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Enantiomer-Specific Bioactivities of Peptidomimetic Analogues of Mastoparan and Mitoparan: Characterization of Inverso Mastoparan as a Highly Efficient Cell Penetrating Peptide Sarah Jones* and John Howl Research Institute in Healthcare Science, School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton, WV1 1LY, United Kingdom ABSTRACT: Retro-inverso transformation has commonly been employed as a strategy both for the synthesis of proteolytically stable peptide analogues and for the detailed investigation of structure activity relationships. Herein, we adopted a similar strategy to probe the structure activity relationships of two biologically active tetradecapeptides. Analogues of the α-helical mastoparan, and the highly potent apoptogenic analogue mitoparan, were synthesized using D-amino acids assembled in both endogenous (inverso) and reverse (retro-inverso) orientations. For a more comprehensive comparison, our studies also included the retro L-enantiomer of both peptides. Contrary to expectation, comparative investigations of cytotoxicity, mast cell degranulation, and cellular penetration demonstrated that, while retro-inverso transformation abrogated the associated biological activities of these helical peptides, inverso homologues retained their bioactivities. Moreover, inverso mastoparan demonstrated the highest translocation efficacy of all analogues with much improved uptake kinetics compared to other cell penetrating peptides (CPPs) including the commonly employed inert vectors penetratin and tat. Data presented herein thus propound the utility of inverso mastoparan as a highly efficient peptide vector. Furthermore, correlation analysis of plasma membrane translocation and intracellular uptake efficacy further supports a two-compartment model of CPP import whereby the intracellular accumulation of polycationic peptides is dependent upon both the efficiency of transport into the cell and their subsequent accretion at distinct subcellular loci.

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contains α-aminoisobutyric acid (Aib), a known helix promoter,14 substituted for Ala at position 10. More recently, a study of the crystal structure of related MP from Polistes jadwagae15 has provided evidence that direct interactions between MP analogues are sufficient to promote helical formation in the absence of biological membranes. Studies of the structure activity relationships of MP analogues identified MitP as a more potent secretagogue and cytotoxic agent.13 Moreover, we have since determined that MitP very efficiently translocates the plasma membrane to localize with mitochondria.16 At this locus, MitP interacts with at least one protein target, the voltage-dependent anionic channel, to initiate the release of cytochrome c and is clearly capable of initiating intrinsic apoptotic events in cultured cells. Thus, the effective targeting of MitP and other MP analogues to diseased cells could provide a very capable therapeutic strategy. The MP sequence has proven value in the design of cell penetrating peptide (CPP) vectors including transportan17 and deletion homologues such as transportan 10.18 Our recent studies16 have indicated that N-terminal extension of MitP with

INTRODUCTION Since the first description of mastoparan1 (MP), a tetradecapeptide (INLKALAALAKKIL) isolated from the venom of Vespula lewisii, many additional analogues of this peptide, both natural and synthetic, have been reported and reviewed elsewhere.2−4 Moreover, MP analogues have proven utility as differential secretagogues,5 components of cell penetrating peptide vectors2,6 and modulators of numerous cellular and pathological processes, some of which may be a downstream consequence of the receptor-independent activation of heterotrimeric G proteins (reviewed in refs 3,4). Additionally, MP and chimeric analogues including galparan2,7 can directly modulate the activities of other membrane-bound protein targets that include Ca2+-ATPases,8 phospholipase D2,9 and the Na+,K+-ATPase of rat brain frontal cortex.7 A major determinant of the biological activity of MP analogues is the formation of an amphiphilic helix.10,11 Moreover, this secondary structural motif is observed when MP and related peptides are bound to either membranes10 or the carboxyl termini of the α-subunits of heterotrimeric G proteins.12 Based upon these and other observations (reviewed ref 4), we utilized a helical wheel projection in the design of mitoparan13 (MitP, [Lys,5,8Aib10]MP), a peptide with enhanced amphiphilicity. MitP presents two additional lysyl side chains in the cationic face and © 2011 American Chemical Society

