Abstract

This study presents a novel computational approach to study molecular recognition and binding kinetics for drug-like compounds dissociating from a flexible protein system. The intermediates and their free energy profile during ligand association and dissociation processes control ligand-protein binding kinetics and bring a more complete picture of ligand-protein binding. The method applied the milestoning theory to extract kinetics and thermodynamics information from running short classical molecular dynamics (MD) simulations for frames from a given dissociation path. High-dimensional ligand-protein motions (3N-6 degrees of freedom) during ligand dissociation were reduced by use of principal component modes for assigning more than 100 milestones, and classical MD runs were allowed to travel multiple milestones to efficiently obtain ensemble distribution of initial structures for MD simulations and estimate the transition time and rate during ligand traveling between milestones. We used five pyrazolourea ligands and cyclin-dependent kinase 8 with cyclin C (CDK8/CycC) as our model system as well as metadynamics and a pathway search method to sample dissociation pathways. With our strategy, we constructed the free energy profile for highly mobile biomolecular systems. The computed binding free energy and residence time correctly ranked the pyrazolourea ligand series, in agreement with experimental data. Guided by a barrier of a ligand passing an αC helix and activation loop, we introduced one hydroxyl group to parent compounds to design our ligands with increased residence time and validated our prediction by experiments. This work provides a novel and robust approach to investigate dissociation kinetics of large and flexible systems for understanding unbinding mechanisms and designing new small-molecule drugs with desired binding kinetics.

Highlights

  • Binding kinetics has become an important topic in molecular recognition because of the importance of fully understanding binding/unbinding and the growing awareness of the correlation between kinetics and drug efficacy.[1−5] Drug binding residence time, which can be estimated by a dissociation rate constant, 1/ koff, is important for determining the efficacy and selectivity of drug candidates

  • Movies S1 and S2 demonstrate the motions of Cyclin-dependent kinase 8 (CDK8)/cyclin C (CycC)-PL2 along PC1 and PC2, respectively

  • Milestones 41 and 58 in Figure S2 have nearly the same center-ofmass distances, even though the milestones capture the conformational change of the αC helix (Figure S2A), whose contribution to unbinding kinetics will be discussed in the sections of the structure-kinetics relationship

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Summary

Introduction

Binding kinetics has become an important topic in molecular recognition because of the importance of fully understanding binding/unbinding and the growing awareness of the correlation between kinetics and drug efficacy.[1−5] Drug binding residence time, which can be estimated by a dissociation rate constant, 1/ koff, is important for determining the efficacy and selectivity of drug candidates. Experiments provide measured binding affinities (ΔG), rate constants (kon and koff), and molecular structures. Protein association and dissociation processes, become an important tool to characterize mechanistic features of binding kinetics and further assist drug development.[6] Features that govern binding kinetics are system-dependent and include ligand properties, conformational fluctuations, intermolecular interactions, and solvent effects.[7−12] the determinants to adjust when optimizing kinetic properties for a drug discovery project are not well understood

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