key: cord-0264847-uxux9037
authors: Chi, L. América; Asgharpour, Somayeh; Correa-Basurto, José; Bandala, Cindy Rodríguez; Martínez-Archundia, Marlet
title: Unveiling the G4-PAMAM capacity to bind and protect Ang-(1-7) bioactive peptide
date: 2022-05-24
journal: bioRxiv
DOI: 10.1101/2022.05.23.493150
sha: 99b4bbafd302d5e3ffd7f72eb040c87075899213
doc_id: 264847
cord_uid: uxux9037

New therapies that allow natural healing processes are required. Such as the endogenous peptide called Angiotensin-(1-7), a safe and eff e drug, which is able to re-balance the Renin-Angiotensin system affected during several pathologies, including the new COVID-19; cardiovascular, renal, and pulmonary disease; diabetes; neuropathic pain; Alzheimer and cancer. However, one of the limiting factors for its application is its unfavorable pharmacokinetic profile. In this work, we propose the coupling of Angiotensin-(1-7) to PAMAM dendrimers in order to evaluate the capacity of the nanocarrier to improve isolated peptide features and to gain insight into the structural as well as the energetic basis of its molecular binding. The In Silico tests were performed in acidic and neutral pH conditions as well as amino-terminated and hydroxyl-terminated PAMAM dendrimers. High-rigor computational approaches, such as molecular dynamics and metadynamics simulations were used. We found that, at neutral pH, PAMAM dendrimers with both terminal types are able to interact stably with 3 Angioteinsin-(1-7) peptides through ASP1, TYR4 and PRO7 key aminoacids, however, there are some differences in the binding sites of the peptides. In general, they bind on the surface in the case of the hydroxyl-terminated compact dendrimer and in the internal zone in the case of the amino-terminated open dendrimer. At acidic pH, PAMAM dendrimers with both terminal groups are still able to interact with peptides either internalized or in its periphery, however, the number of contacts, the percentage of coverage and the number of HBs are lesser than at neutral pH, suggesting a state for peptide release. In summary, amino-terminated PAMAM dendrimer showed slightly better features to bind, load and protect Angiotensin-(1-7) peptides.

The Renin-Angiotensin system (RAS) regulates the blood pressure and also balances ion and other fluids in the human body. For it to function properly, the RAS rests on two opposite axis ( Figure 1 ): 1) in the classical arm, inflammatory, profibrotic, vasoconstrictor and oxidative effects are triggered by the binding of bioactive octapeptide Angiotensin II (Ang-II)

to Angiotensin Type 1 (AT1) cell membrane receptor; meanwhile, 2) in the protective arm, opposite anti-inflammatory, anti-fibrotic, vasodilator and anti-oxidant effects are triggered by the binding of the bioactive heptapeptide Angiotensin 1-7 (Ang-(1-7)) to Mitochondrial Assembly (MAS) proto-oncogene which is a G-protein-coupled receptor (GPCRs). 1,2 There must be a balance between the two main axes, however, under several pathological conditions this balance is shifted towards the classical arm leading to catastrophic effects, as in the case of the worsening of COVID-19 disease. [3] [4] [5] [6] [7] Figure 1: Diagram representing the main metabolic effects driven by the Renin Angiotensin System bioactive peptides (Ang II and Ang-(1-7)) when interacting with its cell receptors AT1 and MAS, respectively. The main proteolytic cleavage of Ang-(1-7) by angiotensinconverting enzyme (ACE) and dipeptidyl peptidase 3 (DPP3), which is associated with Ang-(1-7) rapid degradation and limited therapeutic application. cyclodextrin molecule, 14 liposome-entrapped of angiotensin-(1-7) 15 and an Ang-(1-7) eluting polymer-coated medical device. 16 One of the most promising approaches is the physically entrapment of a drug inside a dendrimer-based nanocarrier using non-covalent interactions. 17, 18 Dendrimers are strong candidates because they offer structural stability, high carrying capacity of molecules, water solubility, modifiable surface functionality, available internal cavities, pH dependence properties and a high capacity to cross cell barriers together with an ease of synthesis or commercial availability. 19 Most of the previous studies have focused on PAMAM, among all dendrimers and hydroxyl-terminated PAMAM dendrimer has been additionally proven to be a non-cytotoxic variant compared with the original amino-terminated PAMAM dendrimer that shows a slight cytotoxic effect. 20 By using the latter strategy, the capacity of PAMAM-OH dendrimer as an Ang(1-7) carrier was previously explored by means of theoretical and experimental studies 21 and main conclusions pointed to: a) one dendrimer could be able to capture two peptides; b) Ang-(1-7) could be protected by the dendrimer in a range between 50-65 %; c) Ang-(1-7)/dendrimer forms stable complexes over 60 ns MD which is confirmed by electrophoretic shift assay; and d) Ang-(1-7)/PAMAM-OH complex had an anti-atrophic effect when administered intraperitoneally in mice. We now extend the strategy to design Ang-(1-7)/PAMAM-(OH/NH) complexes by using Ang-(1-7) NMR structure instead of a molecular model, performing molecular docking taking into account the dendrimer and the peptide flexibility, considering different pH conditions in order to glimpse a possible loading and unloading mechanism and extending sampling of the systems.

