Molecular basis for the targeted binding of RGD-containing peptide to integrin αVβ3

Biomaterials 35 (2014) 1667e1675

Contents lists available at ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Molecular basis for the targeted binding of RGD-containing peptide to integrin aVb3 Yu-Ping Yu a, Qi Wang a, Ying-Chun Liu a, *, Ying Xie b, * a b

Soft Matter Research Center, Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China Department of Pharmaceutics, Peking University, Beijing 100191, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 September 2013 Accepted 27 October 2013 Available online 20 November 2013

Integrin aVb3-targeting peptides with an exposed arginineeglycineeaspartate (RGD) sequence play a crucial role in targeted anticancer drug delivery. The effects of RGD-containing peptide structure and quantity on mechanism of targeted binding of RGD-containing peptide to integrin aVb3 were studied intensively at the molecular level via molecular dynamic simulations. Targeted recognization was mainly driven by the electrostatic interactions between the residues in RGD and the metal ions in integrin aVb3, and cyclic arginineeglycineeaspartateephenylalanineevaline (RGDFV) peptide appeared to be a better vector than the linear RGD-containing peptides. In addition, the optimal molar concentration ratio of RGD peptides to integrin aVb3 appeared to be 2:1. These results will help improve the current understanding on the mechanism of interactions between RGD and integrin aVb3, and promote the application prospects of RGD-based vectors in tumor imaging, diagnosis, and cancer therapy. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: RGD Integrin aVb3 Targeted binding Interaction mechanism Molecular dynamic simulation

1. Introduction RGD-based anticancer strategies are a hot topic in cancer therapy and diagnosis [1]. Tumor angiogenesis is a critical process in tumor growth [2,3], and cell adhesion molecules are involved in tumor angiogenesis. The integrin receptor family presents an important group of cell surface adhesion proteins composed of non-covalently associated a- and b-subunits. These proteins are divalent cation-dependent heterodimeric membrane glycoproteins that play key roles in angiogenesis and tumor metastasis [4e7]. Integrin aVb3 can specifically recognize the peptide motif of arginineeglycineeaspartic (RGD) [8,9]. Inhibition of integrin aVb3 activity by anti-angiogenic drugs modified the RGD-containing peptides can induce endothelial cell apoptosis and inhibits angiogenesis [10]. Thus, targeting of RGD-modified micelles to tumor vasculature is a promising strategy for tumor-targeting treatment. It has attracted significant interest in drug delivery systems. Interaction sites between RGD peptide and integrin aVb3 have been studied extensively [11], and RGD-containing peptides have been widely used to deliver various kinds of cargos including nanoparticles, cytotoxic peptides, low molecular weight drugs, and contrast-enhancing agents [12e15]. However, the dynamic binding

* Corresponding authors. E-mail addresses: [email protected] (Y.-C. Liu), [email protected] (Y. Xie). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.10.072

process and mechanism of RGD-containing peptides interaction with integrin aVb3 under natural conditions or in the RGD-based targeted drug delivery systems remain poorly understood at the molecular level. Under physiological conditions, integrin aVb3 presents a dynamic conformation varying between a low-affinity conformation and a high-affinity one. The RGD peptide can bind to integrin aVb3 in different conformations [16e18], and show conformational changes and integrin aVb3 clustering [19,20]. When the RGD peptide binds to integrin aVb3, a shallow crevice, rather than a deep binding pocket, is formed. Based on its crystal structure, The RGD fits into a crevice between the bpropeller and bA domains of the integrin aVb3 head. The Arg and Asp side chains of RGD come into contact with the bpropeller and bA domains, respectively, contact the bpropeller and bA domains, respectively, pointing in opposite directions. The Arg side chain inserts into a narrow groove on top of the bpropeller domain of integrin aVb3, while the Arg guanidinium group forms two bidentate salt bridges with Asp218 and Asp150 at the bottom of the groove. The Asp side chain is completely buried in the complex, and Asp carboxylate oxygen atoms come into contact with a divalent cation at the “metal ion-dependent adhesion site” (MIDAS) in the bA domain of integrin aVb3 [21,22]. The crystal structures of extracellular aVb3 alone and in a complex with prototypical RGD peptide provide insights into the nature of the tertiary and quaternary changes that accompany the binding of this peptide [23]. Most linear RGD-containing peptides have a short circulation half-life, which results in non-ideal effects on treatment,

