Evaluating Force Field Performance in Thermodynamic Calculations of Cyclodextrin Host–Guest Binding: Water Models, Partial Charges, and Host Force Field Parameters

Computational prediction of noncovalent binding free energies with methods based on molecular mechanical force fields has become increasingly routine in drug discovery projects, where they promise to speed the discovery of small molecule ligands to bind targeted proteins with high affinity. Because the reliability of free energy methods still has significant room for improvement, new force fields, or modifications of existing ones, are regularly introduced with the aim of improving the accuracy of molecular simulations. However, comparatively little work has been done to systematically assess how well force fields perform, particularly in relation to the calculation of binding affinities. Hardware advances have made these calculations feasible, but comprehensive force field assessments for protein–ligand sized systems still remain costly. Here, we turn to cyclodextrin host–guest systems, which feature many hallmarks of protein–ligand binding interactions but are generally much more tractable due to their small size. We present absolute binding free energy and enthalpy calculations, using the attach-pull-release (APR) approach, on a set of 43 cyclodextrin-guest pairs for which experimental ITC data are available. The test set comprises both α- and β-cyclodextrin hosts binding a series of small organic guests, each with one of three functional groups: ammonium, alcohol, or carboxylate. Four water models are considered (TIP3P, TIP4Pew, SPC/E, and OPC), along with two partial charge assignment procedures (RESP and AM1-BCC) and two cyclodextrin host force fields. The results suggest a complex set of considerations when choosing a force field for biomolecular simulations. For example, some force field combinations clearly outperform others at the binding enthalpy calculations but not for the binding free energy. Additionally, a force field combination which we expected to be the worst performer gave the most accurate binding free energies – but the least accurate binding enthalpies. The results have implications for the development of improved force fields, and we propose this test set, and potential future elaborations of it, as a powerful validation suite to evaluate new force fields and help guide future force field development

Attach-Pull-Release Calculations of Ligand Binding and Conformational Changes on the First BRD4 Bromodomain

Bromodomains, protein domains involved in epigenetic regulation, are able to bind small molecules with high affinity. In the present study, we report free energy calculations for the binding of seven ligands to the first BRD4 bromodomain, using the attach-pull-release (APR) method to compute the reversible work of removing the ligands from the binding site and then allowing the protein to relax conformationally. We test three different water models, TIP3P, TIP4PEw, and SPC/E, as well as the GAFF and GAFF2 parameter sets for the ligands. Our simulations show that the apo crystal structure of BRD4 is only metastable, with a structural transition happening in the absence of the ligand typically after 20 ns of simulation. We compute the free energy change for this transition with a separate APR calculation on the free protein and include its contribution to the ligand binding free energies, which generally causes an underestimation of the affinities. By testing different water models and ligand parameters, we are also able to assess their influence in our results and determine which one produces the best agreement with the experimental data. Both free energies associated with the conformational change and ligand binding are affected by the choice of water model, with the two sets of ligand parameters affecting their binding free energies to a lesser degree. Across all six combinations of water model and ligand potential function, the Pearson correlation coefficients between calculated and experimental binding free energies range from 0.55 to 0.83, and the root-mean-square errors range from 1.4–3.2 kcal/mol. The current protocol also yields encouraging preliminary results when used to assess the relative stability of ligand poses generated by docking or other methods, as illustrated for two different ligands. Our method takes advantage of the high performance provided by graphics processing units and can readily be applied to other ligands as well as other protein systems

Computational Calorimetry: High-Precision Calculation of Host–Guest Binding Thermodynamics

We present a strategy for carrying out high-precision calculations of binding free energy and binding enthalpy values from molecular dynamics simulations with explicit solvent. The approach is used to calculate the thermodynamic profiles for binding of nine small molecule guests to either the cucurbit[7]­uril (CB7) or β-cyclodextrin (βCD) host. For these systems, calculations using commodity hardware can yield binding free energy and binding enthalpy values with a precision of ∼0.5 kcal/mol (95% CI) in a matter of days. Crucially, the self-consistency of the approach is established by calculating the binding enthalpy directly, via end point potential energy calculations, and indirectly, via the temperature dependence of the binding free energy, i.e., by the van’t Hoff equation. Excellent agreement between the direct and van’t Hoff methods is demonstrated for both host–guest systems and an ion-pair model system for which particularly well-converged results are attainable. Additionally, we find that hydrogen mass repartitioning allows marked acceleration of the calculations with no discernible cost in precision or accuracy. Finally, we provide guidance for accurately assessing numerical uncertainty of the results in settings where complex correlations in the time series can pose challenges to statistical analysis. The routine nature and high precision of these binding calculations opens the possibility of including measured binding thermodynamics as target data in force field optimization so that simulations may be used to reliably interpret experimental data and guide molecular design