In summary, we coupled Ang- (1) (2) (3) (4) (5) (6) (7) to the most studied kind of dendrimers, PAMAM dendrimers, in order to evaluate their capacity to improve the Ang-(1-7) pharmacokinetic properties and to gain insight into the host-guest (dendrimer/Ang-(1-7)) molecular binding.

Understanding the basis of the host-guest molecular binding is fundamental when a very specific and controlled drug release is needed. It is important to note that the beneficial effect of Ang-(1-7) peptide take place only at low concentrations and overdosing might interfere with its receptor-associated functions. 12 Different pH conditions (acidic and neutral) and two different group terminals (amino and hydroxyl) were tested as they are critical factors for modulating drug delivery and cytotoxicity. Rigorous computational approaches, such as molecular dynamics (MD) and metadynamics (MTD) have been used to evaluate the structural and energetic properties of the Ang-(1-7), dendrimer or the new complexes formed.

Finally, we propose that the best complexes found can be experimentally tested to validate the properties predicted through In Silico approaches, as a potential therapeutic agent.

Molecular models: The NMR structure of Ang-(1-7) peptide (ASP-ARG-VAL-TYR-ILE-HIS-PRO) in solution was retrieved from the protein database with ID: 2JP8; the topology was created using the pdb2gmx module of GROMACS 5.0.7 22 and its parameters were downloaded from the GROMOS-compatible 2016H66 force field; 23 protonation states were assigned according to physical-chemical properties predicted with peptide calculator server 24 and verified by using ProPkA software 25 (zero peptide charge for neutral pH and +2 for acidic conditions) as shown in the Figures S1-S3 of the Supplemental Material.

Two types of PAMAM dendrimers were considered: Generation 4 amino-terminated ethylenediamine (EDA)-cored poly(amidoamine) dendrimer (PAMAM-NH 2 ) and generation 4 hydroxyl-terminated ethylenediamine (EDA)-cored poly(amidoamine) dendrimer (PAMAM-OH) as depicted in Figures S4-S5 . The initial structures and topologies of the dendrimers were created using the newly developed dendrimer topology builder called pyPolyBuilder. 26 The parameters of the dendrimer and its partial charges were obtained from the GROMOScompatible 2016H66 force field. 23 Previously, systematic evaluation of the accuracy of this force field was performed in the simulation of PAMAM dendrimers. 27 Characteristics of the dendrimers building-blocks used in this work can be found in Table S .5-S.17 of the Supporting Information from the work of Ramos et. al. 27 and in Table S1 of this work. Two pH values were considered: acidic pH of 3 and neutral pH of 7. To mimic pH conditions, the protonation states for the PAMAM dendrimers were assigned according to the experimental work by Cakara et al.: 28 at neutral pH, the primary amines were considered protonated and at low pH, both primary and tertiary amines were considered protonated ( Figure S6 ).

Dendrimers and peptide structures were placed separately in a box of SPC water model and then subjected to minimization, equilibration and production of standard MD simulations using GROMACS 5.0.7 22 software, to obtain initial relaxed structures of both types of molecules. Systems were named as: PNH n , POH n , PNH a and POH a , Ang-(1-7) n and Ang-(1-7) a , where letters "n" and "a" correspond to neutral pH and acidic pH, respectively. Abbreviations PNH and POH correspond to amino-terminated PAMAM and hydroxyl-terminated PAMAM dendrimers, respectively.

