Modeling of a C-end rule peptide adsorbed onto gold nanoparticles.

The RPAR peptide, a prototype C-end Rule (CendR) sequence that binds to neuropilin-1 (NRP-1), has potential therapeutic uses as internalization trigger in anticancer nanodevices. Recently, the functionalization of gold nanoparticles with CendR peptides has been proved to be a successful strategy to target the NRP-1 receptor in prostate cancer cells. In this work, we investigate the influence of two gold surface facets, (100) and (111), on the conformational preferences of RPAR using molecular dynamics simulations. Both clustering and conformational analyses revealed that the peptide backbone becomes very rigid upon adsorption onto gold, which is a very fast and favored process, the only flexibility being attributed to the side chains of the two Arg residues. Thus, the different components of RPAR tend to adopt an elongated shape, which is characterized by the pseudo-extended conformation of both the backbone and the Arg side chains. This conformation is very different from the already known bioactive conformation, indicating that RPAR is drastically affected by the substrate. Interestingly, the preferred conformations of the peptide adsorbed onto gold facets are not stabilized by salt bridges and/or specific intramolecular hydrogen bonds, which represent an important difference with respect to the conformations found in other environments (e.g. the peptide in solution and interacting with NRP-1 receptor). However, the conformational changes induced by the substrate are not detrimental for the use of gold nanoparticles as appropriate vehicles for the transport and targeted delivery of the RPAR. Thus, once their high affinity for the NRP-1 receptor induces the targeted delivery of the elongated peptide molecules from the gold nanoparticles, the lack of intramolecular interactions facilitates their evolution towards the bioactive conformation, increasing the therapeutic efficacy of the peptide.

In silico molecular engineering for a targeted replacement in a tumor-homing peptide.

A new amino acid has been designed as a replacement for arginine (Arg, R) to protect the tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) from proteases. This amino acid, denoted (Pro)hArg, is characterized by a proline skeleton bearing a specifically oriented guanidinium side chain. This residue combines the ability of Pro to induce turn-like conformations with the Arg side-chain functionality. The conformational profile of the CREKA analogue incorporating this Arg substitute has been investigated by a combination of simulated annealing and molecular dynamics. Comparison of the results with those previously obtained for the natural CREKA shows that (Pro)hArg significantly reduces the conformational flexibility of the peptide. Although some changes are observed in the backbone...backbone and side-chain...side-chain interactions, the modified peptide exhibits a strong tendency to accommodate turn conformations centered at the (Pro)hArg residue and the overall shape of the molecule in the lowest energy conformations characterized for the natural and the modified peptides exhibit a high degree of similarity. In particular, the turn orients the backbone such that the Arg, Glu, and Lys side chains face the same side of the molecule, which is considered important for bioactivity. These results suggest that replacement of Arg by (Pro)hArg in CREKA may be useful in providing resistance against proteolytic enzymes while retaining conformational features which are essential for tumor-homing activity.

Protonation of the side group in beta- and gamma-aminated proline analogues: effects on the conformational preferences.

This work shows the influence of the side-chain protonation on the conformational properties, relative stabilities, and peptide bond isomerization of four aminoproline isomers. Thus, this research has been useful to define the rules that allow control the conformation of aminoproline with the pH. Comparison of the results obtained using density functional theory calculations for the N-acetyl-N'-methylamide derivatives of the protonated isomers, which differ in the beta- or gamma-position of the substituent and its cis or trans relative disposition, with those reported for the corresponding neutral analogues (J. Phys. Chem. B 2008, 112, 14045) has allowed us to reach the following conclusions: (i) protonation of the amino group produces a reduction of the backbone conformational flexibility and a destabilization of the cis configuration of the amide bond involving the pyrrolidine nitrogen; (ii) the planarity of the peptide bond is broken in some cases to form strong side chain...backbone interactions, which induce a very significant pyramidilization at the amide nitrogen atom; (iii) as was also detected for the neutral analogues, the formation of side chain...backbone intraresidue interactions favor the cis disposition of the substituent; and (iv) protonation of the amino side group increases the energy gaps that separate the different investigated isomers resulting in an enhancement of the destabilization of the dipeptides with the substituent attached in a trans position.

Conformational preferences of beta- and gamma-aminated proline analogues.