1668

Y.-P. Yu et al. / Biomaterials 35 (2014) 1667e1675

imaging, and biological activity. Cyclic RGD is highly suitable in clinical applications because of its higher affinity and tumorspecific intake than the linear RGD [24e26]. All-atom molecular dynamic (MD) studies may provide a better understanding of the targeted binding of RGD peptides to integrin aVb3 at the molecular level. In the present work, the mechanism of the targeted binding of RGD to aVb3 as well as the effects of RGD structure and quantity on this binding mechanism was studied intensively at the molecular level. Understanding the interaction mechanism between RGD peptides and integrin aVb3 in an aqueous solution is crucial for improving the potential application of RGDbased vectors in the early detection of integrin aVb3-positive tumors or therapeutic drugs specifically delivery inside the target cells. 2. Methods The crystal structures of integrin aVb3 were taken from the Protein Data Bank (ID: 1L5G and 3IJE). 1L5G is the extracellular segment of integrin aVb3 in complex with an ArgeGlyeAsp ligand [22], and 3IJE is the complete integrin aVb3 ectodomain plus an a/b transmembrane fragment [27]. As Mn2þ force field parameters were not available, a universally applicable method in which Mn2þ is replaced by Ca2þ was used [28]. The bA domain of integrin aVb3 features three metal ion-dependent adhesion sites: the metal iondependent adhesion site (MIDAS), the adjacent to the MIDAS (ADMIDAS), and the ligand-induced metal-binding site (LIMBS). Ca2þ ions were added to these three sites in the 3IJE structure based on their positions in the 1L5G structure [29], as two ions were not appeared at the MIDAS and ADMIDAS in bA domain of integrin aVb3. A 5-ns MD simulation was then performed to balance the binding of the metal ions. The structures of linear and cyclic RGD-containing peptides were separately selected from the crystal structure of integrin in complex with RGD-containing peptides (PDB ID: 3VI4, 1MOY, 1L5G) [22,30,31]. The nonstandard N-methylvaline and Dphenylalanine in the cyclic RGD ligand were replaced by standard valine and L-phenylalanine, respectively [28]. Multiple RGD peptides bound to multiple integrins aVb3 in the experiments. However, the binding process observed in all-atom MD simulations consumed a great amount of computing resources, and observation of configuration changes in integrin aVb3 could not be accomplished within a limited simulation time. Therefore, this study only focuses on the effects of RGD including its structure and quantity rather than integrin aVb3. As the binding sites are in the headpiece of integrin aVb3 comprising bA from b subunit and bpropeller from a subunit, RGD-containing peptide must reach near the headpiece of integrin aVb3 in the final binding process. Considering to preserve computing efficiency, the RGD peptide was first placed near the headpiece in different spatial orientation. The various systems of the RGD peptide with integrin aVb3 were set in a box filled with water molecules. All-atomistic MD simulations of the RGD peptides and integrin aVb3 were performed using GROMACS-4.5.2 package with Amber 03 [32e35]. All visualizations were done using Visual Molecular Dynamics (VMD) [36]. Temperature and pressure were maintained at 310 K and 101.3 kPa, respectively, using the NoseeHoover temperature control algorithm and the ParrinelloeRahman algorithm [37], respectively. The particle mesh Ewald [38] summation was used to calculate the fullsystem periodic electrostatic interactions, with a cutoff of 12 Å for the separation of the direct and reciprocal space summation. LennardeJones interactions were truncated between 10 Å and 11 Å with a smooth switching of the potential. Covalent bonds involving hydrogen atoms were constrained to their equilibrium lengths through the LINCS algorithms [39], whereas the water geometry was constrained using SETTLE [40]. An integration time step of 2 fs was used for both equilibration and production runs. The atomic

coordinates were saved into the trajectory every 200 ps. After energy minimization, each system was simulated under NVT conditions for 100 ps and under NPT conditions for 100 ps. MD runs were then carried out to simulate the dynamic binding of RGD to integrin aVb3. 3. Results and discussion 3.1. Interaction mechanism of one RGD peptide binding to integrin

aVb3 MD simulations were performed to study the dynamic binding process of a single RGD tripeptide to integrin aVb3. In nearly all of the simulations, spontaneous specific binding of the RGD tripeptide to integrin aVb3 was observed from the trajectory. The binding sites were consistent with the sites in the crystal structure of cyclic RGDFV in integrin aVb3, as shown in Fig. 1. The interaction between the RGD tripeptide and integrin aVb3 during binding mainly involved electrostatic interaction. Two kinds of electrostatic interaction were observed: interactions between residues and metal ions, which contribute to the binding of the RGD tripeptide onto integrin aVb3, and interactions between residues, also called salt bridges. The salt bridge is assessed according to the distance between the donor and acceptor atoms. If the distance between atoms is not over 4.0 Å, the pair is considered a salt bridge [41]. When the geometry is acceptable, salt bridges can coexist with hydrogen bonds. Hydrogen bonds and salt bridges are major contributors to the electrostatic interactions between proteins [42]. The interaction between the linear RGD tripeptide and integrin aVb3 is shown in Fig. 2a. Interactions between AspRGD and the ion at

Fig. 1. Configuration of the combination of the cyclic RGDFV and integrin aVb3. Integrin aVb3 is shown in new cartoon, aV subunit (purple), b3 subunit (yellow). Cyclic RGDFV peptide is shown in VDW. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Y.-P. Yu et al. / Biomaterials 35 (2014) 1667e1675

