Polymer Coiled-Coil Conjugates: Potential for Development as a New

Dec 8, 2010 - Data were acquired using 1024 channels with band-pass filter FL-1 ..... the coiled-coil motif as a therapeutic component of polymer conj...
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Biomacromolecules 2011, 12, 19–27

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Polymer Coiled-Coil Conjugates: Potential for Development as a New Class of Therapeutic “Molecular Switch” Samuel P. E. Deacon,† Bojana Apostolovic,‡ Rodrigo J. Carbajo,§ Anne-Kathrin Schott,§ Konrad Beck,⊥ Marı´a J. Vicent,| Antonio Pineda-Lucena,§ Harm-Anton Klok,*,‡ and Ruth Duncan*,†,# Centre for Polymer Therapeutics, Welsh School of Pharmacy, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, United Kingdom, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Institut des Mate´riaux, and Institut des Sciences et Inge´nierie Chimiques, Laboratoire des Polyme`res, Baˆtiment MXD, Station 12, CH-1015, Lausanne, Switzerland, Structural Biology Laboratory and Medicinal Chemistry Unit, Polymer Therapeutics Laboratory, Centro de Investigacio´n Prı´ncipe Felipe (CIPF), Avda. Autopista del Saler 16-3, E-46012 Valencia, Spain, and Cardiff University School of Dentistry, Heath Park, Cardiff, CF14 4XY, United Kingdom Received July 22, 2010; Revised Manuscript Received October 21, 2010

Polymer therapeutics, including polymeric drugs and polymer-protein conjugates, are clinically established as first-generation nanomedicines. Knowing that the coiled-coil peptide motif is fundamentally important in the regulation of many cellular and pathological processes, the aim of these studies was to examine the feasibility of designing polymer conjugates containing the coiled-coil motif as a putative therapeutic “molecular switch”. To establish proof of concept, we prepared a mPEG-FosWC conjugate by reacting mPEG-maleimide (Mw 5522 g mol-1, Mw/Mn 1.1) with a FosW peptide synthesized to contain a terminal cysteine residue (FosWC). Its ability to form a stable coil-coil heterodimer with the target c-Jun sequence of the oncogenic AP-1 transcription factor was investigated using 2D 15N-HSQC NMR together with a recombinantly prepared 15N-labeled c-Jun peptide ([15N]r-c-Jun). Observation that heterodimerization was achieved and that the polymer did not sterically disadvantage hybridization suggests an important future for this new family of polymer therapeutics.

Introduction Polymer therapeutics including polyethylene glycol (PEG)protein, peptide, and aptamer conjugates are among the most successful first-generation nanomedicines (reviewed in refs 1-3). A growing number of compounds are clinically approved as treatments for both life-threatening diseases and those debilitating diseases prevalent in the aging population (e.g., the PEG-aptamer Macugen and PEG-antitumor necrosis factor-Fab Cimzia see refs 1-3). We have recently begun to explore polymer therapeutics designed to promote tissue repair,4-7 deliver combination chemotherapy,8 and develop polymer therapeutics that localize to newly emerging molecular targets. The main goal is to build on lessons learnt during transfer of the first polymeric anticancer conjugates into clinical development (reviewed in ref 9) to generate practical to develop, clinically useful conjugates. The broad objective of this project is the development of polymer conjugates containing a coiled-coil peptide motif as a putative therapeutic “molecular switch” with the hope of generating a new family of polymer therapeutics. The coiledcoil is an evolutionarily conserved sequence present in 3-5% of all amino acids of proteins and peptides.10,11 It is known that coiled-coil-mediated protein heterodimerization results in * Corresponding authors. E-mails: [email protected], [email protected]. † Welsh School of Pharmacy. ‡ Ecole Polytechnique Fe´de´rale de Lausanne (EPFL). § Structural Biology Laboratory, CIPF. | Polymer Therapeutics Laboratory, CIPF. ⊥ Cardiff University School of Dentistry. # Present address: School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, U.K.

