pharmaceutics articles Molecular Insights on the cyclic Peptide Nanotube-Mediated Transportation of Antitumor Drug 5-Fluorouracil Huifang liu, Jian Chen, Qing Shen, Wei Fu, and Wei Wu School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, P. R. China Received August 19, 2010: Revised Manuscript Received October 11, 2010: Accepted October 21. 2010 Abstract: Self-assembled cyclic peptide nanotubes(CPNs)show a potential use in drug delivery In this study, the CPN composed of (Trp-D-Leu)4-GIn-D-Leu was synthesized and tested for the transport of the antitumor drug 5-fluorouracil(5-FU). CPN-mediated release of 5-FU from liposomes experimentally tested the transportation function of the synthetic CPNs. To explore the transportation mechanism of CPNs, computational studies have been performed on the CPN models stacked by 8 subuints, including conventional molecular dynamics simulations, and steered molecular dynamics(SMD) simulations in the environment of hy dimyristoylphosphatidylcholine(DMPC)lipid bilayer. Our CMD simulations demonstrat the ortho-CPN is the most stable nanotube, in which the GIn residue is in the ortho-position relative to other residues the calculated diffusion coefficient value for inner water molecules as 1.068 x 10-5 cm2.s-1, almost half that of the bulky water and 24 times faster than that of the typical gramicidin A channel. The CPN conserved its hollow structure along the 10 ns CMD simulations, with a tile angle of 50 relative to the normal of DMPC membrane. Results from SMD simulations showed that the 5-FU molecule was transported by hopping through different potential energy minima distributed along subunits, and finally exited the nanotube by escaping from the kink region at the last two subunits. The hopping of 5-FU was driven by switching from interactions of 5-FU with the backbone carbonyl group and amide group of ortho-CPN.The calculated binding free energy profile of 5-FU interacting with the CPn indicated that there was an energy well near the outer end of the nanotube Keywords: 5-fluorouracil; Cyclic peptide nanotube; steered molecular dynamics; drug trans porter; antitumor Introduction tubes are emerging as promising and selective drug trans- Malignant tumor is the most severe disease threatening porters. Our preliminary in vitro and in vivo studies have the health of human beings. More and more antitumor drugs demonstrated that the synthetic decedapeptide tube could have been discovered. However, the biggest problem is that efficiently enhance the antitumor potency of 5-fluorouracil these drugs could not efficiently exhibit their activity in (5-FU)(unpublished data) killing tumors due to the difficulty in their ability to penetrate Cyclic peptide nanotubes(CPNs) are a class of artificial through the cell membrane. Synthetic cyclic peptide nano- channels formed by closed peptide rings which consist of sCorrespondingauthors.E-mail:wfu@fudan.edu.cn(WF) wuwei@fudan.edu.cn(W.W.).Mailingaddress:FudanUni-(1)Adson,A.Burton,PS:Raub,TJ:Barsuhn,CL:Audus versity, School of Pharmacy, 826 Zhangheng Road, Shanghai K. L: Ho, N. F. Passive diffusion of weak organic electrolytes 201203,P.R. China.Fax:+86-21-51980010. Phone:+86- across Caco-2 cell monolayers: uncoupling the contributions of 21-51980010. hydrodynamic, transcellular, and paracellular barriers. J. Pharm 10.1021/mp100274f 2010 American Chemical Society VOL 7, NO. 6, 1985-1994 MOLECULAR PHARMACEUTICS 1985 Published on Web 10/21/2010
Molecular Insights on the Cyclic Peptide Nanotube-Mediated Transportation of Antitumor Drug 5-Fluorouracil Huifang Liu,† Jian Chen,† Qing Shen, Wei Fu,* and Wei Wu* School of Pharmacy, Fudan UniVersity, 826 Zhangheng Road, Shanghai, 201203, P. R. China Received August 19, 2010; Revised Manuscript Received October 11, 2010; Accepted October 21, 2010 Abstract: Self-assembled cyclic peptide nanotubes (CPNs) show a potential use in drug delivery. In this study, the CPN composed of (Trp-D-Leu)4-Gln-D-Leu was synthesized and tested for the transport of the antitumor drug 5-fluorouracil (5-FU). CPN-mediated release of 5-FU from liposomes experimentally tested the transportation function of the synthetic CPNs. To explore the transportation mechanism of CPNs, computational studies have been performed on the CPN models stacked by 8 subuints, including conventional molecular dynamics (CMD) simulations, and steered molecular dynamics (SMD) simulations in the environment of hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer. Our CMD simulations demonstrated that the ortho-CPN is the most stable nanotube, in which the Gln residue is in the ortho-position relative to other residues. The calculated diffusion coefficient value for inner water molecules was 1.068 × 10-5 cm2 · s-1, almost half that of the bulky water and 24 times faster than that of the typical gramicidin A channel. The CPN conserved its hollow structure along the 10 ns CMD simulations, with a tile angle of 50° relative to the normal of DMPC membrane. Results from SMD simulations showed that the 5-FU molecule was transported by hopping through different potential energy minima distributed along subunits, and finally exited the nanotube by escaping from the kink region at the last two subunits. The hopping of 5-FU was driven by switching from hydrophobic interactions between 5-FU and the interior wall of the nanotube to hydrogen bonding interactions of 5-FU with the backbone carbonyl group and amide group of ortho-CPN. The calculated binding free energy profile of 5-FU interacting with the CPN indicated that there was an energy well near the outer end of the nanotube. Keywords: 5-fluorouracil; Cyclic peptide nanotube; steered molecular dynamics; drug transporter; antitumor Introduction Malignant tumor is the most severe disease threatening the health of human beings. More and more antitumor drugs have been discovered. However, the biggest problem is that these drugs could not efficiently exhibit their activity in killing tumors due to the difficulty in their ability to penetrate through the cell membrane.1 Synthetic cyclic peptide nanotubes are emerging as promising and selective drug transporters. Our