Development of an IL-6 antagonist peptide that induces apoptosis in 7TD1 cells

Peptides 24 (2003) 1207–1220

Development of an IL-6 antagonist peptide that induces apoptosis in 7TD1 cells Rossella Manfredini a , Elena Tenedini a , Michela Siena a , Enrico Tagliafico a , Monica Montanari a , Alexis Grande a , Tommaso Zanocco-Marani a , Cristina Poligani a , Roberta Zini a , Claudia Gemelli a , Anna Bergamaschi a , Tatiana Vignudelli a , Francesca De Rienzo b , Pier Giuseppe De Benedetti b , Maria Cristina Menziani b , Sergio Ferrari a,∗ a

Sezione di Chimica Biologica, Dipartimento di Scienze Biomediche, Università degli Studi di Modena e Reggio Emilia, Via Campi 297, 41100 Modena, Italy b Dipartimento di Chimica, Università degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy Received 14 April 2003; accepted 19 June 2003

Abstract Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of proliferation and differentiation of hematopoietic cells and in the pathogenesis of many diseases, including multiple myeloma. This study pursues a way to interfere with IL-6 pathway in an attempt to modulate its biological activity. Here we describe the rational design and biological evaluation of peptides able to antagonize the murine IL-6 activity by interfering with IL-6 Receptor alpha in 7TD1 cells, a IL-6-dependent B-cell line. Of the peptide tested, only Guess 4a is capable of interfering with IL-6 transducing pathway, therefore inducing growth arrest and apoptosis of 7TD1 cells. © 2003 Elsevier Inc. All rights reserved. Keywords: Interleukin-6; Interleukin-6 receptor; Molecular modelling; Computational simulation; Antagonist peptides; Apoptosis; Multiple myeloma

1. Introduction Interleukin-6 (IL-6) is a multifunctional cytokine [1] required, in synergism with other growth factors, for hematopoietic progenitor cells proliferation [36], T and B cells growth and differentiation [47,53], neuronal and macrophage differentiation [12,28], immunoglobulin production [24] and the acute phase response [9]. IL-6, together with tumor necrosis factor-␣ (TNF-␣), IL-1, IL-8, IL-12, IL-18 and interferons, belongs to the proinflammatory cytokines group, which initiates the early inflammatory response [11]. IL-6 is produced by various cell types, including mononuclear phagocytes, fibroblasts, endothelial cells, B and T cells [22,52]. It is also produced by osteoclasts and stimulates the formation of the early osteoclast precursor from bone marrow cells. Moreover, it stimulates, in synergy with other proinflammatory cytokines and transforming growth factor-␤ (TGF-␤), the process of bone-reabsorption [45]. ∗

Corresponding author. Tel.: +39-059-2055400; fax: +39-059-2055410. E-mail addresses: [email protected] (M.C. Menziani), [email protected] (S. Ferrari). 0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.06.005

IL-6 acts through a surface receptor, belonging to the hematopoietin receptor superfamily [23,64], that is composed by two chains: a 80 kDa ligand binding protein (gp80, ␣-chain, IL-6R) and a 130 kDa protein (gp130, ␤-chain) which transduces the signal through the activation of the tyrosine kinases of the Jak family [34]. Extensive studies, including mutagenesis and epitopes mapping with blocking or activating Abs, have demonstrated that three distinct sites constitute the contact points of IL-6 with its receptors [7,61]. Site I interacts with a non-signalling receptor (IL-6 receptor alpha, IL-6R), and is formed by the C-terminal part of helix D and of the AB-loop. This interaction is the prerequisite for the engagement of the signalling gp130 receptor which is recruited by Site II, formed by a limited number of exposed residues on helix A and helix C, and Site III, formed by residues at the amino-terminal end of helix D, spatially flanked by residues in the initial part of the AB-loop [61]. Both IL-6R and gp130 recognize ligands through their cytokine-binding homology region, located in the domains D2 and D3, but gp130 engages an additional N-terminal (D1) Ig-like activation domain in the assembly of the

