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Computational Biology and Chemistry 33 (2009) 451–456

Contents lists available at ScienceDirect

Computational Biology and Chemistry

journa l homepage: www.e lsev ier .com/ locate /compbio lchem

rief communication

omputer-assisted identification of small-molecule Bcl-2 modulators

elanie Füllbecka,∗,1, Nina Gebhardtb,1, Julia Hossbacha, Peter T. Danielb,c, Robert Preissnera

Charité - Universitätsmedizin Berlin, Institute of Molecular Biology and Bioinformatics, Structural Bioinformatics Group, Arnimallee 22, 14195 Berlin, GermanyCharité - Universitätsmedizin Berlin, Department of Hematology, Oncology and Tumor Immunology, Berlin-Buch, GermanyMax Delbrück Center for Molecular Medicine, Berlin, Germany

r t i c l e i n f o

rticle history:eceived 11 May 2009

a b s t r a c t

Apoptosis, the programmed cell death, is a highly regulated process, necessary for normal developmentand homeostasis of the functions of organisms. The Bcl-2 inhibitors BH3I-1 and BH3I-2 were used as

eceived in revised form 2 October 2009ccepted 2 October 2009

eywords:cl-2poptosis

lead compounds to find possible Bcl-2 or Bcl-XL inhibitors by using computer-assisted screening withour in-house database, containing more than four million commercially available molecules. Identifiedcompounds were further investigated regarding their possible application as a drug.

© 2009 Elsevier Ltd. All rights reserved.

nhibitorsomputer-assisted screening

. Introduction

Apoptosis, the programmed cell death, is a physiological pro-ess, necessary for the maintenance of normal development andqually important as cell migration or division for the homeostasisf multicellular organisms (Kerr et al., 1972).

Important regulators of this complex pathway are the proteinsf the Bcl-2 family. Their main function is to control the release ofpoptotic proteins from the mitochondria. Members of the Bcl-2amily interact with a variety of proteins and therefore acceleratehe rupture of the outer membrane or the mitochondria, whicheads to a release of pro-apoptotic proteins and the triggering ofpoptosis (Kim, 2005).

A disregulation of the Bcl-2 family proteins might lead tohe development of cancer, since a failure of the inactivation ofro-apoptotic pathways, or the activation of anti-apoptotic path-ays, might occur in the complex regulation process (Wang et al.,

003).The development of inhibitors against Bcl-2 or Bcl-XL for the use

s anti-cancer drugs might be promising, as there is a real chance

o overcome the cytoprotective functions of these proteins.

∗ Corresponding author. Tel.: +49 30 8445 1665; fax: +49 30 8445 1551.E-mail addresses: mfuellbeck@gmx.de (M. Füllbeck), Nina.Gebhardt@charite.de

N. Gebhardt), Julia.Hossbach@charite.de (J. Hossbach), pdaniel@mdc-berlin.de (P.T.aniel), Robert.Preissner@charite.de (R. Preissner).1 The authors wish it to be known that, in their opinion, the first two authors

hould be regarded as joint first authors.

476-9271/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.compbiolchem.2009.10.001

2. Materials and methods

2.1. Computer-assisted screening

Using our in-house database with more than four million com-pounds, a virtual screening based on 2D and 3D similarities isperformed. Being known structures, BH3I-1 and BH3I-2 could beused as lead compounds. The database enables virtual screeningsfor small-molecules with similar structures (3D similarity) or sim-ilar chemical properties (2D similarity).

To figure out, whether a 2D similarity is available, chemical fea-tures of molecules are compared by using fingerprints. If calculatedfingerprints are available, they can be used to determine the Tani-moto coefficient (Degterev et al., 2001), which describes chemicalsimilarities between two molecules.

In general, a Tanimoto coefficient above 0.85 makes an edu-cated guess, that the investigated chemicals have similar properties(Thimm et al., 2004).

Chemical similarity is not necessarily associated with a similar-ity in biological functions.