1669

Fig. 2. Two experiments on the binding of an RGD-containing peptide to integrin aVb3: (a) Binding sites and (c) interaction energies of the linear RGD tripeptide and integrin aVb3, bA from the b3 subunit (yellow), bpropeller from the aV subunit (purple), the ion at the ADMIDAS (ice-blue), the ion at the MIDAS (ochre), the ion at the LIMBS (violet). (b) Binding sites and (d) interaction energies of the cyclic pentapeptide RGDFV and integrin aVb3 (PDB ID: 1L5G). The specific binding sites obtained from MD simulations coincided with the sites in the crystal structure of cyclic RGDFV in integrin aVb3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the MIDAS become stronger with increasing reaction time, and the final distance was not over 3.0 Å; Salt bridges between ArgRGD and Asp218 and between ArgRGD and Asp150 were formed, and the final distances were not over 4.0 Å. Several hydrogen bonds were also formed. When the electrostatic interaction energy was about 800 kJ/mol and the van der Waals interaction energy was about 60 kJ/mol (Fig. 2c), the interaction energy stabilized such that the binding was also stable. Electrostatic and van der Waals interactions respectively played important and supporting roles in the binding of RGD to integrin aVb3. The crystal structure of cyclic RGDFV in integrin aVb3 may be used to verify the accuracy of the simulation results, as shown in Fig. 2b and d. Based on the sites in the crystal structure, AspRGD binds to a metal ion located at the MIDAS; the ArgRGD guanidinium group is held in place by a bidentate salt bridge to Asp218 at the bottom of the groove and by an additional salt bridge to Asp150 at the rear of Asp218 [22]. Binding sites that remain unchange in the simulation are consistent with the results of linear RGD described above (Fig. 2a and b). The electrostatic interaction energy between the cyclic RGD and integrin aVb3 is about 450 kJ/mol (Fig. 2d), which is lower than that observed for linear RGD and integrin aVb3 (800 kJ/mol). This difference in energy may be due to interactions between two carboxyl groups of AspRGD in linear RGD and ions at the MIDAS in integrin aVb3; these interactions may increase the electrostatic interaction energy. The results obtained prove that simulation of the recognition process of ligands to proteins using MD simulations is both possible and reasonable. It is worth mentioning, except for the above interaction forms, the RGD peptide also forms stable water bridges with some residues in integrin aVb3. In the case of cyclic RGDFV, each residue in

the cyclic RGDFV can form water bridges with integrin aVb3. But ArgRGD forms stable water bridges with Asp150 and Asp218, and the occupancies are up to 65.3% and 70% respectively; AspRGD forms stable water bridges with Asn215 and Ser213 (Fig. 3a), and the occupancies are up to 97.3% and 92.7% respectively. However in the case of linear RGD tripeptide, before the complete combination, AspRGD form water bridges with Asn215, Ser123 and Ala218; GlyRGD can form stable water bridges with Asp218, which is different from the case of cyclic RGDFV (Fig. 3b), and the occupancy is up to 69%, proving that GlyRGD also plays a favorable role in the combination. Some important structure characteristics are found in the sites of bpropeller from the aV subunit and bA from the b3 subunit. Several Asp and Glu residues were close to each other in bpropeller (Fig. 3c), and these structure provide large negative charge, which promotes the combination of RGD peptides with integrin aVb3 by forming salt bridges. As shown in Fig. 3d, there were three Ca2þ ions at the MIDAS, ADMIDAS and LIMBS, and some residues interacted with them. The residues interacting with the Ca2þ ion at the MIDAS moved away from the ion during the binding of RGD to integrin aVb3. Table 1 shows the distance changes (from 0 ns to 80 ns) between the Ca2þ ions and the O atoms of the interacting residues. The distances between the Ca2þ ion at the MIDAS and its interacting residues increased, because of the strong interaction between RGD peptide and the Ca2þ ion. However, the distances between the Ca2þ ion at the ADMIDAS and its interacting residues as well as that between the Ca2þ ion at the LIMBS and its corresponding interacting residues did not change significantly. In contrast to the cases of the residues away from the ions, the O atom (OE1) of Glu220 formed new contacts with the Ca2þ ions at the MIDAS and ADMIDAS (Fig. 3d).

1670

Y.-P. Yu et al. / Biomaterials 35 (2014) 1667e1675

Fig. 3. (a) Water bridges between the cyclic RGDFV and integrin aVb3. (b) Water bridges between the linear RGD and integrin aVb3. (c) Binding sites in bpropeller from the aV subunit. Several Asp and Glu residues, charged negatively, attract ArgRGD to combine with integrin aVb3. Asp218 and Asp150 in the most exposed positions are easiest to combine with ArgRGD. (d) Binding sites of bA from the b3 subunit. The distances between the Ca2þ ion at the MIDAS and its interacting residues increase, because of the strong interaction between RGD peptide and the Ca2þ ion.

During binding, water molecules around the three ions move father away from the three ions. Thus, the ions are exposed, for easy interaction with AspRGD. The linear RGD system shows that AspRGD can interact with the ion at the ADMIDAS, which results in a distance of 2.7 Å (Fig. 2a). Craig et al. [43] reported that AspRGD formed a new contact with the ion at the LIMBS within the first 10 ps of equilibration. This same phenomenon was observed in our simulations. Therefore, ions at the ADMIDAS and the LIMBS can interact with AspRGD when the geometry is acceptable, but the likelihood of such interactions occurring is much lower than that for the ion at the MIDAS. The special structures of integrin aVb3 and RGD peptides determine the binding sites. All the three metal ions are able to interact with the RGD peptide, and the mechanism is the interaction between the residues and the metal ions.

