Ensemble-Based Approaches Ensure Reliability and Reproducibility.

It is increasingly widely recognized that ensemble-based approaches are required to achieve reliability, accuracy, and precision in molecular dynamics calculations. The purpose of the present article is to address a frequently raised question: what is the optimal way to perform ensemble simulation to calculate quantities of interest?

TIES 2.0: A Dual-Topology Open Source Relative Binding Free Energy Builder with Web Portal.

Relative binding free energy (RBFE) calculations are widely used to aid the process of drug discovery. TIES, Thermodynamic Integration with Enhanced Sampling, is a dual-topology approach to RBFE calculations with support for NAMD and OpenMM molecular dynamics engines. The software has been thoroughly validated on publicly available datasets. Here we describe the open source software along with a web portal (https://ccs-ties.org) that enables users to perform such calculations correctly and rapidly.

Identification of Optimal Ligand Growth Vectors Using an Alchemical Free-Energy Method.

In this work, a novel method to rationally design inhibitors with improved steric contacts and enhanced binding free energies is presented. This new method uses alchemical single step perturbation calculations to rapidly optimize the van der Waals interactions of a small molecule in a protein-ligand complex in order to maximize its binding affinity. The results of the optimizer are used to predict beneficial growth vectors on the ligand, and good agreement is found between the predictions from the optimizer and a more rigorous free energy calculation, with a Spearman's rank order correlation of 0.59. The advantage of the method presented here is the significant speed up of over 10-fold compared to traditional free energy calculations and sublinear scaling with the number of growth vectors assessed. Where experimental data were available, mutations from hydrogen to a methyl group at sites highlighted by the optimizer were calculated with MBAR, and the mean unsigned error between experimental and calculated values of the binding free energy was 0.83 kcal/mol.

Computational Fluorine Scanning Using Free-Energy Perturbation.

We present perturbative fluorine scanning, a computational fluorine scanning approach using free-energy perturbation. This method can be applied to molecular dynamics simulations of a single compound and make predictions for the best binders out of numerous fluorinated analogues. We tested the method on nine test systems: renin, DPP4, menin, P38, factor Xa, CDK2, AKT, JAK2, and androgen receptor. The predictions were in excellent agreement with more rigorous alchemical free-energy calculations and in good agreement with experimental data for most of the test systems. However, the agreement with experiment was very poor in some of the test systems, and this highlights the need for improved force fields in addition to accurate treatment of tautomeric and protonation states. The method is of particular interest due to the wide use of fluorine in medicinal chemistry to improve binding affinity and ADME properties. The promising results on this test case suggest that perturbative fluorine scanning will be a useful addition to the available arsenal of free-energy methods.

Assimilating Radial Distribution Functions To Build Water Models with Improved Structural Properties.

The structural properties of three- and four-site water models are improved by extending the ForceBalance parametrization code to include a new methodology allowing for the targeting of any radial distribution function (RDF) during the parametrization of a force field. The mean squared difference (MSD) between the experimental and simulated RDFs contributes to an objective function, allowing for the systematic optimization of force field parameters to reach closer overall agreement with experiment. RDF fitting is applied to develop modified versions of the TIP3P and TIP4P/2005 water models in which the Lennard-Jones potential is replaced by a Buckingham potential. The optimized TIP3P-Buckingham and TIP4P-Buckingham potentials feature 93 and 98% lower MSDs in the OO RDF compared to the TIP3P and TIP4P/2005 models respectively, with marked decreases in the height of the first peak. Additionally, these Buckingham models predict the entropy of water more accurately, reducing the error in the entropy of TIP3P from 11 to 3% and the error in the entropy of TIP4P/2005 from 11 to 2%. These new Buckingham models have improved predictive power for many nonfitted properties particularly in the case of TIP3P. Our work directly demonstrates how the Buckingham potential can improve the description of water's structural properties beyond the Lennard-Jones potential. Moreover, adding a Buckingham potential is a favorable alternative to adding interaction sites in terms of computational speed on modern GPU hardware.

Alchemical Free Energy Estimators and Molecular Dynamics Engines: Accuracy, Precision, and Reproducibility.

The binding free energy between a ligand and its target protein is an essential quantity to know at all stages of the drug discovery pipeline. Assessing this value computationally can offer insight into where efforts should be focused in the pursuit of effective therapeutics to treat a myriad of diseases. In this work, we examine the computation of alchemical relative binding free energies with an eye for assessing reproducibility across popular molecular dynamics packages and free energy estimators. The focus of this work is on 54 ligand transformations from a diverse set of protein targets: MCL1, PTP1B, TYK2, CDK2, and thrombin. These targets are studied with three popular molecular dynamics packages: OpenMM, NAMD2, and NAMD3 alpha. Trajectories collected with these packages are used to compare relative binding free energies calculated with thermodynamic integration and free energy perturbation methods. The resulting binding free energies show good agreement between molecular dynamics packages with an average mean unsigned error between them of 0.50 kcal/mol. The correlation between packages is very good, with the lowest Spearman's, Pearson's and Kendall's tau correlation coefficients being 0.92, 0.91, and 0.76, respectively. Agreement between thermodynamic integration and free energy perturbation is shown to be very good when using ensemble averaging.

