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Design of Acid-activated Antimicrobial Peptide for Tumor Therapy Jingjing Song, Wei Zhang, Ming Kai, Jianbo Chen, Ranran Liang, Xin Zheng, Guolin Li, Bangzhi Zhang, Kairong Wang, Yun Zhang, Zhibin Yang, Jingman Ni, and Rui Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp400052s • Publication Date (Web): 02 Jul 2013 Downloaded from http://pubs.acs.org on July 9, 2013
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Molecular Pharmaceutics
Design of Acid-activated Antimicrobial Peptide for Tumor Therapy
Jingjing Song
†,⊥
, Wei Zhang
†,⊥
, Ming Kai
†⊥
, Jianbo Chen†, Ranran Liang†, Xin
Zheng†, Guolin Li†, Bangzhi Zhang †, Kairong Wang †, Yun Zhang ‡, Zhibin Yang‡, Jingman Ni*,‡, Rui Wang *,†
†
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of
Basic Medical Sciences; Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China ‡
School of Pharmacy, Lanzhou University, 222 South Tianshui Road, Lanzhou
730000, PR China
AUTHOR INFORMATION Corresponding Author * Addressed: School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. Tel.: +86 931 8912567, fax: +86 931 8911255. E-mail addresses:
[email protected] (J.M. Ni),
[email protected] (R.Wang). Author Contributions ⊥
These authors contributed equally to this work.
Notes The authors declare no competing financial interest.
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ABSTRACT Antimicrobial peptides have received increasing attention as potential antitumor drugs due to their new mode of action. However, the systemic toxicity at high concentration always hampers their successful utilization for tumor therapy. Here, we designed a new type of acid-activated antimicrobial peptide AMitP by conjugating antimicrobial peptide MitP to its anionic binding partner MitPE via a disulfide linker. Compared with MitP, AMitP displayed significant antitumor activity at acidic pH and low cytotoxicity at normal pH. The results of MD simulation demonstrate that the changes of structure and membrane binding tendency of AMitP at different pHs played an important role in its pH-dependent antitumor activity. In addition, AMitP showed significant enzymatic stability compared with MitP, suggesting a potential for in vivo application. In short, our work opens a new avenue to develop antimicrobial peptides as potential antitumor drugs with high selectivity.
Kewords: Antimicrobial peptide, Antitumor, Acid-activate, Low toxicity, Enzymatic stability
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INTRODUCTION Currently, long-standing problems in traditional chemotherapy are the severe side effects. However, development of antitumor drugs with high selectivity remains a considerable challenge. Targeted therapy has been developed for an effective approach to improve the therapeutic index of antitumor drugs by using specific cell surface markers.1-4 However, these approaches have achieved limited success against solid tumors, most likely because of heterogeneity in both tumor cell types and cell surface markers.5,6 Thus, it is necessary to develop new therapeutic strategies that exploit other features of tumor physiology that are different from normal tissues. One major difference between many solid tumors and normal tissues is that tumor tissues have a more acidic extracellular environment.7,8 The pH targeting approach has been regarded as a more general strategy than conventional specific tumor cell surface targeting approaches, because the acidic extracellular environment is most common in solid tumors.9 In recent years, several attempts have been made to use the solid tumor acidity to develop new pH-dependent approaches to tumor therapy or drug delivery.10-14 Recently, antimicrobial peptides have received increasing attention as potential candidates not only for antimicrobial agents but also for antitumor drugs.15-19 They preferably attach and insert into tumor cell membranes to form pores, leading to the leakage of intracellular contents and cell death.15 Based on the unique action mechanism, antimicrobial peptides display a certain degree of selectivity to tumor cells and may not encounter chemo-resistance problems compared with the traditional
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chemotherapeutic agents.17 Therefore, antimicrobial peptides might be a major advance in the tumor treatment. However, the damage to normal cells for most antimicrobial peptides at higher concentrations is still one of the major problems that restrict their successful in vivo utilization.20,21 To develop new antimicrobial peptides with high selectivity and low toxicity, we hope to design a new type of antimicrobial peptides that can be activated by tumor acidity; otherwise, it is not active or less active under physiological conditions. To this goal, we proposed a molecular rationale based on selective local unleashing of antimicrobial peptide, as shown in Figure 1. We used antimicrobial peptide mitoparan (MitP) as a framework and synthesized its anionic binding partner peptide (MitPE) by replacing all of lysines with three glutamic acids and two histidines. Subsequently, we synthesized the activatable heterodimer (AMitP) by coupling the anionic peptide MitPE to MitP via a disulfide linker. In this hybrid peptide, MitP can be inactivated at normal pH values due to its binding to MitPE by electrostatic attraction. In contrast, MitP dissociates from its binding partner and recovers its lytic activity when the anionic charges of glutamate residues in MitPE were neutralized at acidic pH values.
