Broadening the Scope of Binding Free Energy Calculations Using a Separated Topologies Approach.

Binding free energy calculations that make use of alchemical pathways are becoming increasingly feasible thanks to advances in hardware and algorithms. Although relative binding free energy (RBFE) calculations are starting to find widespread use, absolute binding free energy (ABFE) calculations are still being explored mainly in academic settings due to the high computational requirements and still uncertain predictive value. However, in some drug design scenarios, RBFE calculations are not applicable and ABFE calculations could provide an alternative. Computationally cheaper end-point calculations in implicit solvent, such as molecular mechanics Poisson–Boltzmann surface area (MMPBSA) calculations, could too be used if one is primarily interested in a relative ranking of affinities. Here, we compare MMPBSA calculations to previously performed absolute alchemical free energy calculations in their ability to correlate with experimental binding free energies for three sets of bromodomain–inhibitor pairs. Different MMPBSA approaches have been considered, including a standard single-trajectory protocol, a protocol that includes a binding entropy estimate, and protocols that take into account the ligand hydration shell. Despite the improvements observed with the latter two MMPBSA approaches, ABFE calculations were found to be overall superior in obtaining correlation with experimental affinities for the test cases considered. A difference in weighted average Pearson () and Spearman () correlations of 0.25 and 0.31 was observed when using a standard single-trajectory MMPBSA setup ( = 0.64 and = 0.66 for ABFE; = 0.39 and = 0.35 for MMPBSA). The best performing MMPBSA protocols returned weighted average Pearson and Spearman correlations that were about 0.1 inferior to ABFE calculations: = 0.55 and = 0.56 when including an entropy estimate, and = 0.53 and = 0.55 when including explicit water molecules. Overall, the study suggests that ABFE calculations are indeed the more accurate approach, yet there is also value in MMPBSA calculations considering the lower compute requirements, and if agreement to experimental affinities in absolute terms is not of interest. Moreover, for the specific protein–ligand systems considered in this study, we find that including an explicit ligand hydration shell or a binding entropy estimate in the MMPBSA calculations resulted in significant performance improvements at a negligible computational cost.

Accurate in silico prediction of protein-ligand binding affinities has been a primary objective of structure-based drug design for decades due to the putative value it would bring to the drug discovery process. However, computational methods have historically failed to deliver value in real-world drug discovery applications due to a variety of scientific, technical, and practical challenges. Recently, a family of approaches commonly referred to as relative binding free energy (RBFE) calculations, which rely on physics-based molecular simulations and statistical mechanics, have shown promise in reliably generating accurate predictions in the context of drug discovery projects. This advance arises from accumulating developments in the underlying scientific methods (decades of research on force fields and sampling algorithms) coupled with vast increases in computational resources (graphics processing units and cloud infrastructures). Mounting evidence from retrospective validation studies, blind challenge predictions, and prospective applications suggests that RBFE simulations can now predict the affinity differences for congeneric ligands with sufficient accuracy and throughput to deliver considerable value in hit-to-lead and lead optimization efforts. Here, we present an overview of current RBFE implementations, highlighting recent advances and remaining challenges, along with examples that emphasize practical considerations for obtaining reliable RBFE results. We focus specifically on relative binding free energies because the calculations are less computationally intensive than absolute binding free energy (ABFE) calculations and map directly onto the hit-to-lead and lead optimization processes, where the prediction of relative binding energies between a reference molecule and new ideas (virtual molecules) can be used to prioritize molecules for synthesis. We describe the critical aspects of running RBFE calculations, from both theoretical and applied perspectives, using a combination of retrospective literature examples and prospective studies from drug discovery projects. This work is intended to provide a contemporary overview of the scientific, technical, and practical issues associated with running relative binding free energy simulations, with a focus on real-world drug discovery applications. We offer guidelines for improving the accuracy of RBFE simulations, especially for challenging cases, and emphasize unresolved issues that could be improved by further research in the field.

