Signal Peptide Mimics Conjugated to Peptide Nucleic Acid - American

Bioconjugate Chem. 2003, 14, 153−157

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Signal Peptide Mimics Conjugated to Peptide Nucleic Acid: A Promising Solution for Improving Cell Membrane Permeability Xiaoxu Li,† Liangren Zhang,† Jingfen Lu,† Yaozu Chen,‡ Jimei Min,† and Lihe Zhang*,† National Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, P. R. China and Department of Chemistry, Lanzhou University, Lanzhou 730000, P. R. China. Received July 24, 2002; Revised Manuscript Received October 8, 2002

The specific binding ability and biostability of PNA (peptide nucleic acid) with DNA or RNA make PNA not only a good tool for the studies of molecular biology but also the candidate for gene-targeting drugs. However, the main obstacle for its potential usage as a therapeutic is the low cell uptake caused by the poor cell membrane permeability. In this paper the hydrophobic pentadecapeptide and two signal peptide mimics, hexa- and decapeptides ending with a positively charged amino acid, were proposed as the linked carrier for the transportation of PNA T10 through the cell membrane; stable spin label was coupled to the peptide-PNA conjugate so that the ESR measurements can be used for the assessment of their transmembrane movements. The syntheses of spin-labeled peptide-PNA conjugates were carried out on MBHA resin with Boc strategy. The cell membrane permeability of the spin-labeled conjugates of peptides and PNA can be determined with ESR, during the incubation of erythrocyte with the samples. According to ESR measurements, the three conjugates exhibit enhanced uptake into erythrocytes. The hexa- and decapeptide-modified PNA showed suitable water solubility. The peptide-PNA conjugates retained their binding ability to complementary DNA. The results suggest that peptide modification of PNA might be a promising solution for improving cell membrane permeability toward PNA.

INTRODUCTION

Peptide nucleic acid (PNA) is a structural DNA mimic in which the entire backbone has been replaced by a polyamide backbone composed of N-(2-aminoethyl)glycine units (1, 2). PNA can hybridize to the complementary DNA and RNA or dsDNA strand with higher affinity than their oligonucleotide counterparts, obeying either Watson-Crick or Hoogsteen base pairing rules (2-5). Upon binding to DNA or mRNA, PNA exerts biological effects including in vitro transcription and translation modulation (6, 7), and PNA is stable toward both nuclease and protease (8). This character of PNA not only makes it a new tool for the studies of molecular biology but also the potential candidate for gene-targeting drugs (9, 10). However, the main obstacle for its potential usage as a therapeutic is the low cell uptake caused by the poor cell membrane permeability (11). Many physical delivery methods such as microinjection and cell treatment by detergent were reported for the studies of antisense or antigene PNA (6, 12). The efforts via chemical modification have also been made to improve cell uptake of PNA. Receptor-mediated cell or tissue internalization mechanism has been proposed for the specific delivery of PNA; for example, lactose (13), dihydrotestosterone (14), and D-peptide analogue of insulin-like growth factor 1 (IGF1) (15) modified PNAs were designed and exhibited rapid and specific entry into the corresponding cells. The transferrin receptor-mediated endocytosis system (16, 17) was also used to transport PNA through the blood-brain barrier in vivo, and gelatin or protein nanoparticles were * To whom correspondence should be addressed. E-mail: [email protected]. † Peking University. ‡ Lanzhou University.

also explored as carriers for PNA (18, 19). On the other hand, PNAs attached with lipophilic groups such as adamantyl (20, 21), phosphonium cation (22), and hydrophobic peptides (23) showed significantly improved membrane penetration in either liposome models or biomembrane systems. Different types of peptides with membrane permeability could be used as a carrier for the membrane transportation of PNA by chemical conjugation. PNA coupled to a retro-inverso delivery peptide was rapidly taken up by cultured cerebral cortex neurons (24); PNA coupled with the cellular transporter peptide, Antennapedia, was efficiently taken up into Bowes cells (25); the cationic peptide (26) linked at the N-end of a PNA pentamer directed at the RNA template of telomerase resulted in enhanced inhibition of telomerase activity; Antennepedia peptide also showed both cell uptake and inhibition of telomerase activity in human melanoma cells (27, 28); SV40 nuclear localization signal (NLS) demonstrated its efficiency for the transportation of PNA as well as the hybridized DNA (29, 30). The accumulated results suggest that peptide conjugation is an attractive strategy for the intracellular delivery of PNA. Signal peptide is an N-terminal extension of premature secretory protein and directs the translocation of protein through the membrane of endoplasmic reticulum of eukaryotes or the inner membrane of prokaryotes. The sequences of most signal peptides have a hydrophobic core consisting of a stretch of hydrophobic amino acids, basic residues near the N-terminal, and small neutral residues (31, 32). The studies strongly support that the hydrophobic interaction between the hydrophobic region (h-region) of the signal peptide and the lipid bilayer of cell membrane is the main cause of transmembrane movement (33-37). Some signal peptides have been used