the formation of either an “active” or an “inactive” multiprotein complex able to turn biological pathways “on” or “off” with a high degree of specificity and that this mechanism is fundamentally important in the regulation of many cellular and pathological processes (reviewed in ref 12). Our first studies used LAEIEAK-based coiled-coil model peptide sequences to examine the effect of primary structure on self-assembly of PEG-b-peptide hybrid block copolymers.13-15 The results obtained highlighted some of the physicochemical challenges for the development of practical therapeutics. Even though the peptides and their conjugates formed coiled-coils, a variety of techniques confirmed the presence of an equilibrium between discrete monomers and dimeric coiled-coil aggregates. To be functional, it is essential that a therapeutic coiled coil conjugate (i) will form a discrete 1:1 heterodimer with its target, (ii) will compete effectively with the natural ligand, and (iii) will not aggregate by forming homodimers. Here we selected the coiledcoil domain of the activator protein 1 (AP-1) transcription factor as a first model to investigate whether a PEG-coiled-coil conjugate could exhibit efficient heterodimeric coiled-coil hybridization. The hypothesis is shown schematically in Figure 1a,b, and the primary aim of these studies was to determine whether a PEG conjugate bearing a coiled-coil peptide could in fact form a heterodimeric coiled-coil with its target. AP-1 was one of the first human transcription factors described.16 Its oncogenic role and contribution to the metastatic, invasive phenotype is documented for many cancers.17-19 As the crystal structure of AP-1 had been described,20 there was certainty that a “coiled-coil” (not simply protein interaction) is essential for function. AP-1 is a dimeric protein complex composed of members of the Jun and Fos protein families. A

10.1021/bm100843e  2011 American Chemical Society Published on Web 12/08/2010

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Figure 1. Schematic showing the hypothesis and synthesis of mPEG-FosWC. (a) Putative target for mPEG-coiled-coil conjugates interaction (protein structure file was obtained from the Protein Data Bank (PDB), DOI:10.2210/pdb1fos/pdb; editing and rendering was conducted using MacPyMOL). (b) Proposed interaction of mPEG-FosWC with c-Jun. (c) Synthesis of mPEG-FosWC. Table 1. AP-1 Target Peptides and Conjugate Characteristicsa pure yield (%)

compound

sequence

c-Jun [15N]r-c-Jun c-Fosc FosWC mPEG-FosWC

ASIARLEEKVKTLKAQNYELASTANMLREQVAQLGA GTASIARLEEKVKTLKAQNYELASTANMLREQVAQLGA ELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEF CASLDELQAEIEQLEERNYALRKEIEDLQKQLEKLGA mPEG-CASLDELQAEIEQLEERNYALRKEIEDLQKQLEKLGA

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theoretical Mw (g mol-1)

b

3988 4156

25 38

4359 10 047

a N.B. All peptides synthesized by Fmoc SPPS used in this study were N- and C-terminally capped by acetylation and amidation respectively. [15N]r-c-Jun was synthesized using a recombinant methodology and therefore was uncapped at both termini. b Not calculated; mass yield for 2 L culture was 3.4 mg. c Not used in this work, included for reference.

c-Fos peptide was chosen for polymer conjugation here (rather than c-Jun) because it does not form homodimeric complexes that mediate transcription.20 A synthetic peptide (FosW)21 with a higher affinity for the basic leucine zipper (bZIP) domain of c-Jun than the wild-type c-Fos was used (Table 1) with the addition of a N-terminal cysteine residue (FosWC) to facilitate site-specific PEGylation using monomethoxypolyethylene glycol (mPEG)-maleimide (Mw 5500 g mol-1) (Figure 1c). The C-terminal proline of FosW and c-Jun was also excluded to aid large-scale synthesis. The ability of the PEG-FosWC conjugate to form a stable coiled-coil heterodimer with the target c-Jun sequence of the AP-1 transcription factor under physiologically relevant conditions was investigated using CD spectroscopy and with a recombinantly prepared 15N-labeled c-Jun peptide ([15N]r-c-Jun) using 2D 15N-HSQC NMR spectroscopy. Preliminary experiments were also conducted using Oregon Green (OG)-labeled FosWC, mPEG, and the mPEGFosWC conjugate to investigate cellular uptake and also to investigate cytotoxicity of both peptide and conjugate.

