|Home | About | Journals | Submit | Contact Us | Français|
Properly designed β3-peptides can inhibit protein•protein interactions, in large part because of their ability to reproduce the side chain presentation of one face of an α-helix.[1–5] This activity, combined with virtually complete resistance to proteolysis[1,6] has led to the prediction that β3-peptides could effectively modulate biological pathways. This promise has been limited by a genuine physical barrier–the plasma membrane–which most β3-peptides cannot traverse. A general strategy to increase cell uptake of β3-peptides would accelerate the widespread application of these molecules as tools or therapeutics. Although polyarginine tags can improve cell uptake of peptides and proteins,[7,8] they can also increase toxicity and diminish protein stability. Moreover, in the context of a β3-peptide dodecamer, a polyarginine tag adds considerable molecular mass.
We reported recently that miniature proteins[10,11] cross the plasma membrane of living mammalian cells and localize in the cytosol when a minimal cationic motif is embedded within their folded structure. Here we report that an analogous approach increases the cell permeability of β3-peptide inhibitors of hDM2 p53 complexation. These molecules we describe thus serve as a starting point for the combinatorial identification of β3-peptides with improved cell uptake and unique biological function. We note that others have previously described cell-penetrating β3-peptides containing multiple arginine side chains[13–15]; however, with the exception of a β3-peptide based on the Tat translocation sequence, these studies all involved β3-peptide homopolymers, none of which possessed biological function.
Our work began with two previously reported 314-helical β-peptides with high affinity for hDM2, β53-8 and β53-12,[5,16] which both upregulate p53 activity when internalized with a commercial transduction reagent (BioPORTERR, Sigma). The 314-helical structure of these molecules presents three distinct faces: an epitope face containing side chains that interact directly with hDM2; a salt bridge face that promotes water solubility and secondary structure; and a structural face that can be varied to fine-tune the helix. We began by asking whether these two molecules would become cell permeable when 2–3 β3-homoarginine (β3hR) residues were embedded within either the “structural” (Strategy 1) or “salt bridge” face (Strategy 2). As negative controls we synthesized variants of β53-3, a well-folded β3-peptide that binds poorly to hDM2, and βNEG, which lacks an hDM2 recognition epitope. As positive controls we synthesized variants of β53-12 and β53-3 containing an N-terminal Arg8 tag (β53-12R8 and β53-3R8, respectively).
We first compared strategies 1 and 2 with respect to their ability to maintain 314-helical structure. The circular dichroism (CD) signature of a 314-helix, in particular the ellipticity minimum at ~ 214 nm, provides a qualitative measure of secondary structure.[2,18] The data indicate that β3-peptides with β3-homoarginine (β3hR) residues embedded on the salt bridge face (β53-12SB2 and β53-12SB3, Strategy 2) retain greater 314-helical character than those modified on the structural face (β53-12R2 and β53-12R3, Strategy 1) (Figures 2a and b). Not surprisingly, β-peptides modified on both epitope faces (β53-12R6-1 and β53-12R6-2) display virtually no 314-helical character in aqueous solution (Figure 2c). Peptides modified with an α-arginine tag (β53-12R8 and β53-3R8) retain structure but are not as 314-helical as the parent peptides (Figure 2d).
Next, we compared the hDM2-affinities of the four sets of β3-peptides using both direct and competition fluorescence polarization assays. We found a direct correlation between 314-helical structure and hDM2 affinity: β3-peptides with β3hR residues embedded within the salt-bridge face (β53-12SB2 and β53-12SB3), which displayed high 314-helix levels by CD (Figure 2b), bound hDM2 well (Kd = 41.7 ± 4.23 and 120 ± 2.00 respectively) (Figure 3b).[4,16] By contrast, β53-8R3, β53-12R3, and β53-12R2 and the highly cationic β53-12R6-1 andβ53-12R6-2, which were all less 314-helical by CD, did not (Figure 3c). The β3-peptides possessing the highest affinity for hDM2 in the direct binding assay (β53-12SB2, β53-12SB3, and β53-12R8) also inhibited the interaction between hDM2 and a p53-derived peptide (p53ADflu) with IC50 values in the low micromolar range, mimicking the activity of the parental β53-12 (Figure 3d). These results indicate that at least in the context of hDM2 recognition, the most successful strategy for maintaining both hDM2 affinity and 314-helical substitutes β3hR for residues on the salt-bridging face, as replacement of other residues leads to a loss in both structure and affinity. Analysis of a computationally-generated model of β53-12 in complex with hDM2 suggests that the “structural” face may be in closer proximity to the hDM2 surface than the “salt-bridge” face, providing one potential explanation for this observation.