Received: June 6, 2011 Revised: December 1, 2011 Published: December 11, 2011 47

dx.doi.org/10.1021/bc2002924 | Bioconjugate Chem. 2012, 23, 47−56

Bioconjugate Chemistry

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serum for 24 h prior to assay. Cells were treated with peptides for 4 h at 37 °C and further incubated with MTT (0.5 mg/mL) for 3 h at 37 °C. Medium was aspirated and the insoluble formazan product was solubilized with DMSO. MTT conversion was determined by colorimetric analysis at 540 nm. Graphical representations of changes in cell viability and LD50 values were calculated using GraphPad Prism 5 software. Quantitative Analyses of Peptide Translocation. The method used to quantitatively assess the uptake of fluorescent peptides was based upon that previously described by Langel’s group.24 This method employed trypsin to remove cell surfaceassociated peptide prior to lysis. U373 MG cells were transferred to 6-well plates and grown to 80% confluence. Immediately prior to the addition of fluorescent MP and MitP analogues, cells were washed with and transferred into 1 mL of phenol red-free DMEM. Cells were incubated with fluorescent peptides at a final concentration of 5 μM for 1 h at 37 °C in a humidified atmosphere of 5% CO2. Cells were then washed four times, detached with 300 μL of 1% (w/v) trypsin at 37 °C, collected by centrifugation and lysed in 300 μL 0.1 M NaOH for two hours on ice. 250 μL of each sample cell lysate were transferred to a black 96-well plate, and analyzed using a ThermoFischer Scientific Fluoroskan Ascent FL fluororescence spectrophotometer (λAbs 544 nm/λEm 590 nm). Live Cell Imaging. U373MG cells were transferred to 35 mm Petri dishes with 12 mm glass bases (IWAKI, purchased from Barloworld Scientific Ltd., Stone, UK) and grown to 90% confluence (conditions as described above). Prior to the addition of fluorescent peptides and/or other materials, cells were washed with and transferred into phenol red-free DMEM. During the period of exposure to peptides, cell layers were maintained at 37 °C in a humidified atmosphere of 5% CO2. Immediately prior to observation, cells were then washed gently (8×) and analyzed with a Carl Zeiss LSM510Meta confocal microscope. All quantitative colocalization analyses were performed using the Carl Zeiss quantitative colocalization analysis software, incorporating an interactive scatter plot and data table linked to the images. For mitochondrial colocalization studies, cells were incubated with 200 nM Mitotracker Green FM (Molecular Probes, Invitrogen, Paisley, UK) alongside fluorescent peptides.16 Overlap coefficients after Manders were used to quantify colocalization. Coefficients generated values between 0 and 1. A value of 0 indicated no colocalization, whereas a value of 1 indicated that all pixels were colocalized. Data were collected from an average of 9 regions of interest, between 50 and 200 μm2 and from 3 independent experiments. For lysosomal colocalization studies, cells were incubated with either 75 nM Lysotacker Green DND-26, a generalized lysosomal stain for acid organelles, or 1 μM Lysosensor Green DND-153, a pH-dependent lysosomal stain (pKa 7.5) that is brightly fluorescent at neutral pH (Molecular Probes, Invitrogen, Paisley, UK). Data were collected from 4 entire images and from 3 independent experiments. Colocalization (coloc) coefficients were calculated for the two different fluorophores according to the following equations: C1 = pixels channel 1 coloc/pixels channel 1 total, C2 = pixels channel 2 coloc/pixels channel 2 total and defined as relative number of colocalizing pixels in channel 1 (designated rhodamine) or 2 (designated fluorescein), respectively, as compared to the total number of pixels above threshold. Colocalization coefficients generated values from 0 to 1.0, whereby a value of 1.0 for channel 1 (designated rhodamine) and a value of 0.4 for channel 2 (designated fluorescein) would

targeting peptides can improve both the cell-type selectivity and potency of chimeric peptides. The in vivo utility of MitP and other MP analogues could, however, be compromised by combined activities of both extracellular and intracellular proteases. Moreover, the synthesis of isomeric peptides is a common approach used to both investigate structure activity relationships and to produce peptides that are more resistant to proteolytic degradation.19,20 Thus, the objective of this study was to compare the intracellular translocation and biological properties of homologues of both MitP and MP containing mostly D-amino acids assembled in both normal (inverso) and reverse (retroinverso) orientations.