Clustering: Representative structures were selected from each MD using clustering by conformations. In the case of dendrimers, the middle structure of the 3 largest clusters was taken as the representative conformation, meanwhile, for peptide, the middle structures in all of the obtained clusters were used unless the cluster had less than 10 members.

Molecular docking: A double molecular docking was performed in order to have initial dendrimer/peptide complexes by using Autodock Vina 29 program. We tested all the conformers of the dendrimer and the peptide previously selected. First, a blind rigid docking was performed separately for all combinations of the conformers (each dendrimer conformer is docked to each peptide conformer). Once the peptide conformer found a binding site, a second flexible local docking was performed in this area for each system allowing a better adaptation in the binding site. According to the number of hydrogen bonds (HBs) and Vina affinity, one peptide conformer was selected for each dendrimer site avoiding overlapping.

We found that peptides can interact with 3 up to 4 different dendrimer sites for both types of terminal groups. Thus, we decided to choose a configuration with 3 peptides because these peptide conformers fulfill better the above-mentioned criteria, it would be interesting for future works to systematically test the number of peptides attached to each dendrimer.

Detailed results are presented in supplemental material (Table S2 ). The double molecular docking approach was developed using our in-house bash script algorithm. 30 MD for complexes: A multi-ligand complex was added to each dendrimer type MD simulation and at each pH value. A total of 4 complexes: [PNH-A] n , [POH-A] n , [PNH-A] a and [POH-A] a , where letters correspond to neutral pH (n), acidic pH (a) and aminoterminated PAMAM (PNH), hydroxyl-terminated PAMAM (POH) and ang-(1-7) peptides (A). Each complex was placed separately in a SPC model water box and then the three steps of energy minimization, equilibration and production of standard MD were performed using GROMACS 5.0.7 software. 22

Metadynamics: In metadynamics (MTD), an external history-dependent bias potential is constructed in the space of a few selected degrees of freedom namely the collective variables (CVs), the effect of the metadynamics bias potential being to push the system away from local minima into exploring new regions of the phase space. 31 In the long time limit, the bias potential converges to minus the free energy 32 (or a proportional quantity in the case of well-tempered metadynamics (WT-MTD)) 33 showing its real flexibility. For Ang-(1-7) metadynamics, we first used the R g as CV, however, it was not enough to describe the peptide behaviour in solution and led to a stucked system in low R g values as depicted in Figure S7 . In our final set up, we used the radius of gyration (R g ) and the total number of donor-acceptor contacts (C d-a ) as collective variables because they are appropriate descriptors of the behavior of flexible molecules in solution. 34 C d-a was calculated using a switching functions inspired by the work of Barducci et al, 35

where r 0 was set to 0.25 nm and n and m were set to 6 and 12, respectively. The sum runs over all of the pairs i, j of Hydrogen, Nitrogen and Oxygen polar atoms and d ij is the distance between each i and j atoms. In order to limit the R g sampling, an upper and a lower harmonical restraint was applied. The restraining potential is in the form:

where k has to be large enough to prevent the peptide to get stuck in an extended/compressed conformation, R g (t) is the value of the R g at time t. R g,max and R g,min are the maximum and minimum values of the R g that we are interested in.

During MD simulations each system was placed in a cubic simulation box such that all dendrimer/peptide/complex atoms were, at least, 1 nm distant from the box edges. Atomic-based pairlist generation was used as implemented in the Verlet algorithm. When needed V-rescale 38 thermostat at 298 K and Parrinello-Rahman 39 barostat at 1 bar were used. This protocol is based on a systematic study that has been recently carried out, confirming that the present scheme is valid. 27 Details on the molecular systems considered in the present work are presented in Table 1 and Table 2 . 

The Root-mean-square deviation (RMSD) of atomic positions is a standard measure of the structural changes mainly used for proteins or non-protein small organic/inorganic molecules.

Nevertheless, due to the large conformational flexibility of dendrimers in aqueous solution,

there is a debate as to whether it is an appropriate molecular descriptor to account for its structural stability. Here, we have employed RMSD analysis to test the convergence of the simulations, in order to obtain equilibrated initial structures for the following steps. As shown in Fig 2a, the PNH n RMSD values undergo large changes from the starting structure in the first 50 ns, afterwards the system seems to reach a stable behavior; meanwhile PNH a RMSD values undergo big changes from the starting structure in the first 20 ns, afterwards the system seems to reach a stable behavior fluctuating around a mean value. The POH n RMSD values undergo a rapid change during the first ns, after that, the RMSD steadily deviates slowly from this new conformation, reaching a new steady-state after 100 ns; meanwhile, POH a RMSD values undergo big changes from the starting structure in the first 20 ns, afterwards the system seems to reach a stable behavior fluctuating around a mean value.

