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

Jean‐françois Gaucher (1) , Marie Reille-Seroussi , Sylvain Broussy (1)

1 CiTCoM - UMR 8038 - Cibles Thérapeutiques et conception de médicaments

Abstract: Macrocyclization constraints are widely used in the design of protein ligands to stabilize their bioactive con- formation 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 differ- ences in thermodynamics arise mainly from the folding Introduction Peptides have gained increased interest as pharmaceuticals. They share many strengths, such as high target selectivity, good efficiency, safety, and tolerability. However, a short plasma half- life, chemical and physical instabilities and low membrane permeability are weaknesses that must be overcome for therapeutic use. Among the possible strategies, a common approach is to constrain peptides, which can reduce suscepti- bility to proteolysis, stabilize their bioactive form and improve both affinity and specificity. However, attempting to correlate constraints to binding thermodynamics is puzzling, if not impossible. While a gain in binding affinity is often observed, inconsistencies in change of entropy and enthalpy upon bind- energy of the peptide upon binding. The systematic reduc- tion in conformational entropy penalty due to helix pre- organization 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 improve- ment in affinity. The precision of the analytical ITC method makes this study a possible benchmark for constrained peptides optimization.

Keywords: peptide folding; helix stabilization; vibrational entropy; Heat Capacity; lactam bridge; macromolecular cristallography; NMR; Isothermal Titration Calorimetry ITC; Enthalpy-entropy compensation; Macromolecular crystallography; Drug design

Domains: Life Sciences [q-bio]; Biochemistry, Molecular Biology; Biophysics; Life Sciences [q-bio]; Biochemistry, Molecular Biology; Structural Biology [q-bio.BM]; Chemical Sciences; Organic chemistry

Jean‐françois Gaucher, Marie Reille-Seroussi, Sylvain Broussy. Structural and ITC Characterization of Peptide‐Protein Binding: Thermodynamic Consequences of Cyclization Constraints, a Case Study on Vascular Endothelial Growth Factor Ligands. Chemistry - A European Journal, 2022, 28 (48), ⟨10.1002/chem.202200465⟩. ⟨hal-03818315⟩