We also compared the four sets of β3-peptides with respect to both cell uptake and cytotoxicity (Figure 4). To assess cell uptake we incubated approximately 500,000 HCT116 colon carcinoma cells with 10 μM fluorescently tagged β-peptide for 1 h, washed the cells with PBS and trypsin, and quantified the resulting mean cellular fluorescence (MCF) using flow cytometry. Under these conditions, the uptake of the α-peptide p53ADflu was low as expected (MCF = 21.4 ± 0.69), whereas the validated cell-penetrating peptides (PRR)3Flu and TatFlu were taken up readily (MCF = 190 ± 10.0 and 1850 ± 182 respectively).[12,19] None of the β3-peptides containing β3hR residues substituted on the structural face (strategy 1, β53-12R2, β53-12R3, β53-8R3) were taken up efficiently (MCF < 50), perhaps because they possess only limited 314-helical structure. By contrast, all of the β-peptides with β3hR residues substituted on the salt bridge face (strategy 2, β53-12SB2,β53-12SB3,β53-8SB3) were taken up with efficiencies that equal or exceed that of (PRR)3Flu (MCF = 139 ± 7.48, 142 ± 3.45, and 282 ± 21.4 respectively). Uptake in this system appears to depend more on total charge than side chain identity, as β53-12SB2Flu was taken up as efficiently as the ornithine-containing analog β53-12Flu, but less efficiently than β53-12SB3Flu and β53-3SB3Flu which each contain three arginines on the salt bridge face. β53-12R6-1Flu and β53-126-2Flu were taken up more efficiently than β53-12Flu but were not pursued because of their low affinity for hDM2. As expected, β3-peptides with an α-arginine tag were taken up well (MCF = 10700 ± 1060 for β53-12R8Flu and 2670 ± 52.5 for β53-3R8Flu). Unlike the α-peptide controls, all of the β3-peptides studied demonstrated increased uptake at a longer time-point (4 h), highlighting their resistance to degradation (see Supporting Information). The relative cytotoxicities of cell-permeable β3-peptides were assessed using a commercially available cell viability assay (CellTiter-Blue™, Promega). β53-12 and β53-12SB2 were minimally toxic even at 10 μM, the concentration used to monitor cell uptake (Figure 4), whereas β3-peptides containing 3 or more β3hR reduced HCT116 cell viability below 80% at this concentration. The toxicity of these latter peptides was even more pronounced at a concentration of 30 μM (see Supporting Information).
Although a trypsin wash was included in the flow cytometry protocol to remove cell surface proteins that might sequester β3-peptide, confocal microscropy was used to confirm internalization and evaluate the sub-cellular locale of β3-peptides taken up by HCT116 cells. All of the cell-penetrating peptides that bound hDM2 (β53-12, β53-12SB2, and β53-12SB3) showed punctate intracellular fluorescence that co-localizes with 10 kDa dextran, suggesting that β3-peptide entry proceeds via a form of endocytosis.
Finally, we performed preliminary experiments to assess whether the cell permeability of β3-peptides β53-12SB2, β53-12SB3, and β53-12R8 was sufficient to measurably antagonize p53•hDM2 complexation in live cells. HCT116 cells were treated with each of these three β-peptides for 8 h along with the control β53-12, and the lysates probed for p53, hDM2, and p21 using Western blots. Previous work has shown that p53•hDM2 antagonists stabilize p53 levels and induce expression of the p53 target genes hDM2 and p21. As shown in Figure 6, both β53-12 and β53-12SB2 increase the levels of p53, albeit modestly, and increase the levels of hDM2 and p21 by ~2-fold. Both β53-12SB3 and β53-12R8 were too cytotoxic at these concentrations to achieve reliable results. While it is certain that further experimentation is necessary to identify the optimal balance between β-peptide sequence, hDM2 affinity, cell permeability, and toxicity, these preliminary results indicate that the minimally cationic β-peptides reported herein represent the critical first step towards a class of protease-resistant peptidomimetics that fulfil the promise of β3-peptides as modulations of intracellular biological pathways.