Ang-(1-7) n and Ang-(1-7) a RMSD values show a more stable behavior from the first ns.

According to these results, we agreed to use the last 100 ns of the simulations as equilibrated data for all the systems. Large changes observed on dendrimer structures are mainly due to the fact that the initial structures were obtained by PyPolyBuilder algorithm which only gives us an initial guess for the dendrimer geometry in vacuum.

The radius of gyration (R g ) might be a more appropriate molecular descriptor to account for the structural stability of dendrimers, it gives a measure for the compactness of a molecular structure. As shown in Figure 2b , the PNH n and PNH a R g values show a more stable behavior from the first nanoseconds around a mean value. POH n and POH a R g values show Table 3 , confirming that GROMOS-compatible 2016H66 force field, together with our chosen methodology were capable to represent the structural properties of the dendrimers used in this work. It can be observed that POH n and POH a has in general a more compact structure (lower R g ) compared with PNH n and PNH a , respectively (Figure 2b ). At neutral pH, this is partly due to the preference of the group terminals to be surrounded by waters (PNH n , Figure 3a) or to interact with internal dendrimer groups (POH n , Figure 3b) , from the total possible interactions between group terminals and waters/internal dendrimer groups, we found that PNH n forms 95 % of the interactions with waters and 5 % with internal groups, meanwhile, POH n forms 85 % of interactions with waters and 15 % with the internal groups. The R g and RMSD fluctuations in the case of PNH n , PNH a and POH a throughout the simulation time, compared to that of POH n , suggests that the formers have a more flexible structure than the latter. This result might be attributed to the continuous movement of the protonated terminal amine groups in PNH n and terminal/internal protonated groups in PNH a and POH a to minimize the unfavorable enthalpic penalty due to electrostatic repulsion on charges of the same sign. To characterize the shape of the dendrimers, an asphericity parameter δ was estimated.

It is important to note that the closer this value is to zero, the more spherical the dendrimer becomes and its calculation is described in detail elsewhere. 27 As shown in Table 4 and Figure 4 , PNH n assume a less spherical structure compared to POH n , meanwhile, PNH a and POH a show an almost spherical behavior. This high asphericity observed in PNH n , might be due to an asymmetry induced by a high degree of charged terminal groups back folding as also reported in the previous works, 27 in contrast, the neutral terminal in POH n seems to be able to accommodate in a more compact way due to its less charge-charge terminal repulsion and its preference of interactions with internal dendrimer groups. In the case of PNH a and POH a , an almost spherical shape might be mainly due to the repulsion between charged tertiary amine groups in the interior of the dendrimers. To characterize how particle density (waters, terminal nitrogens, internal nitrogens and ions) varies as a function of the distance from the dendrimer center, radial distribution functions were measured. As shown in Figure 6a , a higher level of structuration is evidenced by the pronounced peaks on the distributions on PNH n , PNH a and POH a internal nitrogens compared with those of POH n , this is mainly due to a more dense POH n dendrimer compared with less dense, PNH n , being more evident on PNH a and POH a , as evidenced by the height of the peaks. As it can be seen, the dense-core model 49 is confirmed at neutral pH, this model states that the monomer (internal Nitrogen atoms) density distribution is high close to their centers and decays as it approaches the periphery, as evidenced of the height of the peaks from the center to the periphery at neutral pH. However, at acidic pH, dendrimers assume a less dense inner cavity, stretched, open conformation like to the dense-shell model 49 as a consequence of the strong Coulomb repulsions between the charged units. This is in agreement with theoretical models that predicts that the PAMAM-NH 2 core becomes denser upon decreasing the acidity of the medium. Interestingly, this could led to a different drug interaction behaviour and load capacity, probably allowing better drug encapsulation in the big inner cavities at acidic pH ( Figure 5 ).

