Bioconjugate Chem. 2002, 13, 525−529
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Synthesis of Antisense Oligonucleotide-Peptide Conjugate Targeting to GLUT-1 in HepG-2 and MCF-7 Cells Chang-Po Chen,‡ Xiao-Xu Li,‡ Liang-Ren Zhang,‡ Ji-Mei Min,‡ Judy Yuet-Wah Chan,† Kwok-Pui Fung,† Sheng-Qi Wang,# and Li-He Zhang*,‡ School of Pharmaceutical Sciences, Peking University, Beijing 100083, China, Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China, Beijing Institute of Radiation Medicine, Beijing 100850, China. Received August 1, 2001; Revised Manuscript Received January 15, 2002
A simple procedure for the preparation of oligonucleotide-peptide conjugate was developed. p-Hydroxybenzoic acid was used as a linker for the connection of the fragments of peptide and oligonucleotide. It was found that such formed linkage was stable under the conditions of conjugate synthesis. The designed conjugate targeting to GLUT-1 showed up to 50% inhibition of cell proliferation in HepG-2 and MCF-7 cells. Comparing to the results from the expressed antisense RNA in cancer cells, it was proposed that the conjugate of signal peptide mimic and antisense oligonucleotide could improve the permeability of antisense oligonucleotide through cell membrane.
INTRODUCTION
The selective inhibition of expression of specific genes by oligodeoxynucleotides (ODNs) via an antisense or antigene strategy provides an attractive and elegant approach to drug discovery (1). Several requirements must be fulfilled by a potential antisense oligonucleotide including rapid penetration into cells, and stability against the degradation by nuclease (2-4). Antisense oligonucleotides with phosphorothioate backbones exhibit several advantages over other forms, including relatively high nuclease resistance as well as the ability to induce degradation of the target sequence by RNase H (1, 3). On the other hand, chemical modifications with kinds of tethered groups that increase the lipophilic properties of the ODNs have also accumulated. The conjugates of ODNs with liphophilic groups such as cholesterol (5), lipid, psoralen, acridine, or alkyl chain (6-11) which associated with increased activity and in some cases with actual increased intracellular concentration have been described. Recently, studies focused on the increase of both the cell uptake and the biological stability toward nuclease involve peptide modification of antisense ODNs. It was reported that several types of peptides, such as nuclear transport signal sequence, viral fusion peptides, hydrophobic peptides, and signal peptides, have shown intrinsic ability to perturb cell membrane (12-17). It would be interesting to investigate whether such kinds of peptide conjugates could be used for the purpose of improving their cell membrane permeability. It has long been shown that glucose transporter genes (GLUT) are overexpressed in many human cancer cells, providing extra energy for the rapid growth of cancer cells (18), and the antisense oligonucleotides with phosphorothioate backbone (5′-CCA TGG CAG CGC TGC-3′) against GLUT-1 gene exhibited about 25% * To whom correspondence should be addressed. Phone: +8610-62091700. Fax: +86-10-62092724. E-mail: zdszlh@ mail.bjmu.edu.cn. ‡ Peking University. † The Chinese University of Hong Kong. # Beijing Institute of Radiation Medicine.