Figure 1. Molecular rationale for acid-activiated antimicrobial peptide AMitP.
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MATERIALS AND METHODS Synthesis of Peptides. MitP and MitPE with cysteine to serve as N-terminus were synthesized on an MBHA resin using the standard Fmoc-chemistry-based strategy. All crude peptides were purified and analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) on a C18 column, and then characterized by electrospray ionization mass spectrometry (ESI-MS). To synthesis of AMitP, Cys-MitP was reacted with 10 equiv of 2,2′-dipyridyl disulfide in MeOH/H2O (1:1) overnight at RT to generate the thiolpyridine protected peptide (thiolpyr-Cys-MitP). The reaction mixture was purified by RP-HPLC to remove excess 2,2′-dipyridyl disulfide. The purified thiolpyr-Cys-MitP was reacted with 1.5 equiv of purified Cys-MitPE in MeOH/H2O (1:1) overnight at RT to generate AMitP. The reaction mixture was purified by RP-HPLC, and the purified AMitP was then characterized by ESI-MS. Cell Culture. Hela cell line, KB cell line, BEL-7402 cell line, EJ cell line and MCF-7 cell line were cultured in RPMI1640 medium (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) (Hyclone) in a 5% CO2 humidified atmosphere at 37 oC. Circular Dichroism (CD) Measurements. CD spectra of all peptides (50 µM) in 50% TFE/water (V/V) solution adjusted to pH 7.4 or 5.0 were obtained using Olis DSM 1000 CD spectrophotometer (USA). Measurements were performed in a 2 mm path length cell at room temperature. The following parameters were set: 50 nm/min scanning speed, 0.1 nm step size, 0.5 s response time and 1 nm bandwidth. Each spectrum (195-260 nm) was an average of four scans. The α-helical content was
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estimated by previous method.22 Molecular Dynamics Simulations. All simulations reported in this work were performed by using the GROMACS package.23 Temperature control was achieved by using V-rescale thermostat at 315K with 0.1 ps coupling constant, whereas the semi-isotropic Parrinello-Rahman scheme was used to maintain the pressure at 1 atm with the coupling constant of 2.0 ps. The Particle Mesh Ewald (PME) algorithm method was used for the calculation of electrostatic contributions to energies and forces, with grid length of 0.12 nm.24 A cutoff of 1.2 nm was implemented for the Lennard-Jones and a twin cutoff of 1.2 nm was used for the short range interaction. Bond lengths were constrained via the LINCS algorithm.25 A time step of 2 fs was employed, and neighbor list update per 500 steps. All systems were conducted under periodic boundary conditions and electrostatically neutral environment with an appropriate number of Cl-counterions. The first system contained pure-POPC lipid bilayer composed of 128 lipid molecules (64 lipids for each leaflet). The second system contained the same molecules as the first, except that the lipid bilayer comprised a mixture of negatively charged POPG and POPC lipids at a POPG/POPC lipid ratio of 1:3. The α-helical structures of peptides were built with the C-terminus capped with an amide group. The simulations of AMitP in acid environment were performed by the protonation of Glu and His. To observe the structure change of AMitP in lipid bilayer, we performed a series of 50 ns simulations starting with AMitP that completely inserted into the bilayer POPC. The starting configurations of such peptide-bilayer systems were obtained by using the g_membed tool of the
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GROMACS suite.26 In addition, to explore the binding of AMitP to the surface of the POPG/POPC (1:3) lipid bilayer, a system composed of one peptide, 128 lipids per layer, and 6000 water molecules were built. The AMitP with random coil final structure of last simulation was solvated in the water phase close to one of the monolayers of an equilibrated bilayer. This system was simulated for 100 ns. Cytotoxicity Assays. The antiproliferative effects of all peptides were determined by MTT assay. Tumor cells were seeded at 1 × 104 cells/well in 96-well plate 24 h before treatment. After being washed, cells were treated with 100 µL of serum-free medium adjusted to various pH values containing various concentrations of peptides. After 