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EXPERIMENTAL PROCEDURES Materials. N-(9-Fluorenylmethoxy-carbonyl)-D-Isoleucine (Fmoc-D-Ile) was purchased from Sigma-Aldrich (Poole, UK). Other Fmoc-protected amino acids for routine peptide synthesis were purchased from Novabiochem (Beeston, UK). AGTC Bioproducts Ltd. (Hessle, UK) supplied dimethylformamide and dichloromethane for solid-phase peptide synthesis. Culture medium and supplements were purchased from Sigma-Aldrich (Poole, UK), and fetal bovine serum from PAA (Yeovil, UK). Unless otherwise indicated, all other research grade chemicals were purchased from Sigma-Aldrich (Poole, UK). Peptide Synthesis. All peptides utilized in this study were manually synthesized (0.1−0.2 mmol scale) on Rink amide methylbenzhydrylamine (MBHA) resin (Novabiochem, Beeston, UK) employing an N-α-Fmoc protection strategy with O-(6-chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU; AGTC Bioproducts, Hessle, UK) activation. Fluorescent peptides, to be used in confocal live cell imaging and quantitative uptake analyses, were synthesized by amino-terminal acylation of CPP sequences with 6-carboxy-tetramethylrhodamine (Novabiochem, Beeston, UK) as previously described.16 Crude peptides were purified to apparent homogeneity by semipreparative-scale high-performance liquid chromatography, and the predicted masses of all peptides used (average M + H+) were confirmed to an accuracy of ±1 by matrix-assisted laser desorption ionization (MALDI) time-offlight mass spectrometry operated in positive ion mode using α-cyano-4-hydroxycinnamic acid (Sigma) as a matrix.16 Cell Culture. The investigations described herein utilized the cell lines RBL-2H3, a widely studied mast cell model (reviewed in ref 21) and the human glioblastoma-astrocytoma line U373 MG.22 Both lines were routinely maintained in a humidified atmosphere of 5% CO2 at 37 °C in DMEM supplemented with L-glutamine (0.1 mg/mL) 10% (wt/vol) fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL). Secretion Assays. β-Hexoseaminidase, secreted by peptidestimulated RBL 2H3 cells, was assayed in samples of cell medium as previously described.5,13 Five microliter samples were transferred into 96 well plates and incubated with p-nitrophenyl N-acetyl-β-D-glucosamide (20 μL of 1 mM in 0.1 M sodium citrate buffer, pH 4.5) for 1 h at 37 °C. Na2CO3/NaHCO3 buffer (200 μL of 0.1 M, pH 10.5) was then added and β-hexoseaminidase activity determined by colorimetric analyses at 405 nm. Graphical representations and EC50 values were calculated using GraphPad Prism 5 software. Cytotoxicity Assays. Cellular viability was measured by the 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion assay.23 U373MG cells were cultured in 96 well plates and washed and maintained in medium without 48



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indicate that 100% rhodamine pixels colocalized with fluorecein pixels and 40% fluorescein pixels colocalized with rhodamine. Lipid-PAMPA Assay. The parallel artificial membrane permeability assay (PAMPA, Millipore) was used as a non-cell-based assay to assess passive permeability of selected peptides across an artificial membrane. Using a 96-well MultiScreen Permeability plate, the assay measures the ability of compounds to diffuse from a Donor to Acceptor compartment separated by a PVDF membrane pretreated with a lipid-containing organic solvent. Briefly, 4% (w/v) L-α-phosphatidylcholine in dodecane was added at 5 μL/well to donor plates. Within 10 min, 5 μM rhodamine-labeled peptide, dissolved in PBS containing 5% DMSO (v/v), was also added at 150 μL/well to the donor plates. 300 μL PBS containing 5% DMSO was added to each well of the acceptor plates and donor plates placed directly on top. Following 6 h of incubation at room temperature, the contents of the acceptor plates were transferred to a black 96-well plate and analyzed using a ThermoFischer Scientific Fluoroskan Ascent FL fluorescence spectrophotometer (λAbs 544 nm/λEm 590 nm). Apparent permeability (cm/s) was calculated using the following eq:25

Table 1. Peptide Sequences, Abbreviations, and Mass Spectrometric Analysesa

a

L-Amino acids are denoted by capital letters and D-isomers are in lower case. Aib designates the known helix promoter α-aminoisobutyric acid. To enable both quantitative and qualitative uptake analysis, each peptide analogue was N-terminally extended with 6carboxytetramethylrhodamine (TAMRA; TAM). The calculated masses (average M + H+) of all peptides were confirmed to an accuracy of ±1 by MALDI time of flight mass spectrometry (Kratos Kompact Probe operated in positive ion mode).