preliminary in Vitro and in ViVo studies have demonstrated that the synthetic decedapeptide tube could efficiently enhance the antitumor potency of 5-fluorouracil (5-FU) (unpublished data). Cyclic peptide nanotubes (CPNs) are a class of artificial channels formed by closed peptide rings which consist of an even number of alternating D- and L-amino acid residues.2 * Corresponding authors. E-mail: wfu@fudan.edu.cn (W.F.), wuwei@fudan.edu.cn (W.W.). Mailing address: Fudan University, School of Pharmacy, 826 Zhangheng Road, Shanghai, 201203, P. R. China. Fax: +86-21-51980010. Phone: +86- 21-51980010. † These authors contributed equally to this work. (1) Adson, A.; Burton, P. S.; Raub, T. J.; Barsuhn, C. L.; Audus, K. L.; Ho, N. F. Passive diffusion of weak organic electrolytes across Caco-2 cell monolayers: uncoupling the contributions of hydrodynamic, transcellular, and paracellular barriers. J. Pharm. Sci. 1995, 84 (10), 1197–1204. articles 10.1021/mp100274f 2010 American Chemical Society VOL. 7, NO. 6, 1985–1994 MOLECULAR PHARMACEUTICS 1985 Published on Web 10/21/2010
articles By simply adjusting the number and kinds of amino acid Asp-D-Phe )2-] 2 In 1998, studies by polarized attenuated residues, both the internal diameter and external surface total reflectance infrared (ATR-IR)spectroscopy showed that properties can be tailored. Generally, the interior surface of CPNs oriented themselves in a transport-competent mem- such a tube is hydrophilic as indicated by the presence of brane orientation. The cyclic peptide nanotubes could act water molecules, while the exterior surface is hydrophobic, as highly selective and efficient transmembrane channels for permitting facile dissolution in nonpolar solvents. A network ions and small molecules. In 1994, Ghadiri confirmed that of hydrogen bonds between oxygen atoms of the participating the synthetic decapeptide cyclo[-(Trp-D-Leu)4-GIn-D-Leu-I carbonyl groups and amino groups at the backbone of has the function of glucose transportation. Since then adjacent cyclic peptide subunits drives them into a quasi-B- atomic-level theoretical and computational work has helped strand self-assembled tubular structure. The internal diam- to better understand the characteristics of structure, dynamics eter of the nanotube could be adjusted by simply varying and transport activity of the cyclic peptide nanotubes. Engels the size of the peptide ring. Electron diffraction analysis et al. found that cyclic D, L-octapeptide could self-assemble indicated that the tightly hydrogen-bonded rings stacked with as nanotubes. The diffusion of water molecules inside that an average intersubunit distance of 4.7 A. The cyclic D, L- peptide nanotube was much faster than that inside the peptides with appropriate hydrophobic side chains can self- gramicidin A channel. Asthagiri et al. calculated the semble and insert into lipid bilayers, and finally transport solvation free energies of Li, Nat, rb and Cl inside a ons or guest molecules across the membrane self-assembled (D, L)-octapeptide nanotubes using MD-based Studies on CPNs began from the year of 1974 and became perturbation free energy calculations; Tarek et al. found a hot research topic in the area of ion or small molecule that peptide nanotubes could tilt along the normal of lipid transportation. Both experimental and theoretical studies on bilayer after the nanotubes were equilibrated, but the hollow CPNs have been devoted to an understanding of their tubular structure was conserved. Hwang et al. calculated structural and dynamical characteristics, and transportation the free energy barrier for Na and k ions diffusing through properties. In 1993, Ghadiri et al. reported the first well- the cyclic peptide nanotube to be x2. 4 kcal/mol, and found characterized structure of self-assembled cyclic octapeptide that the carbonyl groups of the cyclic peptide were structu subunits cyclo[-(D-Ala-Glu-D-Ala-Gln)2-) in crystalline ally rigid because of the network of hydrogen bonding nanotubular arrays, convincingly establishing that the ring- All these studies confirmed that cyclic peptide readily self- aped subunits can stack through antiparallel B-sheet assembles to be a nanotube with a potential transportation hydrogen bonds to form hollow tubes. Later, Lambert and function. The questions are how the size and kind of peptide co-workers demonstrated their result by the Ir spectrum and determine the properties of a nanotube, and how the morphology of the crystals formed by cyclo[-(Asn-D-Phe- assembled nanotube transports guest molecules like drugs Answers to these questions have great significance and (2)Ghadir, M.R. Self-assembled nanoscale tubular ensembles. Adu. potential applications in the area of drug delivery Mater.1995,7(7),675-677 (3) Buriak, J M; Ghadiri, M. R. Self-assembly of peptide based (12) Polaskova, M. E; Ede, N. J. Lambert. J. N. Synthesis of nanotubes. Mater. Sci. Eng: C 1997, 4(4), 207-212. nanotubule-forming cyclic octapeptides via an Fmoc strategy. Aust (4)Karlstrom, A: Unden, A. Association of cyclic peptides in aqueous J.Chem.1998,51(7),535-540 solution measured by fluorescence quenching Biopolymers 1997 (3) Kim, H. S: Hartgerink, J. D. Ghadiri, M. R. Oriented self- 4(1),1 ssembly of cyclic peptide nanotubes in lipid membranes. J.Am (5)Hartgerink, J. D: Granja, J. R; Milligan, R. A: Ghadiri, M.R. Self-assembling peptide nanotubes. J. A. Chem. Soc. 1996. I (14) Sanchez-Quesada, J: Ghadiri, M.R.: Bayley, H: Braha, O. Cyclic ptides as molecular adapters for a pore-forming protein. J.A. (6) Ghadir, M.R.; Granja, J.R. Buchler, L. K. Artificial Trans- Chem. Soc. 2000, 122(48), 11757-11766. Ashkenasy, G; Ghadir embrane lon Channels from Self-Assembling Peptide Nanotubes. M. R. Boolean logic functions of a synthetic peptide network Nature1994,369(6478),301-304 Am.Chem.Soc.2004,126(36,ll140-11141 (7)Desantis, P ; Morosett, S: Rizzo, R. Conformational-Analysis of (15)Granja, J. R: Ghadiri, M. R. Channel-Mediated Transport of Regular Enantiomeric Sequences. Macromolecules 1974, 7(1) Glucose across Lipid Bilayers. J. Am. Chem. Soc. 1994, 116(23) 52-58. 