10.1021/bc025585w CCC: $25.00 © 2003 American Chemical Society Published on Web 12/12/2002

154 Bioconjugate Chem., Vol. 14, No. 1, 2003

Li et al. Table 1. Properties and Characterization of Spin-Labeled Conjugates of Peptide and PNA, R15T10, R10T10, R6T10, RT10, and Decamer PNA T10 calcd MS found MS Tm (°C) solubility (mg/mL) ESR signal (standard) a

R15T10

R10T10

R6T10

RT10

T10a

4244 4245 --water/DMSO 5 +

2972 2973 78 water >5 +

2806 2808 78 water >5 b

Composed of thymine monomer. b No ESR signal.

Figure 1. Structure of spin-labeled peptide-PNA conjugates.

as a carrier of certain protein domains to bring the whole polypeptide through the cell membrane (38-41). In this paper, the peptide corresponding to the h-region of signal sequences from Kaposi FGF (38, 39) (Figure 1a) and two signal peptide mimics hexa- and decapeptides (Figure 1b,c) were designed as the carriers to conjugate with the model PNA T10 at the N-end. To evaluate the transmembrane behavior of the conceived adducts of peptide and PNA, the stable nitroxyl free radical 3-carboxyl-2,2,5,5-tetramethyl-pyrroline-1-oxyl as reporter group (42) was coupled at the N-end of the PNA moiety (Figure 1). Cell membrane penetration can be exhibited by the changes of the ESR signal during the incubation of erythrocyte with the samples (43, 44). EXPERIMENTAL PROCEDURES

General. The PNA monomer N-(2-Boc-aminoethyl)N-(thymin-1-ylacetyl)glycine1 (45) and the stable free radical 3-carboxyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl (42) are synthesized as described. Boc-protected amino acids MBHA resin are from Pennisular, and DCC is from Aldrich. DCC and DMF are dried with K2CO3 and molecular sieves, respectively, before use; other chemicals are reagent grade from local commercial sources. Solidphase synthesis is conducted with manual apparatus. Standard Ninhydrin test (46) is used for monitoring the coupling reaction. HPLC purification is performed with a Waters system on C-18 reverse phase column (0.8 × 20 mm); mobile phase gradient is B: 20-80% or 0-50% in A within 50 min (A: H2O containing 0.1% TFA; B: acetonitrile containing 0.1% TFA/H2O). UV spectra and Tm are recorded with a Pharmacia LKB Biochrom 4060 spectrophotometer. MALDI-TOF mass spectra are recorded with ZAB-HS, and ESR measurements are conducted with a Bruker ESP 300 ESR spectrometer. Synthesis. Spin-Labeled Pentadecapeptide PNAT10 Conjugate (R15T10). Oligomerization of spin-labeled peptide conjugated PNAT10 was conducted by manual solid-phase peptide synthesis on MBHA resin (213 mg, substitution 0.35 mmol/g) with Boc strategy in the order of peptide synthesis, PNA monomer condensation (45, 47), and in situ spin labeling. The cycle procedure for the solid-phase peptide synthesis was as follows: Boc-deprotection was completed with TFA/DCM (1:2, v/v), 3 mL, 1 × 2 min and 1 × 30 min, and then neutralization by using TEA/DCM (1:10, v/v), 3 mL, 2 × 2 min. Condensation was carried out with DCC (2 equiv) as coupling 1 Abbreviations: Boc, tert-butyloxycarbonyl; DCC, N,N′-dicyclohexylcarbodiimide; DMF, N,N-dimethylforamide; DCM, dichloromethane; ESR, eletron spin resonance; HOBt, 1-hydroxybenzotriazole; MBHA, methylbenzhydrylamine; RP-HPLC, reverse phase high-pressure liquid chromatography; T, thymine; TEA, triethylamine; TFA, trifluoroacetic acid; Z, benzyloxycarbonyl.