By rigid-body structural alignment, two molecules and con-formers thereof, can be compared regarding their 3D structure.For this purpose, the superposition algorithm is used, which wasdeveloped in our group (Thimm et al., 2004).

2.2. Property filtering

To be able to make a statement on the bioavailability of acompound, which might be used as a drug, the Lipinski Rule-of-five is consulted. Compounds that do not achieve the Rule-of-five

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52 M. Füllbeck et al. / Computational B

hould not be considered as candidates for a drug (Lipinski,000).

.3. Docking

Promising candidates were docked in Bcl-XL using the programOLD, which uses a genetic algorithm to explore the full rangef ligand conformational flexibility with partial flexibility of therotein (Jones et al., 1997). It mimics the process of evolution bypplying genetic operators to a collection of putative poses to aingle ligand.

The active site of the protein was defined by a reference lig-nd in a 15 Å radius. For each ligand 50 docking runs and aotal number of 1,000,000 genetic operations were performed.he “early-termination” option was not selected. GoldScore fitnessunction and the �Gbinding were both used as scoring functions.

.4. Experimental methods

.4.1. Cell cultureBjab Bcl-XL transfected (Bjab Bcl-XL), mock vector control cells

Bjab neo, Bjab mock) Jurkat Bcl-XL transfected (Jurkat Bcl-XL) and

Table 1Results from the computer-assisted screening, which was performed with BH3I-1 and BHcorresponding Tanimoto coefficient [9], with their molecular weight, log P, number of hwere not investigated in further in silico experiments.

and Chemistry 33 (2009) 451–456

mock vector control cells (Jurkat mock) were grown in RPMI-1640medium, supplemented with 10% fetal calf serum, 100 U/ml peni-cillin and 0.1 �g/ml streptomycin (Invitrogen, Karlsruhe, Germany)at 37 ◦C fully humidified 5% CO2 atmosphere.

HCT116 wild-type cells (HCT wt), mock vector control cells(HCT116 mock) and their corresponding isogenic knockout sub-lines HCT116 Bax−/−, HCT116 Bak−/− and HCT116Bax−/− Bak−/−(kindly provided by Dr. Bert Vogelstein, John Hopkins CancerCenter, Baltimore, MD, USA) and the HCT116 Bcl-2 and Bcl-XL trans-fected (HCT116 Bcl-2 and HCT Bcl-XL) were cultured in McCoy’s5A medium supplemented with 10% fetal calf serum, 100 U/mlpenicillin and 0.1 mg/ml streptomycin (Invitrogen, Karlsruhe, Ger-many).

2.4.2. Cytostatics for apoptosis assays

BH3I-1 was obtained from Calbiochem, Bad Soden, Germany.

The compounds BH3I-2, 1 and 5 were purchased from Asinex,Moscow, Russia. Compounds 2, 3 and 4 were obtained from Inter-BioScreen, Moscow, Russia and the compounds 6 and 7 werepurchased from Ambinter, Paris, France.

3I-2 as lead compounds. The compounds are displayed as 2D structure, with theirydrogen acceptors and hydrogen donors. The compounds with grey background

iology and Chemistry 33 (2009) 451–456 453

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Table 2Results of the GoldScore and �Gbinding scoring, which were calculated with the dock-ing program GOLD for the lead compounds BH3I-1 and BH3I-2 and the analogues 1,3, 4 and 5. The compounds with a grey background were no promising candidates.