inhibition of proliferation in HL-60 cells (19). We have also reported the synthesis of a spin-labeled conjugate of peptide and peptide nucleic acid (20). In this paper, we described the synthesis of the conjugate of antisense oligodeoxynuleotide (S-oligos) with a signal peptide mimic and discussed its growth inhibition in cancer cells. In most cases, nucleases digest ODNs in a 3′-exo-cleavage manner (21); therefore, it is reasonable to design the conjugate antisense oligonucleotide with a peptide at the 3′-end. The incompatibility of peptide chemistry and nucleic acid chemistry constitutes the major challenge for the synthesis of an intact peptide-ODNs conjugate. The accumulated data and concepts provided a wider range of methods for preparation of this defined structure (1316, 22-33). In this paper, we use p-hydroxybenzoic acid as a linker to connect the fragments of peptide and oligonucleotide, the consecutive assembly of the peptide, and then the ODN on a solid support was applied with much concern for a simple, convenient, and efficient preparative procedure. EXPERIMENTAL SECTION
(1) Synthesis of Peptide on CPG. (a) Hydroxylation of AP-CPG (22). To aminopropyl controlled pore glass (AP-CPG, PE product, pore size 500 Å, 0.5 g, amino group loaded: 0.22 mmol/g), 30 mL of γ-butyrolactone and 13 mg of 4-(dimethylamino)pyridine (DMAP) were added, and the mixture was placed in an incubator at 60 °C for 7 days under shaking. The extent of the reaction was monitored by the disappearance of the primary amino groups by the ninhydrin analysis (34). (b) Loading of the First Amino Acid on CPG. The CPG obtained in the previous step was drained and washed with dichloromethane (DCM), methanol, and DCM and then reacted with N-R-Fmoc-N-ε-Boc-L-lysine (57.4 mg, 0.11 mmol) and DMAP (3 mg) in 2 mL of dimethylformamide (DMF) and DCM (3:1, v/v) for 2 min. To the mixture was added 0.2 mL of dicyclohexylcarbodiimide (DCC) (1 M), and the reaction was performed at 37 °C for 24 h. After the sample was washed with DCM and methanol, a small amount of CPG was deprotected by
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526 Bioconjugate Chem., Vol. 13, No. 3, 2002
trifluoroacetic acid (TFA) to determine the extent of the reaction by ninhydrin assay; the major CPG were treated with acetic anhydride (Ac2O) and triethylamine (TEA) in DCM (3 mL, 1:1:2, v/v) for 20 min to protect the residual hydroxyl groups. The CPG was washed with DCM and methanol alternately and finally by DCM. (c) Elongation of Peptide. The Boc group at the first amino acid on the resulted CPG was removed by treatment with 33% TFA in DCM (v/v) at room temperature for 30 min. The CPG was washed (DCM and MeOH) and neutralized (10% TEA in DCM, v/v 10 min). Further peptide synthesis was carried out on the manual peptide synthesizer using standard Boc chemistry. The coupling reaction was performed sequentially using 2.5-fold molar excess of N-R-Boc protected L-amino acids (A, L, L, A and L) (Penisular products) and DCC with the reaction concentration of 0.1 M in DCM at room temperature for 2 h. The extent of the condensation was determined by ninhydrin assay. The yield of each coupling reaction is over 99%. The retained amino groups were acetylated as before. To determine the component of the peptide, partial resulted CPG was treated by 33% TFA followed by concentrated ammonium hydroxide at 55 °C for 14 h. After the sample was purified on HPLC, the peptide was identified by MALDI-TOF MS and amino acid analysis. MALDI-TOF MS m/z: 628.07 (M + 1), calculated for C30H57N7O7: m/z: 627.43; amino acid analysis: Lys:Ala:Leu ) 1:2:3. (d) Loading of Linker on CPG. Following the described procedure of Boc-deprotection at the terminal amino acid, the CPG was reacted with p-hydroxybenzoic acid (35 mg, 0.25 mmol) in 2 mL of DCM/DMF (1/1, v/v) using DCC (0.25 mmol) as coupling reagent at room temperature for 2 h, and the extent of the reaction was determined by ninhydrin assay using a small amount of washed CPG. After the sample was washed with DCM, MeOH, and DMF alternately, the majority of CPG were drained and dried for the next step. (2) Synthesis of ODNs on CPG. A total of 30 mg of the resultant CPG obtained from previous reaction was served as the solid phase support and submitted to cartridge of Applied Biosystem 931 DNA synthesizer for the synthesis of the desired sequence of S-oligonucleotide. The reaction cycles were carried out in 1-2 µM scale using cyanoethyl protected nucleoside phosphoramidites as coupling blocks, the procedures were set in compliance with the protocols