2 h incubation, 10 µL of MTT (5 mg/ml) was added into each well and incubated for 4 h. The absorbance was determined using microplate reader (Bio-Rad 680) at 570 nm. LDH (lactate dehydrogenase) Leakage Assays. Membrane integrity was measured using the Cytotoxicity Detection KitPLUS (Roche). Hela cells were seeded at 1 × 104 cells/well in 96-well plate 24 h before treatment. After incubation with 100 µL of serum-free medium adjusted to various pH values containing various concentrations of peptides for 1 h, 40 µL of medium was transferred to a 96-well plate and incubated for 15 min with 40 µL of reaction mixture, followed by 20 µL of stop solution. Fluorescence was measured at 490 nm. Untreated cells were defined as no leakage and 100% leakage was defined as total LDH release by lysing cells in 0.2% Triton X-100. Propidium Iodide Uptake Assays. For analysis of the integrity of cell membrane
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after peptide treatment, Hela cells were treated with serum-free medium adjusted to various pH values containing 10 µM of peptides at 37 oC for 30 min. Then, propidium iodide (PI) was added and further incubated for 10 min at room temperature in the dark. The PI uptake was observed with laser confocal scanning microscope. Scanning Electron Microscopy (SEM). Hela cells were treated with serum-free medium adjusted to pH 7.4 or 5.0 containing 10 µM of peptides at 37 oC for 30 min. Cells were washed twice with PBS and fixed with 3% glutaraldehyde at 4 oC. After immediate fixation in glutaraldehyde, all cells were impregnated in 2.5% tannic acid (Sigma) for 2 days. Counter-fixation in 2% osmium tetroxide (Sigma) for 2 h was followed by dehydration in ethanol and drying in a critical point dryer (Ion Tech, Teddington). Cells were coated with gold and analyzed by using a scanning electron microscope (JSM-6380Lv). Hemolysis Assays. Freshly collected mice blood with heparin was centrifuged to remove the buffy coat, and the obtained erythrocytes were washed three times with phosphate buffered saline (PBS), centrifuged for 10 min at 1,000 × g, and resuspended in PBS to 4% (V/V). 100 µL of peptides solution with various concentrations and 100 µL of the erythrocyte suspension were added to the wells of a 96-well plate. PBS and 0.1% Triton X-100 were used as agents for 0 and 100% hemolysis, respectively. Plates were incubated for 1 h at 37 oC and centrifuged at 1,000 × g for 10 min. 100 µL of supernatants were transferred to a 96-well plate, and the release of hemoglobin was determined by using microplate reader at 450 nm. Enzymatic Stability of Peptides. The proteolytic stability of peptides toward
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trypsin and serum was determined by an analytical RP-HPLC assay. To obtain 100% serum, adult Kunming mice were anesthetized, and blood was collected from the carotid with a heparinized syringe. The blood was kept at 4 oC overnight and then centrifuged for 20 min at 1000 g (4 oC). The supernatant was separated and stored at -80 oC. 15 µL of peptide stock solution (10 mM) was added to 285 µL of trypsin solution (10 µg/mL) or 100% serum. Incubations were carried out at 37 oC for 0, 30, 60, and 120 min in triplicate. 40 µL of aliquots were diluted with 80 µL of water-acetonitrile (60:40 v/v) containing 1% TFA, and analyzed by RP-HPLC. The remaining full-length peptide concentration was normalized with respect to the initial concentration. Statistical Analysis. For comparison of two groups, Student’s t tests were used to determine statistical significance. A level of p < 0.05 was considered to be indicative of statistical significance. RESULTS AND DISCUSSION Peptide Design. Mitoparan (MitP), an analogue of antimicrobial peptide mastoparan, could efficiently inhibit the proliferation of tumor cells.27 However, like many other antimicrobial peptides, this peptide at higher concentrations can damage normal cells and consequently narrow its therapeutic index. Thus, improving the specificity to tumor cells is very necessary for the development of MitP as an effective antitumor drug. The electrostatic attraction between anionic components of tumor cell membranes and cationic antimicrobial peptides is believed to play a major role in the strong