3.2. Effect of RGD quantity on the interaction mechanism with integrin aVb3 Experiments on RGD-containing peptide binding to integrin

multiple RGD peptides on the interaction mechanism with integrin

aVb3 was studied by MD simulations. As shown in Fig. 4Ia, two RGD peptides are spontaneously binding to integrin aVb3 and located near the headpiece of integrin aVb3. Fig. 4Ib shows that the two RGD can gather together and form salt bridges. One RGD peptide not only binds to the ion at the MIDAS but also forms salt bridges with Asp150. Another RGD peptide forms only salt bridges with Asp218. Because of interactions between two RGD peptides, they each combine at one site in integrin aVb3 respectively (Fig. 4Ic), which can explain why the RGD multimer always has the higher tumor uptake and longer tumor retention times than its monomer, despite of the distance of the linker among RGD motifs [44e48]. Multiple RGD peptides can increase the probability of combination with integrin aVb3. Two RGD peptides can also bind to the same integrin aVb3: one binds to Asp150 and the other binds to the ion at the MIDAS.

Table 1 The distance changes (from 0 ns to 80 ns) between the Ca2þ ions and the O atoms of the interacting residues (in Å).

aVb3 require a proper concentration of RGD-containing peptides. Multiple RGD peptides are used to have the drug micelle bind to integrin aVb3 specifically. In fact, one or more RGD peptides used to reach near the headpiece of integrin aVb3, because the binding sites of RGD to integrin aVb3 are in this region. One RGD peptide usually binds to one integrin aVb3 via the interaction mechanism described above. However, the interaction mechanism of multiple RGD peptides to integrin aVb3 remains poorly understood. Thus, the effect of

Ca2þ at MIDAS OG (Ser121)a OG (Ser123) OD1 (Asp251) OD2 (Asp119)

0.06 1.93 4.57 2.90

Ca2þ at ADMIDAS OD2 (Asp158) OD2 (Asp217) OD1 (Asn215) O (Pro219)

0.15 0 0.10 0.12

Ca2þ at LIBNS O (Ser123) OD1 (Asp126) OD1 (Asp127) O (Met335)

0.19 0.04 0.02 0.04

a Types of O atoms are marked according to the force field of Amber 03 in GROMACS-4.5.2 package.

Y.-P. Yu et al. / Biomaterials 35 (2014) 1667e1675

1671

Fig. 4. Configurations of the binding of multiple RGD peptides to integrin aVb3: (Ia), (Ib), and (Ic) are for System I (two RGD peptides); (IIa), (IIb), and (IIc) for System II (three RGD peptides); (IIIa), (IIIb), and (IIIc) for System III (four RGD peptides).

Combination with integrin aVb3 does not become better as the local RGD peptide concentration increases. Liu et al. [49,50] reported that two factors, multimeric and high local RGD concentration, contribute to high integrin aVb3 binding affinity, and that a proper linker length among RGD motifs depending on the tumor integrin aVb3 density determines integrin aVb3 binding affinity. To explore the effect of multiple RGD peptides on the interaction mechanism with integrin aVb3, three or more RGD peptides with integrin aVb3 were simulated. Fig. 4IIa shows three RGD peptides near integrin aVb3 interacting with each other and gathering together during the binding process. The third RGD (in violet) binds to the ion at the MIDAS, and the distance between them is 3.7 Å (Fig. 4IIb). However, all the three RGD peptides do not bind to integrin aVb3, orient away from integrin aVb3 and separate finally (Fig. 4IIc). Interactions among three RGD peptides promote difficulties in their binding to integrin aVb3. However, one RGD must eventually bind to integrin aVb3. Fig. 4IIIa shows four RGD peptides near integrin aVb3. As the RGD peptides gather together (Fig. 4IIIb), interactions between them induce difficulties in fitting into the crevice and binding to integrin aVb3 (Fig. 4IIIc). None of RGD peptides bound to integrin aVb3 due to gathering. Therefore, the concentration is not the greater the better, and the distance among RGD had better not be too close. Thus, the linker among RGD motifs, which promotes RGD peptides binding to the adjacent integrin aVb3, plays a key role in avoiding gathering in multiple RGD peptides system [48,51]. The interaction energy between one RGD and integrin aVb3 was about 900 kJ/mol (Fig. 5a). Fig. 5b shows the interaction energy between two RGD peptides and integrin aVb3, energies of about 700 and 300 kJ/mol were observed. In addition, the interaction energy between the first RGD peptide and integrin aVb3 is larger than that