recommended except prolonged 60 s of the capping wait step with the DMAP/Ac2O solution. Stepwise sulfurization was performed with the standard reagent. The yield of each coupling reaction is 95-97%. The reaction was stopped at DMT-on. Normal cleavage and deprotection were completed with aqueous ammonia to obtain the peptide conjugated oligonucleotide (DMT-on). The oligomer was purified using oligonucleotide purification cartridges (OPC, Applied Biosystems). The DMT-on conjugates were purified by HPLC using Delta-Pak C18 column (Waters, 100 Å, 15 µm, 7.8 × 300 mm). The gradient was 0-40% B in 25 min, 40-100% B in 15 min (mobile phase A is 0.1 M triethylammonium acetate (pH 7.0), and mobile phase B is acetonitrile). The purified DMT-on conjugates were detritylated through OPC. After concentration, the hybrid molecules were purified by HPLC using NUCLEOSIL C18 column (Phenomenex, 100 Å, 10 µm, 4.6 × 250 mm). The gradient was the same as above, but here the mobile phase A is 0.02 M triethylammonium acetate (pH 7.0), and the mobile phase B was 50% acetonitrile in water. The resulting product appeared as a white solid (190 µg) after dried, and the sample was identified by MALDI-TOF MS
Chen et al.
or ESI-TOF MS. Antisense oligomer m/z: calculated 5603.33; found: 5603.58; sense oligomer m/z: calculated 5644.87; found: 5643.63. (3) Effects on the Inhibition of Growth of HepG-2 and MCF-7 Cells by Peptide-ODNs Conjugate. Cells were seeded in 96-well culture plates at a concentration of 5000 cells/well (40-50% confluence). After overnight culture, the complete medium was replaced by plain medium. The peptide-ODNs conjugate was dissolved in sterilized phosphate-buffered saline (PBS). Conjugate and liposome complex was prepared (2.5 µM, DNA: liposome ) 1:1) and added into the well. After 5-h incubation at 37 °C, the complex was removed and complete medium was added. For Northern analysis, 6-well culture plate was used instead but the cell confluence, and the conjugate concentration remained unchanged. MTT assay (35): For adherent cells, the medium was first removed before 30 µL of 3-4,5-dimethylthiazol-2,5diphenyl tetrazolium bromide (MTT) (5 mg/mL in PBS) was added in each well. The cells were incubated at 37 °C for 2 h. Then, 100 µL of DMSO was added and the cells were incubated for 5 min. After all crystal was dissolved, optical density was read by an ELISA plate reader at 540 nm. Northern hybridization was described in ref 19. Western hybridization: Total protein was extracted from the cells by lysing the cells with lysis buffer. A total of 30 µg of protein was loaded into each lane of SDSPAGE with 7.5% separating gel and 5% stacking gel. After the protein was transferred to a PVDF membrane, the membrane was blocked by 5% nonfat milk. Then, it was followed by goat anti-human GLUT-1 antibody (Diagostic Research) and HRP-conjugated anti-goat secondary antibody. Finally, the signal was detected by ECL Western blotting detection reagents (Amersham Pharmacia). RESULTS AND DISCUSSIONS
The synthesis of peptide-oligonucleotide conjugate was shown in Scheme 1. The hydroxylation of aminopropyl CPG was completed with γ-butyrolactone and DMAP at a higher temperature. The hydroxyl group can be used to load the first lysine by formation of an ester bond that can also be cleaved under basic conditions in concordance with ordinary ODN synthesis. To fulfill the designed peptide sequence, R-Fmoc-ε-Boc-lysine was selected as the first loaded amino acid on the CPG. The ε-amino group of lysine was used to prolong the peptide sequence, and the R-amino group would play the role of positive charge in the constructed peptide-ODN conjugate. The peptide synthesis was carried out on the CPG with standard solid phase peptide synthesis (SPPS) via Boc strategy. The major concern involved the adverse cleavage of the ester bond between peptide and CPG caused by acidic deprotection procedure. We checked every deprotection and coupling reaction by ninhydrin analysis during the whole process of peptide assembly; fortunately, the adverse cleavage of the ester bond between peptide and CPG did not happen. According to the MALDI-TOF MS, the molecular weight of the final product is concordant with the calculated value of the designed sequence. The hydroxycarboxylic acid is frequently used as a linker for the connection of peptide and oligonucleotides, and the hydroxyl group from the linker can be reacted for the formation of the phosphate diester bond which could connect with 3′-activated nucleoside blocks by the phosphoramidite or phosphotriester chemistry (26, 27).