Quantum mechanical calculations have been used to investigate how the incorporation of an amino group to the Cbeta- or Cgamma-positions of the pyrrolidine ring affects the intrinsic conformational properties of the proline. Specifically, a conformational study of the N-acetyl-N'-methylamide derivatives of four isomers of aminoproline, which differ not only in the beta- or gamma-position of the substituent but also in its cis or trans relative disposition, has been performed. To further understand the role of the intramolecular hydrogen bonds between the backbone carbonyl groups and the amino side group, a conformational study was also performed on the corresponding four analogues of (dimethylamino)proline. In addition, the effects of solvation on aminoproline and (dimethylamino)proline dipeptides have been evaluated using a self-consistent reaction field model, and considering four different solvents (carbon tetrachloride, chloroform, methanol and water). Results indicate that the incorporation of the amino substituent into the pyrrolidine ring affects the conformational properties, with backbone...side chain intramolecular hydrogen bonds detected when it is incorporated in a cis relative disposition. In general, the incorporation of the amino side group tends to stabilize those structures where the peptide bond involving the pyrrolidine nitrogen is arranged in cis. The aminoproline isomer with the substituent attached to the Cgamma-position with a cis relative disposition is the most stable in the gas phase and in chloroform, methanol and water solutions. Replacement of the amino side group by the dimethylamino substituent produces significant changes in the potential energy surfaces of the four investigated (dimethylamino)proline-containing dipeptides. Thus, these changes affect not only the number of minima, which increases considerably, but also the backbone and pseudorotational preferences. In spite of these effects, comparison of the conformational preferences, i.e., the more favored conformers, calculated for different isomers of aminoproline and (dimethylamino)proline dipeptides showed a high degree of consistency for the two families of compounds.

The energy landscape of a selective tumor-homing pentapeptide.

Recently, a potentially powerful strategy based on phage-display libraries has been presented to target tumors via homing peptides attached to nanoparticles. The Cys-Arg-Glu-Lys-Ala (CREKA) peptide sequence has been identified as a tumor-homing peptide that binds to clotted plasmas proteins present in tumor vessels and interstitium. The aim of this work consists of mapping the conformational profile of CREKA to identify the bioactive conformation. For this purpose, a conformational search procedure based on modified simulated annealing combined with molecular dynamics was applied to three systems that mimic the experimentally used conditions: (i) the free peptide; (ii) the peptide attached to a nanoparticle; and (iii) the peptide inserted in a phage display protein. In addition, the free peptide was simulated in an ionized aqueous solution environment, which mimics the ionic strength of the physiological medium. Accessible minima of all simulated systems reveal a multiple interaction pattern involving the ionized side chains of Arg, Glu, and Lys, which induces a beta-turn motif in the backbone observed in all simulated CREKA systems.

Conformational preferences of alpha-substituted proline analogues.

DFT calculations at the B3LYP/6-31+G(d,p) level have been used to investigate how the replacement of the alpha hydrogen by a more sterically demanding group affects the conformational preferences of proline. Specifically, the N-acetyl-N'-methylamide derivatives of L-proline, L-alpha-methylproline, and L-alpha-phenylproline have been calculated, with both the cis/trans isomerism of the peptide bonds and the puckering of the pyrrolidine ring being considered. The effects of solvation have been evaluated by using a Self-Consistent Reaction Field model. As expected, tetrasubstitution at the alpha carbon destabilizes the conformers with one or more peptide bonds arranged in cis. The lowest energy minimum has been found to be identical for the three compounds investigated, but important differences are observed regarding other energetically accessible backbone conformations. The results obtained provide evidence that the distinct steric requirements of the substituent at C (alpha) may play a significant role in modulating the conformational preferences of proline.

Conformations of proline analogues having double bonds in the ring.

The intrinsic conformational preferences of proline analogues having double bonds between carbon atoms in their rings have been investigated using quantum mechanical calculations at the B3LYP/6-31+G(d,p) level. For this purpose, the potential energy surface of the N-acety-N'-methylamide derivatives of three dehydroprolines (proline analogues unsaturated at alpha,beta; beta,gamma; and gamma,delta) and pyrrole (proline analogue with unsaturations at both alpha,beta and gamma,delta) have been explored, and the results are compared with those obtained for the derivative of the nonmodified proline. We found that the double bonds affect the ring puckering and the geometric internal parameters, even though the backbone conformation was influenced the most. Results indicate that the formation of double bonds between carbon atoms in the pyrrolidine ring should be considered as an effective procedure to restrict the conformational flexibility of prolines. Interestingly, we also found that the N-acetyl-N'-methylamide derivative of pyrrole shows a high probability of having a cis peptide bond preceding the proline analogue.