Structure–activity relationships of Bak derived peptides: Affinity and specificity modulations by amino acid replacement

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European Journal of Medicinal Chemistry 43 (2008) 966e972 http://www.elsevier.com/locate/ejmech

Original article

Structureeactivity relationships of Bak derived peptides: Affinity and specificity modulations by amino acid replacement Virginie Frey a,b, Julien Viaud a,b, Guy Subra a,b, Nicolas Cauquil c, Jean-Franc¸ois Guichou a,b, Patrick Casara c, Ge´rard Grassy a,b, Alain Chavanieu a,b,* b

a INSERM, U554, Montpellier, F-34090, France Universite´ Montpellier 1 et 2, CNRS, UMR5048, Centre de Biochimie Structurale, F-34090 Montpellier, France c Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy sur Seine, France

Received 18 April 2007; received in revised form 15 June 2007; accepted 15 June 2007 Available online 6 July 2007

Abstract To study the structureeactivity relationships (SAR) and the binding activity of pro-apoptotic Bak BH3 domain, we synthesised several 16mer peptide analogues corresponding to the region 72-GQVGRQLAIIGDDINR-87. Using different amino acids varying in length, steric and electronic properties, we investigated the role and the nature of physicochemical parameters of residues Val74, Leu78, Ile81 and Ile85, previously identified to be crucial for interactions. With this aim, we measured the affinity of these peptides on two anti-apoptotic proteins Bcl-xL and Bcl-2 by a polarization fluorescence competitive assay. We defined that the most potent peptide on Bcl-xL, which presents a 4.6-fold increase as compared to the parent peptide affinity, was obtained when Ile85 was mutated with a 4-chlorophenylalanine. Finally, assays of eight Bak peptide analogues on Bcl-2 allowed us to postulate that modulations at position 78 could afford peptides with a binding selectivity enhanced for Bcl-xL. These pharmacological and physicochemical parameter data should prove useful for the rational design of non-peptide ligands as potential antagonists of Bcl-2 protein interactions. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Apoptosis; Bcl-xL; Bcl-2; Structureeactivity relationships; Selectivity

1. Introduction Apoptosis, a form of programmed cell death, is a physiological process by which a cell commits suicide. It ensures the balance between cellular proliferation and turnover in nearly all tissues. This balance is delicate: insufficient apoptosis will lead to cancer and autoimmune diseases, whereas too much apoptosis will cause degenerative disorders or strokes [1]. The mechanisms involved in apoptosis are complex. There are mainly two pathways leading to apoptosis: the stress and the death receptor ways. Both of them provoke the activation of caspases, some proteases that will cleave a set of proteins * Corresponding author. INSERM, U554, Montpellier, F-34090, France. Tel.: þ33 4 67 54 86 44; fax: þ33 4 67 41 77 13. E-mail address: [email protected] (A. Chavanieu). 0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2007.06.008

and cause disassembly of the cells. The initiation of apoptosis through caspase activation is tightly controlled by various factors, among which are the Bcl-2 family proteins [2]. The proteins of the Bcl-2 family are key regulators of apoptosis [3,4] which can promote either cell survival (the anti-apoptotic proteins, such as Bcl-2, Bcl-xL, Mcl-1, A1 and Bcl-w) or cell death (the pro-apoptotic proteins such as Bak, Bax, and Bok). All these proteins share at least one of four homology domains, called BH domains. The pro-apoptotic proteins can also be divided into two classes depending on the presence of a unique BH3 a-helical domain (BH3 only proteins, such as Bad, Bid, Bim) or a tandem of BH1, BH2 and BH3 domains (Bax and Bak) [5]. Among the different mechanisms by which Bcl-2 proteins regulate apoptosis, their ability to form homo or heterodimers seems to be crucial [6e8]. Many structural studies of

