochester, NY,
USA).
Biological activity of selected peptide derivatives
Cell culture
MDA-MB-231 (human breast cancer), MCF10A (human,
nontumorigenic mammary gland epithelial cells), A549
(human nonsmall-cell lung cancer), UMUC-3 (human
bladder cancer) and LoVo (human colon cancer) cell lines
were obtained from the American Type Culture Collection
(ATCC; Rockville, Maryland, USA). The MCF-7 cell line
(human breast cancer) was obtained from the European
Collection of Cell Cultures (ECACC; Salisbury, UK).
All cell lines were maintained at the Hirszfeld Institute
of Immunology and Experimental Therapy (HIIET),
Wrocław, Poland. MDA-MB-231 cells were cultured in
RPMI1640 medium (Thermo Fisher Scientific,
Warszawa, Poland) supplemented with 2 mM L-gluta-
mine, 1 mM sodium pyruvate, and 10% fetal bovine se-
rum (all from Sigma-Aldrich, Poznań, Poland). The
MCF10A cell line was cultured in Ham’s F12 medium
with glutamine (Corning) supplemented with 5% (v/v) fe-
tal bovine serum (FBS), 5% (v/v) horse serum, 10 µg/mL

Invest New Drugs
Acknowledgements This work was supported by the National Science
Centre of Poland grant number 2014/13/N/ST5/01582. The authors
would like to thank Łukasz Winiarski for kindly donating 5(6)-carboxy-
fluorescein and Ewa Burchacka and Marcin Skoreński for donating select
α-aminoalkylphosphonate diphenyl esters. The authors would like to
thank Martyna Dawidczyk, Jolanta Sum and Sabina Myśliwiec for their
excellent technical assistance. The authors would like to acknowledge
Tomasz Olszewski for helpful discussions. The authors would like to
acknowledge Adam Lesner and Kamila Sychowska from the University
of Gdańsk for some MS analysis. The authors would also like to thank
Keri Csencsits-Smith from the University of Texas Health Science Centre
at Houston for the critical reading of the manuscript. AŁS, MS, RG, and
JO are thankful to the Wrocław University of Science and Technology for
support (Statute Funds 049U/0096/19).
For the loading control, the membrane was washed with
PBST and incubated with mouse anti-GAPDH IgG (1:1,000
in 0.5% skim milk in PBST; Thermo Scientific, Cat#
MA515738, GA1R). After incubation (1 h, 37 °C), the mem-
brane was washed with PBST (3 times for 5 min, r.t.) and
incubated with anti-mouse HRP-labeled rabbit IgG (1:5,000
in 0.5% skim milk in PBST; Fitzgerald, Acton, MA, USA,
Cat# 43R-1424) for 1 h at 37 °C. After washing, the signal
was developed as described above.
Antiproliferative activity analysis
Funding The work was supported by the National Science Centre of
Poland (grant number 2014/13/N/ST5/01582). AŁS, MS, RG, and JO
are thankful to Wrocław University of Science and Technology for sup-
port (Statute Funds 049U/0096/19).
Antiproliferative activity was assessed in six cell lines
utilizing the sulforhodamine B (SRB) method [60].
Briefly, cells were seeded in 96-well plates (Sarstedt,
Warszawa, Poland) at a density of 5 × 103 cells/well, and
after overnight incubation, the cells were exposed to com-
pounds at various concentrations. After 72 h, the plates
were fixed with 50% (v/v) TCA (all from Sigma-Aldrich),
washed with tap water, and the precipitated proteins were
labeled with 0.1% (w/v) SRB solution in 1% (v/v) AcOH.
Next, the plates were washed with 1% (v/v) AcOH, and
the remaining SRB dye was solubilized with 10 mM un-
buffered TRIS solution. The absorbance was measured
using a Biotek Hybrid H4 reader (BioTek Instruments,
VT, USA) at 540 nm. Crude results were analyzed using
a previously described method [61], and the results are
expressed as the IC50 (compound concentration that re-
duced cell growth by 50%) compared to the vehicletreated
control (0.1% (v/v) DMSO). The samples were analyzed
in triplicate, and each experiment was repeated at least
three times.
Compliance with ethical standards
Conflict of interest All authors declare no conflicts of interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed consent For this type of study, formal consent is not required.
Open Access This article is licensed under a Creative Commons
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long as you give appropriate credit to the original author(s) and the
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Caspase-3 activity assay for apoptosis rate analysis
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