All β3-peptides were synthesized on a 25 μM scale using standard solid-phase Fmoc chemistry, tagged on the N-terminus when necessary, and purified by reverse-phase HPLC as previously described. Fmoc-β3-(L)-amino acids were synthesized from enantiomerically pure α-amino acids via the Arndt-Eistert procedure with the exception of Fmoc-(S)-3-amino-4-(3-trifluoromethylphenyl)butyric acid and Fmoc-(S,S)-trans-2-aminocyclohexane-1-carboxylic acid, which were purchased from AnaSpec, Inc. (San Jose, CA). For characterization of novel β3 peptides used in this study, please see the Supporting Information. Protein overexpression and fluorescence polarization assays were performed as previously described.[4,16] Circular dichroism was performed using a Jasco J-810 spectropolarimeter.
HCT116 cells (American Type Culture Collection, Manassas, VA) were grown in T-75 culture flasks containing McCoy’s 5A Medium supplemented with 10% fetal bovine serum to ~80% confluency, washed twice with 37 °C PBS and incubated with 37 °C PBS-based non-enzymatic cell dissociation solution (10 mL, Chemicon International, Temecula, CA) for 15 minutes. Cells were centrifuged at 500g, resuspended in media, counted by hemocytometer, and diluted to 2200 cells/μL with media. Aliquots of cells (230 μL) were added to fluorescein-labeled peptides (20 μL, 125 μM in PBS). Cells were incubated with peptide for 1–4 hours at 37°C and then washed twice with 37°C PBS (750 μL) to remove extracellular peptide. To ensure removal of any surface-bound peptide, cells were then incubated with 0.25% trypsin (500 μL) at 37°C for 10 minutes, washed once with 4°C media and once with 4°C PBS (750 μL each). Cells were suspended in PBS (500 μL) with propidium iodide (1 μg/mL) and analyzed on a BD FACScan (BD Biosciences, San Jose, CA) equipped with a 488 nm Argon laser. A total of 10,000 events were collected monitoring fluorescein and propidium iodide with 530/30 bandpass and 650 longpass filters, respectively. Events corresponding to cellular debris were removed by gating on forward and side scatter, while dead cells were removed by propidium iodide staining. Geometric means were then calculated from the histogram of fluorescence intensity and corrected for background cellular fluorescence by subtracting the geometric mean of cells treated only with PBS.
HCT116 cells (approximately 105/well) were seeded in 6-well plates containing media (2 mL) and cover glasses. After allowing the cells to adhere for 48 hours, media was removed by aspiration and the cells were washed twice with 37°C PBS. Inverted cover glasses were floated on media containing peptide labeled with fluorescein (200 μL, 20 μM) and/or 10 kDa dextran labeled with AlexaFluor 647 (200 μL, 10 μM, Invitrogen) for 2 hours at 37°C. Cover glasses were then washed with 37°C media and PBS and mounted on microscope slides. Cells were imaged on an LSM 510 Meta (Carl Zeiss MicroImaging, Thornwood, NY) using a 488 nm Ar laser line with a 525/25 nm filter or 633 nm HeNe laser line with a 680/30 nm filter for visualizing fluorescein and AlexaFluor 647, respectively.
HCT116 cells (5000/well) were seeded in 96-well plates and allowed to adhere for at least 24 hours prior to the addition of non-fluorescent β-peptides (solutions prepared in water). After 8 hours incubation with the β-peptides at 37 °C, CellTiter Blue® reagent was added and cells were incubated for another 2 hours at 37 °C. The reduction of rezazurin to resorufin was monitored by fluorescence using an Analyst™ AD 96-384 fluorescence plate reader (LJL Biosystems) using 530/25 excitation and 580/10 emission filters. Cell viability was calculated as the percentage of signal from the β-peptide-treated cells compared to water-treated cells. The mean viability with standard error of three independent experiments, each containing at least three replicates, is reported.