Materials and Methods Materials. 9-Fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids and Rink amide AM resin were obtained from NovaBiochem (Switzerland). Reagents used for peptide synthesis were of peptide grade and were obtained from Iris Biotech (Germany). mPEG-maleimide (Sunbright, ME-050MA, Mn ) 5500 g mol-1, Mn/Mw ) 1.03) was from NOF Corporation (Japan). Ellman’s reagent/5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) was from Sigma-Aldrich (U.K.). MacroCap SP cation-exchange columns (5 mL) were from GE Amersham (U.K.) and Centriprep YM-3 centrifuge tubes were from Amicon (U.K.). General laboratory reagents were of analytical reagent grade and were used as received, Sigma-Aldrich. A cytochrome b5 (cytb5)-r-c-Jun plasmid was assembled by conventional cloning using an in-house modified version of the commercially available pET15b plasmid (Novagen, Spain) containing between the restriction sites NdeI and BamHI an N-terminal fusiontag from the codon-optimized sequence of rat cytb5 followed by a TEV protease recognition site ENLYFQ-KpnI (adapted from ref 22). The c-Jun fragment was obtained by gene synthesis23,24 using the partially complementary oligonucleotides (customized order from Isogen Life

Polymer Coiled-Coil Conjugates Science, Spain) c-Jun forward (5′ ataataggtaccgcctctatcgcgcgtctggaggagaaggtgaaaacgttaaaagcccaaaactatgaattagcgtc 3′), and c-Jun_reverse (5′ tattatggatccttacgcacctaattgggcaacctgctcacgcagcatattggcggtagacgctaattcatagttttgggctt 3′), which were annealed and treated with DNA polymerase. The resulting 138bp segment was amplified by PCR, purified with a gel extraction kit from Qiagen (Spain), restricted with the endonucleases KpnI and BamHI, and ligated into the pET15b-cytb5 plasmid, which had been treated with the same restriction endonucleases. The ligation mixture was transformed into E. coli strain BL21CodonPlus (DE3) obtained from Stratagene (Spain) affording the recombinant strain BL21-CodonPlus (DE3)-pET15B-cytb5-ENLYFQGT-r-c-Jun. Tobacco etch virus (TEV) protease was previously prepared using a recombinant methodology (purified using modified metal affinity chromatography). [15N] ammonium chloride ([15N]H4Cl) was from Cambridge Isotope Laboratories (USA). Complete EDTA-free protease inhibitor tablets and DNase I were from Roche (Spain). TALON metal affinity resin was from BD Biosciences (Spain). Vivaspin 2,000 MWCO and Vivaspin 6 10 000 molecular weight cutoff centrifuge tubes were from Sartorius (Spain). General reagents were of molecular biology grade, and those used for SDS-PAGE were of electrophoresis grade; all were obtained from Merck (Spain). The MCF-7 human breast carcinoma cell line was kindly provided by Tenovus Centre for Cancer Research (Cardiff, U.K.). Peptide Synthesis, Purification, and Characterization. All peptides (Table 1) were synthesized by solid-phase peptide synthesis on Rink Amide AM resin (200-400 mesh, loading 0.71 mmol g-1) using an Fmoc protection strategy25 using an automated peptide synthesizer (Chemspeed PSW 1100). The Fmoc group was removed by the addition of base (20% piperidine in NMP), and each new amino acid was doublecoupled using HOBt and HCTU dissolved in NMP using NMM as the base. The deprotection and coupling times were 5 and 30 min, respectively. Peptides were then capped at the N-terminus using acetic anhydride. To cleave the peptide and remove protective groups, the peptide-loaded resin was treated with a mixture of TFA/TIS/EDT/H2O 94/2/2/2 (v/v) for 3 h. Peptides were then isolated by precipitation in cold diethyl ether, followed by centrifugation (three times) and were then suspended in ddH2O and lyophilized. Peptides were purified by preparative RP-HPLC (Waters 600 automated gradient controller pump module, a Waters Prep Degasser, a Waters 2487 dual λ absorbance detector, and a Waters Fraction Collector III) with a preparative column (Waters Atlantis dC18 OBD 5 µm, 30 × 150 mm) and using a linear AB gradient (A ) ddH2O, B ) ACN, both containing 0.1% v/v TFA) with flow rate of 20 mL min-1 (elution was followed at UV 214 and 274 nm). Purity and MW were then determined by analytical RP-HPLC and ESI-MS in positive ion mode. The mass spectra of each peptide was acquired using a SSQ 710C mass spectrometer (Finnigan MAT, San Jose, CA, USA) equipped with a type ESI source and ICIS software, and the final concentration of peptide was determined in NaH2PO4/Na2HPO4 (10 mM) containing NaF (100 mM) (ε280 nm ) 1480 M-1 cm-1).26 Data were processed with MassLynx V4.0 software (Waters). Synthesis, Purification, and Characterization of mPEG-FosWC. mPEGmaleimide was reacted with FosWC at a 1.5:1 molar ratio in phosphate buffer (pH 7.0) for 2 h (Figure 1c). Typically, peptide (3.7 µmol) was dissolved in 0.1% v/v TFA (0.4 mL) and added to mPEG-maleimide (5.5 µmol) dissolved in degassed phosphate buffer (1.6 mL) (adapted from ref 27). The reaction was monitored using Ellman’s (DTNB) reagent for free thiols.28 Aliquots (20 µL) were taken at time points of 2 min, 1 h, and 3 h. The DTNB solution was prepared as follows; NaOAc 50 mM and DTNB (2 mM in ddH2O) were mixed (50 µL) with Tris pH 8, 1 M (100 µL) ddH2O (840 µL) and added to the sample (10 µL). In the presence of free R-SH, Ellman’s reagent rapidly forms a disulfide bond with the thiol, and the colored thiolate anion released can be monitored at 412 nm. Because of the thioether bond formed between mPEG-maleimide and FosWC, a characteristic blue shift in absorbance can be seen. Finally, the mPEG-FosWC reaction mixture

