Molecular View on the i RGD Peptide Binding Mechanism: Implications for Integrin Activity and Selectivity Profiles

Abstract

Receptor-selective peptides are widely used as smart carriers for specific tumor-targeted delivery. A remarkable example is the cyclic nonapeptide iRGD (CRGDKPGDC, 1) that couples intrinsic cytotoxic effects with striking tumor-homing properties. These peculiar features are based on a rather complex multistep mechanism of action, where the primary event is the recognition of RGD integrins. Despite the high number of preclinical studies and the recent success of a phase I trial for the treatment of pancreatic ductal adenocarcinoma (PDAC), there is little information available about the iRGD three-dimensional (3D) structure and integrin binding properties. Here, we re-evaluate the peptide's affinity for cancer-related integrins including not only the previously known targets αvβ3 and αvβ5 but also the αvβ6 isoform, which is known to drive cell growth, migration, and invasion in many malignancies including PDAC. Furthermore, we use parallel tempering in the well-tempered ensemble (PT-WTE) metadynamics simulations to characterize the in-solution conformation of iRGD and extensive molecular dynamics calculations to fully investigate its binding mechanism to integrin partners. Finally, we provide clues for fine-tuning the peptide's potency and selectivity profile, which, in turn, may further improve its tumor-homing properties.

Chart 1. 2D Chemical Structure of iRGD (1)

Figure 1

Free energy surface (FES) of the folding process of 1 as a function of Dih_cor and H_bond CVs with isosurfaces displayed every 1.5 kJ/mol. The conformation representing the main free energy minimum is shown as the inset.

Figure 2

MD-predicted binding mode of iRGD at the orthosteric binding site of (A) αvβ3, (B) αvβ5, and (C) αvβ6 integrins. The different receptor subunits are depicted as colored surfaces (αv = gray, β3 = red, β5 = cyan, and β6 = green). Amino acids important for peptide binding are highlighted as sticks, while the Mg²⁺ ion in MIDAS is shown as a purple sphere. The ligand is represented as orange ribbons and sticks; nonpolar hydrogens are omitted for the sake of clarity; and H-bonds are shown as black dashed lines.

Figure 3

Ligand’s RMSD plots for the three simulated binding complexes. Prior to the RMSD computation, the trajectories were aligned on the Cα-atoms of the most stable secondary structure elements. Two different ligand’s reference conformations were used: (i) the first frame (equilibrated docking pose) of each MD complex (first row: A–C) and (ii) the average binding pose observed during each MD run (second row: D–F). The RMSD values (bolded lines) are smoothed with a rolling window of 5 ns, while the actual fluctuations are shown with a slight transparency.

Figure 4

iRGD (1) residues RMSF in three binding complexes. The computation was performed on all of the ligand’s heavy atoms.

Figure 5

Possible hints for achieving αvβ3/αvβ5 selectivity. The N-terminus of 1 in its predicted binding modes at αvβ3/αvβ5 is close to a subpocket where three key mutations occur. The αv and β3/β5 subunits are depicted as light and dark gray surfaces, respectively. The key mutations between the two receptors are highlighted as red (β3) and cyan (β5) sticks. The ligand is represented as orange ribbons and sticks; nonpolar hydrogens are omitted for the sake of clarity.

Figure 6

Superposition of the MD-predicted binding pose of iRGD at αvβ3 (A) and αvβ5 (B) with that of [RGD-Chg-E]-CONH₂ at αvβ6. The αv and β subunits are depicted as light and dark gray surfaces, respectively. The key mutations between the three receptors are highlighted as red (β3), cyan (β5), and green (β6) sticks, contoured by transparent surfaces. Peptides 1 and 2 are depicted as orange and magenta sticks, respectively; nonpolar hydrogens are omitted for the sake of clarity.

Figure 7

(A) Hints for increasing αvβ6 affinity and selectivity. A virtual library of nine compounds was designed by mutating Gly⁶ of 1 with natural and non-natural lipophilic amino acids (upper panel). Free energy surfaces (FESs) of the folding process of 4 with isosurfaces were displayed every 1.5 kJ/mol (lower panel). The horseshoe-like conformation of 4 and its superimposition with 1 are shown as insets. (B) Docking-predicted binding mode of compound 4 at the RGD binding site of the αvβ6 integrin. The different receptor subunits are depicted as colored surfaces (αv = gray and β6 = green). Amino acids important for peptide binding are highlighted as sticks, while the Mg²⁺ ion in the MIDAS is shown as a purple sphere. The ligand is represented as cyan ribbons and sticks; nonpolar hydrogens are omitted for the sake of clarity; and H-bonds are shown as black dashed lines.