Compound GoldScore �Gbinding [kJ/mol]

BH3I-1 50.47 −25.591 52.49 −23.493 −122.04 −13.524 −53.88 −19.81BH3I-2 51.33 −38.83

F1

M. Füllbeck et al. / Computational B

.4.3. Measurement of apoptotic cell death by flow cytometryJurkat, Bjab and HCT116 cells were seeded at a density of

05 cells/ml and treated with the indicated concentrations of BH3I-, BH3I-2, 1 and 5. After 72 h, the cells were collected, washed withBS at 4 ◦C and fixed in PBS/2% (v/v) formaldehyde on ice for 30 min.ollowing the fixation the cells were incubated with ethanol/PBS2:1, v/v) for 25 min, pelleted and resuspended in PBS containing0 �g/ml RNase A. Cells were incubated for 30 min at 37 ◦C, pelletednd finally resuspended in PBS containing 50 �g/ml PI. The nuclearNA fragmentation was then quantified by flow cytometric deter-ination of hypodiploid DNA, using a FACScan (Becton Dickinson,eidelberg, Germany). Data were analysed using the CELLQuest-ro software and are given in percentage hypodiploid cells (subG1),hich reflects the number of apoptotic cells.

. Results

.1. Computer-assisted screening

In Table 1, the results of the screening and the propertyrofiling with regard to the Lipinski Rule-of-five are shown. Theanimoto coefficients of all identified compounds are above thehreshold of 0.85, but as the value for 2 (TC: 0.86) is rather low, thisompound will be excluded from further investigations. Moreover,ompounds 6 and 7 will be obviated from the following analyses,ecause of the great number of hydrogen donors (6: 6 hydrogenonors; 7: 7 hydrogen donors), which do not comply with theipinski Rule-of five.

.2. Docking

To make a prediction of the binding affinity for the remain-ng four compounds from the in computer-assisted screening,he molecules were docked into the binding groove of the anti-

poptotic protein Bcl-XL. A peptide of the pro-apoptotic Bak,as used as reference ligand (PDB-code: 1BXL). The docking

esults in Table 2 show, that 1 and 5 possess a higher GoldScorehan the lead compounds, which implies an improved bindingffinity to the target protein, whereas 3 and 4 exhibit a lower

ig. 1. Docking results of (a) BH3I-1, (b) 1, (c) 3, (d) 4 and (e) reference ligand are displaBXL) using the docking program GOLD.

5 53.31 −35.11

GoldScore. Consequentially, 1 and 5 will be investigated in exper-imental results and 3 and 4 will be excluded from the followinganalyses.

The docking results of the lead compounds BH3I-1 and BH3I-2with their corresponding analogues into the binding groove of theanti-apoptotic protein Bcl-XL are shown in Figs. 1 and 2. BH3I-1binds to the upper part of the Bcl-XL binding groove (Fig. 1a),whereas 1 binds to the lower part (Fig. 1b), which is also coveredby BH3I-2 and its analogue (Fig. 2a). Fig. 1c and d shows the bindingof 3 and 4.

Theoretically predicted, potential Bcl-2 inhibitors will be inves-tigated in an apoptosis assay in a variety of cell lines, which havedifferent expression levels of pro- and anti-apoptotic proteins.Fig. 3 gives a survey of the 3D structures of the lead compoundsBH3I-1 and BH3I-2 and the analogues, which have been identi-fied via computer-assisted screening and were tested for theirinhibitory effect.

The compounds 2, 3, 4, 6 and 7 were analysed at a singular con-centration for their inhibitory effect in a DNA fragmentation assay,which verifies the theoretical predictions, as there is no significantbiological effect (data not stated).

Whether the induction of the apoptotic cell death via BH3I-1,BH3I-2 and their corresponding analogues 1 and 5 depends on Bcl-2

or rather on Bcl-XL, was determined by a DNA fragmentation assaywith a number of cell lines, which contain different amounts ofthese anti-apoptotic proteins.

yed. The compounds are docked into the anti-apoptotic protein Bcl-XL (PDB-code:

454 M. Füllbeck et al. / Computational Biology and Chemistry 33 (2009) 451–456

Fig. 2. Docking results of (a) BH3I-2 and (b) 5 are displayed. The compounds are docked into the anti-apoptotic protein Bcl-XL (PDB-code: 1BXL) using the docking programGOLD.