As shown in Fig 6b, showed a conformational equilibrium between the random coil and β-sheet (or a mixture of bend structures), with a bend stabilized by interactions between residues VAL3 and TYR4, this is in very well agreement with our results in Figure 8 , where we can see that the most frequent secondary motif is bent between VAL3 and TYR4. In our classical MD simulations at acidic pH, we found that 60 % of the structures were similar to the NMR solution structure (RMSD < 2 Å when superimposed), meanwhile, at neutral pH, this happened only in 40 % of the frames, thus, this conformation was less frequent at neutral pH ( Figure 9 ). this is a R g in the region spanning from 0.6 to 0.7 nm and contacts ranging between 30 and 35. Interestingly, the structures in the basin showed a persistent HB between ASP1 and TYR4 or between ASP1 and VAL3 (Figure 10b ). This HB was also frequently observed on The one dimensional FES as a funcion of R g is shown in Figure 10c , as can be seen, there are not large free energy barriers (around 1 kT) dividing the metastable states in the main basin A. It is important to note that only 35 % of the structures in basin A were similar to the NMR structure and in agreement with classical MD. This result is coherent with the conformational equilibrium between the random coil and a mixture of bend structures reported experimentally for Ang-(1-7). However, basin A is characterized by a significant degree of flexibility of the N-terminal and C-terminal tails which samples multiple conformations that deviate from the ensemble of the NMR structure as shown in Figure 11 .

Together, the R g , δ and RDFs results validates our chosen methodology and confirms that GROMOS-compatible 2016H66 force field is capable to model the theoretical and experimen- 

The middle structure of the most populated clusters for dendrimers and the peptide were used for the molecular docking of each dendrimer conformer with each of the peptide conformers. Our methodology allowed us to find a suitable binding site for the peptide by using a representative peptide conformation taken from the MD, once the peptide finds a reasonable site, the algorithm allows it to better accommodate into this local site, as can be schematized in Figure 12 .

Final complexes resulted from the docking are shown in Figure 13 . As can be seen, PNH n allows the interaction of the peptides with its inner shells as well as with its surface. POH n , Figure 11 : RMSF of Basin A main clusters as a function of aminoacid backbone atoms. Structures representative of the basin A main clusters are superimposed in the right figure.

Grey represent the NMR structure. Threshold of 0.15 nm is marked to visualize the most flexible atoms. Cluster 1 is marked also with impulses to guide visualization. Figure 12 : Double-docking schematic representation. Each of the peptides conformer is allowed to find a site by using a blind rigid docking, afterwards, once the peptide found a reasonable site, a flexible docking allows it to better accommodate into the site.

only allow the peptide interactions with its surface due to its more compact structure. We can observe that at acidic pH, dendrimers allow a better encapsulation of 3 peptides. Details on the HBs formed between peptides and dendrimers are presented in Tables 5 and 7 . 

To identify global structural changes due to complex formation, the RMSD and the R g at bound state were measured and compared with that of the free structures. It can be seen in Figure 14ac that the binding of peptides stabilizes the dendrimer structure according to RMSD values. Meanwhile, as can be seen in Figure 14bd , the radius of gyration of the dendrimer and the peptides are not importantly affected by the binding. To test complex stability, the distance between the center of mass (COM) of the dendrimer and each peptide was measured. As showed in Figure 15a and Figure 18a , under neutral pH conditions, 2 peptides remained stable bonded during the simulation in the dendrimer internal domain around a distance of 1 nm and a third peptide remained stable bonded at the dendrimer surface around a distance of 3 nm, peptides remain mainly in the same initial sites. The dendrimer kept an open hourglass geometrical shape during the whole simulation time, allowing at least two peptides to be near the core. As shown in Figure 15d , under acidic pH conditions, from the 3 peptides initially at around 1 nm in the dendrimer internal domain, one remained very close to the core during all the simulation time, two went out quickly and stayed near the superficial zone, even leaving the dendrimer at certain points. From the two peptides in the dendrimer surface, one went to the back of the core at the last 50 ns of the simulation, interacting with the other peptide in the core. It appears that for two peptides, transitions from in to out the dendrimer had low energetic barriers or was an unstable state, in such a way that it is easy to get out, meanwhile, the third peptide appears to be in a stable deep minimum that forces it to stay close to the dendrimer core, but further research is required to confirm this hypothesis, due to the system flexibility, a MTD simulation could give us more insight about this process. Whith these results we can conclude that only one peptide remains stably bonded at the core of the dendrimer at acidic pH during the whole MD simulation. To test the protection capabilities of each dendrimer, the percent coverage of peptides due to binding, was measured. The free peptide in solution was taken as a reference, representing This is reasonable considering that 2 peptides are in the superficial zone during most of the simulation time and even leave the dendrimer at certain points. If we consider only the peptide that remains at the core, it still has less coverage percentage compared with the ones at the core at neutral pH, probably because due to the open structure at acidic pH, cavities are also deferentially hydrated. It could mean that the peptide might be a little more accessible to water but due to its localization inside the dendrimer, it is not necessarily accessible to peptides.

