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Enhanced and prolonged cell-penetrating abilities of arginine-rich peptides by introducing cyclic #,#-disubstituted #-amino acids with stapling Makoto Oba, Masayuki Kunitake, Takuma Kato, Atsushi Ueda, and Masakazu Tanaka Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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Bioconjugate Chemistry

Enhanced and prolonged cell-penetrating abilities of arginine-rich peptides by introducing cyclic α,αdisubstituted α-amino acids with stapling Makoto Oba,*† Masayuki Kunitake,† Takuma Kato,† Atsushi Ueda,† Masakazu Tanaka*† †

Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki

852-8521, Japan

ACS Paragon Plus Environment

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ABSTRACT

Cell-penetrating peptides are receiving increasing attention as drug delivery tools, and the search for peptides with high cell-penetrating ability and negligible cytotoxicity becomes a critical research topic. Herein, cyclic α,α-disubstituted α-amino acids were introduced into arginine-rich peptides and an additional staple was provided in the side chain. The peptides designed in the present study showed more enhanced and prolonged cell-penetrating abilities than an arginine nonapeptide due to high resistance to protease and conformationally stable helical structures.


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Arginine (Arg)-rich peptides are some of the most efficient cell-penetrating peptides (CPPs) and deliver membrane-impermeable substances into cells.1,2 Extensive efforts have been made in order to develop Arg-rich peptides and their derivatives with better cell-penetrating abilities.3,4 Stabilization of the secondary structure of oligomers, which have the same design as so-called foldamers,5,6 is one of the promising strategies for efficient CPPs. β-Peptides,7–10 oligoureas,11 and α,α-disubstituted α-amino acids (dAAs)-containing peptides12–18 are known to have the property of stabilizing helical secondary structures and have been reported to possess good cellpenetrating abilities. Furthermore, a hydrocarbon staple at a suitable distance in the peptide side chain enhances the helicities of peptide secondary structures as well as the metabolic stabilities of peptides, and is also an appropriate technique for the design of functional peptides internalizing into intracellular compartments.19–22 We herein investigated the peptide secondary structures and cell-penetrating abilities of Arg-rich peptides by introducing cyclic dAAs and incorporating a staple for the purpose of developing efficient CPPs (Figure 1). We prepared a non-stapled peptide by introducing two cyclic dAAs into an L-Arg nonapeptide (R9 peptide). A stapled peptide was provided by the incorporation of a staple into the side chain of the nonstapled peptide. Peptides containing cyclic dAAs prefer to form helical structures23–25 and have been reported as helical functional peptides such as peptide catalysts26–28 and antimicrobial peptides.29,30 We expected the synergistic effects between the introduction of cyclic dAAs and the staple in Arg-rich peptides to improve their helicities in aqueous solution and cell-penetrating abilities for a long time.


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Figure 1. Design of an R9 peptide, non-stapled peptide, and stapled peptide in the present study. The Fmoc-protected cyclic dAAs 4a and 4b were synthesized according to Scheme 1. Two diastereomers 1a and 1b were prepared from L-malic acid (8 steps, 1a: 22% yield, 1b: 11% yield), as previously reported.31 The Cbz-protecting group was changed to a Boc-protecting group by hydrogenolysis using H2 and Pd/C in the presence of Boc2O (2a: 90% yield, 2b: 91% yield). The treatment of alcohols 2a and 2b with allyl bromide/Ag2O afforded allyl ethers 3a and 3b in yields of 78% and 66%, respectively. The hydrolysis of 3a and 3b under alkaline conditions followed by the TFA treatment gave crude unprotected amino acids, the primary


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amines of which were protected by Fmoc in order to afford the desired cyclic dAAs 4a and 4b in yields of 62% from 3a and 3b in 3 steps. OH S

Cbz-HN

*

CO 2Me

1a: (1S,3S) 1b: (1R,3S)

Boc-HN

*

Allyl bromide Ag2O

CO 2Me

1) NaOHaq. 2) TFA

S

*

S

2a: (1S,3S) 90% 2b: (1R,3S) 91%

O

Boc-HN

OH

H 2, 10% Pd/C, Boc 2O

CO 2Me

3a: (1S,3S) 78% 3b: (1R,3S) 66%

O S

* 3) TMSCl, DIPEA, Fmoc-HN CO 2H Fmoc-Cl 4a: (1S,3S) 62% (3 steps) 4b: (1R,3S) 62% (3 steps)

Scheme 1. Synthesis of Fmoc-protected cyclic dAAs with an allyl group in the side chain. An R9 peptide, non-stapled peptide, and stapled peptide were prepared by the Fmoc solidphase method using COMU, HATU/HOAt, or HBTU/HOBt as the coupling reagents (Supporting Information). In the synthesis of the non-stapled peptide and stapled peptide, Fmocamino acids were introduced into the peptides with double coupling, and capping of the unreacted N-terminal amine was performed using acetic anhydride after each coupling reaction. The staple was performed using a 1st generation Grubbs catalyst on the resins after the Fmocglycine introduction. Carboxyfluorescein (CF) was introduced as a fluorescent label on resins in order to monitor the peptides internalizing into cells.14 Peptides that cleaved from resin were purified with reverse-phase HPLC (RP-HPLC). The homogeneities and purities of the peptides were verified by analytical RP-HPLC and matrix-assisted laser desorption-ionization time-offlight mass (Figure S1).


