Oral disinfectants inhibit protein-protein interactions mediated by the anti-apoptotic protein Bcl-xL and induce apoptosis in human oral tumor cells

Article abstract of DOI:10.1002/anie.201208889

Chlorhexidine and alexidine have long been used as oral disinfectants by humans. Both compounds inhibit protein-protein interactions mediated by the anti-apoptotic protein Bcl-x_L at physiologically relevant concentrations and induce apoptosis in a series of tumor cell lines derived from the tongue and pharynx (see picture). Inhibition of protein-protein interactions is a potential mode of action of drugs in current human use. Copyright

Full text of DOI:10.1002/anie.201208889

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Chemie
DOI: 10.1002/anie.201208889
Protein–Protein Interactions
Oral Disinfectants Inhibit Protein–Protein Interactions Mediated by
the Anti-Apoptotic Protein Bcl-xL and Induce Apoptosis in Human
Oral Tumor Cells**
Martin Grꢀber, Michael Hell, Corinna Grçst, Anders Friberg, Bianca Sperl, Michael Sattler, and
Thorsten Berg*
Significant advances during the last decade in the field of
small-molecule modulators of protein–protein interactions
have raised general awareness of the fact that the modulation
of protein–protein interactions has tremendous potential for
both basic and applied science.[1] Some of the most advanced
orthosteric inhibitors of protein–protein interactions[2–5] are
currently in clinical trials. Here, we demonstrate that the
bisbiguanide oral disinfectants chlorhexidine[6] (the active
ingredient of commercially available Chlorhexamed, Chlo-
rhexal, Periogard, Corsodyl, and Chlorohex) and alexidine[7]
(Esemdent), which have been in human use worldwide for
many years, target cancer-linked protein–protein interactions
between the anti-apoptotic Bcl-2 family protein Bcl-xL and its
pro-apoptotic binding partners. The compounds are shown to
induce apoptosis through the mitochondria-dependent apop-
totic pathway in oral tumor cell lines.
Our study was based on the hypothesis that some small-
molecule drugs already approved for human use against other
targets might display hitherto undiscovered activities against
protein–protein interactions[8] in their therapeutic concentra-
tion range.[9] To test our hypothesis on a protein–protein
interaction target with established relevance for human
health, we decided to focus on the interaction between the
anti-apoptotic Bcl-2 family protein Bcl-xL and its pro-
apoptotic Bcl-2 family binding partner Bad. The interaction
between Bcl-xL and pro-apoptotic Bcl-2 proteins has attracted
significant attention from both cancer biologists and medic-
inal chemists.[2,10–12]
A library of 4088 compounds containing a high proportion
of clinically used small molecules was screened against the
interaction between Bcl-xL and a peptide derived from the
Bad BH3 domain by using a binding assay based on
fluorescence polarization (Z’ = 0.64 Æ 0.11, see Figure S1 in
the Supporting Information).[13] Active compounds in the
primary screen (screening concentration: ca. 50 mm) were
checked for activities against other protein–protein interac-
tions recorded in a previous screening study.[14] These efforts
identified both chlorhexidine (1) and alexidine (2; Figure 1A)
as potentially selective inhibitors of the Bcl-xL/Bad interac-
tion (see Figure S2 in the Supporting Information). Robotics-
based re-analysis of the screening hits confirmed the dose-
dependent inhibition of the interaction between Bcl-xL and
Bad, and also between Bcl-xL and Bak, another protein of the
pro-apoptotic Bcl-2 family, by 1 and 2 with similar activities
(see Figure S3 in the Supporting Information). The interac-
tion of Bcl-xL with Bak, rather than with Bad, was selected for
further analysis because of the larger assay window associated
with the Bcl-xL/Bak assay. Manual validation of the activities
of 1 and 2 against the Bcl-xL/Bak interaction confirmed their
activities in the low micromolar range (IC50: chlorhexidine:
(21.0 Æ 1.0) mm; alexidine: (18.2 Æ 1.6) mm; Figure 1B,C,
Table 1), and the Supporting Information, Table S1). These
activities are well in the concentration range of the oral
[*] Dr. M. Grꢀber,[+] M. Sc. C. Grçst,[+] Prof. Dr. T. Berg
Institute of Organic Chemistry, University of Leipzig
Johannisallee 29, 04103 Leipzig (Germany)
E-mail: tberg@uni-leipzig.de
[$] Current address: Department of Biochemistry
Vanderbilt University School of Medicine
Nashville, TN 37232-0146 (USA)
[+] These authors contributed equally to this work.