10785-10786. (8)Liu, Z. W: Xu, Y Tang, P. Steered molecular dynamics (16) Engels, M: Bashford, D. Ghadir, M.R. Structure and Dynamics Na of Self-Assembling Peptide Nanotubes and the Channel-Mediated J.Phys.Chem.B2006.ll0(25),12789-12795 Water Organization and Self-Diffusion-A Molecular-Dynamics (9)Chen, G J. Su, S.J.; Liu, R. Z. Theoretical studies of monomer Study.J.Am.Chem.soc.1995,17(36),9151-9158 d dimer of cyclo[(-L-Phe'-D-Ala'-)ml and cyclo((-L-Phe' (7)Asthagiri, D; Pratt, L. R: Ashbaugh, H. S. Absolute hydration EN-Ala2-)a](n=3-6).Phys. Chem. B2002,106(7),15 free energies of ions, ion-water clusters, and quasichemical theory chen.Phys.2003,I19(5),2702-2708 (10)Lewis, J. P; Pawley, N. H: Sankey, O. F. Theoretical investigation (18) Tarek, M : Maigret, B: Hipot, C. Molecular dynamics investiga- of the cyclic peptide system cyclo((D-Ala-Glu-D-Ala-GIn)meI-4l tion of an oriented cyclic peptide nanotube in DMPC bilayer J.Phys.Chem.B1997,lO1(49),10576-1058 Biophys.J.2003.85(4),2287-2298 (l) Ghadir, M.R. Granja, J.R. Milligan, R. A: McRee, D. E:(19) Hwang, H: Schatz, G. C: Ratner, M. A. Steered molecular Khazanovich, N. Self-Assembling Organic Nanotubes Based on dynamics studies of the potential of mean force of a Na* or K+ Cyclic Peptide Architecture. Nature 1993, 366 ion in a cyclic peptide nanotube. J. Phys. Che. B 2006. 110 (51),2644826460. 1986 MOLECULAR PHARMACEUTICS VOL 7. NO 6
By simply adjusting the number and kinds of amino acid residues, both the internal diameter and external surface properties can be tailored. Generally, the interior surface of such a tube is hydrophilic as indicated by the presence of water molecules, while the exterior surface is hydrophobic, permitting facile dissolution in nonpolar solvents.3 A network of hydrogen bonds between oxygen atoms of the participating carbonyl groups and amino groups at the backbone of adjacent cyclic peptide subunits drives them into a quasi-- strand self-assembled tubular structure.4 The internal diameter of the nanotube could be adjusted by simply varying the size of the peptide ring. Electron diffraction analysis indicated that the tightly hydrogen-bonded rings stacked with an average intersubunit distance of ∼4.7 Å.5 The cyclic D,Lpeptides with appropriate hydrophobic side chains can selfassemble and insert into lipid bilayers, and finally transport ions or guest molecules across the membrane.6 Studies on CPNs began from the year of 1974 and became a hot research topic in the area of ion or small molecule transportation.7 Both experimental and theoretical studies on CPNs have been devoted to an understanding of their structural and dynamical characteristics, and transportation properties.8-10 In 1993, Ghadiri et al. reported the first wellcharacterized structure of self-assembled cyclic octapeptide subunits cyclo[-(D-Ala-Glu-D-Ala-Gln)2-)] in crystalline nanotubular arrays, convincingly establishing that the ringshaped subunits can stack through antiparallel -sheet hydrogen bonds to form hollow tubes.11 Later, Lambert and co-workers demonstrated their result by the IR spectrum and morphology of the crystals formed by cyclo[-(Asn-D-PheAsp-D-Phe)2-].12 In 1998, studies by polarized attenuated total reflectance infrared (ATR-IR) spectroscopy showed that CPNs oriented themselves in a transport-competent membrane orientation.13 The cyclic peptide nanotubes could act as highly selective and efficient transmembrane channels for ions and small molecules.14 In 1994, Ghadiri confirmed that the synthetic decapeptide cyclo[-(Trp-D-Leu)4-Gln-D-Leu-] has the function of glucose transportation.15 Since then, atomic-level theoretical and computational work has helped to better understand the characteristics of structure, dynamics and transport activity of the cyclic peptide nanotubes. Engels et al. found that cyclic D,L-octapeptide could self-assemble as nanotubes. The diffusion of water molecules inside that peptide nanotube was much faster than that inside the gramicidin A channel.16 Asthagiri et al. calculated the solvation free energies of Li+, Na+, Rb+ and Cl- inside a self-assembled (D,L)-octapeptide nanotubes using MD-based perturbation free energy calculations;17 Tarek et al. found that peptide nanotubes could tilt along the normal of lipid bilayer after the nanotubes were equilibrated, but the hollow tubular structure was conserved.;18 Hwang et al. calculated the free energy barrier for Na+ and K+ ions diffusing through the cyclic peptide nanotube to be ∼2.4 kcal/mol, and found that the carbonyl groups of the cyclic peptide were structurally rigid because of the network of hydrogen bonding.19 All these studies confirmed that cyclic peptide readily selfassembles to be a nanotube with a potential transportation function. The questions are how the size and kind of peptide determine the properties of a nanotube, and how the assembled nanotube transports guest molecules like drugs. Answers to these questions have great significance and (2) Ghadiri, M. R. Self-assembled nanoscale tubular ensembles. AdV. potential applications in the area of drug delivery. Mater. 1995, 7 (7), 675–677. (3) Buriak, J. M.; Ghadiri, M. R. Self-assembly of peptide based nanotubes. Mater. Sci. Eng.: C 1997, 4 (4), 207–212. (4) Karlstrom, A.; Unden, A. Association of cyclic peptides in aqueous solution measured by fluorescence quenching. Biopolymers 1997, 41 (1), 1–4. (5) Hartgerink, J. D.; Granja, J. R.; Milligan, R. A.; Ghadiri, M. R. Self-assembling peptide nanotubes. J. Am. Chem. Soc. 1996, 118 (1), 43–50. (6) Ghadiri, M. R.; Granja, J. R.; Buehler, L. K. Artificial Transmembrane Ion Channels from Self-Assembling Peptide Nanotubes. Nature 1994, 369 (6478), 301–304. (7) Desantis, P.; Morosett, S.; Rizzo, R. Conformational-Analysis of Regular Enantiomeric Sequences. Macromolecules 1974, 7 (1), 52–58. (8) Liu, Z. W.; Xu, Y.; Tang, P. Steered molecular dynamics simulations of Na+ permeation across the gramicidin a channel. J. Phys. Chem. B 2006, 110 (25), 12789–12795. (9) Chen, G. J.; Su, S. J.; Liu, R. Z. Theoretical studies of monomer and dimer of cyclo[(-L-Phe1 -D-Ala2 -)n] and cyclo[(-L-Phe1 -D- MeN-Ala2 -)n] (n ) 3-6). J. Phys. Chem. B 2002, 106 (7), 1570– 