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functional final complex [30,50]. The three dimensional structures of IL-6, gp130 and IL-6R have been determined by X-ray crystallography [7,62,70], however, to date, there are no published reports on structure of higher complexes in which these proteins are involved. The molecular basis by which human IL-6 (hIL-6) activates gp130 is distinct from other members of the hematopoietin receptor family. In fact, experimental evidences, derived by “in vitro” reconstitution and activation of hIL-6 and gp130 complexes [32], show that the IL-6-type signalling complexes are hexamers containing two copies of IL-6, IL-6R and gp130 (2:2:2). It is important to underline that IL-6 plays an important role in all the diseases with an inflammatory component and that in these patients IL-6 serum levels are increased [14,19,25,42,48,55,58,60,71]. Several reports [2,4] suggest that IL-6 is involved in the multiple myeloma (MM) pathogenesis. It has been shown that IL-6 is important for in vivo growth of murine Plasmocytoma and MM [31], suggesting the possible involvement of the cytokine in the generation of this neoplasia as autocrine and paracrine growth factor. In fact, high levels of circulating IL-6 reflect a worse clinical course of the disease [15,21,39]. Therefore, the inhibition or modulation of IL-6 could have profound therapeutic benefits in MM as well as in several other diseases. In fact, anti IL-6 antibodies (Abs) block myeloma cell proliferation in vivo and decrease IL-6 biological activity with an improvement of the patient’s clinical status, but fail due to the activation of the immune response [37,69]. In this study, we present a combined theoretical and experimental approach based on the development of small linear peptides able to interfere, efficiently and specifically, with the murine IL-6 (mIL-6)/IL-6R interaction and therefore to antagonize the mIL-6 biological activity. Molecular modelling and simulation techniques have been used for (a) gaining insights into the pathway of the receptor complex formation and (b) rationalising and interpret the experimental responses. The main goal is to obtain the shortest peptide sequence capable of binding to IL-6, preventing the interaction with its specific receptor. Successful studies on the capacity of synthetic peptides to prevent the IL-6/IL-6R interaction have been previously reported [27,29,43,44,72]. In the absence of crystallographic data for the functional mammalian complex, we utilised the three-dimensional model of the mIL-6 multimeric complex as a working hypothesis for the antagonist design. This model has been obtained by means of the computational procedure previously described for the human complex [46]. Confidence in the IL-6 multimeric three dimensional models is provided by their capability to interpret and predict the ever-increasing number of experimental informations.

The peptides were designed on mIL-6 with the intent of shifting the biological assays in vivo on MM affected mice [3]. The selected peptides were synthesised and tested for biological activity on 7TD1 cells [68], an IL-6-dependent murine cell line, which undergoes growth arrest and apoptosis after IL-6 withdrawal. 2. Materials and methods 2.1. Computational procedure 2.1.1. Homology modelling Sequences of the hormones and receptors were extracted from the EMBL protein sequence database (http://srs. ebi.ac.uk/). The crystallographic structure of the human IL-6 (1ALU.pdb) [62] and of the cytokine-binding region of gp130 (1BQU.pdb) [7] were obtained from the Brookhaven Data Bank [5]. Secondary structure predictions were achieved with the Rost and Sander method [54] at the Internet address http://cubic.bioc.columbia.edu/predictprotein/. The sequence alignments between different species were obtained with the program ClustalW (http://www.ebi.ac.uk/clustalw). The structural restraints derived by the crystal structure of hIL-6 and human gp130 (hgp130), and the 3D hIL-6R model previously derived [46] were exploited for the generation of the corresponding mouse models by the program MODELLER (Rockefeller University) [56]. 2.1.2. Refinement of the 3D structures Energy minimisation and molecular dynamics studies were carried out using the software package QUANTA98 (Molecular Simulations, Inc., Waltham, MA, USA). Molecular mechanics and molecular dynamics calculations were performed using the program CHARMM (Harvard University) [8], according to a procedure described in a previous article [46]. 2.1.3. Molecular simulations of the mIL-6R/IL-6 and peptide–protein interaction The minimisation procedure consisted of 50 steps of steepest descent, followed by a conjugate gradient minimisation until the root mean square (rms) of the gradient of the potential energy was less than 0.001 kcal/mol. The united atom force-field parameters, a 12 Å nonbonded cutoff and a dielectric constant of 80 were used. The minimised coordinates of the hormones, receptors and binary and ternary complexes were used as starting point for dynamics. During dynamics the lengths of the bonds involving hydrogen atoms were constrained according to the SHAKE algorithm [67], allowing an integration time step of 0.001 ps. The ␣-helices conformation was preserved by means of the NOE utility provided by the CHARMM program, which allows the imposition of constraints between the backbone