between the second RGD peptide and integrin aVb3, which further confirms that the electrostatic interactions between residues and metal ions is stronger than that between two residues. The interaction energy between two RGD peptides is larger than that between the second RGD and integrin aVb3 (from 40 ns to 60 ns). Because during that period, the second RGD mainly interacts with the first RGD, so that the second RGD is not far away from the integrin aVb3. When the geometry is acceptable, the second RGD binds to Asp150 in integrin aVb3. The probability of two RGD peptides binding to integrin aVb3 is higher than that for of one RGD binding to integrin aVb3. Fig. 5c shows the interaction energy between a third RGD and integrin aVb3 decreases rapidly at 40 ns, but increases later. The third RGD may bind to integrin aVb3, but interactions between this molecule and the two other RGDs cause it to move away from integrin aVb3. Interaction among RGD peptides hinders RGD binding to integrin aVb3. As illustrated in Fig. 5d, the interaction energies between each RGD peptide and integrin aVb3 are similar to the interaction energies between RGD peptides, and none of the four RGD peptides binds to integrin aVb3, such that no obvious decreasing trend in energy is observed until 100 ns. Compared with one or two RGD peptides, three or four RGD peptides are more difficult to bind to one integrin aVb3 because of interactions and aggregation between RGD peptides. The results indicate that there exists an appropriate concentration of RGD peptides for optimum combination with integrin aVb3. High concentrations of RGD promote peptide gathering, which affects the binding of RGD to integrin aVb3. In the case of RGD multimer, if the linker among RGD motifs is too short, RGD peptides will interact with each other, which decrease the binding affinity of the RGD multimer to integrin aVb3.

1672

Y.-P. Yu et al. / Biomaterials 35 (2014) 1667e1675

Fig. 5. Interaction energies between RGD and integrin aVb3 or other RGD peptides: (a) One RGD peptide with integrin aVb3; (b) System I (two RGD peptides); (c) System II (three RGD peptides); and (d) System III (four RGD peptides). The probability of RGD binding to integrin aVb3 in system I is higher than that of one RGD binding to integrin aVb3. Compared with one or two RGD peptides, three or more RGD peptides are more difficult to bind to integrin aVb3 because of interactions among peptide molecules and their aggregation.

3.3. Effect of RGD structure on the interaction mechanism with integrin aVb3 The binding ability of cyclic RGD-containing peptides to integrin

aVb3 is significantly better than that of the linear RGD-containing peptides. Sutcliffe-Goulden et al. [52] showed that linear RGDcontaining peptides rapidly break down in vivo, resulting in high liver uptake with no specific accumulation in the tumor. However, cyclic RGD-containing peptides appear to be fairly resistant to proteolysis. They are conformationally constrained and trapped in active conformation, which suggests that cyclization improves tumor-targeting efficacy [53]. Bogdanowich-Knipp et al. [54] found a dramatic increase in stability of the cyclic RGD-containing peptides compared with the linear RGD-containing peptides under neutral conditions. This increase in stability is due to decreased structural flexibility imposed by the ring, which prevents the Asp side chain carboxylic acid from orienting itself in an appropriate position for attack on the peptide backbone. However, the dynamic process of RGD-containing peptides is difficult to be observed at the molecular level. Thus, the effect of RGD structure at the molecular level on the interaction mechanism with integrin aVb3 is explored in this section with the aim of understanding RGD-based targeted drug therapy. MD simulation results support the view that cyclic RGD has higher binding affinity to integrin aVb3 than linear RGD. Four sets of systems were simulated. Fig. 6IVa shows that linear RGD is located near the headpiece of integrin aVb3. During the binding process, ArgRGD forms salt bridges with Asp218, with a distance of 2.7 Å; and with AspRGD, with a distance of 2.8 Å (Fig. 6IVb). With increasing simulation time, the salt bridges between ArgRGD and AspRGD broke, while salt bridges between ArgRGD and Asp218 were maintained. The interaction between AspRGD and

the ion at the MIDAS became stronger with decreasing distance (Fig. 6IVaeIVc). Linear RGD overcomes the electrostatic interactions of the salt bridges for binding to the ion at the MIDAS, which proves that the electrostatic interaction between AspRGD and metal ions is stronger than that between salt bridges. Fig. 6Va shows that linear arginineeglycineeaspartate-asparagine (RGDN) is located near the headpiece of integrin aVb3. During the binding process, ArgRGDN and AsnRGDN form salt bridges, the distance between NH1 of ArgRGDN and OD1 of AsnRGDN is 3.4 Å, and the distance between NH1 of ArgRGDN and OD2 of AsnRGDN is 2.8 Å (Fig. 6Vb). Salt bridges are more stable compared with the system IV, because they can persist from 5 ns to 100 ns, which inhibits the binding of linear RGDN to integrin aVb3 (Fig. 6Vc). Fig. 6VIa shows that linear aspartateeserineealanineeprolineeglycineeargininee glycineeaspartate (DSAPGRGD) is located near the headpiece of integrin aVb3. During the binding process, the first AspDSAPGRGD forms salt bridges with ArgDSAPGRGD (Fig. 6VIb), whose interaction leads to the rolling-over of the linear DSAPGRGD configuration and affect its combination with integrin aVb3 (Fig. 6VIc). Linear RGDcontaining peptides in different sequences behave various combinations when binding to the integrin aVb3. Results show that the own combination of linear RGD-containing peptides may occur before binding to integrin aVb3, which leads to low tumor-targeting efficacy. By contrast, combination is not possible for cyclic RGD because of the rigidity of the peptide structure. As showed in Fig. 6VIIa, cyclic RGDFV is located near the headpiece of integrin aVb3. During the binding process, cyclic RGDFV is close to integrin aVb3 and can thus bind to integrin aVb3. Its cyclic configuration is stable, and there is no own backbone combination like the linear RGD (Fig. 6VIIb and VIIc). Cyclic RGDFV has been observed to be 20- to over 100-fold better inhibitors of cel1 adhesion