1575. (10) Lewis, J. P.; Pawley, N. H.; Sankey, O. F. Theoretical investigation of the cyclic peptide system cyclo[(D-Ala-Glu-D-Ala-Gln)m)1-4]. J. Phys. Chem. B 1997, 101 (49), 10576–10583. (11) Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.; Khazanovich, N. Self-Assembling Organic Nanotubes Based on a Cyclic Peptide Architecture. Nature 1993, 366 (6453), 324– 327. (12) Polaskova, M. E.; Ede, N. J.; Lambert, J. N. Synthesis of nanotubule-forming cyclic octapeptides via an Fmoc strategy. Aust. J. Chem. 1998, 51 (7), 535–540. (13) Kim, H. S.; Hartgerink, J. D.; Ghadiri, M. R. Oriented selfassembly of cyclic peptide nanotubes in lipid membranes. J. Am. Chem. Soc. 1998, 120 (18), 4417–4424. (14) Sanchez-Quesada, J.; Ghadiri, M. R.; Bayley, H.; Braha, O. Cyclic peptides as molecular adapters for a pore-forming protein. J. Am. Chem. Soc. 2000, 122 (48), 11757–11766. Ashkenasy, G.; Ghadiri, M. R. Boolean logic functions of a synthetic peptide network. J. Am. Chem. Soc. 2004, 126 (36), 11140–11141. (15) Granja, J. R.; Ghadiri, M. R. Channel-Mediated Transport of Glucose across Lipid Bilayers. J. Am. Chem. Soc. 1994, 116 (23), 10785–10786. (16) Engels, M.; Bashford, D.; Ghadiri, M. R. Structure and Dynamics of Self-Assembling Peptide Nanotubes and the Channel-Mediated Water Organization and Self-DiffusionsA Molecular-Dynamics Study. J. Am. Chem. Soc. 1995, 117 (36), 9151–9158. (17) Asthagiri, D.; Pratt, L. R.; Ashbaugh, H. S. Absolute hydration free energies of ions, ion-water clusters, and quasichemical theory. J. Chem. Phys. 2003, 119 (5), 2702–2708. (18) Tarek, M.; Maigret, B.; Chipot, C. Molecular dynamics investigation of an oriented cyclic peptide nanotube in DMPC bilayers. Biophys. J. 2003, 85 (4), 2287–2298. (19) Hwang, H.; Schatz, G. C.; Ratner, M. A. Steered molecular dynamics studies of the potential of mean force of a Na+ or K+ ion in a cyclic peptide nanotube. J. Phys. Chem. B 2006, 110 (51), 26448–26460. articles Liu et al. 1986 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6
Molecular Insights on Antitumor Drug 5-Fluorouracil articles Scheme 1. Two-Dimensional Structure of the cyclo[-(Trp-D-Leu)4-GIn-D-Leu-] Peptide L H L NH In this work, we synthesized the cyclo[-(Trp-D-Leu)4-GIn- peptide was analyzed by reverse phase high performance D-Leu-] subunit, performed dialysis studies of 5-FU-loaded liquid chromatography(RP-HPLC) in order to determine the liposomes in the environment of cyclic peptide solution, and end point of the cyclization reaction, and the crude product investigated the antitumor activity by means of in vitro was purified by semipreparative RP-HPLC. Finally, the studies Based on our preliminary experimental findings we cyclized peptide was structurally analyzed by ESI-MS and examined the structural and dynamical characteristics of H NMR. CPN-mediated transportation of 5-FU from lipo- CPNS in a fully hydrated dimyristoylphosphatidylcholine somes was studied by an in vitro dialysis method. First, (MPC)lipid bilayer by conventional molecular dynamics 5-FU-loaded liposomes and cyclic peptides in dilute DMF (CMD) simulations. The transportation mechanism of 5-FU solution were sealed in a dialysis bag and dialyzed against by the CPNs is explored by means of steered molecular phosphate buffer saline(pH 7. 3)as the receptor phase(30 dynamics(SMD)simulations. Our results show that synthetic mL, 37C, 100 rpm) within a period of 90 min. The release CPN is able to efficiently transport the drug 5-FU by hopping of 5-FU was tested at 2 mg/mL concentrations of the cyclic the 5-FU molecule through different energy minima along peptides. The amount of the released 5-FU was quantified the nanotube. Our findings are helpful for the design of more at each time interval by HPLC. The mean values of six efficient drug delivery vehicles replicates were reported. Description of the Model Systems. The structural model Experimental Methods of cyclo[-(Trp-D-Leu)4-Gln-D-Leu-] nanotube was built Synthesis of cyclo[-(Trp-D-Leu)-GIn-D-Leu-1 Pep. according to Ghadiri,s model and modified according to the ide and Transportation Test. cyclo[-(Trp-D-Leu)4-Gln X-ray crystallographic structures of related peptide ensem D-Leu-] peptide(as shown in Scheme 1)was synthesized bles by using Sybyl 6.9 software (Tripos Inc, St and cyclized by a traditional solid-phase synthesis strategy. 20 Louis, MO). Although our experiments did not determine Briefly, the linear decapeptide was assembled by standard the exact number of subunits in one CPn nanotube. we Boc chemistry in the solid phase and subsequently cyclized selected the CPN assembly of 8 peptide subunits in order to in solution with high efficiency and reproducibility. L roughly match the thickness of the DMPC bilayer. All the Glutamine was adopted in the amino acid sequence for the side chains of the 8 peptides point outward attributed to their convenience of the peptide synthesis. The synthetic cyclic alternated D and L chirality. As the L-Gln could have three (20) Chen, J. Zhang, B; Xie, C; Lu, Y; Wu, w. Synthesis of a highly (21)Zhu, J. C; Cheng, J; Liao, Z.X.; Lai, Z.H. Liu, B Investigation hydrophobic cyclic decapeptide by solid-phase synthesis of linear of structures and properties of cyclic peptide nanotubes by peptide and cyclization in solution. Chin. Che. Lett. 2010 experiment and molecular dynamics. J Comput.-Aided Mol Des. 4),391-394. 2008,22(1),773-781. VOL 7. NO 6 MOLECULAR PHARMACEUTICS 1987