Homepage: http://www.uni-leipzig.de/~tberg
Dr. M. Grꢀber,[+] Dipl.-Chem. M. Hell,[+] B. Sperl, Prof. Dr. T. Berg
Department of Molecular Biology
Max Planck Institute of Biochemistry, and
Center for Integrated Protein Science Munich (CIPSM)
Am Klopferspitz 18, 82152 Martinsried (Germany)
Dipl.-Chem. M. Hell[+]
Institute of Molecular Health Sciences, ETH Zurich
Schafmattstrasse 18, 8093 Zurich (Switzerland)
Dr. A. Friberg,[$] Prof. Dr. M. Sattler
Institute of Structural Biology, Helmholtz Zentrum Mꢁnchen
Ingolstꢀdter Landstrasse 1, 85764 Neuherberg (Germany)
and
[**] This work was generously supported by the Department of
Molecular Biology (Director: Prof. Dr. Axel Ullrich) at the Max
Planck Institute of Biochemistry (MPIB), the Center for Integrated
Protein Science Munich (CIPSM), the BMBF (NGFN-2, grant
01GS0451 to T.B.), the Deutsche Krebshilfe (grant 108614 to T.B.),
and the Deutsche Forschungsgemeinschaft (grant BE 4572/1-1 to
T.B.). We thank Prof. Ho Sup Yoon (Nanyang Technological
University, Singapore) for the Bcl-xL plasmid and protein backbone
assignments. We extend our thanks to Judith Mꢁller (MPIB) and the
core facility of the MPIB for experimental support, and to Angela
Hollis for critical reading of the manuscript. We are grateful to
Prof. Dr. Wilhelm Krek (ETH Zurich) for supporting the work of
M.H.
Munich Center for Integrated Protein Science
Chair of Biomolecular NMR Spectroscopy
Department of Chemistry, Technische Universitꢀt Mꢁnchen
Lichtenbergstrasse 4, 85747 Garching (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201208889.
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Figure 1. Chlorhexidine and alexidine inhibit protein–protein interactions mediated by Bcl-xL. The deletion mutant Bcl-xL D45–84, which lacks the
flexible loop previously shown to be dispensable for the anti-apoptotic activity of Bcl-xL, was used in these assays.[18,19] A) Structures of
chlorhexidine and alexidine. Activity profile of B) chlorhexidine and C) alexidine against Bcl-xL, XIAP BIR3, and hDLG1 PDZ2. Activity profile of
D) chlorhexidine and E) alexidine against phosphorylation-dependent protein–protein interactions. F) Binding of chlorhexidine to Bcl-xL, as
analyzed by ITC. Parameters deduced from the lower graph: N=(1.28Æ0.0278) sites; K=(9.05Æ1.31)ꢂ104 mÀ1; DH=À(2493Æ78.14) calmolÀ1
;
DS=14.2 calmolÀ1 degÀ1
.