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The conformations of the R9 peptide, non-stapled peptide, and stapled peptide were analyzed by circular dichroism (CD) measurements in 2,2,2-trifluoroethanol (TFE)/H2O (50/50) (Figure 2a) and H2O (Figure 2b). Negative maxima at 205–209 nm (π→π*) and 222–225 nm (n→π*) were diagnostic of a right-handed (P) helical structure, while a negative maximum at 195–200 nm (π→π*) and positive maximum at 217 nm (n→π*) were diagnostic of a random coil structure.32–34 The R9 peptide showed negative maxima at 197–198 nm and positive maxima at 218–219 nm in both solvents, which indicated that the dominant structure of the R9 peptide was a random coil.

On the other hand, the non-stapled peptide and stapled peptide showed

characteristic negative maxima at 205–206 nm and 220–222 nm. The introduction of two cyclic dAAs into Arg-rich peptides led to a marked conformational change from a random coil to a right-handed (P) helical structure. In TFE/H2O solvent (Figure 2a), the non-stapled peptide and stapled peptide showed similar results; however, in H2O solvent (Figure 2b), the intensity of the stapled peptide was higher than that of the non-stapled peptide. These results revealed that the incorporation of the staple besides the introduction of cyclic dAAs enhances the stabilities of the helical structures of Arg-rich peptides in aqueous solution.

(b)

70000

50000

–– R9 peptide –– Non-stapled peptide –– Stapled peptide

30000

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30000

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Figure 2. CD spectra of peptides in TFE/H2O (50/50) (a) and H2O (b). Peptide concentrations: 12.5 µM (a); 25 µM (b). We examined cell-penetrating abilities of peptides into HeLa cells at different incubation times (1, 2, 4, 8, and 24 h) (Figure 3a). The peptide concentration was set at 2 µM, which was the minimum concentration needed to detect fluorescence in all peptides and at all incubation times. After each incubation of HeLa cells with peptides, cells were lysed and the fluorescence intensity of the lysate was measured. The R9 peptide showed strong cellular uptake after the 1-h incubation. On the other hand, the cellular uptake of the non-stapled peptide and stapled peptide was enhanced by an increase in incubation times. The non-stapled peptide and stapled peptide showed significantly stronger cellular uptake than the R9 peptide after the 2-h and 1-h incubations, respectively. Furthermore, the cellular uptake of the stapled peptide was stronger than that of the non-stapled peptide at all incubation times. The stapled peptide showed more than 15- and 2-fold higher internalization than the R9 peptide and non-stapled peptide after the 24-h incubation. The introduction of cyclic dAAs and stapling into the Arg-rich peptide led to enhanced and prolonged cellular uptake. A similar tendency was also observed in Huh-7 cells (Figure S2a) and CHO-K1 cells (Figure S3a). The effect of introduction of cyclic dAAs and stapling on cell-penetrating ability of peptides became prominent by longer incubation. A cell viability assay of HeLa cells (Figure 3b), Huh-7 cells (Figure S2b), and CHO-K1 cells (Figure S3b) treated with peptides at 1, 2, and 4 µM for 2 h revealed the negligible cytotoxicities of peptides.


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Figure 3. (a) Cellular uptake of peptides into HeLa cells. Incubation time-dependency with a peptide concentration of 2 µM.

(b) Cell viability of HeLa cells treated with peptides at

concentrations of 1, 2, and 4 µM for 2 h. Error bars represent the standard deviation, n = 4 (a), n = 6 (b). In order to clarify different cell-penetrating abilities among peptides, the stabilities of peptides against serum and trypsin were examined using HPLC (Figure 4). In general, peptides are easily degraded by proteases in serum, which decrease the cell-penetrating abilities of Arg-rich peptides. The introduction of dAAs into peptides composed of natural α-amino acids is known to increase resistance to enzymatic degradation.12,35–37

The intact R9 peptide incubated in

medium containing 10% serum at 37 ºC was diminished by approximately 40% after the 8-h incubation and reached almost 0% after the 48-h incubation (Figure 4a). On the other hand, 60% and 90% of intact peptides remained, even after the 48-h incubation, for the non-stapled peptide and stapled peptide in medium containing 10% serum, respectively. Figure 4b shows the effects of trypsin, which is one of the serine proteases and cleaves amide bonds at the C terminus of