In this work, we synthesized the cyclo[-(Trp-D-Leu)4-GlnD-Leu-] subunit, performed dialysis studies of 5-FU-loaded liposomes in the environment of cyclic peptide solution, and investigated the antitumor activity by means of in Vitro studies. Based on our preliminary experimental findings we examined the structural and dynamical characteristics of CPNs in a fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer by conventional molecular dynamics (CMD) simulations. The transportation mechanism of 5-FU by the CPNs is explored by means of steered molecular dynamics (SMD) simulations. Our results show that synthetic CPN is able to efficiently transport the drug 5-FU by hopping the 5-FU molecule through different energy minima along the nanotube. Our findings are helpful for the design of more efficient drug delivery vehicles. Experimental Methods Synthesis of cyclo[-(Trp-D-Leu)4-Gln-D-Leu-] Peptide and Transportation Test. cyclo[-(Trp-D-Leu)4-GlnD-Leu-] peptide (as shown in Scheme 1) was synthesized and cyclized by a traditional solid-phase synthesis strategy.20 Briefly, the linear decapeptide was assembled by standard Boc chemistry in the solid phase and subsequently cyclized in solution with high efficiency and reproducibility. LGlutamine was adopted in the amino acid sequence for the convenience of the peptide synthesis. The synthetic cyclic peptide was analyzed by reverse phase high performance liquid chromatography (RP-HPLC) in order to determine the end point of the cyclization reaction, and the crude product was purified by semipreparative RP-HPLC. Finally, the cyclized peptide was structurally analyzed by ESI-MS and 1 H NMR. CPN-mediated transportation of 5-FU from liposomes was studied by an in Vitro dialysis method. First, 5-FU-loaded liposomes and cyclic peptides in dilute DMF solution were sealed in a dialysis bag and dialyzed against phosphate buffer saline (pH 7.3) as the receptor phase (30 mL, 37 °C, 100 rpm) within a period of 90 min. The release of 5-FU was tested at 2 mg/mL concentrations of the cyclic peptides. The amount of the released 5-FU was quantified at each time interval by HPLC. The mean values of six replicates were reported. Description of the Model Systems. The structural model of cyclo[-(Trp-D-Leu)4-Gln-D-Leu-] nanotube was built according to Ghadiri’s model and modified according to the X-ray crystallographic structures of related peptide ensembles11,18,21 by using Sybyl 6.9 software (Tripos Inc., St. Louis, MO). Although our experiments did not determine the exact number of subunits in one CPN nanotube, we selected the CPN assembly of 8 peptide subunits in order to roughly match the thickness of the DMPC bilayer. All the side chains of the 8 peptides point outward attributed to their alternated D and L chirality. As the L-Gln could have three (20) Chen, J.; Zhang, B.; Xie, C.; Lu, Y.; Wu, W. Synthesis of a highly hydrophobic cyclic decapeptide by solid-phase synthesis of linear peptide and cyclization in solution. Chin. Chem. Lett. 2010, 21 (4), 391–394. (21) Zhu, J. C.; Cheng, J.; Liao, Z. X.; Lai, Z. H.; Liu, B. Investigation of structures and properties of cyclic peptide nanotubes by experiment and molecular dynamics. J. Comput.-Aided Mol. Des. 2008, 22 (11), 773–781. Scheme 1. Two-Dimensional Structure of the cyclo[-(Trp-D-Leu)4-Gln-D-Leu-] Peptide Molecular Insights on Antitumor Drug 5-Fluorouracil articles VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 1987
articles different positions relative to other amino acids in the peptide set to 4.5 x 10, 4.5x 10, 4.5x 10-, 0, 0, 0 bar for chain, namely the ortho-position, meta-position and para- xx, yy, z, xy/yx, xzx and ya/zy components for water and position, the 8 cyclic peptides are stacked in three possible DMPC simulations. All bond lengths, including those to low-energy modes(see Figure SI in the Supporting Informa hydrogen atoms, were constrained by the LINCs algorithm. tion). Therefore, three low-energy stacking models of CPn Electrostatic interactions between charged groups within 9 were constructed For each CPN, the interior tube diameter A were calculated explicitly, while long-range electrostatic is 10 A, the side chains of L-Trp and the D-Leu are distributed interactions were calculated using the Particle-Mesh Ewald uniformly, and the center-to-center distance between neigh- method26 with a grid width of 1.2 A and a fourth-order spline boring subunits is 4.75 A. For convenience, the nanotube interpolation. A cutoff distance of 14 A was applied for the subunits are numerically denoted. As displayed in Figure S1 Lennard-Jones interactions. Numerical integration of the equa in the Supporting Information, APR(a-plane region) rep- tions of motion used a time step of 2 fs with atomic coordinates resents the plane of Ca atoms along one peptide subunit, saved every I ps for later analysis. Finally, three 10 ns MD while MPR (midplane region) represents the region between simulations were performed on these systems under the periodic two APRs. The whole length of one CPN is 38.7 A according boundary conditions in the NPT canonical ensemble. to the distance from apri to aprs Constant Velocity SMD Simulations. The SMD has Conventional Molecular Dynamics Simulation. After proved as an effective computational approach to simulate each of the three CPNs (i.e, ortho-CPN, meta-CPN and the transportation process of a small molecule permeating para-CPN) was built, it was inserted into the fully hydrated through a protein channel. In SMD simulations, a guest DMPC bilayer by aligning the axis of Cpn to the normal of molecule or an ion of interest is steered by an imaginary the lipid bilayer. The process of aligning CPN with the atomic force microscopy(AFM) tip, and the time-dependent normal of the DMPC membrane was similar to those used external force is added on the guest molecule to facilitate in our previous membrane protein simulations. When its transportation through the channel. In the present study, solvating the CPN/DMPC system, 42 Na and 42 CI ions we performed SMD simulations in order to explore how the were added in order to simulate the 150 mM physiological 5-FU molecule is transported by the CPn channel. In detail, ion strength. The size of the whole solvated system was 77 5-FU was pulled through the tube of the CPn by employing A X 83 A X 110 A, including one CPN, 189 DMPC an artificial harmonic force on the center of mass(COM)of molecules and 15087 water molecules 5-FU along the longitudinal axis of the CPN (Figure 1). 5-FU Energy minimizations were performed for each of the three molecule was first placed on the top of CPN, 1.27 nm from CPN/DMPC/water systems, first for all water molecules, ther the center of subunit 1, and then the whole system was for the whole system until the maximum force became equilibrated for 1 ns. The molecular topology file for 5-FU smallerthan10.00kcal/mol.a.Theenergy-minimizedCpn/wasgeneratedbytheProDrgserver(http://davapcl DMPC/water system was then subjected to MD simulation. bioch. dundee. ac uk/prodrg). The partial atomic charges of The MD simulations were performed by using the GROMACS 5-FU were determined with the DFT/B3LYP/6-31IG**basis package version 3.3.3 with the GROMOS96 force field. 