V. Frey et al. / European Journal of Medicinal Chemistry 43 (2008) 966e972

complexes between pro- and anti-apoptotic members have been described [9e11]. It has been shown that the anti-apoptotic proteins form an hydrophobic groove via it’s BH1, BH2 and BH3 domains (acting as the receptor domain), in which pro-apoptotic protein, such as Bak, binds via it’s BH3 domain (ligand binding domain) structured in an alpha amphipathic helix. Both the helicity [10] of the BH3 domain and the nature of some of it’s residues [11] have been identified to be crucial for the interaction [12]. The interaction between Bak BH3 peptide and Bcl-2 proteins has been previously described. It has been determined [11] that the minimal sequence of the Bak BH3 peptide necessary for the binding to Bcl-xL was a 16mer peptide corresponding to sequence 72-GQVGRQLAIIGDDINR-87 of the protein. Shorter peptides appeared to be inactive, whereas the activity of longer peptides was not improved significantly. By an ala-scan study, Sattler et al. [11,13] pointed out that the four hydrophobic residues Val74, Leu78, Ile81 and Ile85 were crucial for the binding to Bcl-xL. Moreover, Walensky [13] showed that in the case of BH3 Bid peptides, an increase of the a-helical content obtained through a peptide cyclisation led to an improvement in affinity towards Bcl-2 and pharmacological properties (such as cell-permeable characteristics). From a medicinal chemistry perspective, understanding the mechanism of binding between pro- and anti-apoptotic members of the Bcl-2 family is of utmost importance, particularly to the design of newly derived peptides with enhanced activity. Except peptide cyclisation, no further optimisation has been reported so far. Ultimately, this may favour design of useful non-peptidic drugs. In this work, we investigated further the role of key residues in the interaction between Bak peptide and Bcl-xL or Bcl-2 proteins in order to better define the nature of the physicochemical parameters of crucial residues in terms of qualitative descriptors such as electronic properties, volume, size and hydrophobicity.

967

Fig. 1. NMR structure of Bak peptide in complex with Bcl-xL (PDB code 1BXL). Connoly surface of Bcl-xL reveals the interaction site as a large hydrophobic groove (white ¼ hydrogen; green ¼ carbon; red ¼ oxygen; blue ¼ nitrogen). Cyan ribbon shows the a-helical secondary structure of Bak peptide and crucial hydrophobic residues of Bak Val74, Leu78, Ile81 and Ile85 are shown in sticks.

a fluorescence polarization competition assay as described in Section 3. Results are described in Fig. 2 and EC50 of peptides are given in Table 1. In our test, Bak parent peptide exhibited an EC50 of 174  27 nM to be compared to the KD of 340 nM reported by Sattler based on a test monitoring the fluorescence emission of Trp residues of Bcl-xL [11]. For three of the four positions, our results indicated that increase of the hydrophobic character led to peptides showing a better activity to Bcl-xL. For position 1 (Val74), increasing the aliphatic and steric characters of the lateral chain by a norleucine enhanced the affinity by a factor of 2.5 when compared to the parent peptide. For position 2 (Leu78), the affinity has been improved

1.1. Structureeactivity relationships of Bak derived peptides 1.1.1. First set of mutations The binding surface of interaction between Bcl-xL and Bak peptide is represented in Fig. 1 [11]. The four key residues identified by ala-scan [14] Val74, Leu78, Ile81 and Ile85, are shown in sticks. These residues are buried in different positions of the hydrophobic groove of Bcl-xL and each interacts within a specific environment in terms of neighbourhood residues, mainly hydrophobic, and volume cavities. Our aim was to characterize the role of these crucial residues in order to design optimised peptides. For that purpose, we synthesised peptide analogues mutated in key positions with natural and exotic residues, varying in hydrophobicity, length and steric volume. In a first set of mutations, the four residues were replaced with three amino acids: phenylalanine, norleucine and 2-naphthylalanine. Thus, we changed hydrophobic side chains of original residues (isopropyl, isobutyl) with a pentyl linear chain, a benzyl group and a naphthalene moiety. Peptides were synthesised, and their affinities were evaluated by

Fig. 2. Curves of Bak BH3 mutated and Bak fluoresceinated peptides binding to GST-Bcl-xL quantitated by fluorescence polarization. Parent Bak peptide (Bak) or mutated with a 2-naphthylalanine at position Ile81 (Bak I81 2-Nal) and with a 4-iodophenylalanine at position Ile85 (Bak I85 4-IPhe) were used as competitors in the Bcl-xL/Bak fluorescence polarization assay. Bak fluorescein was used as the fluorescent probe. Binding affinities (n ¼ 2, triplicate) given in tables are EC50, classically measured from the sigmoidal dosee reponse curves.