disinfectant chlorhexidine (1), which is used as a 0.1% and
0.2% (w/v) solution (of the digluconate), corresponding to
1.1 mm and 2.2 mm, respectively. Activity of alexidine (2) as
an disinfecting agent was often shown at 0.035% (w/v; in the
two bisbiguanides (Figure 1B,C). Subsequently, extensive
specificity analysis was carried out against an 11-member
panel of phosphorylation-dependent, structurally diverse
protein–protein interactions.[14] These experiments confirmed
chlorhexidine as a specific inhibitor of binding mediated by
Bcl-xL, and revealed additional, but weaker, activities of
alexidine against other protein–protein interactions (Fig-
ure 1D,E). Further specificity analysis demonstrated that
some of the remaining anti-apoptotic Bcl-2 family proteins
were inhibited by chlorhexidine and alexidine to various
extents (see Figure S4 in the Supporting Information), which
is advantageous given the functional redundancy amongst
proteins of the anti-apoptotic Bcl-2 family.
As a consequence of its narrower selectivity profile, we
focused on chlorhexidine to analyze the thermodynamics of
its binding to Bcl-xL by isothermal titration calorimetry (ITC;
Figure 1F). Consistent with the activity of chlorhexidine in
the competition assay format, ITC indicated a Kd value of
(10.4 Æ 0.7) mm. Binding was mediated by both an enthalpic
component (DH = À(2.25Æ0.25) kcalmolÀ1) and an entropic
component (DS = (16.0 Æ 1.7) calmolÀ1 degÀ1), thus indicating
a combination of electrostatic and hydrophobic interactions.
To gain insight into the binding site of chlorhexidine and
alexidine on Bcl-xL, we carried out 1H,15N-HSQC NMR
experiments with the protein construct originally used in the
NMR-based structural characterization of the Bcl-xL/Bak
interaction.[18] This protein construct lacks the unfolded loop
domain (amino acids 45–84) previously shown to be dispen-
sable for the anti-apoptotic function of Bcl-xL as well as the C-
Table 1: Activities of bisbiguanides 1–3 against the interaction between
Bcl-xL and a Bak-derived BH3-peptide as analyzed in a fluorescence
polarization assay.
Activity
1
2
3
IC50 =(21.0Æ1.0) mm
IC50 =(18.2Æ1.6) mm
(28Æ2)% inhibition at 100 mm
form of the dihydrochloride), which corresponds to 600 mm.[15]
Therefore, the concentrations of chlorhexidine and alexidine
used in mouthwash solutions are likely to be more than
sufficient to inhibit Bcl-xL in surface-exposed cells in the oral
cavity.
As specificity controls, we tested both compounds against
protein–protein interactions mediated by the BIR3 domain of
XIAP[16] and the second PDZ domain of hDLG1.[17] Neither
of the two protein–protein interactions was inhibited by the
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Figure 2. Chlorhexidine (1) and alexidine (2) bind to Bcl-xL D45–84, as shown by 1H,15N-HSQC NMR spectroscopy. A) Chemical shift perturbations
(CSPs) per residue for the binding of 1 (upper panel) and 2 (lower panel) to Bcl-xL. B) Graphical representation of the CSPs for 1 as given in (A)
on the structure of unbound Bcl-xL (PDB entry: 1MAZ).[19] The graphic was generated using PyMOL.[22] C) The amide resonances of Phe146,
Val126, Leu108, Glu129, Asn136, and Gly94 in 15N-labeled Bcl-xL D45–84 (100 mm) in the absence of 1 (black) show strong and dose-dependent
chemical shifts upon addition of 1 (50 mm, blue; 100 mm, magenta; 200 mm, orange; 400 mm, green). D) Overlay of the docking pose of 1 (carbon
atoms shown in pink) with the cocrystal structure of ABT-737 (carbon atoms shown in green) bound to Bcl-xL (PDB entry: 2YXJ).[20] Amino acids
for which CSP are shown in (C) are color coded as in (B).
terminal transmembrane domain.[18,19] Both compounds
caused extensive chemical shift perturbations of a similar
set of amide resonances (see Figure 2A–C and Figures S5 and
S6 in the Supporting Information). The observed spectral
changes are consistent with binding of the small molecules
similar to BH3 peptides to the hydrophobic groove of Bcl-xL.