GLUT-1 Targeting Antisense Oligonucleotide−Peptide Conjugate
Bioconjugate Chem., Vol. 13, No. 3, 2002 527
Scheme 1a
a (i) γ-Butyrolactone, DMP, 60 °C, 7 days; (ii) R-Fmoc-ε-Boc-Lys-OH, DCC, DMAP/DMF/DCM; (iii) Solid-phase peptide synthesis. (a) Deprotection: TFA/DCM, (b) coupling: Boc-Aa-OH, DCC/DCM, (c) capping: Ac2O/TEA/DCM, (d) repeating (a), (b), (c); (iv) (a) TFA/DCM, (b) p-hydroxy benzoic acid, DCC; (v) Standard DNA synthesis of PS oligonucleotide; (vi) (a) Ammonium cleavage, (b) OPC purification; (vii) 5% CF3COOH.
But in some cases the phosphate diester bond derived from aliphatic hydroxyl is unstable in basic condition; β elimination is a common reason for the interruption of the synthesis of peptide-oligonucleotide conjugate (36, 37). An alternative approach is incorporation of thiolcontaining groups that could serve as the linker for the connection of peptide and oligonucleotide. The thioester is more stable, but the preparation seems more complex to accomplish (25, 30). The phosphate diester bond formed from the aromatic hydroxyl, such as the hydroxyl of tyrosine, is much more stable in the basic conditions (38, 39). For this purpose, we suggested that p-hydroxybenzoic acid could be designed for serving as linker with the improved stability. After the condensation of phydroxybenzoic acid on the N-end of peptide, the resultant CPG was submitted to further synthesis of phosphorothioated ODN and showed the high stepwise loading efficiency of nucleoside phosphoramidites same as that of ordinary CPG. The normal basic condition (aqueous ammonia) was adopted to conduct the cleavage of the conjugate from CPG and the deprotection of nucleobase as well as the removing of Fmoc group of lysine. The signal peptide mimic was designed according to the common sequence pattern consisting of a stretch of hydrophobic amino acids and a basic residue (Lys) at the end of peptide; the conformation of peptide bears the highest R-helix tendency according to computer-aided design (Insight II/Discover). Compared to the control sample (the conjugate of peptide-sense oligonucleotide), the designed conjugate showed up to 50% inhibition of cell proliferation in HepG-2 and MCF-7 cells as shown in Figure 1 and Figure 2. Also, the expression of mRNA and protein of GLUT-1 were decreased by the analysis of Northern and Western hybridization (Figure 3, Figure 4). In a previous paper, the same antisense oligonucleotide without peptide conjugated only showed a transient inhibition of HL-60 cell proliferation of about 25%. However, when comparing the efficiency between synthetic antisense oligonucleotides and expressed antisense RNA in inhibiting GLUT-1 gene expression, expressed antisense RNA produced over 50% inhibition of HL-60 cell proliferation (19). Considering all of the data, the efficiency of the designed peptide-antisense oligonucleotide conjugate in inhibiting proliferation of HepG-2 and
Figure 1. The effect of sense and antisense peptide-oligonucleotide on HepG-2 cell proliferation. Sense (2); antisense (4). The results are expressed as mean ( SD (n ) 5).