V [dC r /dt ] Papp = r A ·Cd whereby Vr is the volume of the acceptor well (0.30 cm3), dCr is the change in concentration of the acceptor compartment, dt is the change in time (seconds), A is the area of the filter membrane corrected for porosity (0.24 cm2), and Cd is the concentration of the donor compartment at time 0.

acid side chains are in the same orientation to that of the parent peptide while the carbonyl and amine groups that form the backbone amides are reversed. Such constructs are therefore reputed to retain biological activity owing to the preservation of their side chain orientation, while being resistant to proteolysis.26

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RESULTS Peptide Design. Synthesized in reverse and composed of D-amino acids, retro-inverso transformation ensures that amino

Figure 1. Comparative cytotoxicity of peptides and peptidomimetics. U373MG cells were treated with enantiomeric analogues of MitP (A) and MP (B) for 24 h at the concentrations indicated. Cell viability was measured by MTT conversion and expressed as a percentage of those cells treated with vehicle (medium) alone. Data points are mean ± SEM from 3 experiments performed in quadruplicate. 49



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Table 2. Biological Activities of Enantiomeric Analogues of MP and MitPa Efficacy Indices

Potencies

Peptide

Cytotoxicity

Secretion

Cytotoxicity (LD50)

Secretion (EC50)

Translocation Efficacies

MP iMP rMP riMP MitP iMitP rMitP riMitP

1 0.67 0.44 0.38 1.85 1.66 0.31 0.52

1 1.46 0.25 0.50 4.32 3.18 0.76 0.68

28.67 μM 45.92 μM 95.46 μM 80.40 μM 9.44 μM 13.95 μM >100 μM 67.15 μM

21.54 μM 13.31 μM >100 μM 41.56 μM 7.53 μM 15.30 μM >100 μM >100 μM

15.82 100.0 9.762 7.990 82.21 63.06 14.75 23.23

Efficacy indices express the above basal activities of peptide analogues as a fraction of that of MP determined at a peptide concentration of 30 μM. LD50 values represent the concentration of peptides required to induce 50% cell death of U373MG astrocytoma. EC50 values represent the concentration of peptides required to induce 50% secretion of β-hexoseaminidase from RBL-2H3 cells. Translocation efficacies represent normalized data of mean intracellular fluorescence (minus background) of TAMRA-labelled peptide analogues. a

Figure 2. Comparative secretory activity of enantiomeric analogues. RBL-2H3 cells were treated with analogues of MitP (A) and MP (B) for 15 min at the concentrations indicated. Data points are mean ± SEM from 3 experiments performed in quadruplicate.

an LD50 of 9.44 μM and a cytotoxic efficacy index of 1.85 relative to MP (Table 2). Only a minor reduction in potency and efficacy, to give an LD50 of 13.95 μM and cytotoxic efficacy index of 1.66, was observed for the inverso analogue iMitP (Figure 1A, Table 2). In contrast, retro and retro-inverso transformation dramatically reduced the cytotoxic potencies of rMitP (LD50 > 100 μM) and riMitP (LD50 = 67.15 μM). Thus, the rank order of cytotoxic potencies were MitP > iMitP > riMitP > rMitP. Similarly, cytotoxicity efficacy indices followed the same rank order (Table 2). Though MP has been reported to facilitate mitochondrial permeability transition in cell free systems,27 in living cellular systems the cytotoxic activity of MP is more than likely induced by random membrane perturbations, particularly at higher peptide concentrations. Thus, MP dose-dependently reduced U373MG cell viability with an LD50 of 28.67 μM. Figure 1B and Table 2 show that inverso, retro, and retro-inverso transformation of MP reduced the cytotoxic potencies of the parent compound to give a rank order of potency of MP > iMP > riMP > rMP. Cytotoxicity efficacy indices followed a similar rank order, MP > iMP > rMP > riMP.

Additional peptide homologues of MitP and MP were synthesized in retro (reverse sequence), inverso (all D-amino acids) and retro-inverso (all D-amino acids in reverse sequence) orientations (Table 1). To facilitate quantitative uptake and intracellular localization studies, N-terminal acylation of MP and MitP analogues with 6-carboxytetramethylrhodamine (TAMRA) produced highly fluorescent peptides (Table 1). C-terminally amidated peptides were manually synthesized employing an N-α-Fmoc protection strategy and masses were confirmed by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (Table 1). Comparative Cytotoxicity of Peptides and Peptidomimetics. The highly potent MP analogue MitP translocates plasma membranes to target mitochondria and enhances mitochondria permeability with a subsequent induction of apoptotic events.16 Thus, the cytotoxic effects of retro-inverso, retro, and inverso transformation of MitP were evaluated and MTT conversion used as a measure of cell viability. In accordance with previous reports, MitP, induced a concentration-dependent decrease in U373MG astrocytic tumor cell viability (Figure 1A) to give 50



