Importance of Net Hydrophobicity in the Cellular Uptake of All

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Importance of net hydrophobicity in the cellular uptake of all-hydrocarbon stapled peptides Koki Sakagami, Toshihiro Masuda, Kenichi Kawano, and Shiroh Futaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01130 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 9, 2018

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Molecular Pharmaceutics

Importance of net hydrophobicity in the cellular uptake of all-hydrocarbon stapled peptides

Koki Sakagami, Toshihiro Masuda, Kenichi Kawano and Shiroh Futaki* Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

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

Keywords stapled

peptide,

structurally

constrained

peptide,

cellular

macropinocytosis

1 ACS Paragon Plus Environment

uptake,

endocytosis,

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

All-hydrocarbon stapled peptides are a promising class of protein–protein interaction regulators; their potential therapeutic benefit arises because they have high binding affinity and specificity to intracellular molecules. The cell permeation efficacy of these peptides is a critical determinant of their bioactivity. However, the factors that determine their cellular uptake remain an active area of research. In this study, we evaluated the effect of stapled (or cross-linked) formation on the cellular uptake of six known all-hydrocarbon stapled peptides. We found that the cellular uptake of unstapled peptides (i.e., those bearing olefinic nonnatural amino acids that are not subjected to olefin metathesis) was higher than that for the corresponding stapled peptides. Additionally, the insertion of these olefinic non-natural amino acids into peptide sequences significantly increased their cellular uptake. According to the high-performance liquid chromatography (HPLC) retention times, the overall hydrophobicity of unstapled peptides was greater than that of stapled peptides, followed by the original peptides without olefinic non-natural amino acids. There was not a close correlation between helical content and the cellular uptake of these peptides. Therefore, the increase in overall hydrophobicity resulting from the introduction of non-natural amino acids, rather than the structural stabilization resulting from staple formation, is the key driver promoting cellular uptake. Macropinocytosis, a form of fluid-phase endocytosis, was involved within the cellular uptake of all six peptides.


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Introduction Protein–protein interactions (PPIs) mediate various biological functions and regulatory pathways, including those related to several diseases and pathological conditions. Considerable research has focused on the development of PPI modulators as drug candidates. Protein surfaces involved in PPIs are often large and flat, consisting of hydrophobic amino acids. Molecules that cover large contact surface areas should be preferable to the modulation of PPIs.1 Peptides have ideal features as PPI modulators, as they can attain specific conformations that can cover contact surface areas. They are also capable of binding to their targets with high specificity and potency, resulting in reduced off-target effects and improved safety.2 Despite these advantages, the pharmacological applications of peptides are limited due to their low oral bioavailability, poor metabolic stability, and reduced membrane permeability.3 To overcome these problems and to increase their affinity to the target sites, methods have been developed to employ peptides bearing constrained structures that mimic helical and turn structures.4-11 One of the most promising and frequently employed strategies is to use tethers to stabilize helical peptide structures, including ‘all-hydrocarbon stapled peptides’.12 These all-hydrocarbon stapled peptides are typically constructed by inserting a few olefinterminated, non-natural α,α-disubstituted amino acids. For example, either (S)-2-(4pentenyl)alanine or (R)-2-(7-octenyl)alanine (S5 and R8 respectively) can be inserted into potential helical peptide segments at the (i, i+3), (i, i+4), or (i, i+7) positions (Figure 1). Ruthenium-catalyzed olefin metathesis yields such stapled peptides.13 These stapled structures significantly stabilize the helical peptide while also improving resistance to proteolysis and membrane permeability.14 The successful refinement of peptide sequences and stapling position previously yielded effective inhibitors of tumor-related PPIs in cells, demonstrating their therapeutic promise.15,16 3 ACS Paragon Plus Environment


