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

Abstract

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.

Figure 1

(a) Schematic representation of the system under study showing the position of the particles (black dots) used to define the virtual dihedral angles (see Methods Section). (b) Bioactive conformation proposed for CREKA (according to ref. 5). (c) CREKA analogue constructed by replacing the Arg residue by (Pro)hArg within the bioactive conformation. The Arg and (Pro)hArg side chains are depicted in yellow.

Figure 2

Structure of arginine (Arg) and its homologue containing an additional methylene group (hArg), as well as that of their respective proline-like derivatives considered in this work.

Figure 3

Dihedral angles used to identify the conformations of the N-acetyl-N'-methylamide derivatives of (Pro)Arg (a) and (Pro)hArg (b) studied in this work. The dihedral angles ω₀, φ, ψ and ω are defined using backbone atoms, whereas the endocyclic dihedral angles (χⁱ) and ξ¹, respectively, are given by the atoms of the five-membered ring. In particular, the sequence of atoms used to define φ, χ⁰ and ξ¹ are C(O)-N-C^α-C(O), C^δ-N-C^α-C^β and C^β-C^γ-CH₂-N, respectively.

Figure 4

Minimum energy conformations of Ac-(Pro)Arg-NHMe obtained from B3LYP/6-31+G(d,p) calculations: (a) γ_L[u]g⁺t; (b) γ_L[d]ts^-; (c) γ_L[d]s⁺s⁺; (d) ε_L[d]g^-s⁺; (e) γ_L[d]g^-s⁺ (see Table 1 for geometries). Distances (H···O) and angles (N-H···O) associated with the backbone···backbone and backbone···side chain interactions (dashed lines) are given.

Figure 5

Minimum energy conformations of Ac-(Pro)hArg-NHMe obtained from B3LYP/6-31+G(d,p) calculations: (a) γ_L[d]tg⁺g^-; (b) ε_L[d]g^-g⁺g⁺; (c) γ_L[d]g^-tt (see Table 3 for geometries). Distances (H···O) and angles (N-H···O) associated with the backbone···backbone and backbone···side chain interactions (dashed lines) are given.

Figure 6

Electrostatic parameters determined for the (Pro)hArg residue.

Figure 7

(a) Number of unique minimum energy conformations found for CR*EKA (grey line and diamonds) and natural CREKA (black line and circles) against the number of modified SA-MD cycles used for the conformation search. (b) Distribution of energies for the unique minimum energy conformations of CR*EKA and natural CREKA. Energies are computed relative to the corresponding lowest energy minimum.

Figure 8

Comparison of the distribution of virtual dihedral angles used to define the backbone conformation of CREKA (a) and CR*EKA (b) in each unique minimum energy structure obtained. Color code for the bars: black for dihedral angle values ranging from 0° to 60°, red from 60° to 120°, green from 120° to 180°, blue from 180° to 240°, yellow from 240° to 300° and grey from 300° to 360°.

Figure 9

Ramachandran plot distribution for the five residues of CR*EKA (open circles) and CREKA (filled black circles) considering the more representative minimum energy structures, i.e. those within a relative energy interval of 2 kcal/mol.

Figure 10

Distribution of the unique minimum energy structures generated for CR*EKA (top) and CREKA (bottom) in clusters, which have been grouped on the basis of the formation of hydrogen bonds and salt bridges.

Figure 11

Lowest energy minimum obtained for CR*EKA (a) and CREKA (b) attached to a nanoparticle, and superposition of both structures (c). The surface used to mimic the nanoparticle is represented by a single green ball.