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membrane binding and selective membrane disruption of antimicrobial peptides.20,28 Thus, weakening this electrostatic attraction will significantly decrease the cytotoxicity of antimicrobial peptides. In this regard, some studies focused on the development of new targeting approaches to enhance the specificity of antimicrobial peptides by masking the positive charges or replacing lysines/arginines with histidines.29,11,30 However, these approaches always obtained the specificity concomitant with sacrificing the cytotoxicity of antimicrobial peptides. Therefore, we look forward to a new strategy to increase the specificity of antimicrobial peptides while maintaining their cytotoxicity. Herein, we presented a new targeting strategy based on selective local unleashing of antimicrobial peptides, as shown in Figure 1. Lytic activity of antimicrobial peptide MitP can be largely blocked when coupling it to the anionic binding partner MitPE via a disulfide linker. MitPE was obtained by replacing the lysines in MitP with glutamic acids and histidines, where the purpose of introduction of histidines is to avoid excessive electrostatic attraction between MitPE and MitP and to increase the positive charges of MitPE at acidic pH. The reason for introducing histidines at the positions 5 and 8 is that these positions in MitP are less crucial for its lytic activity.27 The constructed heterodimer can be called activatable MitP (AMitP). When at normal pH, AMitP loses the lytic activity because the cationic charges of MitP are shielding by the anionic MitPE. In contrast, AMitP recovers the lytic activity due to the dissociation of MitP from MitPE at acidic pH. pH-Dependent Structure Changes. Despite the great diversity of antimicrobial
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peptides in sequences, most antibacterial peptides share two essential features in that they are cationic and tend to fold into amphiphilic structures.28,31 The cationic feature favors their binding onto the negatively charged membranes, and the amphiphilicity promotes their incorporation into membranes. The overwhelming majority of antimicrobial peptides become amphipathic only by adopting an appropriate secondary structure (i.e., either α-helix or β-sheet).31 Many structure-activity relationship studies demonstrate that increasing α-helical content can contribute to enhance the membrane-lytic activity of antimicrobial peptides.32 In this study, as shown in Figure 2, the CD results showed that all peptides displayed obvious helical structure in 50% TFE/water either at pH 7.4 or 5.0. However, only the helical content of AMitP at acidic pH (58.2%) was significantly higher than at normal pH (48.5%). This may be because electrostatic attraction effect existing between MitP and MitPE hinder the helix-folding of AMitP at pH 7.4, whereas the lost of electrostatic attraction effect at acidic pH contributes to the dissociation of MitP from MitPE and consequently increase the helical content of AMitP.
Figure 2. CD spectra of peptides in 50% TFE/ water solution at different pHs. To get more details about molecular structure changes of AMitP at different pHs,
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we performed a series of MD simulations in POPC lipid bilayer. As shown in Figure 3A, the cationic MitP in AMitP bound to anionic MitPE at pH 7.4, whereas MitP obviously dissociated from MitPE at pH 5.0, resulting in an increased α-helicity of AMitP (Figure 3B). This data confirmed the result derived from CD assay. The increased α-helicity at acidic pH would be responsible for the enhanced membrane disordering effect of AMitP. It has been reported that the degree of local membrane thinning can be used to reflect the extent of bilayer disordering of antimicrobial peptides.33 As shown in Figure 3C, the bilayer thickness decreases significantly from 2.99 to 2.49 nm after AMitP treatment at acidic pH, suggesting AMitP could induce high membrane disordering at acidic pH. In contrast, the bilayer thickness had no significant change after AMitP treatment at normal pH. Our result suggested that high membrane disordering effect of AMitP at acidic pH would contribute to enhance its membrane-lytic activity.