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Figure 3. Differential intracellular distribution of enantiomeric MitP and MP analogues. U373MG cells were treated with 5 μM TAMRA-labeled peptides with or without 200 nM Mitotracker Green FM for 1 h and viewed by confocal live cell imaging. For MitP analogues, only the inverso analogue TAM-iMitP demonstrated cellular penetration and mitochondrial colocalization (A), whereas TAM-riMitP being restricted here to the outer plasma membrane (B) and TAM-rMitP (C) lacked efficient cell penetrant properties. For MP analogues, TAM-MP (D) appeared to predominantly localize with the plasma membrane, whereas TAM-riMP (E) and TAM-rMP (F) demonstrated minimal staining of the plasma membrane. In contrast when administered at 5 μM, TAM-iMP demonstrated both a high intensity staining of membranes and cytosolic structures (G). Confocal images for B, C, E, and F are presented here merged with images taken under transmission differential interference contrast so as to assist with visualization of subcellular distribution.

Comparative Peptide-Induced Secretion of β-Hexoseaminidase from RBL-2H3 Cells. Secretion of inflammatory mediators from mast cells is a well-defined action of MP and a component of the acute inflammatory response produced by wasp venom.1−4 Thus, secretion of β-hexoseaminidase from the rat basophilic leukemia cell line RBL-2H3, a widely used cellular model for the study of secretory mucosal mast cells,5,28 was used to evaluate the effects of enantiomeric transformation on MP and MitP. We have previously reported13 that MitP demonstrates an enhanced secretory efficacy to that of MP (Table 2). Accordingly, the secretory potency of MitP was enhanced to give an EC50 of 7.53 μM compared to 21.54 μM for MP and a secretory efficacy of 4.32. Similar to the comparative cytotoxic observations for MitP, inverso transformation of MitP produced only minor reductions in secretory potency (15.30 μM) and efficacy (3.18), whereas riMitP and iMitP were biologically inactive (Figure 2A, Table 2). Inverso transformation slightly enhanced the secretory efficacy (1.46) and potency (13.31 μM) of MP, whereas retro and retro-inverso transformation reduced both secretory potency

and efficacy giving a rank order of iMP > MP > riMP > rMP, with rMP being biologically inactive (Figure 2B, Table 2). Comparative Analysis of Qualitative and Quantitative Uptake. The mitochondriotoxic CPP MitP demonstrates an enhanced propensity for cellular penetration compared to wellcharacterized CPP such as penetratin.29 Accordingly, MitP gave a translocation efficacy of 82.21 compared to that of 27.76 for penetratin (Table 2). Given that MitP translocates plasma membranes to colocalize with mitochondria,16 other MitP analogues were evaluated for their cell penetrant ability and degree of mitochondrial colocalization using confocal live cell imaging and quantitative uptake analysis of intracellular fluorescence. Figure 3A shows that following 1 h incubation, only the inverso analogue, TAM-iMitP (5 μM), assumed a distinct colocalization with mitochondrial membranes of U373MG cells (overlap coefficient after Manders = 1.00 ± 0), an observed subcellular localization that is in accordance with (1) the ability of iMitP to induce cell death of U373MG cells with a similar potency and efficacy to that of MitP (Figure 1A, Table 2) and (2) the ability of iMitP to demonstrate a propensity for cellular penetration comparable to that of MitP with an efficacy index of 63.06 51



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this all-D analogue was further characterized by concentrationand time-dependent intracellular uptake studies and compared to the well-characterized CPPs penetratin and tat. Figure 6A demonstrates a concentration-dependent intracellular uptake of iMP into U373MG cells. With an uptake efficacy of 100.00, iMP at a concentration of 5 μM, markedly exceeded that of both penetratin (32.71) and tat (12.24). Moreover, Figure 6B shows a time-dependent intracellular uptake of iMP (5 μM). Data were analyzed using GraphPad Prism 5 software and demonstrated that internalization of iMP, penetratin, and tat occurred with first-order saturable kinetics (F = Fmax × t/t0.5 + t). With a half-life (t0.5) of 10.16 min and a maximum signal intensity (Fmax) of 115.60, iMP demonstrated improved uptake kinetics compared to penetratin (Fmax = 55.20, t0.5 = 27.55) and tat (Fmax = 17.43, t0.5 = 10.30). The parallel artificial membrane permeability assay (PAMPA) was used as a non-cell-based assay to assess passive permeability and direct membrane translocation of selected peptides across an artificial membrane incorporating phosphatidylcholine. iMP (0.1553 × 106 cm/s), MP (0.1165 × 106 cm/s), and MitP (0.0855 × 106 cm/s) demonstrated high apparent permeability values (Papp) compared to those for penetratin (0.0143 × 106 cm/s) and tat (0.0027 × 106 cm/s) (Figure 7). riMP, rMP, rMitP, and riMitP demonstrated no ability for passive diffusion. Pearson correlation analysis (two-tailed, using GraphPad Prism 5) was used to determine whether passive permeability and translocation efficacy in living cellular systems were significantly related. Figure 8 includes Papps of all peptides in this study plotted against their translocation efficacies. When analysis was restricted to analogues of MP and MitP, there was no significant relationship between passive permeability and translocation efficacy (R2 = 0.4316, p = 0.0767). When penetratin and tat were included in the analysis, a positive correlation was observed though an R2 value of 0.4564 indicated that only 45.64% of the variance in Y could be explained by a variation in X (p = 0.0320).

