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

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.


Supporting Information
Convergence was estimated as described in the Materials and Methods section for compound 1.The average exchange acceptance ratio was ≈ 25%.

Figure S1 .
Figure S1.Convergence of PT-WTE calculation on 1. A) Time evolution of the FES during the last 60 ns of simulation.B) Quantitative assessment of the error associated with the FES calculation trough block averages analysis.C) CVs diffusion in the six demuxed (continuous) trajectories.

Figure S2 .
Figure S2.Replica exchange plots of the PT-WTE simulation.A) Replica index found at each selected temperature as a function of time.B) Temperature at which each individual replica is simulated as function of time.The average round trip time with its standard error is 0.573 ± 0.015 ns.

Figure S5 .
Figure S5.A) Ramachandran plot of the avb5 homology model and B) RMSD plot of the secondary structure element (Ca atoms) over the 2 µs long MD simulation of avb5 in complex with iRGD.

Figure S6 .
Figure S6.Docking-predicted binding mode of iRGD at the RGD binding site of avb3, ab5 and avb6 integrins.The different receptors subunits are depicted as colored surfaces (av=grey, b3=red, b5=cyan and b6=green).Amino acids important for peptide binding are highlighted as sticks, while the Mg 2+ ion in the MIDAS is shown as a purple sphere.The ligand is represented as orange ribbon and sticks; nonpolar hydrogens ore omitted for sake of clarity; and H-bonds are shown as black dashed lines.

Figure S7 .
Figure S7.Interatomic distances between the C-ter carboxylic carbon of iRGD's Cys 9 with T315-Og 1 (A) and N317-Cg (B).The bolded lines show values of the distance smoothed with a rolling window of 5 ns, while the actual fluctuations are shown with a slight transparency.

Figure S8 .
Figure S8.3D representation of the upward rotation experienced by iRGD during the first stages of the MD simulation in complex with the avb5 receptor.The grey arrow represents the axis of the rotation.The receptor is depicted as light gray (av subunit) and cyan (b5 subunit) surfaces.The ligand backbone is shown in orange (initial MD frame) and red (final MD frame) cartoons, while the sidechain of Arg 2 and Asp 4 are as shown as sticks to highlight the typical RGD binding pattern.The divalent Mg 2+ cation at the MIDAS is depicted as a purple sphere.

Figure S11 .
Figure S11.A) RMSD plot of the backbone atoms of iRGD in complex with avb3 computed respect to the PT-WTE-predicted conformation of the peptide (B).Stability of the two intramolecular H-bonds (C and D) found in PT-WTE between Arg 2 (C-O)-Gly 6 (N-H) and Arg 2 (N-H)-Pro 7 (C-O), respectively.

Figure S12 .
Figure S12.A) RMSD plot of the backbone atoms of iRGD in complex avb5 computed respect to the PT-WTE-predicted conformation of the peptide (B).Stability of the two intramolecular H-bonds (C and D) found in PT-WTE between Arg 2 (C-O)-Gly 6 (N-H) and Arg 2 (N-H)-Pro 7 (C-O), respectively.

Figure S14 .
Figure S14.3D representation of the unusual Mg 2+ -chelation scheme and binding pattern experienced by iRGD in the avb6 receptor.The receptor is depicted as light gray (av subunit) and green (b6 subunit) surfaces.The ligand backbone is shown in orange (initial MD frame) cartoons, while the sidechain of Arg 2 and Asp 4 are as shown as sticks to highlight the loss of typical RGD binding pattern: the interaction of Arg 2 with (av)-D218 is lost and replaced by a salt-bridge with (av)-D150, while the Mg 2+ cation (purple sphere) is chelated by both the Asp 4 carboxylate and the backbone carbonyl of Gly 2 , leading to a distortion in the backbone conformation of the peptide.

Figure S15 .
Figure S15.Comparison of the dihedral values assumed by iRGD's f-Gly3 and y-Asp 4 in the three MD trajectories (A, B, C) with all the available experimental structures of RGD peptides in complex with RGD-integrin receptors.In each plot, the torsion values observed during the simulations are shown as dots colored based on their timestep.The f-Gly 3 and y-Asp 4 values measured in the experimental structures are depicted as black triangle markers.The list of the PDBs used for the analysis is the following: 2VDM, 2VDN, 2VDO, 2VDP 2VDQ, 2VDR, 3ZDY, 3ZDZ, 3ZE0, 3ZE1, 3ZE2, 4WK4, 4WK2, 4WK0, 3VI4, 4MMZ, 4MMY, 4MMX, 1L5G, 6MK0, 6MSL, 4UM9, 5FFO.

Figure S16 .
Figure S16.A) RMSD plot of the backbone atoms of iRGD in complex with avb6 computed respect to the PT-WTE-predicted conformation of the peptide (B).Stability of the two intramolecular H-bonds (C and D) found in PT-WTE between Arg 2 (C-O)-Gly 6 (N-H) and Arg 2 (N-H)-Pro 7 (C-O), respectively.

Figure S17 .
Figure S17.Schematic representation of the secondary structure elements of the integrins RGD binding site and SDL cavity.av subunit is shown as gray cartoon while a generic b* subunit is shown in beige.

Figure S18 .
Figure S18.3D representation of the RGD binding site of avb3 (A), avb5 (B) and avb6 (C) receptors.The most important mutations occurring at the SDL subpocket were highlighted in sticks.The different receptors subunits are depicted as colored surfaces (av=grey, b3=red, b5=cyan and b6=green).

Figure S19 .
Figure S19.Superposition of the crystal structure of cilengitide at avb3 (PDB code: 1L5G) with the MD-predicted binding pose of iRGD at avb3 (A) and avb5 (B).iRGD is shown as orange sticks and ribbon, while cilengitide is colored in white.The different receptors subunits are depicted as colored surfaces (av=grey, b3=red, b5=cyan).

Figure S20 .
Figure S20.Results of the PT-WTE calculations on the designed compounds 3-7.All the shown FES were computed after 150 ns (per replica) of simulation.As for the parent peptide 1, in all the cases metadynamics converged after about 80-100 ns.Convergence was estimated as described in the Materials and Methods section for compound 1.The average exchange acceptance ratio was ≈ 25%.

Figure S21 .
Figure S21.Results of the PT-WTE calculations on the designed compounds 8-11.All the shown FES were computed after 150 ns (per replica) of simulation.As for the parent peptide 1, in all the cases metadynamics converged after about 80-100 ns.Convergence was estimated as described in the Materials and Methods section for compound 1.The average exchange acceptance ratio was ≈ 25%.