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Figure 3. Snapshots of AMitP in POPC lipids. (A) Structure changes of AMitP at different pHs. Color schemes are as follows: POPC lipid (cyan), water (red), MitP (red), MitPE (blue), disulfide linker (yellow). (B) Secondary structures of AMitP at different pHs as defined by DSSP algorithm. (C) Snapshots of membrane thinning effect. The thickness of membrane was defined as the average phosphate-phosphate distance. Color schemes are as follows: lipid (black), water (red), peptide (green). pH-Dependent Membrane Binding. The binding of antimicrobial peptides to cell membranes is the first and critical step in the process of membrane disruption.20,28 However, it is not easy to show the whole binding process of peptides at molecular levels by traditional experimental methods. Therefore, we used MD simulations to investigate the relative binding tendency of AMitP to membranes at different pHs. Because the membrane potential of tumor cells is negative,15 we used a lipid bilayer comprising 25% negatively charged POPG lipids and 75% zwitterionic POPC lipids to mimic the tumor cell membranes. Initially, AMitP with random coil structure was placed in water phase at pH 7.4 and 5.0 and close to the surface of POPG/POPC lipid bilayer. After 100 ns of simulation, AMitP could rapidly bind to the proximal layer of 14 ACS Paragon Plus Environment
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the lipid bilayer either at pH 7.4 or 5.0 (Figure 4). However, AMitP was prone to form intramolecular hydrogen bonds at pH 7.4, while to form intermolecular hydrogen bonds with POPG/POPC lipid bilayer at pH 5.0 (Table 1), suggesting that the membrane binding tendency of AMitP at pH 5.0 was significantly higher than at pH 7.4. The result derived from MD simulations proved our hypothesis. Taken together, the pH-dependent membrane binding would contribute to enhance the pH-dependent selectivity of AMitP.
Figure 4. Presenting the binding processes of AMitP with POPG/POPC lipids at different pHs. Table 1. Number of hydrogen bonds pH 7.4 pH 5.0 Time POPG/ POPG/ AMitPa AMitPa POPCb POPCb 0 0 0 ns 7 3 9 14 25 ns 8 2 50 ns
9
7
1
14
75 ns
10
9
3
14
7 14 100 ns 11 3 a Hydrogen bond calculated in AMitP. b Hydrogen bond calculated between AMitP and POPG/ POPC lipids.
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pH-Dependent Antitumor Activity. The majority of clinically approved antitumor drugs always cause undesirable severe side effects due to lack selectivity. The tumor acidity can be regarded as a useful target to develop new pH-dependent antitumor drugs with high selectivity. To get a view of the pH-dependent cytotoxicity of AMitP, the antitumor activity of peptides was evaluated at different pHs by MTT assay. Since many antimicrobial peptides are able to kill cells within a very short period of time, we kept the exposure time of cells to peptides for only 2 h in our study. The result derived from MTT assay showed that MitP displayed similar cytotoxicity at pH 7.4, 6.0 and 5.0 (Figure 5A), and MitPE had no antitumor activity at all different pHs (Figure 5B). In contrast, AMitP could kill tumor cells in a dose-dependent manner at acidic pHs, whereas it showed significantly decreased cytotoxicity at pH 7.4 (Figure 5C). However, Figure 5D showed that the mixture of MitP and MitPE in a physical 1/1 displayed no obviously pH-dependent cytotoxicity compared with AMitP, demonstrating that the disulfide linker plays an important role for the pH-dependent cytotoxicity of AMitP. This may be because that the disulfide linker can contribute to the electrostatic attraction of MitP with the anionic MitPE by reducing the entropy of assembly. In addition, we also evaluated the antitumor activity of MitP and AMitP at 10 µM to other tumor cells (such as KB cells, BEL-7402 cells, EJ cells and MCF-7 cells) at different pHs. As shown in Figure 5E, the cytotoxicity of AMitP to other tumor cells also exhibited a significant pH-dependent manner compared with MitP. Taken together, compared with MitP, AMitP exhibited remarkably pH-dependent cytotoxicity toward tumor cells, suggesting that AMitP can be used as a promising
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drug with high pH selectivity for tumor therapy.