Figure 4. Comparative analysis of translocation efficacies of enantiomeric peptide analogues. U373MG cells were incubated with TAMRA-labeled peptides (5 μM) for 1 h at 37 °C. Normalized data are expressed as mean fluorescence (minus background) ± SEM from 3 experiments performed in quadruplicate.

(Figure 4, Table 2). In contrast and under identical experimental conditions, cellular penetration was minimal for rMit (Figure 4, Table 2). Though quantitative uptake analysis (Figure 4, Table 2) demonstrated that riMitP was moderately penetrant, with a translocation efficacy of 23.23, live confocal cell imaging analysis clearly demonstrates that TAM-riMitP is largely confined and restricted to the plasma membrane (Figure 3B). Confocal analyses further confirmed that MP and its related retro and retro-inverso analogues was poorly cell-penetrant and that TAM-MP strongly colocalized with the plasma membrane (Figure 3D). In contrast, following 1 h incubation, iMP demonstrated an enhanced propensity for cellular penetration, exceeding that for MitP (Figure 4, Table 2). However, at an extracellular concentration of 5 μM, TAM-iMP strongly labeled both the plasma membranes and intracellular organelles to a high signal intensity (Figure 3G). U373MG cells were therefore treated with a reduced peptide concentration of 1 μM TAMiMP. Figure 5A clearly shows that TAM-iMP assumed an intracellular distribution, while adopting a punctuate distribution which partly colocalized with lysosomal structures. Indeed, colocalization coefficient analyses further demonstrated that 62% of intracellular TAM-iMP was coincident with these structures. Moreover, the CPP transportan, a chimeric analogue of MP, has been shown30 with time to accumulate in vesicles of nonacidic pH. TAM-iMP was therefore tested for its ability to colocalize with pH neutral lysosomal structures. Figure 5B shows that, following 1 h, a proportion (43%) of TAM-iMP containing vesicles are within pH neutral lysosomes. More significantly, however, after 24 h TAM-iMP assumed a predominant colocalization (81%) with pH neutral lysosomes (Figure 5C). iMP as a Highly Cell Penetrant Peptide. Given that both quantitative and qualitative uptake analysis identified iMP as a highly penetrative sequence and biologically inactive below 3 μM,

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DISCUSSION The major aim of this study was to evaluate the structure activity relationships of enantiomeric analogues of the tetradecapeptides MP and MitP. As indicated below, our findings do not support the contention that retro-inverso transformation is compatible with the bioactives of relatively short helical peptides. Moreover, we have identified iMP as a highly efficient CPP that could have widespread utility for the design of both inert CPP vectors and biologically active peptides. Inverso Transformation Retains the Biological Activities of MitP and MP. To our surprise, complete retro-inverso transformation of MitP abrogated the biological activities associated with this peptide including the induction of apoptotic events and the secretion of inflammatory mediators. Similarly, retro-inverso transformation severely diminished the propensity of MitP to penetrate the plasma membrane. In contrast, the inverso analogue iMitP demonstrated a strong propensity for cellular penetration, a high degree of mitochondrial colocalization, while retaining the apoptogenic activity of its parent analogue. More significantly, all synthesized D-isomers of both peptides, MitP and MP, retained their associated biological activities. iMP like its parent homologue retained its ability, albeit at higher concentrations, to induce secretion of β-hexosaminidase and reduce cell viability, whereas the retro-inverso analogue was largely inactive. Given that iMitP is composed of all D-amino 52



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Figure 5. Accumulation of the highly cell penetrant all D-amino acid MP analogue iMP in lysosomes. U373MG cells were incubated with 1 μM TAMRA-labeled iMP, 75 nM Lysotacker Green DND-26, a generalized lysosomal stain for acid organelles, or 1 μM Lysosensor Green DND-153, a pH-dependent lysosomal stain (pKa 7.5) specific for pH neutral lysosomal structures and visualized by live confocal cell imaging. Following 1 h incubation, TAM-iMP demonstrated a predominant lysosomal distribution (A) with some TAM-iMP containing vesicles being localized to pH neutral structures (B). Following 24 h incubation, intracellular presence of TAM-iMP was still evident with the peptide being predominantly located within pH neutral lysosomes (C).

