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Dipicolylamine/metal complexes that promotes direct cell-membrane penetration of octaarginine Yoshimasa Kawaguchi, Shoko Ise, Yusuke Azuma, Toshihide Takeuchi, Kenichi Kawano, Toan Khanh Le, Junko Ohkanda, and Shiroh Futaki Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00691 • Publication Date (Web): 14 Nov 2018 Downloaded from http://pubs.acs.org on November 18, 2018

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Bioconjugate Chemistry

Bioconjugate Chemistry – A special issue on "Delivery of Proteins and Nucleic Acids: Achievements and Challenges". Article Dipicolylamine/Metal Complexes that Promotes Direct Cell-Membrane Penetration of Octaarginine

Yoshimasa Kawaguchi, a† Shoko Ise, a† Yusuke Azuma, a Toshihide Takeuchi, a Kenichi Kawano, a Toan Khanh Le, a Junko Ohkanda, b and Shiroh Futaki a

a Institute

for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

b Institute

of Agriculture, Shinshu University, Kami-Ina, Nagano 399-4598,

Japan

† These

authors equally contributed to this study.

*Corresponding author: Shiroh Futaki, Ph. D., Professor Institute for Chemical Research, Kyoto University [email protected] ORCID ID: 0000-0002-0124-4002

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Abstract

Marked promotion of membrane permeation of a cell-penetrating peptide, octaarginine (R8), was attained by attachment to a single 2,2′-dipicolylamine moiety (DPA-R8) that forms complexes with metal ions. Studies using giant unilamellar vesicles demonstrated that DPA targets phospholipids and enhances R8 binding to the membranes in the presence of metal ions. While DPA/Zn(II) complex has been most frequently employed to chelate formation with phosphates, Ni(II) had the most prominent effect on membrane binding and penetration of DPA-R8. Facile cytosolic distribution of DPA-R8 was also attained in a few minutes in the presence of Ni(II). Analysis of cellular uptake methods of DPA-R8/Ni(II) suggested the involvement of direct permeation through cell membrane without the use of endocytosis. The applicability of this system to the intracellular delivery of bioactive compounds was exemplified using a peptidomimetic farnesyltransferase inhibitor, FTI277.

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Table of Content graphic

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Introduction

Intracellular delivery of bioactive molecules using cell-penetrating peptides (CPPs) has firmly established its importance in chemical biology and molecular cell biology studies.1-4 CPPs are peptides, with a few to 30 amino acids, that show cell membrane permeability. Chemical conjugation or stable complex formation of these CPPs with biofunctional macromolecules, which inherently have poor membrane permeability (e.g., peptides/proteins, nucleic acid derivatives and various nano-particles), leads to their efficient internalization into cells. This technology has been applied successfully for in-cell imaging/sensing, and for modulation of in-cell molecular interactions.5 CPPs also have been gaining research interests due to its therapeutic potentials to deliver biopharmaceuticals.6

Among CPPs, those being rich in arginine such as the HIV-1 Tat peptide and oligoarginines have been used extensively for intracellular delivery.3,4 The internalization routes of these arginine-rich CPPs include direct translocation through cell membranes (i.e., biophysical methods of permeation) 4


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and via endocytosis (i.e., physiological, vesicular transport systems) (Supporting Information Fig. S1).7-9 The former method may deliver macromolecules of interest immediately to cytosol to exert the expected in-cell functions. In contrast, use of the latter pathway needs an extra step of escaping from vesicular compartments (endosomes) to reach cytosol after engulfment into endosomes.10 Direct penetration is thus more effective. Cell surface accumulation of CPPs is advocated to facilitate the direct membrane translocation.11 Alternatively, extensive interaction of CPP-cargo with membranes not only retain them on either sides of membrane but may also be accompanied by perturbation of membranes and cytotoixicity.12 We thus hypothesized that employing functional groups that can appropriately enhance the membrane interaction may facilitate translocation of R8 (Fig. 1a).

2,2′-Dipicolylamine (DPA) has been reported to form a complex with Zn(II) and can be exploited as a chemosensor to detect phosphorylated peptides and proteins.13 Binuclear DPA-Zn(II) complexes have been frequently employed to increase the recognition ability to anionic phosphate derivatives.14,15 Oligomers

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of a tyrosine derivative bearing a DPA-Zn(II) complex has also been developed as a CPP.16 Via the chelate formation with cell surface carboxylates and phosphates, the oligoDPA-Zn(II) CPPs are taken up into cells via endocytosis. Alternatively, it has been reported that the mononuclear DPA-Zn(II) complex can

Fig. 1. (a) Conceptual scheme for enhancing anchoring of arginine-rich cell-penetrating peptides (CPPs) with cell surfaces, leading to direct translocation through cell membranes. (b) Structure of a DPA-R8-FL/metal complex. (c) Sequences of the peptides used in this study. 6


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bind phosphates although the affinity is lower than that of the binuclear complex.17 Attachment of a DPA moiety to an endosomoytic peptide sufficiently enhanced peptide interaction with cell surfaces in the presence of divalent metals, enhancing endocytic uptake and the endosomolytic activity of the peptide without accompanied by significant cytotoxicity.18 Attachment of a DPA to octaarginine (R8), a representative arginine-rich CPP, may also yield a synergistic increase in the affinity of R8 to cell surface with the help of divalent metals, further facilitating direct membrane translocation of R8 (Fig. 1a). With the expectation in mind, we designed an R8 derivative bearing a DPA unit on the N-terminus (DPA-R8; Fig. 1b).