Figure 5. pH-dependent antitumor activity of peptides at different pHs. (A) MitP. (B) MitPE. (C) AMitP. (D) The mixture of MitP and MitPE in a physical 1/1. (E) Ctyotoxicity of peptides at 10 µM to different tumor cells. Representative of triplicate experiments; bars, SEM. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. indicates no significant difference. pH-Dependent Membrane Disruption Activity. To better understand the killing mechanism of AMitP, LDH leakage assay and propidium iodide (PI) uptake assay were used to monitor the integrity of cell membranes after peptides treatment at different pHs. The LDH leakage assay, used to measure acute membrane disturbance, 17 ACS Paragon Plus Environment
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revealed that only AMitP exhibited obviously pH-dependent membrane-lytic activity compared with MitP and MitPE (Figure 6), supporting the results indicated by the MTT assay. PI is a fluorescent molecule that can be excluded by the membranes of viable cells but can pass through the damaged plasma membranes. To further verify the pH-dependent membrane-lytic activity of AMitP, we studied the change of cell membrane integrity by detecting the uptake of PI in Hela cells after peptides treatment at different pHs. As shown in Figure 7, the positive rate of PI in Hela cells after MitP treatment at pH 7.4, 6.0 and 5.0 have no significant differences. In contrast, the uptake of PI in Hela cells after AMitP treatment at pH 6.0 and 5.0 significantly increased compared with pH 7.4, indicating that the membrane-lytic activity of AMitP is in a pH-dependent manner. Furthermore, SEM was used to examine the subtle morphologic changes of Hela cell membranes after AMitP treatment at pH 7.4 and 5.0, with the results shown in Figure 8. Similarity to the untreated Hela cells, most Hela cells treated with AMitP at pH 7.4 showed plenty of microvilli and adherent smooth surface. Following the AMitP treatment at pH 5.0, the membranes of Hela cells were heavily disrupted and were characterized with significant pore formation and loss of microvilli. The result derived from SEM was consensus to that derived from the LDH leakage assay and PI uptake assay, confirming that AMitP can kill tumor cells at acidic pH via membrane disruption mechanism, just like many naturally occurring antimicrobial peptides. This drastic membrane-lytic activity endows AMitP an excellent merit that it can kill the common multi-drug resistant tumor cells. Furthermore, this unique mechanism makes it more difficult for tumor cells to
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develop resistance to AMitP.
Figure 6. LDH leakage in HeLa cells treated with peptides at different pHs. (A) MitP. (B) AMitP. Representative of triplicate experiments; bars, SEM. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. indicates no significant difference.
Figure 7. PI uptake in HeLa cells treated with MitP and AMitP at different pHs.
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Figure 8. Membrane changes of HeLa cells after AMitP treatment at different pHs. Cytotoxicity to Normal Cells. In the present study, we used hemolysis assay to evaluate the cytotoxicity of AMitP to nomal cells. As shown in Figure 9, AMitP displayed significantly reduced hemolytic activity to red blood cells compared with MitP. The diminished hemolytic activity of AMitP majorly results from the decreased membrane binding ability and membrane disordering effect of AMitP at normal pH. The decreased cytotoxicity of AMitP to normal cells at physiological pH could expand its concentration used in tumor therapy, where more drugs can diffuse into the tumor tissues.
Figure 9. Hemolytic activity of peptides on red blood cells. Representative of triplicate experiments; bars, SEM. *p < 0.05, ***p < 0.001, N.S. indicates no significant difference. Enzymatic Stability. Poor enzymatic stability severely limits the clinical use of natural antimicrobial peptides.34 Trypsin specifically cleaves peptides at the C-terminal amide bonds of lysine and arginine, making it an ideal enzyme to examine the protease stability of our designed antimicrobial peptides. As shown in Figure 10A, AMitP was resistant to degradation by trypsin with about 50% of the peptide 20 ACS Paragon Plus Environment
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remaining in solution after 120 min. In contrast, MitP and MitPE were almost completely hydrolyzed within 30 min. In addition, AMitP exhibited significant stability in serum compared with MitP and MitPE (Figure 10B). The enhanced enzymatic stability of AMitP is mainly because the cleavage sites of AMitP were not easily recognized by the enzymes due to the binding of MitP to the anionic MitPE at normal pH. The stability of AMitP against enzymes would contribute to its in vivo application for tumor therapy.