However, stapled peptides should have cell permeability if they are to be used to modulate intracellular PPIs. Peptides are unable to meet their intracellular target proteins and exert an inhibition effect when the peptide is not delivered into cells, even if the stapled peptide strongly inhibits PPIs in vitro. Thus, efforts have focused on identifying ways to increase the cell permeability of the peptides.15 Constrained, stapled structures may be favorable for membrane translocation from an entropy point of view, assuming that peptides preferentially form helical structures. Alternatively, it has been proposed that a major driver of cellular permeability is the introduction of an all-hydrocarbon staple at the amphipathic boundary to extend the hydrophobic surface. They also proposed that hydrophobicity and helicity are also important factors in cellular uptake.17 Tian et al. examined the effect of cross-linkers in stabilizing helical structures and promoting cellular uptake.18 They concluded that the physicochemical properties of these cross-linkers strongly influence the permeability of peptide into cells, where hydrophobicity is more important than the helical content of amphipathic sequences. These studies led us to hypothesize that, if hydrophobicity was the dominant factor that allows all-hydrocarbon stapled peptides to be internalized into cells, such hydrophobicity could be spurred by the introduction of α−methyl-substituted amino acids rather than constrained structures. Additionally, endocytosis (more specifically, macropinocytosis) has been implicated in the cellular uptake of all-hydrocarbon stapled peptides.19,20 This raises the question of whether all-hydrocarbon stapled peptides that have different physicochemical properties are internalized via the same method. We thus studied how cells uptake all-hydrocarbon stapled peptides and compared this mechanism to that of the corresponding unstapled peptides (those bearing olefinic nonnatural amino acids that were not subjected to olefin metathesis). Six peptides with different physicochemical properties (including amino acid length, hydrophobicity, amphiphilicity, and helicity) were selected to study the impact of these properties on cellular uptake (Table 1).20-25


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We found that unstapled peptides had a higher cellular uptake than stapled peptides for all six peptides. There was no strong correlation between helicity and cellular uptake, confirming that hydrophobicity is the dominant factor determining cellular uptake for the peptides studied. We additionally confirmed that macropinocytosis is involved in the cellular uptake of all six peptides.



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Materials and Methods Peptide synthesis. Peptides were synthesized using Fmoc-solid-phase synthesis on a rink amide resin, as reported previously.20-26 Peptides with non-natural, α,α-disubstituted amino acids bearing olefin tethers were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) chemistry using a 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate

(HCTU)/1-hydroxybenzotriazole

(HOBt)/diisopropylethylamine

(DIEA) coupling system. We used a coupling time of 120 min for the olefin-bearing nonnatural amino acids, (S)-N-Fmoc-2-(4-pentenyl)alanine and (R)-N-Fmoc-2-(7-octenyl)alanine (S5 and R8 respectively) (Sigma-Aldrich). Ring closing metathesis was performed using 20 mol (%) of a Hoveyda–Grubbs second-generation catalyst (Sigma-Aldrich) in dichloroethane (DCE) for 2 h at 50ºC under argon.27,28 The reaction solution was then drained for 15 min, and the resin washed with DCE and dimethylformamide (DMF). Fluorescence labeling was performed using a fluorescein isothiocyanate (FITC) 5-isomer and γ-aminobutyric acid (GABA) as a spacer.29 N-terminal acetylation was performed using acetic anhydride in the presence of DIEA.30 Deprotection and detachment from the resin were performed using trifluoroacetic acid (TFA)–ethanedithiol (EDT) (95:5) at 25°C for 3 h, followed by purification via reverse phase high-performance liquid chromatography (RP-HPLC). The peptide mass was confirmed by matrix-assisted laser desorption ionization time-of flight mass spectroscopy (MALDI-TOFMS) (Table S1 in Supporting Information). Cell culture. HeLa cells (human cervical epitheloid carcinoma, Riken BRC Cell Bank) were cultured in an α-minimum essential medium (α-MEM) supplemented with 10% (v/v) heat-inactivated bovine serum (BS) [α-MEM(+)]. Cells were maintained at 37°C in a humidified 5% CO2 incubator. Flow Cytometry. HeLa cells (8.0 × 105) in α-MEM(+) were cultured in 24-well microplates (Iwaki) overnight at 37ºC. Cells were pre-incubated for 30 min in the presence or 6 ACS Paragon Plus Environment