Water often plays a key role in mediating protein-ligand interactions. Understanding contributions from active-site water molecules to binding thermodynamics of a ligand is important in predicting binding free energies for ligand optimization. In this work, we tested a non-equilibrium switching method for absolute binding free energy calculations on water molecules in binding sites of 13 systems. We discuss the lessons we learned about identified issues that affected our calculations and ways to address them. This work fits with our larger focus on how to do accurate ligand binding free energy calculations when water rearrangements are very slow, such as rearrangements due to ligand modification (as in relative free energy calculations) or ligand binding (as in absolute free energy calculations). The method studied in this work can potentially be used to account for limited water sampling via providing endpoint corrections to free energy calculations using our calculated binding free energy of water.

Key to the fragment optimisation process within drug design is the need to accurately capture the changes in affinity that are associated with a given set of chemical modifications. Due to the weakly binding nature of fragments, this has proven to be a challenging task, despite recent advancements in leveraging experimental and computational methods. In this work, we evaluate the use of Absolute Binding Free Energy (ABFE) calculations in guiding fragment optimisation decisions, retrospectively calculating binding free energies for 59 ligands across 4 fragment elaboration campaigns. We first demonstrate that ABFEs can be used to accurately rank fragment-sized binders with an overall Spearman’s r of 0.89 and a Kendall τ of 0.67, although often deviating from experiment in absolute free energy values with an RMSE of 2.75 kcal/mol. We then also show that in several cases, retrospective fragment optimisation decisions can be supported by the ABFE calculations. Comparing against cheaper endpoint methods, namely Nwat-MM/GBSA, we find that ABFEs offer better ranking power and correlation metrics. Our results indicate that ABFE calculations can usefully guide fragment elaborations to maximise affinity.

We determined the effectiveness of absolute binding free energy (ABFE) calculations to refine the selection of active compounds in virtual compound screening, a setting where the more commonly used relative binding free energy approach is not readily applicable. To do this, we conducted baseline docking calculations of structurally diverse compounds in the DUD-E database for three targets, BACE1, CDK2 and thrombin, followed by ABFE calculations for compounds with high docking scores. The docking calculations alone achieved solid enrichment of active compounds over decoys. Encouragingly, the ABFE calculations then improved on this baseline. Analysis of the results emphasizes the importance of establishing high quality ligand poses as starting points for ABFE calculations, a nontrivial goal when processing a library of diverse compounds without informative co-crystal structures. Overall, our results suggest that ABFE calculations can play a valuable role in the drug discovery process.

Apoptosis signal-regulating kinase 1 (ASK1), a key component of the mitogen-activated protein kinase (MAPK) cascades, has been identified as a promising therapeutic target owing to its critical role in signal transduction pathways. In this study, we proposed novel pyridin-2-yl urea inhibitors exhibiting favorable physicochemical properties. The potency of these compounds was validated through in vitro protein bioassays. The inhibition (IC50) of compound 2 was 1.55 ± 0.27 nM, which was comparable to the known clinical inhibitor, Selonsertib. To further optimize the hit compounds, two possible binding modes were initially predicted by molecular docking. Absolute binding free energy (BFE) calculations based on molecular dynamics simulations further discriminated the binding modes, presenting good tendency with bioassay results. This strategy, underpinned by BFE calculations, has the great potential to expedite the drug discovery process in the targeting of ASK1 kinase.