Fig. 3. 3D structure of (a) BH3I-1, (b) 1, (c) BH3I-2 and (d) 5. The atoms are coloured as fogreen: chlorine, light blue: iodine.

Fig. 4. Amount of hypodiploid cells in Bjab neo/mock cells and Bjab Bcl-XL cells after72 h treatment with (a) BH3I-1, (b) 1, (c) BH3I-2 and (d) 5. Apoptosis was determinedby measuring fragmentation of genomic DNA (hypodiploid cells) by flow cytometry.The values represent means ± s.d. (n = 3).

llows: grey: carbon, purple: bromine, yellow: sulphur, red: oxygen, blue: nitrogen,

3.3. Bcl-XL dependent induction of apoptosis by BH3I-1, BH3I-2and its analogues in Bjab cells

The induction of apoptosis (shown as number of hypodiploidcells) is increased by adding the lead compounds to Bjab neo/mockand Bjab Bcl-XL cells (Fig. 4a and c).

The BH3I-2 analogue shows a higher percentage of apoptoticcells at lower concentrations compared to the lead compound inBjab Bcl-XL cells, but a reduced number of apoptotic events in thecontrol vector cell line (Fig. 4d).

Compared to the mock cells, the Jurkat Bcl-XL cells showdecreased apoptosis, when they are treated with BH3I-2 and thecorresponding analogue 5 (Fig. 5) whereas the BH3I-2 analogueshows an increased number of apoptotic cells compared to the leadcompound (Fig. 5b).

3.4. Induction of apoptosis by BH3I-2 and its analogue isindependent of Bcl-XL and Bcl-2 in HCT116 cells

The number of hypodiploid events in cells, treated with the leadcompound BH3I-2 and its analogue, is not significantly different

(Fig. 6).

Furthermore, the influence of the pro-apoptotic proteinsBax and Bak on the induction of apoptosis via BH3I-1, BH3I-2, 1 and 5 was investigated with a variety of knockout celllines.

M. Füllbeck et al. / Computational Biology and Chemistry 33 (2009) 451–456 455

Fig. 5. Flow cytometry analysis of DNA fragmentation in Jurkat mock and Bcl-XL

cells treated with BH3I-2 and its analogue. (a) The lead structure BH3I-2 and (b) 5for 72 h. The results were given in percentage of hypodiploid cells, which reflectsthe number of apoptotic cells. The values represent means ± s.d. (n = 3).

Fig. 6. Amount of hypodiploid cells in HCT116 cells (mock, Bcl-XL and Bcl-2) treatedwith BH3I-2 and its analogue compound 5. (a) BH3I-2 and (b) 5 for 72 h. Apoptosiswas determined by measuring fragmentation of genomic DNA (hypodiploid cells)by flow cytometry. The values represent means ± s.d. (n = 3).

Table 3Calculated IC50 values for BH3I-1, BH3I-2 and the novel analogues 1 and 5.

Compound IC50 [�M]

BH3I-1 293.95

Fig. 7. Flow cytometry analysis of DNA fragmentation in HCT116 cells (wt, Bak−/−; Bak(a) BH3-I, (b) 1, (c) BH3I-1 and (d) 5 for 72 h. The results were given in percentage of hymeans ± s.d. (n = 3).

Compound 1 81.23BH3I-2 67.26Compound 5 36.11

3.5. Slight Bax and Bak dependency in the induction of apoptosisin HCT116 cells by BH3I-2 and analogue, but not by BH3I-1 and 1

In Fig. 7a and b, it becomes obvious that the presence or absenceof Bak or Bax has no significant influence on the amount of apoptoticevents induced by BH3I-1 and its analogue. Unlike BH3I-1, BH3I-2and its analogue shows slight effects in the increase of hypodiploidcells, dependent on the presence or absence of Bax and Bak (Fig. 7cand d).

After treatment with BH3I-2, the HCT116wt shows the highestrate of apoptosis, followed by Bak−/− and Bak−/− Bax−/−. Cellswithout Bax have the lowest amount of hypodiploid cells (Fig. 7c).For both compounds, the IC50 value was calculated (Table 3).

