Structural and ITC Characterization of Peptide‐Protein Binding: Thermodynamic Consequences of Cyclization Constraints, a Case Study on Vascular Endothelial Growth Factor Ligands

Abstract

Macrocyclization constraints are widely used in the design of protein ligands to stabilize their bioactive conformation and increase their affinities. However, the resulting changes in binding entropy can be puzzling and uncorrelated to affinity gains. Here, the thermodynamic (Isothermal Titration Calorimetry) and structural (X-ray, NMR and CD) analysis of a complete series of lactam-bridged peptide ligands of the vascular endothelial growth factor, and their unconstrained analogs are reported. It is shown that differences in thermodynamics arise mainly from the folding energy of the peptide upon binding. The systematic reduction in conformational entropy penalty due to helix preorganization can be counterbalanced by an unfavorable vibrational entropy change if the constraints are too rigid. The gain in configurational entropy partially escapes the enthalpy/entropy compensation and leads to an improvement in affinity. The precision of the analytical ITC method makes this study a possible benchmark for constrained peptides optimization.

Dimer Peptide Ligands of Vascular Endothelial Growth Factor: Optimizing Linker Length for High Affinity and Antiangiogenic Activity

“…In a previous article comparing monocyclic and bicyclic peptides, including M1 , we measured an average ratio ⟨ K d,37°C / K d,20 °C ⟩ equal to 4.7 (S.D. 0.7) . For the current dimer series, this ratio was 3.2 (S.D.…”

“…The change in binding affinity imparted by the intramolecular nature of the second step can be described by the effective molarity defined as M eff = K d inter / K d intra . Hence: Similarly, since monomer peptides M1 – M3 have two receptor sites on VEGF The absence of any cooperativity between the two VEGF binding sites leads to K d monomer, 2nd binding = 2 K d inter and (unit M 2 ). ,, …”

“…These five linkers were used as provided by the supplier, with controlled average mass ranges and dispersity values Đ smaller than 1.1. 23 The monomer peptide M1 was synthesized as previously described 19 but with a mutation n L6K and an acetylation of the N-terminus to avoid side reactions during dimer formation. Lys 6 of the monomer was either acetylated to give M2 or reacted with half-stoichiometric amounts of PEG linkers to give the dimers.…”

“…Besides, performing ITC assays at different temperatures can give access to the variations of heat capacity upon binding, using the equation 24 For each peptide, both direct and reverse titrations were performed to improve the accuracy, according to the analytical method previously described. 19 For instance, Figure 2 shows the titrations of dimer D6 binding with VEGF homodimer at 20 and 37 °C analyzed with the software SEDPHAT (one direct titration and one reverse titration are shown). 25 The final thermodynamic results were derived from the global analysis normalized on the VEGF concentration, with all direct and reverse titrations in SEDPHAT at confidence level P = 95% (Table SI).…”

“…15−17 We shortened this peptide to 15 amino acids 18 and introduced a helix-stabilizing lactam bridge. 19 The resulting peptide has an affinity of 38 nM for VEGF and has a molecular weight of 2 kDa. Dimerization of this monomer peptide with small linkers could lead to ligands of less than 5 kDa.…”