significantly, at a concentration of 3 μM or below, iMP is devoid of the biological activity of MP such as mast cell degranulation and perturbation of biological membranes. Moreover, even after 24 h of exposure and at an application dose of 3 μM iMP, both mitochondrial integrity and cellular viability of U373MG cells were unaffected. These data thus propound the utility of iMP as a potential CPP. As previously stated, the CPP transportan is a chimeric analogue of MP and temporal analysis of its intracellular destination has shown that a significant percentage of this peptide accumulates in vesicles of nonacidic pH.30 Data presented herein also demonstrate that, following 1 h incubation, 43% TAM-iMP colocalize with pH neutral lysosomes, and after 24 h incubation, TAM-iMP assumes a predominant colocalization (81%) with pH neutral lysosomes. As a point of conjecture, CPPs that avoid complete entrapment within acidic vesicles are of great utility. If we assume that endocytotic uptake, particularly at low concentrations of CPP, is indeed the predominant mechanism of

acids, it would therefore not be presumptuous to conclude that iMitP is a potential protease-resistant apoptogenic peptide. It is noteworthy that analysis of quantitative uptake data alone could be misleading when evaluating the penetrative features of MP analogues. Trypsinization merely removes membrane-bound peptide. Since MP embeds within the outer cell membrane, trypsinization alone is insufficient to remove this nonpenetrant peptide. This phenomenon is further confounded by the fact that D-amino acid containing peptides are resistant to trypsinzation. This anomaly is typified by the observation that riMitP, while giving a modest uptake efficacy of 23.23, is sequestered at the plasma membrane, an observation that only live confocal cell imaging can determine. iMP as a Highly Efficacious and Protease-Resistant CPP. Of all analogues tested, iMP demonstrated the highest translocation efficacy, exceeding even that of MitP. Moreover, iMP displayed much improved uptake kinetics compared to the commonly employed CPP vectors penetratin and tat. More 53



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Figure 6. iMP as a highly penetrant peptide. (A) Concentration-dependent uptake of iMP as compared to the well-characterized CPP penetratin and tat. U373MG cells were treated with TAMRA-labeled peptides for 1 h at 37 °C at the concentrations indicated. (B) Temporal intracellular accumulation of iMP as compared to penetratin and tat. U373MG cells were incubated at 37 °C with TAMRA-labeled peptides (5 μM) for the times indicated. For both parts A and B, normalized data are expressed as mean fluorescence (minus background) ± SEM from 3 experiments performed in triplicate.

Figure 8. Correlation analysis between passive permeability and translocation efficacy. Data points are riMP (open circle), rMP (closed circle), tat (inverted open triangle), rMitP (inverted closed triangle), MP (open square), riMitP (closed square), penetratin (open diamond), iMitP (closed diamond), MitP (closed triangle), and iMP (star).

Figure 7. Apparent permeability (Papp) of selected peptides using the parallel artificial membrane permeability assay (PAMPA). Data points are mean ± SEM from 5 experiments performed in quintuplicate.

intracellular accumulation, then a pH-dependent lysosomal environment is imperative not only to the longevity of the CPP, but also to its escape and final desired destination. Such a hypothesis was proposed by Raagel et al.30 in a study that investigated the temporal destination of 3 commonly used CPPs, poly Arg, tat, and transportan. Unlike observations with other CPPs investigated, a significant percentage of pH neutral lysosomes contained transportan. Given that acidic lysosomal compartments are detrimental to the future utility of CPP owing to inevitable proteolysis and degradation, Raagel et al.30 proposed that peptide accumulation in pH neutral lysosomes may offer a

feasible “escape route” for transportan-based CPPs. Data presented indicate that iMP not only escapes entrapment within acidic organelles, but being composed of all D-amino acids is also spared from proteolysis, as evidenced by its intracellular distribution 24 h after exogenous application. It is noteworthy that, in part, intracellular uptake of transportan and its deletion analogue TP10 is mediated by caveolin-dependent endocytosis,31 an apt finding since caveolar-mediated endocytosis, entailing 54



