Multistate Method to Efficiently Account for Tautomerism and Protonation in Alchemical Free-Energy Calculations

26 March 2024, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


The majority of drug-like molecules contain at least one ionizable group, and many common drug scaffolds are subject to tautomeric equilibria. Thus, these compounds are found in a mixture of protonation and/or tautomeric states at physiological pH. Intrinsically, standard classical molecular dynamics (MD) simulations cannot describe such equilibria between states, which negatively impacts the prediction of key molecular properties in silico. Following the formalism described by de Oliveira and co-workers (J. Chem. Theory Comput. 2019, 15, 424–435) to consider the influence of all states on the binding process based on alchemical free energy calculations, we demonstrate in this work that the multistate method replica-exchange enveloping distribution sampling (RE-EDS) is well suited to describe molecules with multiple protonation and/or tautomeric states in a single simulation. We apply our methodology to a series of eight inhibitors of factor Xa with two protonation states and a series of eight inhibitors of glycogen synthase kinase 3β (GSK3β) with two tautomeric states. In particular, we show that given a sufficient phase-space overlap between the states, RE-EDS is computationally more efficient than standard pairwise free-energy methods.


Binding free energy
Free energy calculation

Supplementary materials

Supporting Information
Full description of the inhibitors and their representation following a hybrid topology; Additional details of the (RE-)EDS simulations performed in this work (parameter optimization); In-depth discussion of the setup and results of the application of net charge correction factors for the FXa dataset; Supplementary figures allowing to further interpret the results of the simulations presented in the main text.


Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.