23 set by using the CHelpG method implemented in GAMESS The solvent(water and DMPC)molecules of each initial program. To avoid large fluctuation in the position, a stiff the solute(CPNs)at 300 K for 20 ps. Then the Cpn was 5-FU. It should be pointed out that the pulling velocity (Pull) equilibrated for 5 ps while the solvent molecules were Is an important parameter in our SMD simulations. Higher constrained at 10, 50, 100, 200, and 298 K. Afterward, each pulling velocity may lead to remarkable nonequilibrium effects, system was equilibrated for 500 ps without any constraints. resulting in obvious errors of the simulation results. Very low To maintain the systems at a constant temperature of 300 velocity will make the SMD simulations extremely time- K, the Berendsen thermostat was applied using a coupling consuming, thus computationally not doable. To find an time of 0. I ps for the bulk water and DMPC. The pressure appropriate pulling velocity, five SMD simulations were was maintained by coupling to a reference pressure of 1 bar. performed using different pulling velocities(0. 1 A ps-,5 x The values of the anisotropic isothermal compressibility were (24)Berendsen, H. J. C. Postma, J. P. M: Vangunsteren, W.F.: Dinola. A: Haak, J. R. Molecular d (22) Fu. W: Shen, J. H: Luo, X. M.: Zhu, w. L: Cheng. J. G: Yu. external bath. J. Chem. Phys. 1984. 81(8). 3684-3690. K. Q; Briggs, J. M. Jin, G. Z; Chen, K. X: Jiang, H. L.(25)Hess, B: Bekker, H: Berendsen, H J C. Fraaije, J. LINCS: A Dopamine DI receptor agonist and D2 receptor antagonist effects linear constraint solver for molecular simulations. J. Comput of the natural product(-)-stepholidine: Molecular Modeling and Chem.1997,l8(12),1463-1472. dynamics Simulations. Biophys J. 2007, 93(5),1431-1441 (26) Darden, T: York, D; Pedersen, L. Particle Mesh Ewald-an (23)Vandrunen, R. Vanderpoel, D. Berendsen, H J C. GROMACS-a N Log(N) Method for Ewald Sums in Large Systems. J. Chem. software package and parallel computer for molecular dynamics Phys.1993,98(12,10089-10092. In Abstracts of Papers, 209th National Meeting of the American (27)Schuettelkopf, A. W. van Aalten, D M. F. PRODRG-a tool for Chemical Society: American Chemical Society: Washington, DC, high-throughput crystallography of protein-ligand complexes. Acta 1995:P49-COMP. Daura, X: Mark, A E; van Gunsteren, w.F. Crystallogr. 2004, D60, 1355-1363 Par arametrization of aliphatic CHn united atoms of GROMOS96 (28)Bode, B. M: Gordon, M. S. MacMolPlt: A graphical user interface force field. J. Comput. Chem. 1998,19(5),535-547 for GAMESS. J. Mol Graphics Modell. 1998. 16(3). 133 1988 MOLECULAR PHARMACEUTICS VOL 7. NO 6
different positions relative to other amino acids in the peptide chain, namely the ortho-position, meta-position and paraposition, the 8 cyclic peptides are stacked in three possible low-energy modes (see Figure S1 in the Supporting Information). Therefore, three low-energy stacking models of CPN were constructed. For each CPN, the interior tube diameter is 10 Å, the side chains of L-Trp and the D-Leu are distributed uniformly, and the center-to-center distance between neighboring subunits is 4.75 Å. For convenience, the nanotube subunits are numerically denoted. As displayed in Figure S1 in the Supporting Information, APR (R-plane region) represents the plane of CR atoms along one peptide subunit, while MPR (midplane region) represents the region between two APRs. The whole length of one CPN is 38.7 Å according to the distance from APR1 to APR8. Conventional Molecular Dynamics Simulation. After each of the three CPNs (i.e., ortho-CPN, meta-CPN and para-CPN) was built, it was inserted into the fully hydrated DMPC bilayer by aligning the axis of CPN to the normal of the lipid bilayer. The process of aligning CPN with the normal of the DMPC membrane was similar to those used in our previous membrane protein simulations.22 When solvating the CPN/DMPC system, 42 Na+ and 42 Cl- ions were added in order to simulate the 150 mM physiological ion strength. The size of the whole solvated system was 77 Å × 83 Å × 110 Å, including one CPN, 189 DMPC molecules and 15087 water molecules. Energy minimizations were performed for each of the three CPN/DMPC/water systems, first for all water molecules, then for the whole system until the maximum force became smaller than 10.00 kcal/mol ·Å. The energy-minimized CPN/ DMPC/water system was then subjected to MD simulation. The MD simulations were performed by using the GROMACS package version 3.3.3 with the GROMOS96 force field.23 The solvent (water and DMPC) molecules of each initial system were equilibrated with CPN structures by constraining the solute (CPNs) at 300 K for 20 ps. Then the CPN was equilibrated for 5 ps while the solvent molecules were constrained at 10, 50, 100, 200, and 298 K. Afterward, each system was equilibrated for 500 ps without any constraints. To maintain the systems at a constant temperature of 300 K, the Berendsen thermostat24 was applied using a coupling time of 0.1 ps for the bulk water and DMPC. The pressure was maintained by coupling to a reference pressure of 1 bar. The values of the anisotropic isothermal compressibility were set to 4.5 × 10-5 , 4.5 × 10-5 , 4.5 × 10-5 , 0, 0, 0 bar-1 for xx, yy, zz, xy/yx, xz/zx and yz/zy components for water and DMPC simulations. All bond lengths, including those to hydrogen atoms, were constrained by the LINCS algorithm.25 Electrostatic interactions between charged groups within 9 Å were calculated explicitly, while long-range electrostatic interactions were calculated using the Particle-Mesh Ewald method26 with a grid width of 1.2 Å and a fourth-order spline interpolation. A cutoff distance of 14 Å was applied for the Lennard-Jones interactions. Numerical integration of the equations of motion used a time step of 2 fs with atomic