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cationic amino acids (Arg or lysine), on the stabilities of Arg-rich peptides. The R9 peptide was easily degraded by trypsin and less than 10% of the intact peptide remained after the 0.5-h incubation, whereas the non-stapled peptide and stapled peptide showed longer half-lives of approximately 0.5 h and 1.5 h than the R9 peptide (ca 0.15 h). These results imply that the synergistic effects of the introduction of cyclic dAAs and stapling elevates the stabilities of Argrich peptides against protease digestion, which may lead to enhanced and prolonged cellpenetrating abilities.

(a)

(b) 100

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–– R9 peptide –– Non-stapled peptide –– Stapled peptide 80

60


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Intact peptide (%)

80

Intact peptide (%)

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60

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0

0 0

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Time (h)

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Figure 4. Stabilities of peptides against serum (a) and trypsin (b). The intracellular distribution of the R9 peptide, non-stapled peptide, and stapled peptide (green) in HeLa cells (Figures 5 and S4), Huh-7 cells (Figure S5), and CHO-K1 cells (Figure S6) was investigated using confocal laser scanning microscopy (CLSM) after staining late endosomes/lysosomes with LysoTracker Red (red) and nuclei with Hoechst 33342 (blue). HeLa cells were observed after 2-h, 8-h, and 24-h incubations with peptides. Figures 5a and S4a showed the same locations under different experimental conditions for the observation of green


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signals. Figure 5a was observed under the same conditions as those for the detection of green signals, while Figure S4a was observed under optimal conditions for each peptide and incubation time. The intensities of the green signals in Figure 5 were consistent with the cellular uptake of peptides in Figure 3. Small green or yellow spots were observed in cells treated with each peptide, which suggests that the three peptides examined were internalized into cells via any endocytosis, and not direct diffusion through the cell membrane.

We quantified the

colocalization between peptides and LysoTracker Red using Pearson’s correlation coefficient (Figure 5b) and the colocalization ratio based on the equation shown in Supporting Information (Figure S4b).38,39 Three peptides exhibited similar values at all incubation times and were distributed in late endosomes/lysosomes with an increase in incubation times. In the case of Huh-7 and CHO-K1 cells, CLSM observations were only conducted after the 2-h incubation and the colocalization ratio of peptides with LysoTracker Red was quantified (Figures S5 and S6). Similarly, no significant difference was observed in the colocalization ratio among the three peptides. The CLSM observations imply that the non-stapled peptide and stapled peptide newly designed in the present study were internalized into cells by the same mechanism as the R9 peptide. In order to gain further information on the cellular uptake mechanisms of peptides, inhibitory experiments were conducted using specific inhibitors of endocytosis (amiloride: macropinocytosis;

sucrose:

clathrin-mediated

endocytosis;

filipin:

caveolae-mediated

endocytosis) (Figure S7). The uptake of the three peptides was decreased by the treatment with sucrose, indicating that the three peptides passed through the cell membrane via clathrinmediated endocytosis.


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(a)

2h

8h

24 h

(b) 0.5

Non-stapled peptide

Pearson’s correlation coefficient

R9 peptide

— R9 peptide — Non-stapled peptide — Stapled peptide

Stapled peptide

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0.4

0.3

0.2

0.1 0

8

16

24

Time (h)

Figure 5. (a) Intracellular distribution of the R9 peptide, non-stapled peptide, and stapled peptide (green) with late endosomes/lysosomes (red) and nuclei (blue) stained using LysoTracker Red and Hoechst 33342, respectively.

Scale bars represent 20 µm.

(b)

Quantification of Pearson’s correlation coefficients between peptides and LysoTracker Red. Error bars represent the standard deviation, n = 24. Taken together, these results indicate that the greater cell-penetrating ability of the stapled peptide is derived from increased stability against proteases in serum and not changes in entry mechanisms. The introduction of cyclic dAAs and stapling elevated the stabilities of Arg-rich peptides in medium containing 10% serum and led to enhanced and prolonged cell-penetrating abilities. However, the stapled peptide showed two-fold greater permeability than the R9 peptide and non-stapled peptide against HeLa cells, even after the 1-h incubation (Figure 3a), in which


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similar levels of intact peptides incubated with medium containing 10% serum may remain (Figure 4a).