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was lyophilized and purified by cation-exchange chromatography (Macrocap SP) using a linear AB gradient (A ) sodium citrate buffer, pH 3.2, 20 mM, B ) A + NaCl, 1M) and flow rate of 5 mL min-1. An ¨ ktaPrime system with a fixed wavelength detector (280 nm) was used. A The final product was redissolved in water and desalted by ultracentrifugation (Centriprep YM-3, 3000g); the supernatant was washed and centrifuged three times with ddH2O. The peptide content was determined as above for free peptides, and aliquots were stored at -80 °C. MALDI-TOF MS using an adaptation of the method by Meier and Schubert29 was used to confirm the molecular weight of the monoPEGylated conjugate. Recombinant Expression and Purification of [15N]r-c-JUN. The methods used for preparation and characterization of [15N]r-c-Jun are shown schematically in Figure 2. An expression plasmid was prepared that contained the cytb5-r-c-Jun DNA. It contained an N-terminal cytb5 fusion tag with a TEV protease cleavage site to facilitate soluble expression of c-Jun (method adapted from ref 22). Sequencing of clone no. 5 (cytb5-r-c-Jun(5)) confirmed the expected sequence, the TEV protease cleavage site, the His tag, and the start and stop codons. The plasmid was then transformed into Escherichia coli BL21CodonPlus (DE3). Cells were grown in M9 media containing [15N]H4Cl, and expression was induced by the addition of isopropyl-β-D-thiogalactosidase (1 mM, final concentration) when the cells reached an optical density of 0.6 at 600 nm. After induction, the bacteria were further incubated for 5 h at 37 °C and harvested by centrifugation (3500g, 4 °C, 15 min). They were resuspended in lysis buffer (Tris HCl buffer, pH 8.0, 20 mM) and then ultrasonicated (Misonix Microson XL2000 equipped with a Microprobe P3 at an output control setting of 15; Misonix Inc., distributed by Hucoa-Erloss Spain) eight times with 30 s pulses and 30 s intervals. The crude lysate was centrifuged (10 000g, 4 °C, 30 min) and purified using low-pressure, metal-affinity chromatography with TALON resin using standard protocols. In brief, a slurry (12 mL 50% w/v) was prepared in ddH2O, and the media were equilibrated by washing with five-column volumes of ddH2O, followed by five-column volumes of freshly prepared Tris HCl buffer, pH 8.0, 20 mM. The medium was then transferred to a polypropylene container (ca. 250 mL) and incubated with the crude lysate supernatant at 4 °C for 30 min, agitating every 5-10 min. The media and lysate suspension were transferred back to the purification column and washed with fivecolumn volumes (30 mL) of Tris HCl buffer containing imidazole, 10 mM. Stepwise elution with increasing imidazole concentrations was performed: 50 mM × 30 mL, 250 mM × 6 mL, 500 mM × 9 mL, 1000 mM × 6 mL. Fractions were collected corresponding to each imidazole concentration and aliquots (20 µL) taken for analysis by SDSPAGE. Fractions containing pure cytb5-r-c-Jun were pooled, and the cytb5 tag was cleaved by incubating with TEV protease at 4 °C for 24 h. TEV protease and cytb5 were removed using a 10 000 MWCO centrifugation step (4000g, 12 °C, 10 min). The eluate containing cleaved [N15]r-c-Jun was concentrated using a 2000 MWCO centrifugation step (8000g, 4 °C, 11 min). This step was repeated until the volume of supernatant remaining was ∼600 µL; excess salt and imidazole were then removed by washing with ddH2O three times. The final peptide was lyophilized and stored dry; SDS-PAGE electrophoresis and MALDI-TOF MS were used to confirm purity and MW, respectively. NMR and CD Spectroscopy. NMR spectra were recorded on a Bruker Avance Ultrashield Plus 600 spectrometer equipped with 5 mm single-axis gradient TCI cryoprobe. All data were processed using the program Topspin 1.3 (Bruker GmbH, Karlsruhe, Germany). Samples were prepared to a peptide concentration of 75 µM in NaH2PO4/ Na2HPO4 (10 mM, pH 7.4) containing NaF (100 mM) in ddH2O containing D2O (5% v/v). 1H NMR spectra were acquired with 16 K complex points and a spectral width of 8.4 kHz. The total number of scans was 256, with a repetition delay of 1.5 s. A Watergate scheme was used to suppress the water signal. Two-dimensional 15N-HSQC heteronuclear experiments were acquired with spectral widths of 8 kHz (1H dimension) and 2.2 kHz (15N dimension), 40 scans, and a repetition