Figure 10. Enzymatic stability of peptides. (A) Stability of peptides against trypsin. (B) Stability of peptides in serum. Representative of triplicate experiments; bars, SEM. ***p < 0.001. CONCLUSIONS In this study, we developed a new type of acid-activated antimicrobial peptide AMitP by coupling MitP with its anionic peptide MitPE via a disulfide linker. The pH-dependent cytotoxicity makes AMitP a promising drug targeting tumor tissues 21 ACS Paragon Plus Environment
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with an acidic extracellular environment. This is a preliminary study, further studies on applications of this type of acid-activated antimicrobial peptide are underway in our laboratory. Nevertheless, our work opens a new avenue to design antimicrobial peptides that preferentially kill cells in acidic solid tumors, with reduced toxicity to normal tissues. ACKNOWLEDGMENT We are grateful for the grants from the National Natural Science Foundation of China (Nos. 91213302, 20932003, 81273440 and 81202400), and the Key National S&T Program “Major New Drug Development” of the Ministry of Science and Technology of China (2012ZX09504-001-003). We also thank the Gansu Supercomputing Center of Cold and Arid Environment and Engineering Research Institute of Chinese Academy of Sciences for providing computational resource.
REFERENCES (1) Venditto, V. J.; Simanek, E. E. Cancer therapies utilizing the camptothecins: a review of the in vivo literature. Mol. Pharm. 2010, 7, 307-349. (2) Aina, O. H.; Liu, R.; Sutcliffe, J. L.; Marik, J.; Pan, C. X.; Lam, K. S. From combinatorial chemistry to cancer-targeting peptides. Mol. Pharm. 2007, 4, 631-651. (3) Ojima, I. Guided molecular missiles for tumor-targeting chemotherapy-case studies using the second-generation taxoids as warheads. Acc. Chem. Res. 2008, 41, 108-119. (4) Low, P. S.; Henne, W. A.; Doorneweerd, D. D. Discovery and development of
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Page 23 of 26
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folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc. Chem. Res. 2008, 41, 120-129. (5) Scholler, N.; Fu, N.; Yang, Y.; Ye, Z.; Goodman, G. E.; Hellström, K. E.; Hellström, I. Soluble member(s) of the mesothelin/megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 11531-11536. (6) Fox, E. J.; Salk, J. J.; Loeb, L. A. Cancer genome sequencing-an interim analysis. Cancer Res. 2009, 69, 4948-4950. (7) Vaupel, P.; Kallinowski, F.; Okunieff, P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 1989, 49, 6449-6465. (8) Gerweck, L. E.; Seetharaman, K. Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer. Cancer Res. 1996, 56, 1194-1198. (9) Lee, E.S.; Gao, Z.; Bae, Y. H. Recent progress in tumor pH targeting nanotechnology. J. Control. Release 2008, 132, 164-170. (10) Tannock, I. F.; Rotin, D. Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res. 1989, 49, 4373-4384. (11) Makovitzki, A.; Fink, A.; Shai, Y. Suppression of human solid tumor growth in mice by intratumor and systemic inoculation of histidine-rich and pH-dependent host defense-like lytic peptides. Cancer Res. 2009, 69, 3458-3463. (12) Na, K.; Lee, E. S.; Bae, Y. H. Self-organized nanogels responding to tumor
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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
Page 24 of 26
extracellular pH: pH-dependent drug release and in vitro cytotoxicity against MCF-7 cells. Bioconjug. Chem. 2007, 18, 1568-1574. (13) An, M.; Wijesinghe, D.; Andreev, O. A.; Reshetnyak, Y. K.; Engelman, D. M. pH-(low)-insertion-peptide (pHLIP) translocation
of membrane
impermeable