coordinates saved every 1 ps for later analysis. Finally, three 10 ns MD simulations were performed on these systems under the periodic boundary conditions in the NPT canonical ensemble. Constant Velocity SMD Simulations. The SMD has proved as an effective computational approach to simulate the transportation process of a small molecule permeating through a protein channel.8 In SMD simulations, a guest molecule or an ion of interest is steered by an imaginary atomic force microscopy (AFM) tip, and the time-dependent external force is added on the guest molecule to facilitate its transportation through the channel. In the present study, we performed SMD simulations in order to explore how the 5-FU molecule is transported by the CPN channel. In detail, 5-FU was pulled through the tube of the CPN by employing an artificial harmonic force on the center of mass (COM) of 5-FU along the longitudinal axis of the CPN (Figure 1). 5-FU molecule was first placed on the top of CPN, 1.27 nm from the center of subunit 1, and then the whole system was equilibrated for 1 ns. The molecular topology file for 5-FU was generated by the PRODRG server (http://davapc1. bioch.dundee.ac.uk/prodrg/).27 The partial atomic charges of 5-FU were determined with the DFT/B3LYP/6-311G** basis set by using the CHelpG method implemented in GAMESS program.28 To avoid large fluctuation in the position, a stiff spring (280 pN·Å) rather than a soft spring was assigned to 5-FU.19 It should be pointed out that the pulling velocity (Vpull) is an important parameter in our SMD simulations. Higher pulling velocity may lead to remarkable nonequilibrium effects, resulting in obvious errors of the simulation results. Very low velocity will make the SMD simulations extremely timeconsuming, thus computationally not doable. To find an appropriate pulling velocity, five SMD simulations were performed using different pulling velocities (0.1 Å· ps-1 , 5 × (22) Fu, W.; Shen, J. H.; Luo, X. M.; Zhu, W. L.; Cheng, J. G.; Yu, K. Q.; Briggs, J. M.; Jin, G. Z.; Chen, K. X.; Jiang, H. L. Dopamine D1 receptor agonist and D2 receptor antagonist effects of the natural product (-)-stepholidine: Molecular Modeling and dynamics Simulations. Biophys. J. 2007, 93 (5), 1431–1441. (23) Vandrunen, R.; Vanderspoel, D.; Berendsen, H. J. C. GROMACSsa software package and parallel computer for molecular dynamics. In Abstracts of Papers, 209th National Meeting of the American Chemical Society; American Chemical Society: Washington, DC; 1995; p 49-COMP. Daura, X.; Mark, A. E.; van Gunsteren, W. F. Parametrization of aliphatic CHn united atoms of GROMOS96 force field. J. Comput. Chem. 1998, 19 (5), 535–547. (24) Berendsen, H. J. C.; Postma, J. P. M.; Vangunsteren, W. F.; Dinola, A.; Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81 (8), 3684–3690. (25) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. LINCS: A linear constraint solver for molecular simulations. J. Comput. Chem. 1997, 18 (12), 1463–1472. (26) Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewaldsan N.Log(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98 (12), 10089–10092. (27) Schuettelkopf, A. W.; van Aalten, D. M. F. PRODRGsa tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. 2004, D60, 1355–1363. (28) Bode, B. M.; Gordon, M. S. MacMolPlt: A graphical user interface for GAMESS. J. Mol. Graphics Modell. 1998, 16 (3), 133. articles Liu et al. 1988 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6
Molecular Insights on Antitumor Drug 5-Fluorouracil articles Time(min) Figure 2. Transportation profiles of 5-FU mediated by cyclic peptides. The transportation of 5-FU is represented Figure 1. Representative of initial CPN/DMPC/water in terms of the accumulated transport percentage of drugs system for SMD simulation. The front half of the bilayer as a function of time. The concentration of the cyclic and subunits are sliced away to display a view of the inner peptides was 35 uM(; the molar ratio of cyclic peptides wall of the tube, and the tube molecular surface of each to total phosphatidylcholine and cholesterol was 1: 600) subunit is shown in a different color 5-FU is shown as The control group was without cyclic peptides. The stick, which was pulled into the tube along the tube axis results were presented as raw data through a harmonic potential (spring and arrow).The nitrogen and oxygen of the choline and phosphorate are Table 1. The First- order Rate Constant k(min ) and shown as blue and orange balls; the other atoms of the lipid as well as water molecules are represented as sticks. concn of CP (uM) k(min) v(nmol/min) 0.0004 1209±2.06 10-2Aps-1,1×10-2Aps-,5×10-3Aps-1,2.5×10-3 0.0114 0.56±1945 A.-). Our testing results showed that a SMD simulation with the pulling velocity in the range of 2.5 x 10-3A. The concentration of cyclic peptide in its solvent DMF, whose ps to 0. 1 final molar ratio to total phosphatidylcholine and A.- produced a similar force profile. Herein, the trajectories released system is 1: 600 at minimum velocity 2.5 X 10-3A ps- was adopted to illus- (Table 1). The presence of a small amount of DMF has no trate the CPN-mediated transportation mechanism of 5-FU significant effect on 5-FU transportation rate. The observed Results and discussion linear relation between transport rate and 5-FU concentration strongly supports a simple transmembrane channel-mediated Experimentally Observed 5-FU Transportation by transportation process. Moreover, studies on three kinds of CPN Nanotube cyclo[-(Trp-D-Leu) -GIn-D-Leu-]subunits carcinoma cell lines in vitro and the mice inoculated with were synthesized by a two-step solid-phase/solution synthesis S180 solid tumor in vivo demonstrated that the administration strategy with a purity of over 98% and were structurally of cyclic peptide nanotubes efficiently enhanced the antitu characterized by H NMR and mass spectrometry. The mor activity of 5-FU (unpublished data). All of these absorption, fluorescence spectrophotometry and gel perme- experiments demonstrate that the synthetic cyclic peptides ation studies have shown that the synthetic cyclic peptides stack and self-assemble into tubes, followed by