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absence of endocytosis inhibitors in serum-free MEM [α-MEM(–)]. After pre-incubation, the cells were treated with FITC-labeled stapled or unstapled peptides (1 µM each) in α-MEM(–) for 1 h at 37ºC. After washing with phosphate-buffered saline (PBS), cells were incubated with 0.01% trypsin in PBS for 5 min at 37ºC. Cells were then centrifuged at 800 × g for 5 min, washed twice with PBS, and subjected to flow cytometry analysis using an Attune NxT Flow Cytometer (Thermo Fisher). Each sample was analyzed over 10000 events. HPLC. To evaluate the relative hydrophobicity of the peptides, the retention times of acetylated peptides were recorded on a Cosmosil 5C4 column (4.6 × 150 mm) using a linear gradient of 5–95% acetonitrile containing 0.1% TFA in 0.1% aqueous TFA over 40 min (detection at 220 nm). Endocytosis inhibitor assay. HeLa cells (8.0 × 105) in α-MEM(+) were cultured in 24well microplates until they were 80−90% confluent. The cells were pre-incubated for 30 min in α-MEM(–) in the presence or absence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) (100 µM), wortmannin (1 µM), pitstop2 (30 µM), and nystatin (50 µM). After pre-incubation, cells were washed with PBS and were then treated with 1 µM of FITC-labeled stapled peptides for 1 h at 37ºC in α-MEM(–) in the presence or absence of these pharmacological inhibitors at the same concentrations. Cells were subjected to flow cytometry analysis after washing with PBS. To examine whether stapled peptides were internalized via endocytosis, HeLa cells were pre-incubated at 4ºC for 30 min, followed by a 30-min incubation with FITC-labeled stapled peptides. After cells were washed with PBS, the cellular localization and cellular uptake amount of the peptides were analyzed using a confocal laser scanning microscope (CLSM; FV1000, Olympus) and flow cytometry. Peptide cellular localization. HeLa cells in α-MEM(+) were cultured in 35-mm glassbottomed dishes until they were 80−90% confluent. The medium was replaced with fresh αMEM(–) containing FITC-labeled peptides after washing with α-MEM(–). Cells were then



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incubated at 37ºC, and the peptide cellular localization was analyzed by CLSM. The peptide concentration and incubation period were determined via previously published procedures; specifically, ATSP6935 (5 µM, 4 h), SAHp53 (2 µM, 2 h), SAHB (2 µM, 2 h), STAD (2 µM, 1 h), WAHM (5 µM, 6 h), and SAHM (5 µM, 4 h). Dextran uptake. HeLa cells in α-MEM(+) were cultured in 35-mm glass-bottomed dishes until they were 80−90% confluent. The cells were cultured in α-MEM(–) for 18 hours after washing with α-MEM(–). To analyze the amount of dextran taken up by the cells, cells were incubated with 1 mg/mL FITC-dextran (70 kDa) in the presence of 5 µM acetylated peptides in α-MEM(–) for 15 min at 37°C. Cells were then collected and washed with PBS and subjected to flow cytometry analysis. To demonstrate the cellular localization of peptides and dextran, cells were incubated with 500 µg/mL of dextran labeled with tetramethylrhodamine (TMR; Invitrogen) in the presence of FITC-labeled peptides in α-MEM(–) at 37 °C; cells were then analyzed using CLSM. We used peptide concentrations and incubation times as per previous studies; this included ATSP6935 (5 µM, 4 h), SAHp53 (2 µM, 2 h), SAHB (2 µM, 2 h), STAD (2 µM, 1 h), WAHM (5 µM, 6 h), and SAHM (5 µM, 4 h). Large Unilamellar Vesicles (LUVs). A lipid film was formed by rotary evaporation of 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