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oxygen atoms of residue i and the backbone nitrogen atoms of residue i + 4, whereas the ␤-sheet structure was maintained by applying NOE constraints between opposite strands. The structures were thermalised to 300 K with 5 ◦ C rise per 6000 steps by randomly assigning individual velocities from the Gaussian distribution. After heating, the systems were allowed to equilibrate until the potential energy versus time was approximately stable (34 ps). Velocities were scaled by a single factor. An additional 10 ps period of equilibration with no external perturbation was run. Time-averaged structures were then determined over the last 100 ps of each simulation. Data were collected every 0.5 ps. Interaction energies at the monomer–monomer interfaces and at the receptor binding site were computed by using a dielectric constant ε = 4r. 2.2. Peptide synthesis Briefly, peptide synthesis was carried on at 0.1 mM scale (ABI model 433A synthesizer, Applied Biosystems, Inc., Foster City, CA, USA) using standard Fmoc chemistry. Peptides were cleaved from the resin by anhydrous fluorhydric acid (HF) and purified by preparative reverse phase HPLC (Perkin-Elmer Corporation, Norwalk, CT, USA) using a 10 nM × 250 nM Vydac C18 (Vydac, Hesperia, CA, USA) and acetonitrile gradients in aqueous 0.1% TFA (trifluoracetic acid). Peptide molecular weights were determined by MALDI-TOF mass spectrometry (matrix associated laser desorption and ionisation time-of-flight). Prior to cell assay the peptides were dissolved in cell culture medium. 2.3. Cell cultures 7TD1 cell line (a kind gift from Dr. Van Snick) is a hybridoma derived from cell fusion of LPS activated B lymphocytes and SP2/0-AG14 murine plasmacytoma cell line [68]. 7TD1 cells are IL-6 growth and survival dependent. In fact, when these cells are cultured in the absence of IL-6 undergo growth arrest and apoptosis in 24–48 h. The number of the IL-6 receptor on 7TD1 cells is 690/cell [16]. 7TD1 cells were cultured in Iscove’s modified Dulbecco medium (Gibco-BRL, Paisley, UK) supplemented with 20% heat inactivated FBS, 2 mM l-glutamine, 0.55 mM l-arginine, 0.24 mM l-asparagine, 50 ␮M 2-mercaptoethanol, 0.1 mM hypoxanthine, 16 ␮M thymidine and 2 ng/ml (87 pM) of mIL-6 (R&D Systems Inc., Minneapolis, MN, USA). 2.4. Peptide treatment 7TD1 cells were washed three times with Iscove’s medium and seeded at 6 × 104 cells/ml in the above described culture conditions for 5 h without IL-6; the different peptides were added at the concentration of 10, 30, 60 and 120 ␮M, respectively, in dose-dependent experiments (Fig. 4), and of 30 ␮M in all other experiments; after 3 h

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the IL-6 was restored. Peptide treatment was carried out for 3 days, monitoring proliferation and apoptosis every 24 h. Each set of experiments was performed using different control cultures: (1) 7TD1 cells supplemented with IL-6 (positive control); (2) 7TD1 cells without IL-6 (negative control); (3) 7TD1 cells with IL-6 and IL-6 inhibitory peptides; (4) 7TD1 cells with IL-6 and IL-6 scrambled peptides. 2.5. Apoptosis analysis 2.5.1. Morphological analysis Morphology was analysed on cytospin preparations after May–Grunwald–Giemsa staining in the time course experiments. 2.5.2. Propidium iodide (PI) staining and flow cytometric analysis To evaluate the percentage of apoptotic cells with hypodiploid DNA content 5 × 105 cells were pelletted and resuspended in 500 ␮l of fluorescent hypotonic solution (50 ␮g/ml PI, 0.1% sodium citrate and 0.1% Triton X-100), incubated at 4 ◦ C for at least 15 min and then analysed [49] with Epics XL flow cytometer (Coulter Electronics Inc., Hialeah, FL, USA) equipped with a single 488-nm argon laser and XL2 software (Coulter Electronics Inc.). To avoid counting of apoptotic bodies as apoptotic cells and so overestimating apoptotic cell percentage, in preliminary experiments we have compared the apoptosis percentage in the same sample using both the fluorescent hypotonic solution by Nicoletti et al. and the standard method for DNA staining in apoptosis. 2.5.2.1. Alcohol-fixation method. Briefly, 5 × 105 cells were pelletted, resuspended in 1 ml of cold 70% ethanol and put in ice for 30 min. After washing with 1 ml of PBS, the cells were stained with the fluorescent hysotonic solution (20 ␮g/ml PI, 0.2 mg/ml A in PBS), incubated at room temperature for at least 30 min and then analysed [66]. 2.5.3. DNA fragmentation analysis DNA extraction was performed using a modification of the technique described by Gross-Bellard et al. [26]. Briefly, the cells were pelleted and washed twice in PBS. The pellet was suspended in 0.4 ml of SEDTA (0.1 M NaCl, 50 mM Na2 EDTA, pH 7.8) containing 33 ␮l of 10 mg/ml Proteinase K and 40 ␮l of 10% SDS. The lysate was incubated for at least 4 h at 37 ◦ C, then extracted once with 1 ml of SEDTA saturated phenol and 1 ml of CIA (2% isoamyl alcohol in chloroform). The supernatant was precipitated overnight at −20 ◦ C with 40 ␮l of Na acetate 3 M, pH 5, 15 ␮g of E. Coli tRNA and 2.5 ml of cold 100% ethanol. The pellet was suspended in 0.4 ml of 1×TE buffer (10 mM Tris, 1 mM Na2 EDTA, pH 8), then 1 ␮l of 10 mg/ml DNase-free RNase was added and the suspension was incubated for 15 min at 37 ◦ C. A second digestion with Proteinase K was

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carried out for 1 h at 37 ◦ C with 100 ␮l of Proteinase K in SEDTA (1 ml of 10 mg/ml Proteinase K and 9 ml of SEDTA) after addition of 10 ␮l of 10% SDS. The DNA suspension was then extracted once with 0.5 ml of 1:1 phenol/CIA, and the supernatant was precipitated as before. The pellet, after washing with 70% ethanol, was vacuum dried and suspended in 50 ␮l of 1 × TE buffer. To evaluate the internucleosomal DNA fragmentation, DNA was electrophoresed on agarose gel [41].