4. Discussion

The Bcl-2 protein family plays a pivotal role in the regulationof apoptosis (Burlacu, 2003). Bcl-2 and Bcl-XL, two anti-apoptoticmembers of the Bcl-2 protein family, do not only contribute to can-cer progression by inhibiting apoptosis, but are also responsiblefor the resistance of cancer cells against current cancer treat-

ments (Reed, 2006; Wang et al., 2003). Therefore, Bcl-2 proteinsare promising new targets in cancer therapy.

Degterev et al. (2001) showed, that apoptosis induced by thecompounds BH3I-1 and BH3I-2, is similar to the cell death caused byan overexpression of pro-apoptotic Bcl-2 family members, but does

−/− and Bax−/−) after the treatment with BH3I-1 and the analogue compound 1.podiploid cells, which reflects the number of apoptotic cells. The values represent

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56 M. Füllbeck et al. / Computational B

ot lead to Bax insertion into mitochondrial membranes. They con-luded, that BH3I-1 and BH3I-2 induce apoptosis by inhibiting theeterodimerisation of Bcl-XL/Bcl-2 and by releasing pro-apoptoticcl-2 family members, which in turn initiate downstream apoptoticvents (Degterev et al., 2001).

Using BH3I-1 and BH3I-2 as lead compounds for a computer-ssisted screening, we identified seven compounds. By applicationf a variety of bioinformatical methods, the compounds 1 and 5howed best properties which could be verified by apoptosis assaysn a variety of cell systems. Experimental results of 2, 3, 4, 6 and 7alidated the theoretical predictions, which specified these com-ounds to be no promising anti-cancer agents. To compare 1 andwith the properties of the lead compounds BH3I-1 and BH3I-2,

ells, overexpressing Bcl-XL proteins, were used and it revealed,hat the lead compounds as well as their analogue, show Bcl-XLependency (Figs. 4 and 5).

In cells, overexpressing Bcl-XL, a decreased amount of apoptoticells is detectable after treatment with 1 and 5 as these cells containore anti-apoptotic Bcl-XL. BH3I-1 and its analogue do not show

ny Bax dependency, from which it can be concluded, that neitherhe lead structure nor compound 1 can induce a conformationalhange in Bax, which supports the thesis that both BH3Is directlynteract with Bcl-2.

BH3I-2 shows similar properties as BH3I-1, referring to thenduction of Bcl-2 dependent apoptosis. Between the lead struc-ure and its analogue, no significant difference in the amountf hypodiploid cells can be seen, although the analogue showsmproved apoptosis, inducing abilities compared to BH3I-2 in otherell lines. Influencing the Bcl-2 induced apoptosis seems to bempossible in Bcl-2 and Bcl-XL expressing cell lines (Fig. 6). Espe-

ially, it should be pointed out, that 5 shows a higher induction ofpoptosis in Bak−/−, Bax−/− and Bak−/− Bax−/− cells comparedo BH3I-2, and it seems that 5 can lead to a heterodimerisation ofax (Fig. 7). This shows that an improvement of binding abilities

s possible and that this might even lead to a different mechanism

and Chemistry 33 (2009) 451–456

of the induction of apoptosis, compared to the original structures.5 seems to be able to induce apoptosis by Bax insertion into themitochondrial membrane, an ability that the lead structure BH3I-2does not exhibit.

Here we can show that computer-assisted screening is an effec-tive tool to identify improved Bcl-2 inhibitors with an increasedbinding affinity (Table 3). The combination of 2D and 3D similarityscreening, leads to the identification of compounds that can inhibitthe activation of anti-apoptotic proteins and induce apoptosis incells overexpressing Bcl-2 family proteins.

Acknowledgements

This work was supported by Deutsche Krebshilfe and Sonder-forschungsbereich 449.

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