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Figure 2. Preparation and characterization of [15N]r-c-Jun. (a) Protocol for the expression and purification of [15N]r-c-Jun. The SDS-PAGE gel confirmed the purity of the peptide to be >90%. (b,c) Comparison of the MALDI-TOF MS spectra for unlabeled r-c-Jun and [15N]r-c-Jun, respectively.

delay of 1s. Two-dimensional 1H, 1H-NOESY experiments were acquired with 150 ms mixing time. One-dimensional 1H NMR was used to confirm peptide and conjugate identity and purity. Initially, 2D 15N-HSQC NMR experiments were conducted at a 1:1 ratio ([15N]rc-Jun:FosWC or mPEG-FosWC). Experiments were then conducted to investigate the effect of increasing the concentration of either FosWC or mPEG-FosWC on heterodimerization. The concentration of c-Jun in all experiments was 75 µM; FosWC or mPEG-FosWC was then added such that the final concentration of each was 75 or 150 µM (1:1 and 1:2 ratios, respectively). Because it was not possible to amidate and acetylate the N- or C-termini of [15N]r-c-Jun selectively because side-chain modification

would also have occurred, unlabeled, synthetic c-Jun was used for the CD experiments. (These were performed under the same conditions as those used for NMR experiments.) CD spectra were acquired using an AVIV CD215 spectropolarimeter with 0.2 mm quartz cells at 3 nm/ min with a bandwidth of 1 nm, and buffer baselines were subtracted. In all cases, the dynode voltage was well below 500 V, and mean residue ellipticities [θ]MRW were calculated on the basis of concentrations determined by UV spectroscopy, assuming ε280 ) 1480 M-1 cm-1 for the single Tyr residue.26 Uptake by MCF-7 Cells. FosWC-OG and mPEG-FosWC-OG conjugates (∼20 µg OG/mg conjugate) were synthesized as described in the Supporting Information. MCF-7 cells were seeded in six-well