3. Results 3.1. Modelling of the mouse IL-6R/IL-6/gp130 complex The structural restraints derived from the crystal structure of hIL-6, hgp130 and the 3D hIL-6R model previously published [46] were exploited for the generation of the corresponding mouse models, according to the sequence alignments reported in Table 1. The ternary human complex

Table 1 Sequence alignment of human and murine IL-6

(a) Helical secondary structure elements are shown as boxes enclosing the appropriate residues. The underlined residues were not resolved in the X-ray hIL-6 structure. (b) The cytokine binding domains of the specific human and murine IL-6R, ␤-sheet elements predicted by the Rost and Sander method [54] are highlighted. (c) The cytokine binding domains of the specific human and murine gp130 receptors. ␤-Sheet elements, as detected from the X-ray structure are shown as boxes enclosing the appropriate residues.

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Fig. 1. Overimposition of the model of the trimeric murine complex IL-6R/IL-6/gp130 (grey) and the experimental structure of the (viral)IL-6/(human)gp130 binary complex (black). The N-terminal Ig-like domain (D1) of gp130, and the cytokine-binding domains (D2 and D3) are highlighted for both IL-6R and gp130 receptors.

described in a previous article [46], and based on the growth factor complex paradigm, was taken as a template for the assembly of the mouse complex. The mutual orientation of the three components was adjusted in order to maximize the complementarity of the electrostatic potentials computed on mIL-6 and mIL-6R for the binary complex, and on mIL-6/mIL-6R and murine gp130 (mgp130) for the ternary complex, and the final complex underwent energy refinement and molecular dynamics in order to optimize the intermolecular interactions. The crystal structure of the complex between viral IL-6 and human gp130 has been published [13]; unlike human IL-6, viral IL-6 directly activates gp130 without the requirement for a specific receptor (IL-6R) [32]. However, superimposition of the theoretical model (of the ternary complex) and the experimental structure (of the binary complex) shows that the docking orientations of the mouse and viral cytokines on gp130 are almost identical (Fig. 1). On the other hand, the contact residues seen in the structure of the viral complex are in the same positions as human IL-6/gp130 contact residues previously mapped by mutagenesis, although the overall sequence homology is very low (25%) and the core of the interface is different, being dominated by hydrophobic interactions in the case of the viral species, while the human and murine species involve more polar interactions. 3.2. Design of murine IL-6 antagonists The structural regions of the IL-6 specific receptor and of gp130 that participate in the binding and signal transduction processes were identified on the basis of molecular simulations carried out on the three dimensional model of the mIL-6 multimeric complex (Fig. 2). The main contribute to the total interaction energy (IE) of the trimeric complex

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(IEIL-6R/IL-6/gp130 = −318.79 kcal/mol) is furnished by the IL-6R/IL-6 interaction (IEIL-6R/IL-6 = −139.27 kcal/mol). In particular, a detailed analysis of the important regions of Site I protein–protein interactions reveals that the B2–C2 loop, composed by the stretch of residues 129–139, and the F2–G2 loop, composed by the stretch of residues 182–189, of the IL-6 specific receptor contribute significatively to the total IE of the dimer (IL-6R/IL-6) in the final ternary complex (Fig. 2). Moreover, several intermolecular interactions observed in these zones involve residues identified to be important for binding by site directed mutagenesis studies on the human species. Therefore these domains were used as templates in our synthetic design. In order to identify the minimal effective sequence capable of preventing the interaction of IL-6 with its receptors, the importance of structural constraints provided by the framework for conferring specific binding activity for the target molecule has been evaluated. Several linear peptides of different length were built and insights into their conformation and relative flexibility due to different number of constituent residues were obtained by dynamic simulated studies. On this basis, peptides Guess 3b, Guess 4a, Guess 5a and Guess 2c (Table 2) were selected as potential IL-6 antagonist peptides, while peptide Guess 4b (Table 2) was chosen as a negative control since, being part of the ␤-sheet core of the receptor, according to our model it should not interact with IL-6. 3.3. Computational simulations of synthetic peptides interacting with mIL-6 A description of the main interactions established during the dynamics simulation between Loop B2–C2 and Loop F2–G2 of the IL-6R and IL-6 in the ternary complex is given in Table 3. Several intermolecular interactions involve residues (in bold) identified to be important for binding and dimerisation activity by site directed mutagenesis studies on the hIL-6 and hIL-6R [46]. It is worth noting that, although the hydrophilic groups are required to give specificity, the overall IL-6R/IL-6 interaction in the ternary complex is

Table 2 IL-6 antagonist peptides Peptide

Sequence

Region

Guess 3b

182 KEELDLGQ189

Guess 4a

180 RGKEELD186

Guess 2c

131 WDPSYYL137

Guess 5a

180 RGKEELDLGQ189

Guess 4b Guess 4a-SC

136 YLLQFQLRYR145 EDLGREK

Loop F2–G2/IL-6R (Site I of interaction IL-6/IL-6R) Loop F2–G2/IL-6R (Site I of interaction IL-6/IL-6R) Loop B2–C2/IL-6R (Site I of interaction IL-6/IL-6R) Loop F2–G2/IL-6R (Site I of interaction IL-6/IL-6R) ␤-Sheet C2/IL-6R

Sequences designed by computational simulation.