agent. Two equivalents of Boc-protected amino acid or PNA monomer was dissolved in DCC/DMF (1:1, v/v), giving the final concentration of 0.1 M of Boc amino acid or PNA monomer; after the coupling reaction, the unreacted amino groups were blocked by acetylation with a mixture of 2.5 mL of Ac2O/DCM (1:1, v/v) and 2.5 mL of TEA/DCM (1:1, v/v) for 20 min. The coupling reaction was allowed to proceed for 2 h with stirring at rt. The Kaiser test (46) was used to monitor the completion of Bocdeprotection and coupling reaction, respectively. For the peptide synthesis, the first Boc-Ala (0.1 mmol) was loaded on MBHA resin, and the coupling was done sequentially with Boc-protected Leu, Leu, Ala, Leu, Leu, Val, Ala, Pro, Leu, Leu, Ala,Val, Ala, and Ala. The PNA monomer condensation was conducted continuously after the completion of amino acid coupling cycles. Following the 10th PNA monomer coupling cycle, 2.5 equiv of 3-carboxy2,2,5,5-tetramethyl-3-pyrroline-1-oxyl (final concentration was 0.1 M in DCM) was added to the reaction vessel and stirred with PNAT10-peptide-resin in the presence of DCC (2.5 equiv, 0.1 M). The coupling reaction was completed within 2 h according to the Kaiser test. After capping and wash procedures, the product-resin was dried and treated with anhydrous HF (at 0 °C, 1 h) for cleavage of the oligmer from the resin, and the crude product was purified on C-18 RP-HPLC (20-80% B containing 0.1% TFA in A within 50 min, detected at 260 nm). The final product revealed the standard nitroxyl free radical signals under ESR spectrum measurement (three peaks, g ) 2.0029, aN ) 15.8 G, ∆H0 ) 2.5 G). MALDITOF MS result: 4245, calculated: 4244. Spin-Labeled Decapeptide PNAT10 Conjugate (R10T10). The procedure of oligomerization, cleavage, and purification is the same as spin-labeled pentadecapeptide PNAT10 conjugate, except the coupling order of amino acids is K, A, L, L, A, L, P, L, L, A; MALDI-TOF MS found: 3848, caculated: 3848; ESR measurement gives the standard three-peak signal as above. Spin-Labeled Hexapeptide PNAT10 Conjugate (R6T10). The procedure of oligomerization, cleavage, and purification is the same as spin-labeled pentadecapeptideconjugated PNA, except the coupling order of amino acids is K, A, L, L, A, L; MALDI-TOF MS found: 3454, caculated: 3453; ESR measurement gives the standard three-peak signal as above. Spin-Labeled PNAT10 (RT10). The procedure of oligomerization and cleavage is the same as the described in spin-labeled pentadecapeptide-conjugated PNAT10, but only K is coupled on the resin before condensation of the PNA monomers. HPLC purification is carried out with B: 0-50% in A within 50 min. MALDI-TOF MS found: 2972, caculated: 2973; ESR measurement gives the standard three-peak signal as above. PNA T10 (T10). The procedure for synthesis of PNA T10 is the same as described above without the spin labeling. MALDI-TOF MS found: 2808, calculated: 2806.

Spin-Labeled Peptide−PNA Conjugates

Bioconjugate Chem., Vol. 14, No. 1, 2003 155 Table 3. Molecular Total Energy Calculated by the Computer Simulating Method for the r Helix of Nanopeptides

Figure 2. ESR signal strength of spin-labeled conjugates of peptide and PNA R15T10 (1), R10T10 (2), R6T10 (3), and spinlabeled PNA RT10 (4) incubated with erythrocyte. The strength is presented as the percentage of the initial peak height at low field. Table 2. Molecular Total Energy Calculated by the Computer Simulating Method for the r Helix of Pentapeptides sequence

total energy (kcal/mol)

sequence

total energy (kcal/mol)