The peptide protection is especially relevant for bonds susceptible to hydrolytic attack by endopeptidasas, as schematized in Figure 17 . are between internal branches of PNH n with ASP1 side chain in the peptide, but with much low occupations than in the case of neutral pH. According to Figure 19a , at neutral pH, TYR4 and ILE5 residues, interacted preferably with internal dendrimer groups rather than with the terminal groups, which was expected due to its hydrophobic/partially hydrophobic nature. On the other hand, ASP1 and PRO7 residues, interacted preferably with terminal dendrimer groups rather than with the internal groups, which was expected due to its negative charged side-chain/C-terminal groups. ARG2, VAL3 and HIS6 interacts similarly with terminal or internal dendrimer groups but in a less frequent way. As expected, arginine residue was more exposed to solvent due to its charged aminoacid side chain. According to Figure 19b , at acidic pH, ASP1, ARG2, TYR4 and HIS6 keep in contact preferably with internal dendrimer groups. Due to a more open dendrimer cavities, ASP1 and ARG2 are able to enter and interact with internal polar groups compared with the neutral case. Due to its hydrophobic/partially hydrophobic nature TYR4 keeps in contact preferably with internal dendrimer as in the neutral case but less frequently. HIS6

at acidic pH is considered as positively charged, while at neutral pH is neutral, this change modify its preference to be in contact with internal dendrimer groups instead with positively charged terminal groups. Finally, VAL3, ILE5 and PRO7 hydrophobic residues keep almost no contact with the dendrimer, here C-terminal is neutral compared with the neutral case.

Taking into account the number of peptides stably bonded near the core, the higher percent coverage of peptides from water, the major number of HBs between dendrimer and peptides during the whole simulation time, the frequency in which this HBs occur and the peptide and dendrimer frequency of contacts, it is likely that amino terminated PAMAM dendrimer can act as a better encapsulator near neutral pH and as a release agent under acidic pH. However, further studies are needed in this direction.

We measured RMSD as a kind of first indicator of stability upon complex formation. It can be seen in Figure 20ac that the binding of peptides slightly stabilizes the dendrimer structure at neutral pH but, it does not happen at acidic pH. Besides that, the structure of the peptides are not greatly affected by the binding to the dendrimer. For their part, the R g of either the dendrimer or the peptide are not importantly affected by the binding. Higher fluctuations on dendrimer RMSD and R g at acidic pH correspond to the continuous movement of the protonated groups which makes it difficult to be stabilized by ligand binding.

To further evaluate complex stability, the distance between the Center Of Mass (COM) of the dendrimer and each peptide was measured. As shown in Figure 21a , under neutral pH conditions, all the three Ang-(1-7) peptides remained at a stable distance of 1.6 ± 0.1 nm from the dendrimer COM, this is, mostly on the dendrimer surface taking into account that the R g is of 1.46 nm, as also illustrated in the timeline evolution of frames in Figure 23a .

A closer view into the simulations shows that peptides in [POH-A] n , explore the nearby area around its initial binding site, which could be due to the symmetry of the dendrimer groups. Moreover, the spherical compacted shape of the dendrimer appears to avoid peptide's complete internalization. The above results might imply that the energetic barriers between superficial binding sites are low, allowing the peptide to go from one local minimum to another, in contrast, the energy barrier needed to leave the dendrimer appears to be high enough, keeping the peptides interacting with the dendrimer as shown in Figure 22a compared with its formation free in solvent (40 % of the simulation time), this imply that these intra peptide HBs are at least equally broken but this is not necessarily in favor of a peptide/dendrimer interaction (Table 7) , it is also because part of the time the residue is solved exposed and interacts with waters. According to Figure 24a , at neutral pH, TYR4 interacts preferably with internal dendrimer groups rather than with the terminal groups, which was expected due to its partially hydrophobic aromatic nature which prefers to be buried in a hydrophobic core and its po- Particularly, the inclusion of the TYR residue is favored in both dendrimer types, as in the case of β-cyclodextrin complexes. 14 In the other hand, ASP1, ARG2, VAL3 and PRO7 residues, stay in contact slightly more with terminal dendrimer groups rather than with the internal groups, in case of negative charged ASP1 and C-terminal PRO7 with polar hydroxil terminal group in the dendrimer, arginine residue was in contact with terminal groups in the dendrimer and exposed to solvent due to its charged aminoacid side chain. ILE5 and HIS6