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Fig. 2. Ribbon structure representation of the minimised average structure of the trimeric murine complex IL-6R/IL-6/gp130 (top right). The total interaction energy (IE) of the trimer and the contributors due to the IL-6R/IL-6, IL-6/gp130 and IL-6R/gp130 interactions is reported. Details of Site I show the energy contribution of the B2–C2 and F2–G2 loops of IL-6R to the IL-6R/IL-6 interaction. The location of the designed peptides onto the B2–C2 and F2–G2 loops of the IL-6R is showed (left).

dominated by the hydrophobic forces; in fact, more than 50% of the total IE between the hormone and the IL-6R (IE = −139.27 kcal/mol) (Fig. 2), computed on the minimised average structure of the binary complexes, is due to the dispersion forces as represented by the van der Waals (vdW) component of the total IE (IEvdW = −76.59 kcal/mol, IEEl = −33.28 kcal/mol, IEHB = −29.39 kcal/mol). Loops B2–C2 and F2–G2 are two of the main contributors to the total IL-6R/IL-6 IE with −34.74 and −50.95 kcal/mol, respectively (see Table 4 and Fig. 2). The interaction between residues belonging to Loop B2–C2 and IL-6 is mainly dispersive (IEvdW = −24.07 kcal/mol), while the electrostatic (El) component dominates the Loop F2–G2/IL-6 interaction (IEEl = −21.64 kcal/mol, IEHB = −12.69 kcal/mol). Peptide Guess 2c achieves a total IE with IL-6 more stable than its corresponding loop in the ternary complex (Table 4) but, visual inspection of the dynamic trajectory shows that it moves away from the original place and interacts with residues at the interface between helix A and helix D. Peptides Guess 4a, Guess 3b and Guess 5a establish very good intermolecular interactions with residues of the C-terminal portion of mIL-6 helix D, the total IE being always at least double compared to that achieved by the original F2–G2 loop

of the receptor (Table 4). However, a slow reorganisation of the backbone conformation of the peptides was observed during the warm-up and equilibration phases. This reorganisation resulted in a remarkable increase of intermolecular contact points, but the IE contributed by the same mIL-6 residues involved in the original interaction with the F2–G2 loop of the receptor (see Table 3) decreased constantly. This is represented by the IE/number of residues (nres) descriptor (Table 4), which takes into account only the IE between the peptides and the IL-6 residues L15, E154, L157, K158, L161, R162 and R165, listed in Table 3. This descriptor is normalised by the nres in the peptide. More importantly, the dynamic behaviors of the peptides Guess 3b, Guess 4a and Guess 5a interacting with mIL-6 pointed out the specificity of the Guess 4a–IL-6 interaction. In fact, as it is showed by the solvent accessible surface (SAS) descriptor listed in the last column of Table 4, R165 is a completely buried residue in the IL-6 ternary complex, and Guess 4a is the only peptide which reproduces this situation. During the earliest steps of dynamics the Guess 4a peptide moves from its original position and undergoes conformational changes that optimize its interaction with the C-terminal portion of helix D, as it is showed in Fig. 3,

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Table 3 Interaction energy (IE) (kcal/mol) between IL-6 and the stretch of amino acids belonging to Loops B2–C2 and F2–G2 of the specific IL-6 receptor

The van der Waals (vdW), electrostatic (El) and hydrogen bonding (HB) components are also listed. The peptides designed after the mIL-6R sequence are represented on the right. Residues identified to be important for binding and dimerisation activity by site directed mutagenesis studies on the hIL-6 and hIL-6R are in bold.

where the minimised average structure of the Guess 4a–IL-6 complex is reported.

B), with extensive cell shrinkage, pyknosis and karyorrexis (Fig. 5, Panel B).

3.4. IL-6 dependence of 7TD1 cells

3.5. Treatment of 7TD1 cells with IL-6 antagonist peptides

7TD1 is a murine hybridoma cell line with a doubling time of approximately 15 h if cultured as described in Section 2 [68]. 7TD1 cells represent a good model for the assay of IL-6 biological activity, being IL-6 growth and survival dependent. In fact, after IL-6 withdrawal the cells undergo rapidly growth arrest and apoptosis, as demonstrated by hypodiploid DNA content and morphological features. These analyses show that after 96 h without IL-6 the 7TD1 cells presents about 90% of hypodiploid nuclei (Fig. 4, Panel

7TD1 cells were treated with synthetic peptides as described in Section 2. All the synthesised peptides reported in Table 2 are unable to inhibit the IL-6 biological activity, except for Guess 4a. In fact, the Guess 4a peptide, designed on Site I of interaction IL-6/IL-6R (Loop F2–G2/IL-6R) is effective in inducing growth arrest and apoptosis in 7TD1 cells. In dose-dependent experiments we observed no difference in efficacy among Guess 4a peptide concentrations of 30,

