Adapting free energy perturbation simulations for large macrocyclic ligands: how to dissect contributions from direct binding and free ligand flexibility.

Kerstin Wallraven,Fredrik L Holmelin,Andrey I Frolov,Tom N Grossmann,Adrian Glas,Sven Hennig

doi:10.1039/c9sc04705k

Abstract

Large and flexible ligands gain increasing interest in the development of bioactive agents. They challenge the applicability of computational ligand optimization strategies originally developed for small molecules. Free energy perturbation (FEP) is often used for predicting binding affinities of small molecule ligands, however, its use for more complex ligands remains limited. Herein, we report the structure-based design of peptide macrocycles targeting the protein binding site of human adaptor protein 14-3-3. We observe a surprisingly strong dependency of binding affinities on relatively small variations in substituent size. FEP was performed to rationalize observed trends. To account for insufficient convergence of FEP, restrained calculations were performed and complemented with extensive REST MD simulations of the free ligands. These calculations revealed that changes in affinity originate both from altered direct interactions and conformational changes of the free ligand. In addition, MD simulations provided the basis to rationalize unexpected trends in ligand lipophilicity. We also verified the anticipated interaction site and binding mode for one of the high affinity ligands by X-ray crystallography. The introduced fully-atomistic simulation protocol can be used to rationalize the development of structurally complex ligands which will support future ligand maturation efforts.

Highlights

Selective ligands are the basis for most strategies aiming at the elucidation or modulation of biological processes.[1]
Macrocyclic peptide 1 comprises 11 amino acids and harbors an R- and an S-con gured a-methyl, a-alkyl amino acid at position 3 and 6, respectively (X(Me)R3 and X(Me)S6, Fig. 1B). Both amino acids are connected via their alkyl side chains forming an eight membered hydrocarbon crosslink. This hydrophobic crosslink contributes to binding by engaging in direct interactions with the target protein 14-3-3 and by stabilizing the bioactive conformation of the free ligand.[34]
We report the structure-guided optimization of a macrocyclic peptide ligand targeting the protein binding groove of human adaptor protein 14-3-3

Summary

Introduction

Selective ligands are the basis for most strategies aiming at the elucidation or modulation of biological processes.[1]. This hydrophobic crosslink contributes to binding by engaging in direct interactions with the target protein 14-3-3 and by stabilizing the bioactive conformation of the free ligand.[34] Given the importance of the central macrocycle, we consider peptide 1 a good starting point for the structure-based design of smaller peptide ligands with high binding affinity.

Results

Conclusion