V. Frey et al. / European Journal of Medicinal Chemistry 43 (2008) 966e972

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Table 1 Binding affinity on Bcl-xL (nM) for the first set of single mutated peptides (n ¼ 2)

WT Norleucine Phenylalanine 2-Naphthylalanine Alaninea

Val74

Leu78

Ile81

Ile85

174  26 71  8b 304  36 350  7 15 000

174  26 318  137 405  55 127  17b 270 000

174  26 655  152 2141  70 1297  377 17 000

174  26 118  46b 54  8b 125  12b 93 000

a

Data from Sattler’s publication. Data shown in bold correspond to enhanced affinities compared to the Bak parent peptide. b

by a factor of 1.4 using a 2-naphthylalanine residue bearing consequent hydrophobic and aromatic components. Then, an improvement of the EC50 has been obtained for position 4 (Ile85) whatever be the hydrophobic residues used, with a preference for a phenylalanine residue that induced a decrease of the EC50 to 54  8 nM. Interestingly, none of the mutations on position 3 (Ile81) allowed us to enhance the affinity of the peptides, suggesting this position to be very sensitive to mutations. This first approach indicated that particular parameters e mainly hydrophobic and aromatic criteria e control the interactions at each of the four positions pointed out by Sattler. This led us to modulate more specifically these positions with residues bearing various physicochemical parameters. However, considering the less crucial role of Val74 [15e17] and the improvement already obtained with norleucine, we decided to focus our study on the region of the Bak peptide delineated by Leu78 and Ile85.

to an improvement of affinity. Thus, only the 2-naphthylalanine, which enhanced the affinity by a factor of 1.4 (Table 1), has a positive effect on peptide affinity. Interestingly, peptide with a 1-Nal induced a drastic loss of activity. 1.1.3. Replacement of Ile81 In a moderate manner Ile81 has also been described as a crucial residue for the binding since the affinity was reported to decrease by a factor of 50 when replaced by an alanine [11] (a factor of 100 in our study). In the first set of mutations, none of the three amino acids used led to an improvement of affinity (Table 1); we so selected six new hydrophobic residues with different steric volumes and electron-rich groups. As illustrated in Table 3, neither rigid hydrophobic residues such as naphthylalanine, tryptophane and more flexibles such as biphenylalanine nor phenylalanine substituted with an electronwithdrawing group in position 3 or 4 (nitrophenylalanine, methyltyrosine, chlorophenylalanine) exhibited higher affinity than the parent peptide.

1.1.2. Replacement of Leu78 According to Sattler [11], Leu78 was described to be the most important residue in the 16mer peptide as it’s binding activity was reported to decrease by a factor of 800 when replaced by an alanine (a factor of 1500 in our study). As seen in our primary results, strong hydrophobic and aromatic components enhanced the affinity of the peptide. We so synthesised new related peptides using homophenylalanine, 1-naphthylalanine, 3pyridinylalanine and 3-chlorophenylalanine to assess aromatic moieties with different steric and electronic parameters. Peptides with a cyclohexylalanine as a non-aromatic residue and a methionine as an aliphatic amino acid were also synthesised. As shown in Table 2, none of the new hydrophobic residues led

1.1.4. Replacement of Ile85 From the three amino acids selected at first to replace Ile85, it appeared this position was more tolerant to mutations and led to enhanced peptides (Table 1). To better define physicochemical parameters that lead to stronger interaction, ten new mutated peptides were synthesised (Table 4). Adding polar amino acids such as histidine and glutamic acid markedly decreased the affinity to Bcl-xL, confirming that the hydrophobicity of Ile85 was the key factor of the interaction. Compared to the parent peptide, six peptides with hydrophobic residues exhibited an improved EC50. Several aromatic residues as well as a non-aromatic cyclohexylalanine residue led to significant improvement of affinity. Bulky aromatic residues such as naphthylalanine as well as an aliphatic norleucine residue also resulted in an increase of the affinity. Lastly, adding electron-withdrawing group in position 3 or 4 on the phenylalanine residue led to more potent peptides. Finally the substitutions at position 85 were in preferential order: 4-chlorophenylalanine > 4iodophenylalanine > phenylalanine > 3-chlorophenylalanine > cyclohexylalanine > 1-naphthylalanine > norleucine > 2naphthylalanine > tyrosine > isoleucine, and the EC50 was improved by a factor of 4 (Ec50 ¼ 38  11 nM) when Ile85 was mutated with a 4-chlorophenylalanine.