The strongest perturbations were observed in helices a2–a5,
which form the binding groove for BH3-domain-derived
peptides or small molecules, thus suggesting that the com-
pounds bind to the hydrophobic BH3 binding pocket of Bcl-xL
(see Figure S7 in the Supporting Information).[18] These data
are consistent with the displacement of BH3 domain peptides
from Bcl-xL, as observed in the fluorescence polarization
assays (see Figure 1B,C and Figure S3 in the Supporting
Information). Amino acids with strong chemical shift pertur-
bations included Phe146, Val126, Glu129, Leu108, Asn136,
and Gly94 (see Figure 2C and Figure S6 in the Supporting
Information). The first four of these amino acids are part of
a subpocket to which the Bcl-xL inhibitor ABT-737[11] (see
Figure S7C in the Supporting Information) binds, as deter-
mined by X-ray crystallography (Figure 2D).[20] Intriguingly,
molecular docking experiments using AutoDock Vina[21]
suggested that one of the 4-chlorophenyl rings of chlorhex-
idine occupies the same hydrophobic subpocket of Bcl-xL as
the 4-chlorobiphenyl moiety of ABT-737.
The binding mode suggested by molecular docking was
further interrogated by chemical synthesis and biochemical
testing of analogues. Removal of the chlorine atom of
chlorhexidine (1), as realized in deschlorohexidine (3), led
to a strong reduction in binding affinity (Table 1). Combined
structure–activity data indicate the requirement for appro-
priate hydrophobic interactions mediated by the outer sub-
stituents on the biguanide moieties for binding of the
compounds to Bcl-xL, and show that shorter linker units
between the two biguanide moieties are well-tolerated (see
Table S1 in the Supporting Information).
Since inhibition of Bcl-xL is expected to increase the
apoptotic rate of tumor cells, which often overexpress Bcl-xL,
we chose to investigate the effects of 1 and 2 on the apoptotic
rate of tumor cells derived from oral tissues. Deschlorohex-
idine (3) served as a negative control compound because of its
high structural similarity to the more-specific inhibitor 1 but
significantly lower activity in vitro. Three tongue squamous
cell carcinoma cell lines (SCC-4, SCC-9, SCC-15) and two
pharynx carcinoma cell lines (FaDu and Detroit 562) were
exposed to the compounds. Apoptosis, as characterized by
irreversible degradation of DNA, was visualized by a TUNEL
assay (Figure 3A). Both 1 and 2, but not the negative control
compound 3, induced DNA strand breaks characteristic of
apoptosis in all five cell lines (see Figure 3B,C and Figure S9
in the Supporting Information).
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Figure 4. Chlorhexidine (1) and alexidine (2) induce mitochondrial
membrane depolarization in tumor cell lines derived from the tongue
and pharynx. A) Principle of the assay. B) Mitochondrial accumulation
of the fluorescent dye tetramethylrhodamine ethyl ester (TMRE) is
strongly reduced in cells exposed to 1 and 2 for 24 h, but only to
a significantly lesser extent in cells exposed to 3. Single cells are
sorted according to TMRE content (x axis) and cell size (forward
scatter, y axis) by flow cytometry. Experiments were carried out in
triplicate; representative flow cytometry images are shown. C) Quan-
tification of the experiments as shown in (B) and Figure S10 in the
Supporting Information. Data represent mean values and standard
deviations from triplicate experiments. * p<0.05; ** p<0.01; ***
Figure 3. Chlorhexidine (1) and alexidine (2) induce apoptosis in
tumor cell lines derived from the tongue and pharynx, as analyzed by
the TUNEL assay. A) Principle of the TUNEL assay. B) DNA strand
breaks characteristic of apoptosis induced by treatment of cells with
the compounds for 24 h are visualized (red). Cell nuclei are stained
with DAPI (blue). Representative fluorescence images are shown. Scale
bar: 10 mm. C) Quantification of the experiments as shown in (B) and
Figure S9 in the Supporting Information. Data represent mean values
and standard deviations from triplicate experiments. At least 100 cells
were analyzed in each replicate experiment under each treatment
condition. ** p<0.01; *** p<0.001 (Student’s t-test against DMSO
control).
p<0.001 (Student’s t-test of compounds against DMSO control). ###
+++
p<0.001 (Student’s t-test of 30 mm 1 against 30 mm 3).