Thermodynamic and structural insights into the repurposing of drugs that bind to SARS-CoV-2 main protease.

Although researchers have been working tirelessly since the COVID-19 outbreak, so far only three drugs - remdesivir, ronapreve and molnupiravir - have been approved for use in some countries which directly target the SARS-CoV-2 virus. Given the slow pace and substantial costs of new drug discovery and development, together with the urgency of the matter, repurposing of existing drugs for the ongoing disease is an attractive proposition. In a recent study, a high-throughput X-ray crystallographic screen was performed for a selection of drugs which have been approved or are in clinical trials. Thirty-seven compounds have been identified from drug libraries all of which bind to the SARS-CoV-2 main protease (3CLpro). In the current study, we use molecular dynamics simulation and an ensemble-based free energy approach, namely, enhanced sampling of molecular dynamics with approximation of continuum solvent (ESMACS), to investigate a subset of the aforementioned compounds. The drugs studied here are highly diverse, interacting with different binding sites and/or subsites of 3CLpro. The predicted free energies are compared with experimental results wherever they are available and they are found to be in excellent agreement. Our study also provides detailed energetic insights into the nature of the associated drug-protein binding, in turn shedding light on the design and discovery of potential drugs.

The performance of ensemble-based free energy protocols in computing binding affinities to ROS1 kinase.

Optimization of binding affinities for compounds to their target protein is a primary objective in drug discovery. Herein we report on a collaborative study that evaluates a set of compounds binding to ROS1 kinase. We use ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) and TIES (thermodynamic integration with enhanced sampling) protocols to rank the binding free energies. The predicted binding free energies from ESMACS simulations show good correlations with experimental data for subsets of the compounds. Consistent binding free energy differences are generated for TIES and ESMACS. Although an unexplained overestimation exists, we obtain excellent statistical rankings across the set of compounds from the TIES protocol, with a Pearson correlation coefficient of 0.90 between calculated and experimental activities.

An Open Drug Discovery Competition: Experimental Validation of Predictive Models in a Series of Novel Antimalarials.

The Open Source Malaria (OSM) consortium is developing compounds that kill the human malaria parasite, Plasmodium falciparum, by targeting PfATP4, an essential ion pump on the parasite surface. The structure of PfATP4 has not been determined. Here, we describe a public competition created to develop a predictive model for the identification of PfATP4 inhibitors, thereby reducing project costs associated with the synthesis of inactive compounds. Competition participants could see all entries as they were submitted. In the final round, featuring private sector entrants specializing in machine learning methods, the best-performing models were used to predict novel inhibitors, of which several were synthesized and evaluated against the parasite. Half possessed biological activity, with one featuring a motif that the human chemists familiar with this series would have dismissed as "ill-advised". Since all data and participant interactions remain in the public domain, this research project "lives" and may be improved by others.

Optimization of Protein-Ligand Electrostatic Interactions Using an Alchemical Free-Energy Method.

We present an explicit solvent alchemical free-energy method for optimizing the partial charges of a ligand to maximize the binding affinity with a receptor. This methodology can be applied to known ligand-protein complexes to determine an optimized set of ligand partial atomic changes. Three protein-ligand complexes have been optimized in this work: FXa, P38, and the androgen receptor. The sets of optimized charges can be used to identify design principles for chemical changes to the ligands which improve the binding affinity for all three systems. In this work, beneficial chemical mutations are generated from these principles and the resulting molecules tested using free-energy perturbation calculations. We show that three quarters of our chemical changes are predicted to improve the binding affinity, with an average improvement for the beneficial mutations of approximately 1 kcal/mol. In the cases where experimental data are available, the agreement between prediction and experiment is also good. The results demonstrate that charge optimization in explicit solvent is a useful tool for predicting beneficial chemical changes such as pyridinations, fluorinations, and oxygen to sulfur mutations.

Quality of life after ileoanal pouch: a comparison of J and W pouches.

INTRODUCTION: Standard treatment for ulcerative colitis and prevention of malignancy is total proctocolectomy with a neoileal pouch. The ideal configuration of the pouch has been debated. We hypothesized that there was no difference in quality of life between the J pouch and the W pouch. MATERIAL AND METHODS: We retrospectively reviewed the medical records of all patients undergoing ileoanal anastomosis with pouch construction at a single community-based teaching hospital over an 11+-year period. We collected demographic, operative, and postoperative data and then developed and distributed a survey designed to assess patient quality of life following pouch construction. The data of patients who had J pouches were then compared with those of patients who had W pouches. Forty-nine patients were identified; 30 had J pouches and 19 had W pouches. RESULTS: The groups did not differ significantly in age, sex, or indication for surgery. Significant differences were detected in readmission rates (J = 63%, W = 21%; p = 0.004) and length of follow-up (J = 61 months, W = 117 months; p = 0.001). Complication rates, length of stay, and conversion to end ileostomy rates were similar between groups. Self-reported health status, activity restrictions, urgency, seepage, protective pad use, and number of bowel movements at night were also similar. A significant difference existed in number of bowel movements per day (J = 6, W = 4.5, p = 0.041). No difference in quality of life was found between groups. Subgroup analysis of ulcerative-colitis-only patients had no effect on results. CONCLUSION: Because the J pouch is less technically demanding, it should be the preferred configuration.