Polymer Coiled-Coil Conjugates (1 mL) plates at 5 × 105 cells mL-1 in clear complete media and incubated for 24 h at 37 °C, 5% CO2. Probes were added at an OG concentration of 1.5 µg mL-1, and the cells were incubated at 37 °C for 1 h. They were then washed, and fresh medium was added before incubation for a further 1 h. At the end of the incubation period, the plates were placed on ice, and the cells were washed (three times) with cold (4 °C) PBS, pH 7.4 (1 mL). An aliquot (1 mL) of PBS, pH 7.4 was then added to each well, and the adherent cells were scraped from the plate surface and transferred to Falcon tubes. The samples were centrifuged at 4 °C for 5 min at 1000g. The cells were resuspended in cold (4 °C) PBS, pH 7.4 (200 µL) and analyzed using a Becton Dickinson FACS Calibur cytometer equipped with an argon laser (488 nm) and emission filter (550 nm). Data were acquired using 1024 channels with band-pass filter FL-1 (530 ( 15 nm) and collected with 10 000 cell counts in the gated region per sample and processed using CellQuest version 3.3 software. To account for autofluorescence, control cells incubated with only clear complete media were also assessed. Additionally, at the end of the experiment, an aliquot of the cell culture medium was assessed using SEC (using a PD-10 column) to determine the stability of the OG-conjugates. Cytotoxicity against MCF-7 Cells. The cytotoxicity of FosWC and mPEG-FosWC (with and without the transfection reagent Tfx-50) was assessed (72 h) using MCF-7 cells (seeded in 96-well plates at a density of 40 000 cells mL-1). Test compounds (100 µL) (n ) 6) were added, and after a 67 h of incubation, MTT (20 µL; 5 mg mL-1 in PBS) was added. After a further 5 h of incubation, the MTT solution was removed and DMSO (100 µL) was added, and after 30 min at 37 °C, the absorbance was measured at 550 nm using a plate-reader (Sunrise, Tecan). Cell growth was expressed as a percentage relative to the control cells.

Results and Discussion Over the past decade, coiled-coil-based protein biomimetic materials have been proposed as new biomaterials and defined matrices for tissue engineering,30,31 as bioresponsive hybrid hydrogels for controlled drug delivery,32,33 as coiled-coilantibody fragment conjugates,34 and as epitope displays for vaccination.35 Recently, we have also proposed coiled-coil peptide linkers as a new tool for creation of bioresponsive polymer-drug conjugates.36 Remarkably, with the exception of the recently described pro-apoptotic, polymer-coiled-coil antibody cross-linkers,37 there has been relatively little interest in the use of coiled-coil-peptide conjugates as therapeutics.19,38-40 This can be attributed to both the complexity of synthesis and characterization and the difficulty of proving a heterodimeric coiled-coil interaction in a biological setting. Moreover, medicinal chemists often point to challenges such as lack of oral bioavailability, rapid proteolytic degradation, and the biological barriers to efficient target-specific delivery as a motivation to pursue rational design of low-molecular-weight drugs targeting pathways regulating coiled-coils.41 Such delivery challenges are not insignificant, but given the increasing clinical success of polymer-protein, peptide, and aptamer conjugates, it seems likely that coiled-coil conjugates could have real potential for use as innovative therapeutics for use as treatments of diseases where current options are limited. Even though the challenges for efficient delivery of macromolecular therapeutics to the cytosol/nucleus are well-documented, the oncogenic AP-1 transcription was chosen here as a first target to establish the feasibility of target hybridization of a PEG-coiled-coil conjugate because it was known that a “coiled-coil” interaction is essential for AP-1 function,20 and thus it provides an ideal model. Both c-Jun and FosWC peptides were synthesized using solid-phase synthesis with a purity of >95% with typically a 20-25% yield (Table 1). Mass was