(Student’s t-test of 10 mm 2 against 10 mm 3).
p<0.001
Anti-apoptotic Bcl-2 family proteins such as Bcl-xL inhibit
the intrinsic apoptosis pathway by binding to and sequestering
pro-apoptotic proteins of the Bcl-2 family. Small-molecule-
mediated release of the inhibition by anti-apoptotic proteins
of the Bcl-2 family leads to oligomerization of the pro-
apoptotic Bcl-2 proteins Bak and Bax located in the
mitochondrial membrane, thereby causing depolarization of
the mitochondrial membrane and induction of the intrinsic
apoptosis pathway. To investigate whether apoptosis induced
by chlorhexidine and alexidine is triggered by the intrinsic
pathway we carried out mitochondrial membrane depolari-
zation assays using the same five tumor cell lines as used in the
TUNEL assay. This assay type exploits the fact that the
cationic dye tetramethylrhodamine ethyl ester (TMRE)
rapidly accumulates in cells with intact, active mitochondria,
but fails to do so in apoptotic cells with depolarized
membranes (Figure 4A). Both 1 and 2 led to a strong
reduction of TMRE uptake, consistent with depolarization
of the mitochondrial membrane leading to the subsequent
induction of apoptosis by these agents (see Figure 4B,C and
Figure S10 in the Supporting Information). In contrast,
deschlorohexidine (3), which lacks strong activity against
Bcl-xL in vitro, had a significantly weaker effect on TMRE
uptake.
To provide further evidence that the induction of apop-
tosis in tumor cells is mediated, at least in part, by inhibition
of the Bcl-xL/Bak interaction, we performed co-immunopre-
cipitation assays in FaDu cells (see Figure S11 in the
Supporting Information). These experiments confirmed that
both 1 and 2 interfere with binding between Bcl-xL and Bak,
while the negative control compound 3 only had a minor
effect. Combined cell-based assay data demonstrate the
potent effects of 1 and 2 in inducing apoptosis in tumor cell
lines derived from human oral tissue through the intrinsic
apoptosis pathway, and suggest that this effect is mediated, at
least in part, by the inhibition of Bcl-xL. Our data are in line
with previous reports on the induction of cell death by 1 in
L929 fibroblasts,[23] and the apoptosis-inducing activity of
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In summary, we have identified the anti-apoptotic protein
Bcl-xL as a target of two oral disinfectants. To our knowledge,
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nistic evidence that compounds in current widespread, every-
day household use in the industrialized world can exert an
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Received: November 6, 2012
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Published online: && &&, &&&&
Keywords: antitumor agents · apoptosis · drug discovery ·
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inhibitors · protein–protein interactions
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Communications
Protein–Protein Interactions
M. Grꢁber, M. Hell, C. Grçst, A. Friberg,
B. Sperl, M. Sattler,
T. Berg*
&&&& — &&&&
Oral Disinfectants Inhibit Protein–Protein
Interactions Mediated by the Anti-
Apoptotic Protein Bcl-xL and Induce
Apoptosis in Human Oral Tumor Cells
Chlorhexidine and alexidine have long
been used as oral disinfectants by
humans. Both compounds inhibit pro-
tein–protein interactions mediated by the
anti-apoptotic protein Bcl-xL at physio-
logically relevant concentrations and
induce apoptosis in a series of tumor cell
lines derived from the tongue and phar-
ynx (see picture). Inhibition of protein–
protein interactions is a potential mode
of action of drugs in current human use.
6
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ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 6
These are not the final page numbers!