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Table 4 Energies (kcal/mol) for the interactions of IL-6 with Loops B2–C2 and F2–G2 of the specific IL-6 receptor, and with selected linear peptides Peptide

IE

IE vdW

El

HB

IE/nres

SAS (Å2 )

Loop B2–C2 2c

−34.74 −81.54

−24.07 −46.17

−2.44 −10.80

−8.23 −24.46

−4.9 –

0.0 75.0

Loop F2–G2 4a 3b 5a

−50.95 −123.95 −106.18 −107.50

−16.69 −59.22 −54.14 −76.32

−21.64 −27.07 −23.53 −9.48

−12.69 −37.65 −28.50 −21.69

−10.1 −6.0 −4.3 −5.3

0.0 0.0 53.0 40.0

The total interaction energy (IE), and the van der Waals (vdW), electrostatic (El) and hydrogen bonding (HB) are listed. IE/nres accounts for the interaction energy between the peptides and the IL-6 residues L15, E154, L157, K158, L161, R162 and R165, normalised by the number of residues in the peptide. SAS is the solvent accessible surface of the IL-6 residue R165.

60 and 120 ␮M, while 10 ␮M concentration is less effective (Fig. 6). In fact, the results obtained by cytofluorimetrical analysis of DNA content show that after 96 h of treatment with 30 ␮M Guess 4a, the 7TD1 cells show a high percentage of apoptosis, as demonstrated by hypodiploid DNA content in the 88.2% of the cells (Fig. 4, Panel C). Otherwise the positive control shares high proliferative capacity and good viability (Fig. 4, Panel A), with less than 10% of apoptotic cells. Guess 4a scrambled (SC) treated cells (30 ␮M) show the same characteristics of proliferation and viability of untreated cells (Fig. 4, compare Panels A and D): this obser-

Fig. 3. Ribbon structure representation of the minimised average structure of the complex between mIL-6 and peptide Guess 4a. The residue R165, which is completely buried by the peptide, is represented explicitly. The position of the peptide before (input structure, in red) and after dynamics (minimised average structure, in yellow) is showed at the bottom.

Fig. 4. Flow cytometric analysis of peptide treated 7TD1 cells. DNA fluorescence of PI-stained 7TD1 cells under different experimental conditions. The percentage of apoptotic cells are indicated on each panel. Results representative of five independent experiments are reported. Mean values ± 2 S.E.M. (confidence range 95%) resulted to be—Panel A: positive control, 7.9 ± 6.9; Panel B: negative control, 84.3 ± 10.2; Panel C: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a, 85.02 ± 15.4; Panel D: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-SC, 6.5 ± 3.8; Panel E: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4b, 6.6 ± 7.4; Panel F: 7TD1 cells treated for 96 h with 30 ␮M of Guess 5a, 6.04 ± 4.6.

vation validates the experimental data obtained with Guess 4a peptide and excludes possible aspecific toxic effects. The other peptides listed in Table 2 are clearly ineffective in inducing apoptosis in 7TD1 cells: in fact the amount of apoptotic cells ranges from 3.7% in Guess 4b (Fig. 4, Panel E) to 6.2% in Guess 5a treated cells (Fig. 4, Panel F). Superimposible results are obtained with the Guess 3b and Guess 2c peptides (data not shown). To avoid over-estimation of apoptotic cells, that is if isolated apoptotic bodies are counted as apoptotic cells, we compared the apoptosis percentage in the same sample using both the fluorescent hypotonic solution by Nicoletti et al. [49] and the “alcohol-fixation method” for DNA staining in apoptosis [66] (see Section 2). In our hands, both hypotonic staining solution and the alcohol-fixation method produced the same quantitative results, indicating that apoptotic cells and debris are appropriately discriminated also by the quick method by Nicoletti et al. (data not shown).

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Table 5 Guess 4a mutagenesis Peptide

Sequence

Region

Guess 4a

RGKEELD

Guess 4a-D Guess 4a-F Guess 4a-N

RGKEEDD RGKEEFD RGKEEND

Loop F2–G2/IL-6R (Site I of interaction IL-6/IL-6R) – – –

Sequences designed by computational analysis simulation. In each sequence, the mutated amino acid is in bold.

72 h the percentage of apoptotic cells markedly increases (50.4% of cells, Fig. 7, Panel D) and reaches the 81.5% after 96 h of treatment (Fig. 7, Panel E). Morphological analysis reproduces this kinetic of apoptosis induction (Fig. 8): in fact, whereas the positive control (Fig. 8, Panel A) and the Guess 4a-SC treated cells (Fig. 8, Panel F) show a very good viability, the Guess 4a treated cells start to present cell shrinkage, pyknosis and karyorrhexis after only 48 h (Fig. 8, Panel C); these alterations appear more obvious after 72 and 96 h (Fig. 8, Panels D and E, respectively). 3.6. Treatment of 7TD1 cells with Guess 4a variant peptides

Fig. 5. Morphological analysis after May–Grunwald–Giemsa staining of peptide treated 7TD1 cells—Panel A: positive control; Panel B: negative control; Panel C: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a; Panel D: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-SC; Panel E: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4b; Panel F: 7TD1 cells treated for 96 h with 30 ␮M of Guess 5a. Magnification: 1000×.