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caveolae “pinch off” and the formation of nondegrative endocytic organelles, provides a nonacidic nonproteolytic means for intracellular delivery.32−34 However, data presented herein indicate that iMP is still largely confined to lysosomal structures, albeit those of neutral pH. Thus, a very realistic application for this highly penetrant MP analogue could be its substitution for the MP component of TP10 in an attempt to enhance the penetrative propensity of this deletion analogue of transportan.18 More specifically, iMP could be a useful component of stearylated TP10 analogues that avoid endosomal entrapment and have recently been shown to facilitate the delivery of oligonucleotides35 and plasmids.36 Two-Compartment Model of CPP Accretion. The membrane mimetic PAMPA assay was used as a simple approach to initially distinguish those CPP that were capable of direct membrane translocation from those that required interaction with membrane-associated moieties such as glucoseaminoaglycans.37 Compared to penetratin and even more so tat, MP and its analogues iMP and MitP demonstrated relatively high apparent permeability values (Papp). However, this is perhaps not so surprising owing to their adopted amphipathic α-helical structure within lipid environments.38,39 Furthermore, confocal analyses with live cells (Figure 3D) indicated that MP adopts a predominant plasma membrane localization and is a more true depiction of its cellular destination since MP is widely reported to incorporate into plasma membranes to target heterotrimeric G proteins.10,12 Only a weak correlation was calculated between uptake efficacy in living cellular systems and Papp across an artificial membrane composed of phosphatidyl choline. These data, and a predominance of published observations, indicate that two relatively independent processes determine the accumulation of polycationic CPPs at intracellular sites. First, uptake across the plasma membrane (PM) is dependent upon direct translocation, as is mostly evident with amphipathic α-helical structures and endocytosis, as reported for nonhelical polycationic CPP such as tat, or a combination of both mechanisms. Second, the consequence of CPPs binding to intracellular targets, or accumulating within intracellular organelles, facilitates a more discrete accretion and a distinct intracellular localization. This hypothesis is no more exemplified than in the case of MP, which adopts an amphipathic α-helical structure in membrane environments. Thus, in a pure membrane mimetic environment such as the PAMPA assay, MP demonstrates a relatively large Papp. However, in living cells MP incorporates into the PM but subsequently binds heterotrimeric G proteins and other protein targets on the cytosolic side of the PM. This accounts for observations of MP’s relatively low uptake efficacy into live cells and a final destination mostly confined to the PM as determined by confocal live cell imaging analyses. MitP is clearly capable of direct membrane translocation owing to its relatively high Papp. Moreover, and unlike MP, MitP subsequently redistributes to specifically bind mitochondria.6 Similarly and as a point of conjecture, iMP, demonstrating a high propensity for passive diffusion, translocates the PM to bind and colocalize within membrane-associated cholesterol-rich compartments that progress to form caveosomes of neutral pH. This model of CPP uptake provides an interesting caveat and feasible rationale to explain why CPP-bioconjugates often adopt different uptake mechanisms from that of the CPP vector alone. For example, tat predominantly enters the cell through endocytotic mechanisms, and endosomal entrapment has proven particularly problematic for tat-mediated delivery of oligonucleotides.

However, tat-mediated delivery of nuclear-targeting proapoptogenic peptides has demonstrated a certain degree of success40,41 and therefore suggests that CPP-bioconjugates may facilitate differential protein targeting to that of the CPP and thus escape endosomal entrapment. Moreover, there are numerous examples of successful modulation of intracellular biology using CPP bioconjugates that have escaped endosomal confinement, which can be no more exemplified by the TP10mediated delivery of bioactive peptide cargoes for the successful intracellular modulation of protein kinase C and p42/44 mitogen active protein kinases.42,43 Many more examples are evident but beyond the scope of this paper. In summary, all D-isomers of MP and MitP retain their associated biological activities, while retro-inverso transformation renders these peptides inactive. During the course of this SAR study, iMP demonstrated an enhanced propensity for cellular penetration and thus propounds its utility as either a novel CPP or as a component of pre-existing CPP constructs.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 01902 321130.

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ABBREVIATIONS:

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REFERENCES

CPP, cell penetrating peptide; Fmoc, N-(9-Fluorenylmethoxycarbonyl); MALDI, matrix-assisted laser desorption ionization; MP, mastoparan; iMP, inverso MP; rMP, retro MP; riMP, retro-inverso MP; MitP, mitoparan; iMitP, inverso MitP; rMitP, retro MitP; riMitP, retro-inverso MitP; MTT, 3-(4,5dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide; PAMPA, parallel artificial membrane permeability assay; PM, plasma membrane; TAMRA (TAM), 6-carboxytetramethylrhodamine; TP10, transportan 10

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