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Challenges Encountered Applying Equilibrium and Non-Equilibrium Binding Free Energy Calculations

revised on 12.04.2021, 23:05 and posted on 13.04.2021, 07:45 by Hannah Baumann, Vytautas Gapsys, Bert L. de Groot, David Mobley
Binding free energy calculations have become increasingly valuable to drive decision making in drug discovery projects.
However, among other issues, inadequate sampling can reduce accuracy, limiting the value of the technique.
In this paper we apply absolute binding free energy calculations to ligands binding to T4 lysozyme L99A and HSP90 using equilibrium and non-equilibrium approaches. We highlight sampling problems encountered in these systems, such as slow side chain rearrangements and slow changes of water placement upon ligand binding. These same types of challenges are likely to show up in other protein-ligand systems as well and we propose some strategies to diagnose and test for such problems in alchemical free energy calculations. We also explore similarities and differences in how the equilibrium and the non-equilibrium approaches handle these problems. Our results show the large amount of work still to be done to make free energy calculations robust and reliable and provide insight for future research in this area.


Computational alchemy for molecular design and optimization

National Institute of General Medical Sciences

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Advancing predictive physical modeling through focused development of model systems to drive new modeling innovations

National Institute of General Medical Sciences

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Email Address of Submitting Author


University of California, Irvine


United States

ORCID For Submitting Author


Declaration of Conflict of Interest

DLM is a member of the Scientific Advisory Board of OpenEye Scientific Software and an Open Science Fellow with Silicon Therapeutics.

Version Notes

Updated version with edits, improvements, and additional calculations