their insertion tend to self-assemble and diffuse into lipid bilayers due to into the liposome membrane and transportation of antitumor the hydrophobic interactions of their side chains with the drug 5-FU across the liposome membrane hydrophobic chains of lipid when it is distributed into The ortho.cpn Is the most Stable self-Assembled aqueous suspension of liposomes. Our dialysis experiments anotube. The conventional CMd simulations for 3 model test such an assembling and insertion process. The release systems were conducted to investigate the structural and percentage of 5-FU from drug-loaded liposomes after adding dynamical properties of synthesized CPN. The 10 ns CMD cyclic peptides is shown in Figure 2. It can be seen that only simulations showed that ortho-CPN, meta-CPN and para 5% of 5-FU diffused from liposomes without cyclic peptides. CPN behaved differently in explicitly hydrolyzed DMPC In contrast, nearly 70%o of 5-FU was released into the solution bilayer. As shown in Figure S2 in the Supporting Informa when the concentration of cyclic peptide increased to 2 mg/ tion, meta-CPN sustained the hollow structure at the begin mL. A similar profile was found for the diffusion of glucose ning, but it began to bend at 2.6 ns and kept the curving across the CPNs. Similarly, the first-order transport rate posture in the rest of simulation For para-CPN, the tube constant k and average transport rate v in 90 min were much started to collapse during the first 1 ns simulation and higher than that of the control group(without cyclic peptides) completely collapsed at the end, demonstrating para-CPN VOL 7. NO 6 MOLECULAR PHARMACEUTICS 1989
10-2 Å· ps-1 , 1 × 10-2 Å· ps-1 , 5 × 10-3 Å· ps-1 , 2.5 × 10-3 Å·ps-1 ). Our testing results showed that a SMD simulation with the pulling velocity in the range of 2.5 × 10-3 Å· ps to 0.1 Å· ps-1 produced a similar force profile. Herein, the trajectories at minimum velocity 2.5 × 10-3 Å · ps-1 was adopted to illustrate the CPN-mediated transportation mechanism of 5-FU. Results and Discussion Experimentally Observed 5-FU Transportation by CPN Nanotube. cyclo[-(Trp-D-Leu)4-Gln-D-Leu-] subunits were synthesized by a two-step solid-phase/solution synthesis strategy with a purity of over 98% and were structurally characterized by 1 H NMR and mass spectrometry.20 The absorption, fluorescence spectrophotometry and gel permeation studies6 have shown that the synthetic cyclic peptides tend to self-assemble and diffuse into lipid bilayers due to the hydrophobic interactions of their side chains with the hydrophobic chains of lipid when it is distributed into aqueous suspension of liposomes. Our dialysis experiments test such an assembling and insertion process. The release percentage of 5-FU from drug-loaded liposomes after adding cyclic peptides is shown in Figure 2. It can be seen that only 5% of 5-FU diffused from liposomes without cyclic peptides. In contrast, nearly 70% of 5-FU was released into the solution when the concentration of cyclic peptide increased to 2 mg/ mL. A similar profile was found for the diffusion of glucose across the CPNs.15 Similarly, the first-order transport rate constant k and average transport rate V in 90 min were much higher than that of the control group (without cyclic peptides) (Table 1). The presence of a small amount of DMF has no significant effect on 5-FU transportation rate. The observed linear relation between transport rate and 5-FU concentration strongly supports a simple transmembrane channel-mediated transportation process. Moreover, studies on three kinds of carcinoma cell lines in Vitro and the mice inoculated with S180 solid tumor in ViVo demonstrated that the administration of cyclic peptide nanotubes efficiently enhanced the antitumor activity of 5-FU (unpublished data). All of these experiments demonstrate that the synthetic cyclic peptides stack and self-assemble into tubes, followed by their insertion into the liposome membrane and transportation of antitumor drug 5-FU across the liposome membrane. The ortho-CPN Is the Most Stable Self-Assembled Nanotube. The conventional CMD simulations for 3 model systems were conducted to investigate the structural and dynamical properties of synthesized CPN. The 10 ns CMD simulations showed that ortho-CPN, meta-CPN and paraCPN behaved differently in explicitly hydrolyzed DMPC bilayer. As shown in Figure S2 in the Supporting Information, meta-CPN sustained the hollow structure at the beginning, but it began to bend at ∼2.6 ns and kept the curving posture in the rest of simulation. For para-CPN, the tube started to collapse during the first 1 ns simulation and completely collapsed at the end, demonstrating para-CPN Figure 1. Representative of initial CPN/DMPC/water system for SMD simulation. The front half of the bilayer and subunits are sliced away to display a view of the inner wall of the tube, and the tube molecular surface of each subunit is shown in a different color. 5-FU is shown as stick, which was pulled into the tube along the tube axis through a harmonic potential (spring and arrow). The nitrogen and oxygen of the choline and phosphorate are shown as blue and orange balls; the other atoms of the lipid as well as water molecules are represented as sticks. Figure 2. Transportation profiles of 5-FU mediated by cyclic peptides. The transportation of 5-FU is represented in terms of the accumulated transport percentage of drugs as a function of time. The concentration of the cyclic peptides was 35 µM ([; the molar ratio of cyclic peptides to total phosphatidylcholine and cholesterol was 1:600). The control group (9) was without cyclic peptides. The results were presented as raw data. Table 1. The First-Order Rate Constant k (min-1) and Average Transport Rate v (nmol/min) of 5-FU in 90 min concn of CP (µM) k (min-1) v (nmol/min) 0 0.0004 12.09 ( 2.06 35a 0.0114 140.56 ( 19.45 a The concentration of cyclic peptide in its solvent DMF, whose final molar ratio to total phosphatidylcholine and cholesterol in the released system is 1:600. Molecular Insights on Antitumor Drug 5-Fluorouracil articles VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 1989