LVALL VLLAL AVALL

49.49 49.49 59.92

AVLLA LALLA AAVAL

61.44 40.20 71.60

Computer Simulation. The molecular total energy was calculated on an SGI indy workstation using the Biopolymer module in INSIGHT II 95.0 (Biosym). Observation of Membrane Permeability by ESR Measurement. Healthy adult’s erythrocytes were washed three times with isoosmotic phosphate buffer solution (PBS, pH 7.0) by centrifugation (10 min × 2000 rpm) (43, 44). The erythrocytes were suspended with PBS, and the density of cell in the suspension was adjusted to 50 million/mL and was incubated at 37 °C for 15 min prior to use. The spin-labeled peptide-conjugated PNA or spinlabeled PNA (2 mM) in 200 µL PBS (containing 2% DMSO) was mixed with 200 µL erythrocyte suspension. The mixture was incubated at 37 °C. The same volume of sample was picked out from the mixture at beginning of incubation, and thereafter periodically, and submitted to ESR measurement on a Bruker ESP 300 ESR spectrometer, respectively, and the spectra as well as signal strength at the same field was recorded. The percentage of ESR signal strength was calculated from the changes of ESR measurements before and after the incubation of erythrocyte with samples (Figure 2). RESULTS AND DISCUSSION

The mechanism of signal peptide in transmembrane movement is complex, but in most cases, the hydrophobic region composed of 5-15 amino acid residues is critical to the hydrophobic interaction with the phospholipids on the membrane. Studies also showed that the polarity and the conformation adopted by the signal sequence played an important role to facilitate the crossing membrane behavior: with the basic residue near the N-end, and most of the h-region in the signal sequence, it more readily formed an R-helix (31, 32). To design the peptide sequences used for PNA modification, computer-aided modeling was used to search the ideal sequence with the lowest systemic energy in simulation of the R-helix. According to the data of the simulation, two new sequences LALLAK and ALLPLALLAK with the system energy of 40.20 kcal/mol and 90.65 kcal/mol respectively (Table 2, 3) were proposed as the signal peptide mimics for the modification of PNA. A positively charged lysine was selected as a polarity enhancer which can be appended at the end of peptide, and the lysine would also

sequence

total energy (kcal/mol)

sequence

total energy (kcal/mol)

ALLPAVLLA ALLPLALLA VALLPAVLL VALLPALLA AVALLPAVL AVALLPLLA

112.9 90.65 124.67 112.80 134.56 112.39

AAVALPAVL AAVALPLLA AAVAPAVLL AAVAPALLA AAVPAVLLA AAVPLALLA

146.20 124.08 144.02 132.35 142.47 120.12

contribute to its solubility, as well as the possible role of directing the adduct to access the partial polar cell membrane surface (49, 50). To assess the membrane permeability of the conceived peptide-linked PNA, we designed nitroxyl free radical as a report group so that the permeability of the spin-labeled adduct of peptide and PNA could be detected by the measurement of the ESR signal changes. Therefore, three spin-labeled peptide PNA conjugates (R15T10, R10T10, and R6T10) and the control sample, spin-labeled PNA (RT10), were designed and synthesized by standard solid-phase peptide chemistry. After cleavage with HF treatment, the products show the correct molecular weights by MALDI-TOF MS and the ESR signal with standard nitroxyl free radical parameters: three peaks, g ) 2.0029, aN ) 15.8 G, ∆H0 ) 2.5 G. The results also demonstrate that the selected spin label molecule cannot be chemically influenced through a series of peptide synthesis conditions including HF treatment (Table 1). Tm values of R15T10, R10T10, R6T10, and RT10 measured with the complementary sequence dA10, respectively, show that such modification has only little influence on its hybridizing behavior (Table 1). Stable nitroxyl free radical can be readily reduced to hydroxylamine by reductive agent. In erythrocyte the concentration of the reductive agent is 2-3 mM which can reduce the free radical and dramatically quench the ESR signals inside the cell (43, 44). In our experiments, the erythrocyte suspension in isoosmic buffer was free of plasma via a washing procedure so that the changes of ESR signal were only due to the intracellular chemical conditions. The results (Figure 2, curve 1) showed that the ESR signal strength decreased significantly after 3 h incubation of the pentadecapeptide-conjugated PNA (R15T10) with erythrocyte suspension, and no change was observed in the case of control sample (RT10) at the same condition (Figure 2, curve 4). It indicated that the free radical of R15T10 was reduced by the reductive agents inside the cells. The signal changes after 1.5 h incubation was also observed and showed the same as that determined after 3 h. It may infer that peptidePNA conjugate R15T10 can penetrate the cell membrane rapidly and reach an equilibrium within 1.5 h. The same ESR measurements were used to assess the cell membrane permeability of hexapeptide- and decapeptideconjugated PNAs, respectively. R10T10 and R6T10 showed almost the same decrease of the ESR signal strength after the incubation with erythrocyte for over 4 h (Figure 2, curve 2 and 3). After 4 h of incubation, no more decrease of ESR signal was exhibited. It means that the two peptide-PNA conjugates bear a similar extent of cell uptake. Although pentadecapeptide-PNA conjugate R15T10 appears to have a faster penetrating rate than that of the hexa- and decapeptide modifiers, the pentadecapeptide modification makes the whole PNA T10 adduct difficult to dissolve in water. Lower water solubility may constitute another disadvantage for the feasiblity of