Table 2 Binding affinity on Bcl-xL (nM) for single mutated peptides at position Leu78 (n ¼ 2)

Table 3 Binding affinity on Bcl-xL (nM) for single mutated peptides at position Ile81 (n ¼ 2)

Residue

EC50  SEM

Residue

EC50  SEM

Leucine Cyclohexylalanine Homophenylalanine 3-Chlorophenylalanine Methionine 3-Pyridinalanine 1-Naphthylalanine

174  26a 219  2 225  14 359  23 1431  79 9740  716 >10 mM

Isoleucine 3-Chlorophenylalanine 1-Naphthylalanine Tryptophane Biphenylalanine 4-Nitrophenylalanine Methyltyrosine

174  26a 200  68 299  17 375  12 1246  235 1466  660 3615  739

a

Data shown in bold correspond to the affinity of Bak parent peptide.

a

Data shown in bold correspond to the affinity of Bak parent peptide.

V. Frey et al. / European Journal of Medicinal Chemistry 43 (2008) 966e972 Table 4 Binding affinity on Bcl-xL (nM) for single mutated peptides at position Ile85 (n ¼ 2) Residue

EC50  SEM

4-Chlorophenylalanine 4-Iodophenylalanine 3-Chlorophenylalanine Cyclohexylalanine 1-Naphthylalanine Tyrosine Isoleucine Biphenylalanine 4-Nitrophenylalanine Histidine Glutamic acid

38  11 45  3 56  7 58  20 77  1 159  37 174  26a 267  24 594  190 5122  1472 >10 mM

See also Table 1 for the replacement with a phenylalanine. a Data shown in bold correspond to the affinity of parent peptide.

1.1.5. Double residue replacements and intra peptide cyclisation Because modifications of Leu78 and Ile85 allowed an increase of affinity of Bak peptides, we then investigated the effects of double replacement on these positions (Table 5). With a 2-naphthylalanine on position 78 and a phenylalanine or a 4-chlorophenylalanine on position 85, peptide affinities (54  4 nM and 53  12 nM, respectively) were enhanced by a factor of 3 when compared to the parent peptide (EC50 ¼ 174  26 nM) but not for mono-substituted peptide on position 85 (54  8 nM and 38  11 nM, respectively). These results suggest that an alternative approach should to be used to further enhance peptide affinities. A previous report by Walensky et al. [13] indicated that peptide helix stabilisations of synthetic Bid BH3 domain induced a higher affinity of binding to Bcl-2 as well as a better cell penetration. To achieve such stabilisation Walensky et al. performed an intra peptide cyclisation through a strategy using disubstituted non-natural amino acids containing olefin-bearing tethers to generate an all-hydrocarbon staple. In this context, we synthesised an intra cyclic analogue of Bak (BakCyclm) with a 4-chlorophenylalanine at position 85, which corresponds to the most effective amino acid replacement obtained so far. However, in our case the intra cyclisation was obtained through a side chain amide bond using an AllocLys and an AllylGlu at positions 80 and 84, respectively. As a control, a cyclic Bak peptide with an isoleucine at position 85 (BakCycl) was also synthesised. In presence of TFE, all linear Bak peptides tested, in particular peptides bearing an isoleucine or a 4-chlorophenylalanine at position 85, presented a helical content of around 16% and thus predominantly existed as a random coil. Whereas for BakCycl and BakCyclm peptides, Table 5 Binding affinity on Bcl-xL (nM) for the double amino replacement at positions Leu78 and Ile85 (n ¼ 2) Position Leu78

Position Ile85

EC50  SEM

2-Naphthylalanine 2-Naphthylalanine

Phenylalanine 4-Chlorophenylalanine

54  4 53  12

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this content was found to be 33% and 32%, respectively, which indicates that a-helix secondary structure is reinforced. These data are in good agreement with previous reports [13,18] on Bak peptide analogues bearing lactam bridges. Tested by fluorescence polarization competition on Bcl-xL (data not shown), the BakCycl peptide exhibited an EC50 of 67  9 nM, which is 2.6 lesser than the linear Bak peptide. This result confirms the correlation between helical content and activity enhancement observed for synthetic BH3 domains of Bcl-2 proteins. However, the cyclic mutated BakCyclm presented an EC50 of 28  1 nM, which is in the same range as the linear mutated analogue (EC50 ¼ 38 nM  11 nM). Thus, as previously shown with BH3 peptides [15,18,19], the helical content roughly correlates with the biological activity but the peptide sequence is also critical, particularly for peptides with a strong binding activity.