Flow cytometrical data were confirmed by morphological analysis. In fact, as shown in Fig. 5, the 96 h, 30 ␮M treatment with Guess 4a causes apoptosis in the large majority of the cells, as demonstrated by cell shrinkage, pyknosis and karyorrhexis (Fig. 5, Panel C). On the contrary, the treatments performed with Guess 4b (Fig. 5, Panel E), Guess 5a (Fig. 5, Panel F) or with the control peptide Guess 4a-SC (Fig. 5, Panel D), are unable to interfere with the proliferative capacity or the viability of 7TD1 cells. The same results were obtained with Guess 3b and Guess 2c peptides (data not shown). Therefore, we have demonstrated that the Guess 4a peptide is able to antagonize IL-6 biological activity; furthermore, Guess 4a shows a very high efficacy, since its action is exerted also at lower concentration (30 ␮M) in respect to other evidences [27]. The flow cytometrical analysis after PI staining (Fig. 7) demonstrates that Guess 4a starts to induce apoptosis after a short treatment (48 h) (20.6% of cells, Fig. 7, Panel C); after

Mutations on the native sequence of peptide Guess 4a were considered in order to test sequence specificity. Twenty-five single mutants were designed and theoretically evaluated for their ability to interact with mIL-6. They provided interaction energies to mIL-6 worse than, or equal to the native one, however, some of them were selected for biological evaluation and are reported in Table 5. The peptides Guess 4a-D, Guess 4a-F and Guess 4a-N (Table 5) were tested under the above described conditions on 7TD1 cells. All the mutagenised peptides are unable to inhibit IL-6 biological activity: in fact, flow cytometric analysis for the DNA content shows that the cells treated with the variant peptides do not undergo apoptosis, maintaining a high proliferative capacity and good viability also after 96 h of 30 ␮M peptide treatment (Fig. 9, Panels C, D and E, respectively) and at higher peptide concentration (120 ␮M) (data not shown). In addition, the characteristic DNA ladder pattern is evident in DNA extracted from Guess 4a treated cells (Fig. 10, Lane 4), while cells treated with Guess 4a variant peptides (Fig. 10, Lanes 5–7) and other peptides (data not shown) present a high molecular weight DNA.

4. Discussion Comparative modelling and computational simulations of mIL-6, mIL-6/mIL-6R and mIL-6/mIL-6R/mgp130 have allowed the identification and the quantitative analysis of

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Fig. 6. Dose-dependent efficacy of Guess 4a peptide. Concentration response of the Guess 4a peptide on the viability of 7TD1 cells in time-course experiment. The percentage of apoptotic cells were derived from flow cytometrical data after PI staining. Average results from a quintuplicate experiment are shown together with the corresponding S.E.M. values.

the molecular determinants for structure stabilisation and ligand–receptor recognition. On these basis were chosen the domains to be used as templates in the synthetic design, by the following strategy: (a) identification of the region of the receptors mainly involved in the interactions by means of computational simulations of the mIL-6 multimeric complex; (b) evaluation of the minimal effective sequence capable of binding to IL-6, preventing its interaction with the specific receptor; (c) selection of the peptide candidates for synthesis and biological evaluation on the basis of theoretical screening of the peptide–target interaction properties. We focussed our attention essentially on loops B2–C2 and F2–G2 of the IL-6 specific receptor which contribute significantly to the ligand–receptor interaction. Among the several peptides designed and screened “in silico” we selected the peptides reported in Table 2 for synthesis and biological evaluations. According to our model peptides Guess 4a, Guess 3b and Guess 5a should retain some of the IL-6R features determinant for IL-6 recognition, while peptide Guess 4b should not be active, being part of the ␤-sheet core of the receptor.

In our study, among the tested peptides, only Guess 4a showed the ability to interfere with proliferation and survival of 7TD1 cells. In fact, morphological, flow cytometrical and molecular analyses showed that Guess 4a treated 7TD1 cells undergo growth arrest and apoptosis. By acting as a receptor, Guess 4a is able to inhibit IL-6 biological activity in 7TD1 cells. Different concentrations of the corresponding scrambled peptide (Guess 4a-SC) showed no interference with 7TD1 proliferation or survival. These results demonstrate that Guess 4a is a specific IL-6 competitive inhibitor. The IL-6 receptor concentration in 7TD1 cells is 690 molecules/cell [16]. In this light, the molar ratio receptor/Guess 4A is largely favourable to the competitor peptide. The computational simulations of the peptide–protein interactions provided a rationalisation of the in vitro experiments. Peptide specificity seems to be conferred by the achievement of strong interactions with amino acid residues located at the C-terminal portion of IL-6 helix D. More important, a correlation is observed between the antagonistic activity and the ability of the peptide to interact and completely bury the R165 residue. Point mutants of peptide