(POPC)

in

chloroform

for

circular

dichroism (CD) measurements and tryptophan binding assays.31 The lipid film was then hydrated with PBS after vacuum-drying overnight. The suspension was subjected to three freeze–thaw cycles, and LUVs were generated through two 0.1 µm Nucleopore polycarbonate filters (100 nm pore size) using a LiposoFast extruder system (AVESTIN) to give LUVs (diameter = 100 nm). The concentration of LUVs was determined using a LabAssay Phospholipid kit (Wako). Circular Dichroism. CD spectra were recorded using a Jasco 820 UV-VIS spectropolarimeter. Spectra were obtained over a wavelength range of 200–260 nm with a spectral


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bandwidth of 1 nm, a time response of 0.5 s, a scan speed of 50 nm/min, and a step resolution of 0.5 nm. Each spectrum was expressed over an average of five spectra. Spectra were measured for a 20-µM peptide solution dissolved in PBS in the presence or absence of 2 mM POPC LUVs (100 nm) at 25°C. The molar ellipticity was expressed per decimal residue. Tryptophan Binding Assay. Binding between peptides and LUVs was assessed based on changes in tryptophan fluorescence upon titration with increasing concentrations of POPC LUVs (100 nm); this was done as reported previously.32 A solution of peptides (1 µM) in 10 mM MES containing 150 mM NaCl (pH 7.4) was titrated with 4 mM of LUVs in 10 mM MES containing 150 mM NaCl (pH 7.4) to a total lipid concentration of 600 µM. The intensity of tryptophan fluorescence at 325 nm was monitored from 310 to 380 nm at an excitation of 280 nm (Shimadzu RF-5300 fluorescence spectrometer). The resultant data were fit to a Langmuir-type isotherm using the following equation (KaleidaGraph 4.1): ‫ܨ = ܨ‬௠௔௫

[௅] [௅]భൗ ା[௅]

,

మ

where F is the fluorescence intensity at a given added lipid concentration, Fmax is the saturating intensity at high lipid concentrations, [L] is the lipid concentration, and [L]1/2 is the lipid concentration that corresponds to binding half-saturation at F=Fmax/2.33 Giant Unilamellar Vesicles (GUVs). GUVs were prepared by hydrating hybrid films of agarose and lipids as reported previously.34,35 Briefly, a 1% agarose solution in Milli-Q water (20 µL) was spread onto a 12-mm glass-bottomed dish (Iwaki) and incubated at 40°C for 1 h. A solution of POPC in chloroform (2 mg/mL) containing 0.3% (w/v) rhodamine-1,2dioleoyl-sn-glycero-3-phosphoethanolamine (rhodamine-PE) was overlaid onto the agarose and dried under vacuum for 30 min. A 0.1-M sucrose solution in PBS (150 µL) was then applied to the glass-bottomed dish as an inner GUVs buffer; this solution was then incubated at 25°C for 3 h in a dark environment. The supernatant was then gently removed, followed by the addition of a 0.1-M glucose solution in PBS (150 µL) as the outer buffer for the GUVs. A 9 ACS Paragon Plus Environment


FITC-labeled peptide solution in PBS was then added to the GUVs suspension. GUVs were then analyzed by CLSM after incubation at room temperature for 15 min.


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Results and Discussion Peptides. To evaluate the effect of structural constraints (created upon staple formation) on cellular uptake, we selected six all-hydrocarbon stapled peptides (ATSP6935, SAHp53, SAHB, STAD, WAHM, and SAHM) (Table 1). We selected these peptides as they all have cellular bioactivity and are actually delivered into cytosol by crossing the cellular membrane (except for ATSP6935; see Footnote 1). [Footnote 1: Previously, the structure of ATSP6935 was further modified by substituting leucine-to-cyclobutylalanine (Cba) and adding two alanines to the C-terminus to yield ATSP7041. The applicability of this form as a cancer therapy was studied in depth.21 However, except for the non-natural α,α-disubstituted amino acids that are involved in staple cross-link formation (e.g., S5 and R8), we selected peptides composed of natural, proteinogenic amino acids. This was done to simplify our analysis of cellular uptake methods based on physicochemical properties. Thus, we chose to study ATSP6935 rather than ATSP7041.] Additionally, the differences in chain length, hydrophobicity, amphiphilicity, net charge, and helical content between these peptides may provide information regarding relationships among the physicochemical properties, cellular uptake efficacy, and pathways by which uptake occurs. We labeled the N-termini of peptides using either fluorescein via a spacer (such as GABA) or acetylation and amidated the Ctermini to eliminate possible charge effects to compare peptide cellular uptake efficacy. Peptide chains were constructed by Fmoc-solid-phase peptide synthesis (SPPS) on a Rink amide resin. Both S5 and R8 were incorporated into the peptide sequences. After peptide chain construction and N-terminal modification, all-hydrocarbon staples were formed by ruthenium-catalyzed olefin metathesis. Unstapled peptides bearing intact S5 and R8 that were not subjected to olefin metathesis were also prepared to evaluate how staples impact peptide cytosolic distribution and cellular uptake efficiency.