1.2. Affinities of several peptides on Bcl-2 Another interesting question concerning the Bak BH3 domain is it’s specificity towards different members of antiapoptotic proteins of the Bcl-2 family. We then investigated the effect of eight peptide mutations on the binding to Bcl-2, a key protein in the apoptotic process. For each crucial position of the Bak peptide, one mutation with a noxious effect and one with a positive impact were selected. For each peptide, the ratio EC50(Bcl-2)/EC50(Bcl-xL) is indicated to reflect the specificity of the peptide for Bcl-xL. As shown in Table 6 and in accord with Petros et al. [14], Bak BH3 parent peptide binds to Bcl-2 with an EC50 of 3020  973 nM, which represents a reduction by a factor of 17 when compared to Bcl-xL. Effect on binding affinity to Bcl-2 of mutations at positions 74 and 85 was in good correlation with those observed on Bcl-xL. Moreover, an increase in term of selectivity for Bcl-xL was observed when Ile85 was replaced with a 4-chlorophenylalanine. At position 78, the two mutations selected, a 2-naphthylalanine and a methionine, were deleterious for the binding to Bcl-2 by a factor of 3 and 5.5, respectively. Interestingly, the residue 2-naphthylalanine led to a higher specificity towards Table 6 Binding affinity on Bcl-2 (nM) of eight single mutated peptides (n ¼ 2) EC50  SEM Bak Position 74 Norleucine 2-Naphthylalanine Position 78 2-Naphthylalanine Methionine Position 81 Tryptophane Biphenylalanine Position 85 4-Chlorophenylalanine 4-Nitrophenylalanine a

Ratioa

3020  973

17

757  192 6129  263

11 17

16 970  980 29 875  1765

133 21

2796  212 20 745  2295

7 17

1135  57 8018  30

30 13

Ratio ¼ EC50 (Bcl-2)/EC50 (Bcl-xL).

970

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Bcl-xL with a ratio of 133. By contrast, an Ile to Trp mutation at position 81 was found to be the only amino acid replacement to slightly improve the binding to Bcl-2 but not to Bcl-xL. 2. Discussion Proteins of the Bcl-2 family have been identified as major modulators of apoptosis, a crucial phenomenon for cell viability and development. Although the precise molecular mechanism by which these Bcl-2 proteins regulate apoptosis is still under investigation [20], it appears that the anti-apoptotic proteins and the pro-apoptotic proteins of the Bcl-2 family modulate their opposing functions through heterodimerization [21] and that the ratio of pro- to anti-apoptotic proteins could be associated with the progress or not with the apoptotic process [22,23]. Thus, an imbalance between pro- and anti-apoptotic proteins leads to the accumulation of cells or to an excessive apoptosis which can lead to several human diseases such as cancers, autoimmune syndromes, neurodegenerative disorders. Structural studies have determined that a hydrophobic groove on the surface of anti-apoptotic proteins form a binding site for the BH3 domain of pro-apoptotic proteins [22] and peptides derived from the BH3 domain of Bax, Bid, Bad and Bak inhibit heterodimerization in in vitro assays and induce cellular effects [24e27]. Also, several small organic active compounds have been identified that bind into the binding groove of Bcl-2 or Bcl-xL and promote apoptosis [12,16,18,28e33] although it was demonstrated that only one of these, ABT-737, was a BH3 domain. Because proteineprotein interactions are a key regulator mechanism of this cellular process, the understanding of interactions could be useful for the design of peptides or peptidomimetics with an enhanced activity, as well as for the discovery of small organic compounds that could modulate formulation of dimers. The binding of Bak BH3 peptides to Bcl-xL has already been studied by Sattler et al. through an ala-scan experiment that has clearly demonstrated the importance of several hydrophobic or charged residues. In this study, using a set of peptides corresponding to the Bak BH3 domain, we first investigated more precisely the nature of physicochemical parameters of critical hydrophobic residues that interact with the bottom of the binding groove (Fig. 1). Our results confirm that hydrophobic interactions are the key parameter for the four critical residues; however, more detailed analysis revealed that each local cavity of the groove, wherein one critical residue lies, presents a specific behaviour in terms of complementarities and capability to adapt an amino acid replacement. In the case of the Val74, which mainly interacts with Val126 and Leu130 on Bcl-xL, we showed that replacement with aromatic residues led to peptides with a correct EC50 value (<500 nM) but the affinity is only increased with the aliphatic norleucine. For Leu78, the most important residue for the interaction with Bcl-xL [11], we determined that the local cavity is relatively tolerant to amino acid replacements as five peptide analogues among nine synthesised