In this report, we show the effect of enhancing membrane anchoring of R8 by the introduction of a single DPA moiety that considerably promotes direct cell membrane translocation of the peptide. While DPA-Zn(II) complex is most frequently employed to chelate formation with phosphates and carboxylates, DPA-Ni(II) complex had the most prominent promotion effect on the membrane translocation. The applicability of this system to the intracellular delivery of

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bioactive molecules was exemplified through the delivery of a peptidomimetic inhibitor of farnesyltransferase (FTase), FTI277, leading to inhibition of farnesylation-dependent processing of HDJ2, a co-chaperone of mammalian heat shock cognate 70 protein.

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Results and Discussion

Preparation of DPA-R8 peptides

To evaluate the effect of DPA moiety on the membrane translocation of R8, DPA-R8 bearing a fluorescein moiety on the C-terminus (DPA-R8-FL) was prepared (Fig. 1b and c), where the fluorescein moiety can also serve as a model of

low-molecular

weight

compounds.

Preparation

of

DPA-R8-FL

was

summarized on Fig. 2. Briefly, a peptide chain was constructed using an Fmoc-solid-phase peptide synthesis where the Mtt (=4-methyltrityl) group19 was employed as a protecting group of lysine. After the N-terminus was acetylated, the

Mtt

group

was

removed

by

the

treatment

with

1,1,1,3,3-hexafluoroisopropanol (HFIP)/dichloromethane (DCM) (1:4).20 The peptide resin was successively treated with 2-pyridinecarboxaldehyde and NaBH(AcO)3 to convert the ε-amino moiety of lysine to DPA as previously reported.21 After cleavage of peptide from the resin and deprotection, a fluorescein moiety was introduced onto the thiol group of the cysteine to yield DPA-R8-FL. For reference, R8 without bearing the DPA moiety (R8-FL) and 9



DPA-R4-FL having the less number of arginines in the sequences were also prepared (Fig. 1c).

Fig. 2. Preparation of DPA-R8-FL. Peptide chain of DPA-R8-FL was constructed on a Rink amide resin (TGS-RAM) resin. Abbreviations denote: Ac, acetyl; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Mtt, 4-methyltrityl; Trt, trityl; DIEA, diisopropylethylamine; HFIP; 1,1,1,3,3-hexafluoroisopropanol; DCM, dichloromethane; TFA, trifluoroacetic acid; EDT, 1,2-ethanedithiol.

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Phospholipid targeting and enhancement of R8 binding to the membranes by DPA–metal complexes

To evaluate whether appending the DPA unit to the R8 actually benefits R8 membrane binding, confocal microscopic imaging of giant unilamellar vesicles (GUVs) treated with DPA-R8-FL in the presence of metals was conducted (Fig. 3). In addition to Zn(II), which has been employed most frequently for complex formation with a DPA unit, the effects of Ni(II) and Co(II) were also studied. Considering that plasma membranes are rich in zwitterionic (neutral) lipids, including phosphatidylcholine,22 GUVs comprised of 100%

dioleoyl-sn-glycero-3-phosphocholine

(DOPC)

were

employed.

DPA-R8-FL was treated with DOPC GUVs for 1 h in the presence of metal prior to confocal microscopic observation. Although the DPA-Zn(II) complex was expected to have the highest affinity to phosphate,21 no significant DPA-R8-FL signals were observed on GUV membranes in the presence of Zn(II) (Fig. 3, upper panels; see also Fig. S2 for wide field images). Instead, DPA-R8-FL in the presence of Ni(II) yielded significant membrane labeling.

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Fig. 3. Confocal microscopy images of giant unilamellar vesicles (GUVs) composed of DOPC (= dioleoyl-sn-glycero-3-phosphocholine) (upper panels) or DOPC/DOPS (= 1,2-dioleoyl-sn-glycero-3-phospho-L-serine) (4:1) (lower panels), treated with DPA-R8-FL (1.5 µM) in the presence of metal ions for 1 h (1.5 µM). Scale bars, 10 µm. The panels under these images represent the intensity profiles of fluorescein signals along the lines on the confocal images. High fluorescence intensity in the areas corresponding to GUV membranes indicated membrane accumulation of DPA-R8-FL.

Incremental net negative charges of the membranes led to increased membrane interactions of DPA-R8-FL/metal complexes, as previously suggested for both DPA16 and R8.23 When DPA-R8-FL was treated with GUVs comprised of DOPC and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) (4:1), which is a negatively charged phospholipid, significant DPA-R8-FL signals were observed in the presence of the metals (Fig. 3, lower panels, and Fig. S2). No significant membrane labeling was observed in either 100% DOPC or 12


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DOPC/DOPS (4:1) GUVs by R8-FL, which lacked the DPA moiety (Fig. 1c and Fig. S2). These results clearly demonstrated that DPA targets phospholipids and enhances R8 binding to the membranes with the help of these metals, especially Ni(II).

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To confirm if the DPA unit attached to R8 forms complexes with

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metals, an isothermal titration calorimetry (ITC) study was conducted. Metal ions

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were instilled into DPA-R8 [Ac–K(DPA)GRRRRRRRR–amide] or R8 [Ac–

16


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RRRRRRRR–amide] at 25°C (Table 1 and Fig. 4). Whereas metal addition to R8 did not yield significant increases or decreases in temperature (data not shown),

Fig. 4. Isothermal titration calorimetry (ITC) measurement of the DPA-R8 complex with (a) Ni(II), (b) Co(II), and (c) Zn(II). The peptide concentration was 25.2 µM for Co(II) or Zn(II) titration and 75.6 µM for Ni(II) titration. Note the differences in the time scales between (a), (b), and (c).

temperature increases were observed after the addition of metal to DPA-R8 solution, suggesting that complex formation occurred with metals. The resulting binding isotherms were best fitted to a 1:1 association model. The dissociation constant (Kd) between DPA and a metal is in the order of Co(II) < Zn(II)

Metal Complexes that Promote ... - ACS Publications

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