Cellular uptake of stapled and unstapled peptides. To evaluate the contribution of stapled, cross-linked structures to peptide cellular uptake and cytosolic translocation, the cellular uptake of all-hydrocarbon stapled and unstapled peptides was analyzed by confocal laser scanning microscopy (CLSM) after treating the cells with FITC-labeled peptides (Figure 2A). Significant FITC-signals were observed in cells treated with peptides with stapled structures, as reported previously. Such punctate signals may suggest that peptides are trapped in endosomes. Diffuse cellular staining suggests that the peptides are localized within the cytosol, which is important for their intracellular activity (i.e., PPI inhibition; Figure 2A, upper panels). Unstapled peptides also yielded an intracellular distribution similar to that of their corresponding stapled peptides, suggesting that peptide cytosolic translocation occurs even without stapled structures (Figure 2A, lower panels). We further analyzed the amount of peptide cellular uptake using flow cytometry. Of particular interest, the amount of cellular uptake for unstapled peptides was 10–40% greater than that for stapled peptides, regardless of their other physicochemical properties (Figure 2B). This suggests that stapled structures did not affect the cytosolic distribution of the peptides. We then examined the effect of helical peptide structures on their cellular uptake efficacy. Outer leaflet of cell membranes is predominantly composed of neutral lipid including phosphatidylcholine.36 We thus employed large unilamellar vesicles (LUVs) consisting of 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a model of cell membranes and determined the circular dichroism (CD) spectra of stapled and unstapled N-terminally acetylated peptides in the presence of POPC LUVs in phosphate-buffered saline (PBS) (pH 7.4). The CD spectra of these peptides were suggestive of helical structures (Table 2 and Figure S1 in Supporting Information). Molecular ellipticity at 222 nm ([θ]222) has been used as a measure of helical content.37 The helical content of stapled peptides was greater than that


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of unstapled peptides for ATSP6935, SAHp53, SAHB, and WAHM. Additionally, STAD and SAHM showed slightly greater helicity for unstapled peptides versus stapled peptides. Therefore, the amount of cellular uptake for each peptide did not correspond to the helical content in the presence of liposomes. Additionally, we analyzed the structure of these peptides in an aqueous solution in the absence of LUVs. Although the peptide helical content was generally slightly smaller than it was in the presence of LUVs, we observed varying effects of cross-linking on helical tendencies (Table 3 and Figure S1 in Supporting Information). Although increases in helical content were observed upon cross-link formation for SAHp53, SAHB, and STAD, the unstapled peptides ATSP6935, WAHM, and SAHM had higher helicity. These trends are not in accord with the presence of POPC LUVs (Footnote 2). [Footnote 2: The possible hydrophobic interaction between R8 and S5 (for ATSP6935) and between S5 (for WAHM and SAHM) in PBS may have contributed to this increase in helicity. The possible aggregation of more than one unstapled molecule in aqueous solution may also result in higher helicity. In the presence of POPC LUVs, interactions between peptides with membranes may become a dominant factor driving the formation of helical structures. These factors could result in differences between how staple structures impact peptide formation in the absence and presence of membranes.] Ruthenium-catalyzed olefin metathesis results in the elimination of ethylene. Stapled peptides may thus be less hydrophobic than unstapled ones. This was confirmed by the HPLC retention times collected using a C4 column, where each unstapled peptide had a longer retention time (i.e., a higher hydrophobicity) than the corresponding stapled peptide (Figure 2C). It has also been reported that hydrophobicity is the most important factor determining the internalization efficacy of stapled peptides into cells.17 We thus plotted relative peptide cellular uptake (shown in Figure 2B) against HPLC retention time (Figure 2C). These data demonstrate a relationship between peptide cellular uptake and HPLC retention time