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confirmed by ESI-TOF MS, and the peptide characteristics are given in the Supporting Information (Figures S1 and S2). The recombinantly prepared [15N]r-c-Jun peptide also had a high level of purity (>90%), and the expected MW was confirmed by MALDI-TOF MS (Figure 2b,c). The cytb5 tag proved to be a convenient tool because its red color gives a visual quality check at each step of expression and purification. It should be noted that a N-terminal GT sequence remained following TEV protease cleavage. The CD spectra of c-Jun and [15N]r-c-Jun at 37 °C were, however, very similar, and the spectra suggested a typical random-coil (Figure 3d). PEG was selected as the polymer platform here because PEGylation is already a well-established method for modulating plasma half-life, improving biological stability, and reducing immunogenicity of bound proteins (reviewed in refs 2 and 3). A thioether linkage was chosen to link PEG and the coiled-coil peptide because this has demonstrated good physiological stability in the clinically approved drug Cimzia. The use of FosWC (N-terminal cysteine) enabled 1:1 conjugation with mPEG-maleimide (maleimides react 1000 times faster with thiols than with amines), and the Ellman’s assay for free thiols showed that a 1.5 molar excess of mPEG-maleimide resulted in an almost complete reaction within minutes (Figure 3a). The cation-exchange HPLC method developed for conjugate purification clearly resolved all reactants (Figure 3b), and MALDITOF MS confirmed the molecular weight of the conjugate to within the theoretical range given the polydispersity of the mPEG-maleimide reagent (Figure 3c). As anticipated, mPEGmaleimide showed no structure by CD, whereas the mPEGFosWC conjugate revealed a typical coiled-coil peptide conformation (Figure 3). The purity and folding state of the c-Jun and FosWC peptides was investigated using 1D and 2D 1H NMR and CD spectroscopy under conditions chosen to model the physiological environment, that is, at 37 °C and pH 7.4. The c-Jun peptide displayed a limited number of peaks in the NH/aromatic region (6.0-10.0 ppm) with a very low dispersion, indicating the presence of a random coil peptide (Supporting Information, Figure S3). Similarly, the [15N]r-c-Jun peptide 15N-HSQC spectrum showed a limited number of backbone 1H-15N correlations (Figure 4a) with a very low dispersion (6.7 to 8.3 ppm) indicative of a disordered peptide. The NMR observations agreed with the CD spectroscopy. Conversely, the FosWC and mPEG-FosWC peptides showed higher dispersion of resonances and the presence of R-helical NH-NH connections in the 2D 1 H NOESY spectra, providing evidence of homodimeric coiledcoil formation (Supporting Information, Figure S3). (Additional studies using 1D 1H NMR to determine the effect of temperature on the conformation of c-Jun, FosWC, and mPEG-FosWC are also reported in Figure S4 of the Supporting Information). The addition of an equimolar amount of FosWC to [15N]r-cJun produced a dramatic change in the 15N-HSQC spectrum (Figure 4b). Obviously, the unlabeled FosWC will not generate signals, so the new peaks visible are attributable to structural changes in [15N]r-c-Jun, due to the formation of a structured hybrid. A 2D NOESY spectrum of [15N]r-c-Jun:FosWC showed characteristic NH-NH NOE peaks that were indicative of an R-helical structure. Similarly, the addition of the mPEG-FosWC conjugate to the [15N]r-c-Jun peptide at a 1:1 ratio led to a significant increase in the number of peaks observed (Figure 4d). Importantly, spectral overlay (Figure 4f) showed that the pattern observed following the addition of mPEG-FosWC to [15N]r-c-Jun was almost identical to that seen for the FosWC: [15N]r-c-Jun hybrid. Far UV CD spectra acquired for mixtures

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Figure 3. Synthesis and characterization of mPEG-FosWC and associated peptides. (a) Use of Ellman’s reagent (DNTB) to monitor mPEGmaleimide reaction with FosWC. (b) Cation-exchange chromatography purification of mPEG-FosWC. (c) MALDI-TOF MS analysis of mPEGFosWC in positive ion, linear mode. (d-f) CD spectroscopy of peptides at 37 °C for the individual reagents and the c-Jun:FosWC and the c-Jun:mPEG-FosWC complexes (75 µM peptide each, 1:1 ratio).