Figure 2. The effect of sense and antisense peptide-oligonucleotide on MCF-7 cell proliferation. Sense (2); antisense (4). The results are expressed as mean ( SD (n ) 5).
Figure 3. Northern blot analysis of the effect of sense and antisense peptide-oligonucleotide on GLUT-1 gene expression in HepG-2 and MCF-7 cells. Negative control (lane 1); sense (lane 2); antisense (lane 3).
MCF-7 cells is same as the expressed antisense RNA in HL-60 cells. It seems that the designed peptide sequence could improve the properties of antisense oligonucleotide for the penetration through the cell membrane.
528 Bioconjugate Chem., Vol. 13, No. 3, 2002
Figure 4. Western blot analysis of the effect of sense and antisense peptide-oligonucleotide on GLUT-1 gene expression in HepG-2 cells. Negative control (lane 1); sense (lane 2); antisense (lane 3).
Signal peptide is an N-terminal extension of premature secretary protein and directs the translocation of protein through the membrane of endoplasmic reticulum of eukaryotes or the inner membrane of prokaryotes. They typically consist of a hydrophobic core consisting of a stretch of hydrophobic amino acids with high tendency of R-helix, basic residues near the N-terminal, and some neutral residues at the C-terminal (40, 41). The cellular mechanism involved in translocation of signal peptide is complex, but it is hypothesized that the transportation of the polypeptide chain is related to the hydrophobic property, conformation, and charge nature of the signal peptide sequence, with the cooperative function of signal peptide on the lipid bilayer membrane (16, 17). It was reported that the long h-region sequence which had been applied as a carrier of certain protein domain enables the whole polypeptide to get through the cell membrane (41), and some kind of amphiphilic anionic peptide with over 20 amino acids shows the great potential of mediating cytosol and nuclear delivery of coincubated oligonucleotides with its disturbance effect on the membrane (42). In this paper, a short peptide antisense oligonucleotide conjugate was designed, and it may provide a simple method for the modification of antisense oligonucleotides. In conclusion, a short signal peptide mimic was designed, and a simple procedure for the preparation of peptide-oligonucleotide conjugate was developed. It was found that the designed conjugate could inhibit the proliferation of HepG-2 and MCF-7 cells, and the efficiency is same as the expressed antisense RNA. ACKNOWLEDGMENT
This study was supported by National Natural Science Foundation of China, and by National Key Project for Basic Research (G1998051103) awarded by The Ministry of Science and Technology, People’s Republic of China, and supported by an earmarked grant from Research Grants Council (ref. CUHK 4148/98M), Hong Kong, China. LITERATURE CITED (1) Uhlman, E., and Peyman, A. (1990) Antisense oligonucleotides: a new therapeutic principle. Chem. Rev. 90, 543-548. (2) Wagner, R. W. (1994) Gene inhibition using antisense oligonucleotides. Nature 372, 333-335. (3) Crooke, S. T. (1992) Oligonucleotide therapy. Curr. Opin. Biotechnol. 3, 656-661. (4) Vlasov, V. V., Balakireva, L. A., and Yakubov, L. A. (1997) Transport of oligonucleotides across natural and model membrane. Biochim. Biophys. Acta 1197, 95-108. (5) Krieg, A. M., Tonkinson, J., Matson, S., Zhao, Q., Saxon, M., Zhang, L., Bhanja, U., Yakubov, L. and Stein, C. A. (1993) Modification of antisense phosphodiester oligodeoxynucleotides by a 5′-cholesteryl moiety increases cellular association and improved efficacy. Proc. Natl. Acad. Sci. U.S.A. 90, 10481052. (6) MacKellar, C. Graham, D., Will, D. W., Burgess, S., and Brown, T. (1992) Synthesis and physical properties of antiHIV antisense oligonucleotides bearing terminal lipophilic groups. Nucleic Acids Res. 20, 3411-3417.
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