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Fig. 7. Apoptosis kinetics by flow cytometric analysis of Guess 4a treated 7TD1 cells. DNA fluorescence of PI-stained 7TD1 cells under different experimental conditions. The percentage of apoptotic cells are indicated on each panel. Results representative of five independent experiments are reported. Mean values ± 2 S.E.M. (confidence range 95%) resulted to be—Panel A: positive control, 8.4 ± 3.8; Panel B: negative control, 81.5 ± 10.4; Panel C: 7TD1 cells treated for 48 h with 30 ␮M of Guess 4a, 23.5 ± 9; Panel D: 7TD1 cells treated for 72 h with 30 ␮M of Guess 4a, 46 ± 11.8; Panel E: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a, 81.3±17.2; Panel F: 7TD1 cells treated for 96 h with 30 ␮M of Guess 3a-SC, 8.5 ± 3.8.

Guess 4a, which retain most of the interactions realised by the native sequence but without shielding completely R165, do not manifest antagonistic characteristics, as demonstrated by biological assays. Therefore, at least in this case, the exact mimicry of the surface involved in molecular recognition seems to be unnecessary for antagonistic characteristics and only some of the features found in the original receptor have to be retained. It is worth noting that these findings were in contradiction with previously reported data on antagonist peptides of hIL-6. A deca-peptide with the human sequence YRLRFELRYR, corresponding to peptide Guess 4b was reported to be able to inhibit the IL-6-dependent growth of B9 and XG-1 cells [27], while the synthetic linear hepta-peptide RGKEEVD, overlapping with the esa-peptide Guess 4a, was described as unable to interfere with the receptor binding. Structural constraints provided by the protein framework

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Fig. 8. Morphological analysis after May–Grunwald–Giemsa staining of Guess 4A treated 7TD1 cells: Panel A: positive control; Panel B: negative control; Panel C: 7TD1 cells treated for 48 h with 30 ␮M of Guess 4a; Panel D: 7TD1 cells treated for 72 h with 30 ␮M of Guess 4a; Panel E: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a; Panel F: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-SC. Magnification: 1000×.

were claimed to be important for conferring specific binding activity to this peptide, since by inserting the stretch of amino acids of the human specific IL-6R sequence GKEEVD into a minibody, antagonistic potency was retained [43,44]. Our design differs from others [6,17,18,20,33,38,51, 57,63,65] because it targets the IL-6 and not the IL-6R; moreover, in respect to other experiments performed with the IL-6 antagonist peptides [27,29], our strategy presents the advantage of a higher efficiency being the active concentration about 10-fold lower. This property is particularly significant for a future possible therapeutical development. We believe that the construction of IL-6 antagonist peptides represents a promising strategy to modulate IL-6 activity in vivo. IL-6 is involved in the pathogenesis of many diseases, and particularly in MM, by supporting the proliferation activity and the survival of myeloma cells by paracrine/autocrine mechanism [15,21]: IL-6 inhibits apoptosis in myeloma cells

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Fig. 10. Analysis by agarose gel electrophoresis of DNA recovered from 7TD1 cells treated with Guess 4a variants: Lane 1: molecular weight marker 100 bp DNA ladder (Gibco-BRL, Paisley, UK); Lane 2: positive control; Lane 3: negative control; Lane 4: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a; Lane 5: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-D; Lane 6: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-F; Lane 7: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-N; Lane 8: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-SC. Fig. 9. Flow cytometric analysis of 7TD1 cells treated with Guess 4a variants peptides. DNA fluorescence of PI-stained 7TD1 cells under different experimental conditions. The percentage of apoptotic cells are indicated on each panel. Results representative of five independent experiments are reported. Mean values ± 2 S.E.M. (confidence range 95%) resulted to be—Panel A: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a, 82.2 ± 14.8; Panel B: negative control, 82.1 ± 17.6; Panel C: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-D, 8.9 ± 3.6; Panel D: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-F, 9.4 ± 6.6; Panel E: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-N, 6.8 ± 6.3; Panel F: 7TD1 cells treated for 96 h with 30 ␮M of Guess 4a-SC, 8 ± 8.2.

by increasing the ratio of antiapoptotic versus pro-apoptotic proteins [40,59] via constitutive activation of the Stat3 transcription factor [10]. Since IL-6 is a powerful growth factor for myelomatous cells, the idea of interfering with its pathway in an attempt to establish a new therapeutic tool can be interesting. It has already been shown that the therapeutical approach with anti IL-6 Ab blocks myeloma cells proliferation in vivo and inhibits the serum IL-6 activity, but activates the immune response within few weeks [37,69]. In this study, we have developed a mIL-6 antagonist peptide capable, at least in vitro, to reduce mIL-6 biological activity. This kind of approach provides the advantage of interfering selectively with the signal transduction pathway of IL-6, excluding the generic interferences with the natural immunity mechanisms that other drugs such as dexamethasone and thalidomide have [35].

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