Reliable Oligonucleotide Conformational Ensemble Generation in Explicit Solvent for Force Field Assessment Using Reservoir Replica Exchange Molecular Dynamics Simulations

Molecular dynamics force field development and assessment requires a reliable means for obtaining a well-converged conformational ensemble of a molecule in both a time-efficient and cost-effective manner. This remains a challenge for RNA because its rugged energy landscape results in slow conformational sampling and accurate results typically require explicit solvent which increases computational cost. To address this, we performed both traditional and modified replica exchange molecular dynamics simulations on a test system (alanine dipeptide) and an RNA tetramer known to populate A-form-like conformations in solution (single-stranded rGACC). A key focus is on providing the means to demonstrate that convergence is obtained, for example, by investigating replica RMSD profiles and/or detailed ensemble analysis through clustering. We found that traditional replica exchange simulations still require prohibitive time and resource expenditures, even when using GPU accelerated hardware, and our results are not well converged even at 2 μs of simulation time per replica. In contrast, a modified version of replica exchange, reservoir replica exchange in explicit solvent, showed much better convergence and proved to be both a cost-effective and reliable alternative to the traditional approach. We expect this method will be attractive for future research that requires quantitative conformational analysis from explicitly solvated simulations

Consensus Conformations of Dinucleoside Monophosphates Described with Well-Converged Molecular Dynamics Simulations

Dinucleoside monophosphates (DNMPs) have been described using various experimental approaches as flexible molecules which generate ensembles populating at least a small set of different conformations in solution. However, due to limitations of each approach in its ability to delineate the ensemble of conformations, an accurate and quantitative description of certain conformational features has not been performed for all DNMPs. Here, we apply a temperature replica-exchange molecular dynamics approach to fully and quickly converge conformational distributions of all RNA DNMPs immersed in the TIP3P water model using the AMBER ff14 force field. For a selection of DNMPs, the conformational ensembles were also generated when immersed in the OPC water model using alternative AMBER and CHARMM force fields. The OPC water model and other force field choices did not introduce new conformational classes but shifted the populations among existing conformations. Except for pyrimidine–pyrimidine dinucleosides, all other DNMPs populated four major conformations (which are defined in the main text and labeled A-form, Ladder, Inverted, and Sheared), in addition to an Extended form. Pyrimidine–pyrimidines did not generate the Sheared conformation. Distinguishing features and stabilizing factors of each conformation were identified and assessed based on the known experimental interpretations. The configuration of the glycosidic bond and the nonbonding interactions of hydrogen bond acceptors with the 2′-hydroxyl group were found to play determining roles in stabilizing particular conformations which could serve as a guide for potential force field modifications to improve the accuracy. Additionally, we computed stacking free energies based on the DNMP conformational distributions and found significant discrepancies with a previous study. Our investigation determined that the AMBER force field was incorrectly implemented in the previous study. In the future, this simulation approach can be used to quickly analyze the effects of new force field modifications in shifting the conformational populations of DNMPs, and can can be further applied to foresee such effects in larger RNA motifs including tetranucleotides and tetraloops

Bind3P: Optimization of a Water Model with Host-Guest Binding Data

We report a water model, Bind3P (Version 0.1), which was obtained by using sensitivity analysis to readjust the Lennard-Jones parameters of the TIP3P model against experimental binding free energies for six host-guest systems, along with pure liquid properties. Tests of Bind3P against >100 experimental binding free energies and enthalpies for host-guest systems distinct from the training set show a consistent drop in the mean signed error, relative to matched calculations with TIP3P. Importantly, Bind3P also yields some improvement in the hydration free energies of small organic molecules, and preserves the accuracy of bulk water properties, such as density and the heat of vaporization. The same approach can be applied to more sophisticated water models that can better represent pure water properties. These results lend further support to concept of integrating host-guest binding data into force field parameterization