Polymer Coiled-Coil Conjugates

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Figure 4. Investigation of mPEG-FosWC:[15N]r-c-Jun target hybridization using 2D 1H, 15N-HSQC NMR spectroscopy at 37 °C. (a) [15N]r-c-Jun (75 µM). (b) [15N]r-c-Jun and FosWC (both 75 µM). (d) [15N]r-c-Jun and mPEG-FosWC (both 75 µM peptide concentration). The overlaid spectra (c) (a + b), (e) (a + d), and (f) (b + d).

of c-Jun:FosWC and c-Jun:PEG-FosWC at 37 °C revealed a [θ]208/[θ]222 ratio that was indicative of the formation of a coiledcoil for both complexes. (A ratio of the CD intensities for dimeric coiled-coils [θ]208/[θ]222 ≈ 1 is indicative of tertiary interactions between helices.) Raising the ratio of [15N]r-c-Jun:FosWC/mPEG-FosWC from 1:1 to 1:2 produced no change in the signal chemical shifts seen in the 15N HSQC spectra (Supporting Information, Figure S5), indicating that heterodimerization was preferred over homodimerization. Furthermore, the volumes of the peaks were

unchanged, indicating that heterodimeric hybridization was saturated at a 1:1 ratio and all of the [15N]r-c-Jun peptide was part of a heterodimeric complex. Such observations strongly support the conclusion that the heterodimeric state is favored under physiological conditions. For a mPEG-FosWC conjugate to be an effective therapeutic agent, efficient cytosolic/nuclear delivery is essential. PEG is not known as an endosomolyic polymer, so an additional “helper” formulation would be needed. Because previous studies reported that the transfection reagent Tfx-50 was able to deliver

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Biomacromolecules, Vol. 12, No. 1, 2011

Figure 5. Uptake of FosWC-OG, FosWC-OG:Tfx-50, mPEG-FosWCOG, mPEG-FosWC-OG:Tfx-50 and Tfx-50 alone over the first 2 h of the transfection protocol. Data show cell-associated fluorescence determined by flow cytometry and represents the mean (n ) 6) ( SD of two separate experiments. Inset shows the uptake of FosWC and mPEG-FosWC without Tfx-50 on a magnified y axis. Time (h) is shown above each bar. Statistical significance determined using a two-way ANOVA with Bonferroni post hoc test. N.S. represents no significant difference, * ) p < 0.05, ** ) p < 0.01, *** ) p < 0.001. Black shows difference ( Tfx-50, blue shows difference between FosWC-OG:Tfx-50 and mPEG-FosWC-OG:Tfx-50 complexes.

c-Fos-derived peptides in vitro,42 preliminary biological experiments were conducted to investigate the uptake and cytotoxicity of mPEG-FosWC ( Tfx-50 using the human breast cancer cell line MCF-7 as a model. In terms of cell association over 1 h (Figure 5), FosWC-OG showed a higher cell association than that seen for the PEG conjugate. Both compounds are probably taken up by endocytosis following the pathway recently described for mPEG-OG in MCF-7 cells.43 After the 1 h “chase” phase, that is, at t ) 2 h, the FosWC-OG cell-associated fluorescence did not significantly change, whereas that of mPEG-FosWc fluorescence decreased by ∼40% (Figure 5), suggestive of exocytosis. This phenomenon has been reported for other polymer conjugates,43 but clearly, the precise entry mechanism of mPEG-FosWC and its subsequent fate requires further investigation, and the kinetics require quantification. The addition of the Tfx-50 transfection reagent markedly increased (∼25-fold) FosWC-OG and mPEG-FosWC-OG cellassociated fluorescence after 1 h (Figure 5). In this case, after the 1 h “chase” period, FosWC-OG cell-associated fluorescence doubled, whereas once again the mPEG-FosWC-OG fluorescence decreased by ∼40%. Determination of free OG levels in the incubation medium revealed a slight increase (rising from