(Spearman’s correlation ρ= 0.008), suggesting that hydrophobicity rather than rigid structure is important for promoting the cellular uptake of peptides.

The importance of hydrophobicity in the cellular uptake of stapled peptides. The greater hydrophobicity of unstapled peptides versus stapled peptides may result in increased interactions with membranes. We investigated this through a tryptophan binding assay using ATSP6935. ATSP6935 contains tryptophan in the middle of its structure (between stapled S5 and R8). Binding of the peptide to the lipid should result in an increase in fluorescence intensity along with a blue shift to 325 nm (excitation at 280 nm) as reported previously.32 Titration with POPC LUVs yielded a rapid increase in fluorescence intensity from the unstapled peptide versus the stapled peptide. Although this increase in fluorescence was almost saturated at a lipid/peptide (L/P) ratio of ~300 in the case of unstapled ATSP6935 (uns-ATSP6935), a ratio of ~600 was necessary for binding saturation between the stapled ATSP6935 (sta-ATSP6935) and LUVs (Figure 3A). ATSP3848, which was used as the starting peptide in the design of ATSP6935 that lacks R8 and S5 in its sequence (Table 4), demonstrated significantly lower cellular uptake than sta-ATSP6935 (Figure 3B). Its HPLC retention time (Rt) under the same conditions as those depicted in Figure 2C was 20.2 min (Figure 3C), suggesting that its hydrophobicity is lower than sta-ATSP6935. These results suggest that the hydrophobicity of R8 and S5 increases the binding of these peptides to membranes and thus their eventual cellular uptake. However, the CD spectrum of ATSP3848 in 50% trifluoroethanol (TFE, which mimics the conditions under which peptides cross the membrane core), suggests the deficiency of a helical, constrained structure (Figure 3D). The importance of hydrophobicity rather than constrained structures in determining the internalization of stapled peptides was confirmed using an analog of ATSP6935, where R8 and S5 were substituted for α-aminoisobutyric acid (Aib) (Aib–ATSP6935) (Table 4). Aib has 14 ACS Paragon Plus Environment

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a pronounced tendency to induce α-helical conformation.38 The CD spectrum of Aib– ATSP6935 was similar to that of sta- and uns-ATSP6935, confirming the impact of Aibs in yielding an enhanced helical structure in 50% TFE (Figure 3D) and in PBS (Figure S2 in Supporting Information). HPLC analysis of Aib–ATSP6935 (Rt = 24.0 min) suggests that its hydrophobicity is between that of sta-ATSP6935 (Rt = 28.1 min) and ATSP3848 (Rt = 20.2 min) (Figure 3C). The cellular uptake of Aib–ATSP6935 was considerably lower than that of sta-ATSP6935 (Figure 3B and C). In addition to hydrophobicity, previous studies have suggested that amphiphilicity is also important to the internalization of stapled peptides.17 To confirm this, we synthesized a scrambled peptide of ATSP6935 (scr-ATSP6935, Table 4). This peptide was designed such that hydrophobic amino acids in ATSP6935 are less clustered on one side of the helix (and are thus less amphiphilic), while retaining the positions of R8 and S5 (Figure S3A in Supporting Information). The peptide hydrophobicity was determined by HPLC retention times, as shown in Figure 3C; these times can be ordered as ATSP3848 < Aib–ATSP6935

Importance of Net Hydrophobicity in the Cellular Uptake of All

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