Y.-P. Yu et al. / Biomaterials 35 (2014) 1667e1675

1673

Fig. 6. Configurations of different RGD-containing peptides to integrin aVb3 during the binding process: (IVa), (IVb), and (IVc) are for System IV (the linear tripeptide RGD); (Va), (Vb), and (Vc) for System V (the linear tetrapeptide RGDN); (VIa), (VIb), and (VIc) for System VI (the linear octapeptide DSAPGRGD); and (VIIa), (VIIb), and (VIIc) for System VII (the cyclic pentapeptide RGDFV). Linear RGD-containing peptides interact with their own backbone easily; however, such interaction is impossible for the cyclic RGD-containing peptide.

to vitronectin and/or laminin fragment Pl compared with a linear variant or GlyeArgeGlyeAspeSer [55]. The superiority of cyclic RGD in integrin aVb3 binding affinity and tumor-specific intake can be further proved from the trends of interaction energies and RMSD. The energies of the interaction of cyclic RGD with integrin aVb3 decrease faster and are generally lower than those of linear RGD (Fig. 7a). As well, RMSD studies show that configurational changes in linear RGD are greater than those of the cyclic form (Fig. 7b). Because of the flexible structure of linear RGD, its ability to combine with integrin aVb3 is not markedly better than that of cyclic RGD. The structure of RGD-containing peptides is important for the targeted binding of RGD-containing peptides to integrin aVb3. The tumor-homing cyclic peptide iRGD was recently reported to be a tumor-targeting and tumor-penetrating vector for antitumor therapy. In addition, iRGD homes and spreads within tumor tissue far more efficiently than linear CRGDC [56e58]. Its cyclic structure contributes much to the superiority of iRGD. Because of the flexible structure of the linear, ArgRGD in linear RGD can form salt bridges with Glu, Asp, and the two carboxyl oxygen atoms of the C-terminus.

AspRGD in linear RGD can form salt bridges with Lys, Arg, His and the amide N of the N-terminus around. Thus, when considering the synthesis of RGD monomers or polymers, that these residues interact with Arg or Asp should be noted and care must be taken to avoid adding these residues to the sequence of RGD-containing peptides. Cyclic RGD is better than the linear RGD for use in targeted delivery of anti-angiogenic therapy at the molecular level. Interactions between Asp and the metal ions determine the binding ability of RGD-containing peptides to integrin aVb3. 4. Conclusion Molecular dynamics simulations were performed to study the dynamic process of RGD-containing peptides binding to integrin aVb3, and the effects of RGD structure and quantity on this interaction mechanism with integrin aVb3 were analyzed at the molecular level. Specific binding activity was mainly driven by the electrostatic interactions between the residues and the metal ions. In addition, water bridges and the van der Waals interactions also

1674

Y.-P. Yu et al. / Biomaterials 35 (2014) 1667e1675

Fig. 7. (a) Interaction energies between different RGD-containing peptides and integrin aVb3. (b) RMSD of the different RGD-containing peptides. The interaction energy between cyclic RGDFV and integrin aVb3 is lower and more stable than that between linear RGD and integrin aVb3. And the configurational changes in linear RGD are greater than those of the cyclic form.

played a supporting role in the combination of RGD to integrin aVb3. Studies on the effect of RGD quantity on the interaction mechanism indicated that a proper concentration of RGD peptides is required to achieve optimum binding. Two RGD peptides are best for binding to the same integrin aVb3. Cyclic RGD-containing peptides behave higher binding affinity to integrin aVb3 than linear RGD. These results provide a deeper understanding of the interaction mechanism between RGD peptides and integrin aVb3 at the molecular level, which is significant in improving the application prospects of RGDbased vectors in tumor imaging and targeted drug delivery of antiangiogenic therapy. Acknowledgments

[15]

[16] [17] [18]

[19]

[20] [21]

This work was financially supported by the National Natural Science Foundation of China (Nos. 21273200 and J1210042). The authors would like to thank scientific computing cloud platform of Beijing computational science for one month free to share computing resources.

