Rational cyclization-based minimization of entropy penalty upon the binding of Nrf2-derived linear peptides to Keap1: A new strategy to improve therapeutic peptide activity against sepsis.

Nrf2 is a critical regulator of innate immune response and survival during sepsis, which is constitutively degraded through binding to the Keap1 adapter protein of E3 ubiquitin ligase. Two linear peptides DLG and ETG derived from, respectively, the low-affinity and high-affinity motifs of Nrf2 binding site exhibit self-binding affinity to Keap1 central hole (active pocket); they can be exploited as therapeutic self-inhibitory peptides to disrupt the Nrf2-Keap1 interaction. Molecular dynamics simulation and binding energetics decomposition reveal that the two peptides possess large flexibility and intrinsic disorder in unbound free state, and thus would incur a considerable entropy penalty upon binding to Keap1. In order to improve Keap1-peptide binding affinity (or free energy ΔG), instead of traditionally increasing favorable enthalpy contribution (ΔH) we herein describe a rational peptide cyclization strategy to minimize unfavorable entropy penalty (ΔS) upon the binding of Nrf2-derived linear peptides to Keap1. Crystal structure analysis impart that the native active conformations of DLG and ETG peptides bound with Keap1 are folded into U-shape and hairpin configurations, respectively, and adopt their turning head to insert into the central hole of Keap1. Here, cyclization is designed by adding a disulfide bond across the two arms of DLG U-shape or ETG hairpin, which would not influence the direct intermolecular interaction between Keap1 and peptide as well as desolvation effect involved in the interaction, but can effectively constrain the conformational flexibility and disorder of the two peptides in free state, thus largely minimizing entropy penalty upon the binding. Both free energy calculation and binding affinity assay substantiate that the cyclization, as might be expected, can moderately or considerably enhance peptide binding potency to Keap1, with affinity (dissociation constant Kd) increase by 1.4-7.5-fold for designed cyclic peptides relative to their linear counterparts.