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therapeutics. However, the hexa- and decapeptide modification both improve the cell membrane permeability and in the meantime bear acceptable water solubility (Table 1). To support the ESR results, an antisense oligonucleotide-peptide conjugate containing LALLAK was synthesized, and the antisense activities targeting GLUT-1 in HepG-2 and MCF-7 cells were investigated (51). It was found that the synthetic antisense oligonucleotide-peptide conjugate showed up to 50% inhibition of cell proliferation in HepG-2 and MCF-7 cells. Compared to the previous paper, the same antisense oligonucleotide without peptide conjugated only showed a transient inhibition of HL-60 proliferation of about 25% and the expressed antisense RNA produced over 50% inhibition (52). Therefore, it seems that the efficiency of the synthetic antisense oligonucleotide peptide conjugate is the same as the expressed antisense RNA in the intact cells. In conclusion, the hydrophobic pentadecapeptide and two signal peptide mimics, hexa and decapeptides ended with a positively charged amino acid, were proposed as the linked carrier for the transportation of PNA T10 through the cell membrane. Stable spin label was coupled to the peptide-PNA conjugate so that the ESR measurements can be used for the assessment of their transmembrane movements. The syntheses of spin-labeled peptidePNA conjugates were carried out on MBHA resin with Boc strategy. According to the measurement of ESR, the three conjugates, R15T10, R10T10, R6T10, exhibit an enhanced rate of uptake across the erythrocyte membrane. The hexa- and decapeptide modified PNAs, R10T10 and R6T10, show suitable water solubility. The peptidePNA conjugates retain their binding ability to the complementary DNA. The results suggest that peptide modification of PNA might be a promising solution for improving cell membrane permeability toward PNA. ACKNOWLEDGMENT

This work is financially supported by the National Natural Science Foundation of China and the research grant G 1998051103 awarded by The ministry of Science and Technology, People’s Republic of China. LITERATURE CITED (1) Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1991) Sequence- selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500. (2) Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., Berg, R. H., Kim, S. K., Norden, B., and Nielsen, P. E. (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogenbonding rules. Nature 365, 566-568. (3) Leijon, M., Gra¨slund, A., Nielsen, P. E., Buchardt, O., Norde´n, B., Kristensen, S. M., and Eriksson, M. (1994) Structural characterization of PNA-DNA duplexes by NMR. Evidence for DNA in a B-like conformation. Biochemistry 33, 9820-9825. (4) Brown, S. C., Thomson, S. A., Veal, J. M., and Davis, D. G. (1994) NMR Solution structure of a peptide nucleic acid complexed with RNA. Science 265, 777-780. (5) Cherny, D. Y., Belotserkovskii, B. P., Frank-Kamenetskii, M. D., Egholm, M., Buchardt, O., Berg, R. H., and Nielsen, P. E. (1993) DNA unwinding upon strand- displacement binding of a thymine-substituted polyamide to doublestranded DNA. Proc. Natl. Acad. Sci. U.S.A. 90, 1667-1670. (6) Hanvey, J. C., Peffer, N. J., Bisi, J. E., Thomson, S. A., Cadilla, R., Josey, J. A., Ricca, D. J., Hassman, C. F., Bonham, M. A., Au, K. G., Carter, S. G., Bruckenstein, D. A., Boyd, A.

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