[22]

[23] [24]

References [25] [1] Danhier F, Breton AL, Prèat V. RGD-based strategies to target alpha (v) beta (3) integrin in cancer therapy and diagnosis. Mol Pharma 2012;9:2961e73. [2] Kerbel RS. Antiangiogenic therapy: a universal chemosensitization strategy for cancer? Science 2006;312:1171e5. [3] Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 2007;6:273e86. [4] Arnaout MA. Integrin structure: new twists and turns in dynamic cell adhesion. Immunol Rev 2002;186:125e40. [5] Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in angiogenesis and lymphangiogenesis. Nat Rev Cancer 2008;8:604e17. [6] Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nat Rev Cancer 2002;2:91e100. [7] Amaout MA, Mahalingam B, Xiong JP. Integrin structure, allsotery, and bidirectional signaling. Annu Rev Cell Dev Biol 2005;21:381e410. [8] Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding to integrins. J Biol Chem 2000;275:21785e8. [9] Ruoslahti E. RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 1996;12:697e715. [10] Hynes RO. A reevaluation of integrins as regulators of angiogenesis. Nat Med 2002;8:918e21. [11] Dedhar S, Ruoslahti E, Pierschbacher MD. A cell surface receptor complex for collagen type I recognizes the Arg-Gly-Asp sequence. J Cell Biol 1987;104: 585e93. [12] Temming K, Schiffelers RM, Molema G, Kok RJ. RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature. Drug Res 2005;8:381e402. [13] Dubey PK, Mishra V, Jain S, Mahor S, Vyas SP. Liposomes modified with cyclic RGD peptide for tumor targeting. J Drug Target 2004;12:257e64. [14] Haubner R, Weber WA, Beer AJ, Vabuliene E, Reim D, Sarbia M, et al. Noninvasive visualization of the activated aVb3 integrin in cancer patients

[26]

[27]

[28]

[29] [30]

[31]

[32]

[33] [34]

[35]

by positron emission tomography and [18F] Galacto-RGD. PLoS Med 2005;2: 244e52. Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G, et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res 2004;32:e149. Askari JA, Buckley PA, Mould AP, Humphries MJ. Linking integrin conformation to function. J Cell Sci 2009;122:165e70. Luo BH, Carman CV, Springer TA. Structural basis of integrin regulation and signaling. Annu Rev Immunol 2007;25:619e47. Mould AP, Barton SJ, Askari JA, McEwan PA, Buekley PA, Craig SE, et al. Conformational changes in the integrin bA domain provide a mechanism for signal transduction via hybrid domain movement. J Biol Chem 2003;278: 17025e35. Cluzel C, Saltel F, Lussi J, Paulhe F, Imhof BA, Wehrle-Haller B. The mechanisms and dynamics of aVb3 integrin clustering in living cells. J Cell Biol 2005;171: 383e92. Gottschalk KE, Kessler H. A computational model of transmembrane integrin clustering. Structure 2004;12:1109e16. Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scitt DL, et al. Crystal structure of the extracellular segment of integrin aVb3. Science 2001;294: 339e45. Xiong JP, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, et al. Crystal structure of the extracellular segment of integrin alpha v beta 3 in complex with an Arg-Gly-Asp ligand. Science 2002;296:151e5. Arnaout MA, Goodman SL, Xiong JP. Coming to grips with integrin binding to ligands. J Cell Biol 2002;14:641e51. Gurrath M, Muller G, Kessler H, Aumailley M, Timpl R. Conformation/activity studies of rationally designed potent anti-adhesive RGD peptides. Eur J Biochem 1992;210:911e21. Dechantsreiter MA, Planker E, Mathä B, Lohof E, Holzemann G, Jonczyk A, et al. N-methylated cyclic RGD peptides as highly active and selective aVb3 integrin antagonists. J Med Chem 1999;42:3033e40. Haubner R, Gratias R, Diefenbach B, Goodman SL, Jonzyk A, Kessler H. Structural and functional aspects of RGD-containing cyclic pentapeptides as highly potent and selective integrin aVb3 antagonists. J Am Chem Soc 1996;118: 7461e72. Xiong JP, Mahalingham B, Alonso JL, Borrelli LA, Rui XL, Anand S, et al. Crystal structure of the complete integrin aVb3 ectodomain plus an a/b transmembrane fragment. J Cell Biol 2009;186:589e600. Chen W, Lou J, Hsin J, Schulten K, Harvey SC, Chen Z. Molecular dynamics simulations of forced unbending of integrin aVb3. PLoS Comput Biol 2011;7: e1001086. Dong X, Mi LZ, Zhu J, Wang W, Hu P, Luo BH, et al. aVb3 integrin crystal structures and their functional implications. Biochemistry 2012;51:8814e28. Nagae M, Re S, Mihara E, Nogi T, Sugita Y, Takagi J. Crystal structure of a5b1 integrin ectodomain: atomic details of the fibronectin receptor. J Cell Biol 2012;197:131e40. Trong IL, McDevitt TC, Nelson KE, Staytou PS, Stenkamp RE. Structural characterization and comparison of RGD cell-adhesion recognition sites engineered into streptavidin. Acta Crystallogr Sect D 2003;59:828e34. Berendsen HJC, Van der Spoel D, Van Drunen R. GROMACS: a message-passing parallel molecular dynamics implementation. Comput Phys Commun 1995;91:43e56. Sorin EJ, Pande VS. Exploring the helix-coil transition via all-atom equilibrium ensemble simulations. Biophysical 2005;88:2472e93. DePaul AJ, Thompson EJ, Patel SS, Haldeman K, Sorin EJ. Equilibrium conformational dynamics in an RNA tetraloop from massively parallel molecular dynamics. Nucleic Acids Res 2010;38:4856e67. Duan Y, Wu C, Chowdhury S, Lee MC, Xiong G, Zhang W, et al. A point-charge force field for molecular mechanics simulations of proteins based on