Not only high resistance to proteases, but also other factors including a

conformationally stable helical structure appear to contribute to the efficient cell-penetrating ability of the stapled peptide. In conclusion, we designed three Arg-rich peptides, the R9 peptide, non-stapled peptide, and stapled peptide, and assessed their conformations in solution and cell-penetrating abilities in the cultured cells. The introduction of cyclic dAAs into the R9 peptide (non-stapled peptide) and additional stapling (stapled peptide) stabilized the helical secondary structures of Arg-rich peptides and increased resistance to protease degradation in serum, which led to enhanced and prolonged cell-penetrating abilities. The results of the present study provide a strategy for the further development and design of novel CPPs.

The cell-penetrating abilities of peptides

containing dAAs according to secondary structures are under investigation by our group and will be reported elsewhere in the near future. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental details and additional data. (PDF) AUTHOR INFORMATION Corresponding Authors *M. O. E-mail: [email protected] *M. T. E-mail: [email protected]


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Author Contributions This manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work was financially supported in part by JSPS KAKENHI Grant Number 25713008 (for M.O.) and by a Grant from the Takeda Science Foundation (for M.O.). REFERENCES 1. Wender, P. A., Mitchell, D. J., Pattabiraman, K., Pelkey, E. T., Steinman, L., Rothbard, J. B. (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters. Proc. Natl. Acad. Sci. USA 97, 13003–13008. 2. Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., Sugiura, Y. (2001) Arginine-rich peptides. J. Biol. Chem. 276, 5836–5840. 3. Wender, P. A., Galliher, W. C., Goun, E. A., Jones, L. R., Pillow, T. H. (2008) The design of guanidinium-rich transporters and their internalization mechanism. Adv. Drug Deliv. Rev. 60, 452–472. 4. Nakase, I., Takeuchi, T., Tanaka, G., Futaki, S. (2008) Methodological and cellular aspects that govern the internalization mechanism of arginine-rich cell-penetrating peptides. Adv. Drug Deliv. Rev. 60, 598–607.


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35. Nachman, R. J., Isaac, R. E., Coast, G. M., Holman, G. M. (1997) Aib-containing analogues of the insect kinin neuropeptide family demonstrate resistance to an insect angiotensinconverting enzyme and potent diuretic activity. Peptides 18, 53–57. 36. Yamaguchi, H., Kodama, H., Osada, S., Kato, F., Jelokhani-Niaraki, M., Kondo, M. (2003) Effect of α,α-dialkyl amino acids on the protease resistance of peptides. Biosci. Biotechnol. Biochem. 67, 2269–2272. 37. Zikou, S., Koukkou, A.-I., Mastora, P., Sakarellos-Daitsiotis, M., Sakarellos, C., Drainas, C., Panou-Pomonis, E. (2007) Design and synthesis of cationic Aib-containing antimicrobial peptides: conformational and biological studies. J. Pept. Sci. 13, 481–486. 38. Oba, M., Aoyagi, K., Miyata, K., Matsumoto, Y., Itaka, K., Nishiyama, N., Yamasaki, Y., Koyama, H., Kataoka, K. (2008) Polyplex micelles with cyclic RGD peptide ligands and disulfide cross-links directing to the enhanced transfection via controlled intracellular trafficking. Mol. Pharmaceutics 5, 1080–1092. 39. Oba, M., Kato, T., Furukawa, K., Tanaka, M. (2016) A cell-penetrating peptide with a guanidinylethyl amine structure directed to gene delivery. Sci. Rep. 6, 19913.


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TABLE OF CONTENTS


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Figure 1. Design of an R9 peptide, non-stapled peptide, and stapled peptide in the present study. 185x232mm (300 x 300 DPI)


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Scheme 1. Synthesis of Fmoc-protected cyclic dAAs with an allyl group in the side chain. 75x43mm (300 x 300 DPI)



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Figure 2. CD spectra of peptides in TFE/H2O (50/50) (a) and H2O (b). Peptide concentrations: 12.5 µM (a); 25 µM (b). 254x190mm (200 x 200 DPI)


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Figure 3. (a) Cellular uptake of peptides into HeLa cells. Incubation time-dependency with a peptide concentration of 2 µM. (b) Cell viability of HeLa cells treated with peptides at concentrations of 1, 2, and 4 µM for 2 h. Error bars represent the standard deviation, n = 4 (a), n = 6 (b). 254x190mm (200 x 200 DPI)



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Figure 4. Stabilities of peptides against serum (a) and trypsin (b). 254x190mm (200 x 200 DPI)


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Figure 5. (a) Intracellular distribution of the R9 peptide, non-stapled peptide, and stapled peptide (green) with late endosomes/lysosomes (red) and nuclei (blue) stained using LysoTracker Red and Hoechst 33342, respectively. Scale bars represent 20 µm. (b) Quantification of Pearson’s correlation coefficients between peptides and LysoTracker Red. Error bars represent the standard deviation, n = 24. 705x470mm (72 x 72 DPI)


Enhanced and prolonged cell-penetrating abilities ... - ACS Publications

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