keep in contact similarly with terminal or internal dendrimer groups. At acidic pH, ASP1, ARG2, VAl3 and TYR4 interact preferibly with internal dendrimer groups. ILE5, HIS6 and PRO7 interact equally with internal and terminal groups but in a less frequent manner.

Taking into account the number of peptides stable bonded at the surface, the higher per- This complex was evolved in an MD during 200 ns, evolution of the peptides is showed in Figure S7 . As can be seen, initially, peptides are close from the core in the internal voids of the dendrimer, eventually the dendrimer becomes more and more compact and finally, the peptides remain interacting at the dendrimer surface. This give us an idea of what could happen if the encapsulation is previously done, the complex at neutral pH evolve to a situation similar to the presented previusly (Figure 24a ), dendrimer prefers a compact structure and peptides interact mostly with external surface. This is the case of reference, 21

where dendrimer initial open structure for complex formation appears to be taken from a non-equilibrated structure, eventually dendrimer becomes more and more compact, even releasing one of the peptides, but at the end of the 60 ns MD productions, simulations does not necessarily reach convergence and could show a meta-stable state where peptides are partially encapsulated. Here, we show that in the last 100 ns of the MD production, most part of the peptides are interating with external dendrimer parts whether it started with the peptides in or out, with slightly differences. Details are showed in Figure S8 .

Structural parameters such as the radius of gyration, the asphericity and the radial distribution function confirmed that the GROMOS-compatible 2016H66 force field, together with our chosen methodology are capable to model the theoretical and experimental structural features of PAMAM dendrimers evaluated in this work.

The classical MD simulations of PAMAM dendrimers in solution showed that hydroxylterminated PAMAM dendrimer at neutral pH has a more compact, less structured, more spherical, less hydrated and less flexible structure than amino-terminated PAMAM dendrimer. These results emphasises how a single change in terminal group could lead to differential behaviour of dendrimer structure and eventually in Ang-(1-7) binding. Also, this structural information is envisioned to prove useful for the encapsulation of other drugs.

The classical MD simulations of Ang-(1-7) in solution yielded to conformations in which a bend secondary structure is stabilized by interactions between residues VAL3 and TYR4, which is in agreement with NMR experimental studies. It was also found that at acidic pH, 60 % of the Ang-(1-7) conformations were similar to the NMR solution structure, meanwhile, at neutral pH, this also happened but in a slightly less frequent percentage of conformations.

In the accelerated MD metadynamics simulations, Ang-(1-7) free energy surface showed a unique broad free energy minimum on the radius of gyration and donor-acceptor contact phase space, confirming the Ang-(1-7) flexibility due to its peptide nature. One dimensional free energy surface as a function of radius of gyration showed that free energy barriers between metastable states in the main basin are on the order of 1 kT, thus easily passed.

At neutral pH, PAMAM dendrimers with both terminal types are able to interact with at least 3 peptides in a stable way, with a coverage of up to 70%. However, hydroxyl-terminated PAMAM dendrimer only partially encapsulate the peptides due to its compact structure.

At acidic pH, PAMAM dendrimers with both terminal groups are still able to interact with positevely charged Ang-(1-7) peptides, either internalized or in its periphery, however, contacts, coverage and HBs are lesser than at neutral pH, suggesting a state for peptide release.

With the results found so far, it appears that amino-terminated PAMAM dendrimer at neutral pH posses slightly better features to load and protect Ang-(1-7) peptides than hydroxyl-terminated PAMAM dendrimer. However, either hydroxyl-terminated PAMAM dendrimer as well as amino-terminated PAMAM dendrimer are able to bind Ang-(1-7) peptides in a differential form, suggesting that hydroxyl-terminated PAMAM dendrimer is able to act as nanocarrier/nanotransporter more than nanoprotector of this peptide.