showed an EC50 lower than 500 nM. In fact, the binding pocket for Leu78, which is formed by Tyr101, Leu108, Val126, and Phe146 of Bcl-xL accepts aromatic or aliphatic amino acids. However, the affinity was only improved with the bulky aromatic 2-naphthylalanine residue. Moreover, the replacement with a 1-naphthylalanine abolished the binding activity, despite the same hydrophobic steric volume than the 2-naphthylalanine, which suggests that the conformation and orientation of the side chain of the residue to be critical. By contrast, Ile81 seems a less tolerant position to mutation either to maintain or increase the affinity on Bcl-xL as three peptides among nine presented an EC50 lower than 500 mM. At this binding pocket, the 1-naphthylalanine was well accepted but not the 2-naphthylalanine isomer. Whatever be the physicochemical parameters assessed (aromaticity, steric volume, electronic properties) affinity remained lower than that of the parent peptide, which suggests this part of the binding groove in Bcl-xL is relatively restrictive. Finally, most hydrophobic mutations at position Ile85 led to more potent peptides (from two to four times as compared to the parent peptide). Indeed, nine out of eleven Bak peptide analogues exhibited higher affinities than the parent peptide. Interestingly, a phenylalanine substituted with electron-withdrawing groups such as halogens led to higher active analogues of our set of peptides. When bound to Bcl-xL in a distant pocket from the three preceding residues, Ile85 seems to be located in an environment which could allow more additional interactions that those observed with the parent Bak peptide. Besides the structureeactivity relationship of critical residues for the interaction to Bcl-xL, we then determined if amino acid replacement could orient to a binding selectivity towards another member of the anti-apoptotic sub-family, Bcl-2. Our results clearly indicated that mutations on positions Val74, Ile81 and Ile85 led to peptides with a ratio EC50(Bcl-2)/ EC50(Bcl-xL) only slightly modified which indicated that no change in selectivity was observed. On the other hand with a 2-naphthylalanine at position Leu78, peptide analogue appears to be four times more potent on Bcl-xL when compared to the parent Bak peptide. These results seem to indicate that the binding pocket for Leu78 could be the local region that could afford more selective peptides or organic compounds to Bcl-xL. In conclusion, mutated peptides and a-helix stabilisation allowed us to identify peptides with a 4.5e6-fold increase of the affinity. However, neither multi residue replacements nor intra peptide cyclisation gave us the possibility to increase a threshold of activity (around 30 nM). Taken together, these results demonstrated that because no or minor improvements were obtained with analogues mutated at positions Leu78 and Ile81, Ile85 and to a lesser extent, Val74 appear to be the key residues to improve the affinity on Bcl-xL. Whereas position Leu78 appears as a key location to explore and optimise related Bak peptides with a higher specificity. This information could certainly be useful in the discovery and optimisation of pharmacological peptides or small organic compounds that block the interaction between Bak and Bcl-xL [31].