Bind3P: Optimization of a Water Model Based on Host–Guest Binding Data

We report a water model, Bind3P (Version 0.1), which was obtained by using sensitivity analysis to readjust the Lennard-Jones parameters of the TIP3P model against experimental binding free energies for six host–guest systems, along with pure liquid properties. Tests of Bind3P against >100 experimental binding free energies and enthalpies for host–guest systems distinct from the training set show a consistent drop in the mean signed error, relative to matched calculations with TIP3P. Importantly, Bind3P also yields some improvement in the hydration free energies of small organic molecules and preserves the accuracy of bulk water properties, such as density and the heat of vaporization. The same approach can be applied to more sophisticated water models that can better represent pure water properties. These results lend further support to the concept of integrating host–guest binding data into force field parametrization

Bind3P: Optimization of a Water Model Based on Host–Guest Binding Data

We report a water model, Bind3P (Version 0.1), which was obtained by using sensitivity analysis to readjust the Lennard-Jones parameters of the TIP3P model against experimental binding free energies for six host–guest systems, along with pure liquid properties. Tests of Bind3P against >100 experimental binding free energies and enthalpies for host–guest systems distinct from the training set show a consistent drop in the mean signed error, relative to matched calculations with TIP3P. Importantly, Bind3P also yields some improvement in the hydration free energies of small organic molecules and preserves the accuracy of bulk water properties, such as density and the heat of vaporization. The same approach can be applied to more sophisticated water models that can better represent pure water properties. These results lend further support to the concept of integrating host–guest binding data into force field parametrization

Structural and Energetic Analysis of 2‑Aminobenzimidazole Inhibitors in Complex with the Hepatitis C Virus IRES RNA Using Molecular Dynamics Simulations

Despite the many biological functions of RNA, very few drugs have been designed or found to target RNA. Here we report the results of molecular dynamics (MD) simulations and binding energy analyses on hepatitis C virus internal ribosome entry site (IRES) RNA in complex with highly charged 2-aminobenzimidazole inhibitors. Initial coordinates were taken from NMR and crystallography studies that had yielded different binding modes. During MD simulations, the RNA–inhibitor complex is stable in the crystal conformation but not in the NMR conformation. Additionally, we found that existing and standard MD trajectory postprocessing free energy methods, such as the MM-GBSA and MM-PBSA approaches available in AMBER, seem unsuitable to properly rank the binding energies of complexes between highly charged molecules. A better correlation with the experimental data was found using a rather simple binding enthalpy calculation based on the explicitly solvated potential energies. In anticipation of further growth in the use of small molecules to target RNA, we include results addressing the impact of charge assignment on docking, the structural role of magnesium in the IRES–inhibitor complex, the entropic contribution to binding energy, and simulations of a plausible scaffold design for new inhibitors

Bridging Calorimetry and Simulation through Precise Calculations of Cucurbituril–Guest Binding Enthalpies

We used microsecond time scale molecular dynamics simulations to compute, at high precision, binding enthalpies for cucurbit[7]­uril (CB7) with eight guests in aqueous solution. The results correlate well with experimental data from previously published isothermal titration calorimetry studies, and decomposition of the computed binding enthalpies by interaction type provides plausible mechanistic insights. Thus, dispersion interactions appear to play a key role in stabilizing these complexes, due at least in part to the fact that their packing density is greater than that of water. On the other hand, strongly favorable Coulombic interactions between the host and guests are compensated by unfavorable solvent contributions, leaving relatively modest electrostatic contributions to the binding enthalpies. The better steric fit of the aliphatic guests into the circular host appears to explain why their binding enthalpies tend to be more favorable than those of the more planar aromatic guests. The present calculations also bear on the validity of the simulation force field. Somewhat unexpectedly, the TIP3P water yields better agreement with experiment than the TIP4P-Ew water model, although the latter is known to replicate the properties of pure water more accurately. More broadly, the present results demonstrate the potential for computational calorimetry to provide atomistic explanations for thermodynamic observations