For the PAMAMNH(OH)/Ang-(1-7) complexes, the therapeutic mechanism could be encapsulate or capture the peptide at near neutral pH and deliver them at acidic pH, where repulsions between protonated primary and/or tertiary amines give rise to a open and hydrated environment, allowing positively charged peptides to be solvated and released easily.

Finally, we propose that the complexes studied here should be experimentally tested to validate the properties predicted through In Silico approaches, as potential therapeutic agents.

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Angiotensin-(1-7) with thioether bridge: an angiotensin-converting enzyme-resistant, potent angiotensin-(1-7) analog

Study of angiotensin-(1-7) vasoactive peptide and its b-cyclodextrin inclusion complexes: Complete sequence-specific NMR assignments and structural studies

Long-lasting cardiovascular effects of liposome-entrapped angiotensin-(1-7) at the rostral ventrolateral medulla

Angiotensin-(1-7) eluting polymercoated medical device to reduce restenosis and improve endothelial cell function

In silico search, chemical characterization and immunogenic evaluation of amino-terminated G4-PAMAM-HIV peptide complexes using three-dimensional models of the HIV-1 gp120 protein

Complexation of peptide epitopes with G4-PAMAM dendrimer through ligand diffusion molecular dynamic simulations

How to study dendrimers and dendriplexes III. Biodistribution, pharmacokinetics and toxicity in vivo

PAMAM dendrimer-cell membrane interactions

The complex of PAMAM-OH dendrimer with Angiotensin (1-7) prevented the disuse-induced skeletal muscle atrophy in mice

GROMACS: fast, flexible, and free

A GROMOS-compatible force field for small organic molecules in the condensed phase: The 2016H66 parameter set

com: a set of web utilities for the calculation of peptide and peptoid properties and automatic mass spectral peak assignment

Graphical analysis of pH-dependent properties of proteins predicted using PROPKA

Automated Preparation of Molecular Topologies and Initial Configurations for Molecular Dynamics Simulations of Arbitrary Supramolecules

Molecular dynamics simulations of PAMAM and PPI dendrimers using the GROMOS-Compatible 2016H66 Force Field

Microscopic protonation equilibria of poly (amidoamine) dendrimers from macroscopic titrations

AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading

Double-docking-approach

Wiley Interdisciplinary Reviews: Computational Molecular Science

Metadynamics: a method to simulate rare events and reconstruct the free energy in biophysics, chemistry and material science

Well-tempered metadynamics: a smoothly converging and tunable free-energy method. Physical review letters

Combining metadynamics simulation and experiments to characterize dendrimers in solution

Free-energy landscape of protein oligomerization from atomistic simulations

LINCS: a linear constraint solver for molecular simulations

Particle mesh Ewald: An Nlog(N) method for Ewald sums in large systems

Canonical sampling through velocity rescaling

Polymorphic transitions in single crystals: A new molecular dynamics method

Peptide folding: when simulation meets experiment

A time-independent free energy estimator for metadynamics

Dendrimer building toolkit: Model building and characterization of various dendrimer architectures

Association of nicotinic acid with a poly (amidoamine) dendrimer studied by molecular dynamics simulations

Structural investigation of PAMAM dendrimers in aqueous solutions using small-angle neutron scattering: effect of generation

Dynamics of star-burst dendrimers in solution in relation to their structural properties

Atomistic computer simulations on multi-loaded PAMAM dendrimers: A comparison of amine-and hydroxyl-terminated dendrimers

Host-guest complexation of curcumin and coumarin 6 with PAMAM-OH: Insight from fluorescence spectroscopy and molecular dynamics simulations

Association of a weakly acidic anti-inflammatory drug (ibuprofen) with a poly (amidoamine) dendrimer as studied by molecular dynamics simulations

Dendrimers in solution: insight from theory and simulation

vasoactive peptide and its β-cyclodextrin inclusion complexes: complete sequencespecific NMR assignments and structural studies

Peptide Dynamics and Metadynamics: Leveraging Enhanced Sampling Molecular Dynamics to Robustly Model Long-Timescale Transitions

Metabolism of angiotensin-(1-7) by angiotensin-converting enzyme

Converting enzyme determines plasma clearance of angiotensin-(1-7)