phalloidin toxin inhibits cancer cell proliferation. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 20246-20250. (14) Zhang, W.; Song, J. J.; Zhang, B. Z.; Liu, L. W.; Wang, K. R.; Wang. R. Design of acid-activated cell penetrating peptide for delivery of active molecules into cancer cells. Bioconjug. Chem. 2011, 22, 1410-1415. (15) Hoskin, D. W.; Ramamoorthy, A. Studies on anticancer activities of antimicrobial peptides. Biochim. Biophys. Acta 2008, 1778, 357-375. (16) Schweizer, F. Cationic amphiphilic peptides with cancer-selective toxicity. Eur. J. Pharmacol. 2009, 625, 190-194. (17) Wang, K. R.; Yan, J. X.; Zhang, B. Z.; Song, J. J.; Jia, P. F.; Wang, R. Novel mode of action of polybia-MPI, a novel antimicrobial peptide, in multi-drug resistant leukemic cells. Cancer Lett. 2009, 278, 65-72. (18) Zhang, W.; Li, J.; Liu, L. W.; Wang, K. R.; Song, J. J.; Yan, J. X.; Li, Z. Y.; Zhang, B. Z.; Wang, R. A novel analog of antimicrobial peptide Polybia-MPI, with thioamide bond substitution, exhibits increased therapeutic efficacy against cancer and diminished toxicity in mice. Peptides 2010, 31, 1832-1838. (19) Chen, C.; Hu, J.; Zhang, S.; Zhou, P.; Zhao, X.; Xu, H.; Zhao, X.; Yaseen, M.; Lu, J. R. Molecular mechanisms of antibacterial and antitumor actions of designed
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surfactant-like peptides. Biomaterials 2012, 33, 592-603. (20) Matsuzaki, K. Control of cell selectivity of antimicrobial peptides. Biochim Biophys. Acta 2009, 1788, 1687-1692. (21) Eckert, R. Road to clinical efficacy: challenges and novel strategies for antimicrobial peptide development. Future Microbiol. 2011, 6, 635-651. (22) Magzoub, M.; Kilk, K.; Eriksson, L. E.; Langel, U.; Gräslund, A. Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles. Biochim. Biophys. Acta 2001, 1512, 77-89. (23) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. J. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 2008, 4, 435-447. (24) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: an N. log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993, 98, 10089-10092. (25) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. J. LINCS: a linear constraint solver for molecular simulations. J. Comp. Chem. 1997, 18, 1463-1472. (26) Wolf, M. G.; Hoefling, M.; Aponte-Santamaría, C.; Grubmüller, H.; Groenhof, G. g_membed: efficient insertion of a membrane protein into an equilibrated lipid bilayer with minimal perturbation. J. Comput. Chem. 2010, 31, 2169-2174. (27) Jones, S.; Howl, J. Charge delocalisation and the design of novel mastoparan analogues: enhanced cytotoxicity and secretory efficacy of [Lys5, Lys8, Aib10]MP. Regul. Pept. 2004, 121, 121-128. (28) Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 2002, 415,
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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
389-395. (29) Rozema, D. B.; Ekena, K.; Lewis, D. L.; Loomis, A. G.; Wolff, J. A. Endosomolysis by masking of a membrane-active agent (EMMA) for cytoplasmic release of macromolecules. Bioconjug. Chem. 2003, 14, 51-57. (30) Tu, Z.; Volk, M.; Shah, K.; Clerkin, K.; Liang, J. F. Constructing bioactive peptides with pH-dependent activities. Peptides. 2009, 30, 1523-1528. (31) Yeaman, M. R.; Yount, N. Y. Mechanisms of antimicrobial peptide action and resistance. Pharmacol. Rev. 2003, 55, 27-55. (32) Dathe, M.; Wieprecht, T. Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells. Biochim. Biophys. Acta 1999, 1462, 71-87. (33) Leontiadou, H.; Mark, A. E.; Marrink, S. J. Antimicrobial peptides in action. J. Am. Chem. Soc. 2006,128, 12156-12161. (34) Marr, A. K.; Gooderham, W. J.; Hancock, R. E. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr. Opin. Pharmacol. 2006, 6, 468-672.
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