V. Frey et al. / European Journal of Medicinal Chemistry 43 (2008) 966e972

3. Material and methods 3.1. Materials All Fmoc-amino acid derivatives, resins, reagents and solvents used in the peptide synthesis were purchased from Applera, France. TFA and phenol were purchased from SDS. All chemicals were reagent grade and used without further purification. Palladium tetrakis was synthesised as described [34]. 3.2. Peptides synthesis Using Fmoc-chemistry, the solid phase synthesis of Bak peptide analogues was carried out on Fmoc-PAL-PEG-PS resins at a 0.2 mmol scale using a continuous flow apparatus (Pioneer Workstation, Applera, France) as previously described [35]. All Fmoc-amino acids (4 eq.) were coupled by in situ activation with O-(benzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium tetrafluoroborate (TBTU, 4 eq.) and diiso-propylethylamine (DIEA) in N,N-dimethylformamide for a minimum of 30 min. Amino side chain were protected as follows: Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, FmocGlu(OtBu)-OH, Fmoc-Asn(Trt)-OH. Double couplings were only performed for Arg76 and Gln77. After completion of the chain assembly, the resins were extensively washed with isopropanol and dried overnight under vacuum. Deprotection and cleavage were achieved by treatment with a mixture of trifluoroacetic acid (TFA) (88%), water (5%), phenol (5%) and triisopropylsilane (2%) then peptides were precipitated in ether, filtered and dried under vacuum. The crude peptides were then purified by preparative-reverse phase high performance liquid chromatography (rp-HPLC) on a Delta Pack C18 (300 A) 1.9  30 cm column, using a water (0.1% TFA)eacetonitrile (0.8%TFA) gradient during 60 min. The homogeneity (>95%) was confirmed by analytical rp-HPLC with a C18 column and ESI-MS on a Q-Star (Applera France). Cyclic peptides were synthesised as described above, replacing Ile80 by AllocLys and Asp84 by AllylGlu in the Bak sequence; after completion of the chain assembly, the resins were extensively washed with isopropanol and dried overnight under vacuum. AllocLys and AllylGlu were deprotected using palladium tetrakis as follows: in a reactor under mechanical agitation, resin was washed with CHCl3 then swelled in CHCl3 (35 mL per gram of resin) for 10 min. Resin was then placed under a nitrogen atmosphere during 10 min. Acetic acid (0.5 mL per gram of resin), N-methylmorpholine (2 mL per gram of resin) and palladium tetrakis (6 eq.) were added to the resin. Resin was stirred during 3 h under a nitrogen atmosphere. At the end of the reaction, resin was washed three times with CHCl3. Cyclisation between the two lateral chains was executed directly after their deprotection, as follows: resin was rinsed with DMF, then suspended in DMF (40 mL per gram of resin); HATU (4 eq.) and DIEA (9 eq.) were added and the mixture was stirred during 3 h. At the end of the reaction, resin was washed three times with DMF, and dried under vacuum.

971

Cyclisation was checked using a Kaiser test. Finally deprotection, cleavage and purification were achieved as for linear peptides.

3.3. Preparation of Bcl-xL and Bcl-2 Recombinant GST-Bcl-xL or Bcl-2 (pGEX 5X vector, Amersham) deleted of the c-terminal 21 amino acids were produced in Escherichia coli (BL21) and purified on gluthathionesepharose 4 Fast Flow 3 resin (Amersham). GST-Bcl-xL and GST-Bcl-2 were then dialysed against binding buffer (20 mM phosphate buffer, pH ¼ 7.4, 1 mM EDTA, 50 mM NaCl).

3.4. Fluorescence polarization assays Fluorescence polarization (FP) assays were performed essentially as described [36]. Briefly, for competition experiments, fluorescent Bak BH3 peptide (15 nM), labelled at it’s amino terminus with fluorescein (Neosystem), and GSTBcl-xL (100 nM) or GST-Bcl-2 (1 mM) were mixed and 10 mL tested peptide (first diluted in 10 mM DMSO then in binding buffer and pluronic acid 0.05% for final concentration from 109 to 104 M) in a final volume of 40 mL. The plate was mixed on a shaker for 1 min and incubated at room temperature for an additional 30 min. Fluorescence polarization in millipolarization units (mP) was measured with a FusionÔPackard equipment at room temperature with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. All experimental data were analyzed using Prism 3.0 software (Graphpad Software Inc., San Diego, CA), and EC50 values were generated by fitting the experimental data using a sigmoidal doseeresponse nonlinear regression model.

3.5. Circular dichro€ısm Circular dichroism (CD) measurements were carried out on a Chirascan spectrophotometer (Applied Photophysics). Measurements were taken using a 1 mm path length. All spectra were recorded in 1.0 nm wavelength increments with a 0.5 s time constant. Each spectrum is the average of five scans corrected for background solvent effects by subtraction of the appropriate buffer blank. Bak and it’s mutants in either 40 mM sodium phosphate, pH 7.2, or 40 mM sodium phosphate (30% TFE), pH 7.2, were diluted to a concentration of 0.2 mg/ml, respectively. Spectra were scanned in the far-UV from 260 to 185 nm. The mean residue molecular weight used in the calculation of the mean residue ellipticity was calculated for each peptide. The spectrum was analyzed using CDNN. CDNN is a trained neural network circular dichroism deconvolution program written by Gerald Bo¨hm, Institut fu¨r Biotechnologie, Martin-Luther-Universita¨t Halle-Wittenberg, Germany [37].

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Acknowledgements This study was supported by Laboratoire Coope´ratif Servier-CNRS-UM1.We thank Dr. John Hickman, Dr. Olivier Geneste and Dr. Yannick Bessin for scientific discussions and Mr. Terry Page for comments on the manuscript. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

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