Y.-P. Yu et al. / Biomaterials 35 (2014) 1667e1675

[36] [37] [38] [39] [40] [41] [42] [43]

[44]

[45]

[46]

[47]

condensed-phase quantum mechanical calculations. Comput Chem 2003;24: 1999e2012. Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph Model 1996;14:33e8. Lindahl E, Hess B, Van Der Spoel D. GROMACS 3.0: a package for molecular simulation and trajectory analysis. J Mol Model 2001;7:306e17. Darden T, York D, Pedersen L. Particle mesh ewald e an n$log(n) method for ewald sums in large systems. J Chem Phys 1993;98:10089e92. Hess B, Bekker H, Berendsen HJC, Fraaije J. LINCS: a linear constraint solver for molecular simulations. J Comput Chem 1997;18:1463e72. Miyamoto S, Kollman PA. Settle: an analytical version of the shake and rattle algorithm for rigid water models. J Comput Chem 1992;13:952e62. Barlow DJ, Thornton JM. Ion-pairs in proteins. J Mol Biol 1983;168:867e85. Xu D, Tsai CJ, Nussinov R. Hydrogen bonds and salt bridges across proteinprotein interfaces. Protein Eng 1997;10:999e1012. Craig D, Gao M, Schulten K, Vogel V. Structural insights into how the MIDAS ion stabilizes integrin binding to an RGD peptide under force. Structure 2004;12:2049e58. Chen XY, Liu S, Hou Y, Tohme M, Park R, Bading J, et al. MicroPET imaging of breast cancer aV-integrin expression with 64Cu-labeled dimeric RGD peptides. Mol Imaging Biol 2004;6:350e9. Poethko T, Thumshirn G, Hersel U, Rau F, Haubner R, Schwaiger M, et al. Improved tumor uptake, tumor retention and tumor/background ratios of pegylated RGD multimers. J Nucl Med 2003;44:46P. Poethko T, Schottelius M, Thumshirn G, Herz M, Haubner R, Henriksen G, et al. Chemoselective pre-conjugate radiohalogenation of unprotected mono- and multimeric peptides via oxime formation. Radiochim Acta 2004;92:317e27. Thumshirn G, Hersel U, Goodman SL, Kessler H. Multimeric cyclic RGD peptides as potential tools for tumor targeting: solid-phase peptide synthesis and chemoselective oxime ligation. Chem Eur J 2003;9:2717e25.

1675

[48] Poethko T, Schottelius M, Thumshirn G, Hersel U, Herz M, Henriksen G, et al. Two-step methodology for high yield routine radiohaligenation of peptides: 18 F-labeled RGD and octreotide analogs. J Nucl Med 2004;45:892e902. [49] Liu S. Radiolabeled multimeric cyclic RGD peptides as integrin aVb3 targeted radiotracers for tumor imaging. Mol Pharm 2006;3:472e87. [50] Liu S. Radiolabeled cyclic RGD peptides as integrin aVb3-targeted radiotracers: maximizing binding affinity via bivalency. Bioconjug Chem 2009;20: 2199e213. [51] Wu Y, Zhang XZ, Xiong ZM, Cheng Z, Fisher DR, Liu S, et al. MicroPET imaging of glioma aV-integrin expression using 64Cu-labeled tetrameric RGD peptide. J Nucl Med 2005;46:1707e18. [52] Sutcliffe-Goulden JL, O’Doherty MJ, Marsden PK, Hart IR, Marshall JF, Bansal SS. Rapid solid phase synthesis and biodistribution of 18F-labelled linear peptides. Eur J Nucl Med 2002;29:754e9. [53] Ogawa M, Hatano K, Oishi S, Kawasumi Y, Fujii N, Kawaguchi M, et al. Direct electrophilic radiofluorination of a cyclic RGD peptide for in vivo aVb3 integrin related tumor imaging. Nucl Med Biol 2003;30:1e9. [54] Bogdanowich-Knipp SJ, Chakrabarti S, Williams TD, Dillman RK, Siahaam TJ. Solution stability of linear vs. cyclic RGD peptides. J Pept Res 1999;53:530e41. [55] Aumailley M, Gurrath M, Müller G, Calvete J, Timpl R, Horst K. Arg-Gly-Asp constrained within cyclic pentapoptides strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1. FEBS Lett 1991;291:50e4. [56] Su S, Wang H, Liu X, Wu Y, Nie G. iRGD-coupled responsive fluorescent nanogel for targeted drug delivery. Biomaterials 2013;34:3523e33. [57] Ye Y, Zhu L, Ma Y, Niu G, Chen X. Synthesis and evaluation of new iRGD peptide analogs for tumor optical imaging. Bioorg Med Chem Lett 2011;21: 1146e50. [58] Sugahara KN, Teesalu T, Karmali PP, Kotamraju VR, Agemy L, Girard OM, et al. Tissue-penetrating delivery of compounds and nanoparticles into tumors. Cancer Cell 2009;16:510e20.