Carbohydrates are key biological mediators of molecular recognition and signaling processes. In this case study, we explore the ability of absolute binding free energy (ABFE) calculations to predict the affinities of a set of five related carbohydrate ligands for the lectin protein, concanavalin A, ranging from 27-atom monosaccharides to a 120-atom complex-type N-linked glycan core pentasaccharide. ABFE calculations quantitatively rank and estimate the affinity of the ligands in relation to microcalorimetry, with a mean signed error in the binding free energy of -0.63 ± 0.04 kcal/mol. Consequently, the diminished binding efficiencies of the larger carbohydrate ligands are closely reproduced: the ligand efficiency values from isothermal titration calorimetry for the glycan core pentasaccharide and its constituent trisaccharide and monosaccharide compounds are respectively -0.14, -0.22, and -0.41 kcal/mol per heavy atom. ABFE calculations predict these ligand efficiencies to be -0.14 ± 0.02, -0.24 ± 0.03, and -0.46 ± 0.06 kcal/mol per heavy atom, respectively. Consequently, the ABFE method correctly identifies the high affinity of the key anchoring mannose residue and the negligible contribution to binding of both β-GlcNAc arms of the pentasaccharide. While challenges remain in sampling the conformation and interactions of these polar, flexible, and weakly bound ligands, we nevertheless find that the ABFE method performs well for this lectin system. The approach shows promise as a quantitative tool for predicting and deconvoluting carbohydrate-protein interactions, with potential application to design of therapeutics, vaccines, and diagnostics.

Absolute binding free energy calculations with explicit solvent molecular simulations can provide estimates of protein-ligand affinities, and thus reduce the time and costs needed to find new drug candidates. However, these calculations can be complex to implement and perform. Here, we introduce the software BAT.py, a Python tool that invokes the AMBER simulation package to automate the calculation of binding free energies for a protein with a series of ligands. The software supports the attach-pull-release (APR) and double decoupling (DD) binding free energy methods, as well as the simultaneous decoupling-recoupling (SDR) method, a variant of double decoupling that avoids numerical artifacts associated with charged ligands. We report encouraging initial test applications of this software both to re-rank docked poses and to estimate overall binding free energies. We also show that it is practical to carry out these calculations cheaply by using graphical processing units in common machines that can be built for this purpose. The combination of automation and low cost positions this procedure to be applied in a relatively high-throughput mode and thus stands to enable new applications in early-stage drug discovery.

Recent advances in relative protein-ligand binding free energy calculations have shown the value of alchemical methods in drug discovery. Accurately assessing absolute binding free energies remains a challenging endeavour, mostly limited to small model cases. We demonstrate accurate absolute binding free energy estimates for 128 pharmaceutically relevant ligands across 7 proteins using a highly parallelizable non-equilibrium method. These calculations also provide detailed physical insight into the structural determinants of binding, identifying subtle rotamer rearrangements between protein apo and holo states that are crucial for binding. The challenge behind absolute binding free energy calculations stems in large part from the need to explicitly account for the protein’s apo state. In this work we present several approaches to obtain apo state ensembles, including a novel rigorous method to generate protein-ligand ensembles for the ligand in its decoupled state. Altogether, we present an effective open-source protocol for prospective application in drug discovery.

Absolute binding free energy calculations for molecules binding to the GPCR GCGR at the protein/bilayer/water interface.

Accurate estimation of protein-ligand (PL) binding free energies is a crucial task in medicinal chemistry and a critical measure of PL interaction modeling effectiveness. However, traditional computational methods are often computationally expensive and prone to errors. Recently, deep learning (DL)-based approaches for predicting PL interactions have gained enormous attention, but their accuracy and generalizability are hindered by data scarcity. In this study, we propose LumiNet, a versatile PL interaction modeling framework that bridges the gap between physics-based models and black-box algorithms. LumiNet utilizes a subgraph transformer to extract multiscale information from molecular graphs and employs geometric neural networks to integrate PL information, mapping atomic pair structures into key physical parameters of non-bonded interactions in classical force fields, thereby enhancing accurate absolute binding free energy (ABFE) calculations. LumiNet is designed to be highly interpretable, offering detailed insights into atomic interactions within protein-ligand complexes, pinpointing relatively important atom pairs or groups. Our semi-supervised learning strategy enables LumiNet to adapt to new targets with fewer data points than other data-driven methods, making it more relevant for real-world drug discovery. Benchmarks show that LumiNet outperforms the current state-of-the-art model by 18.5% on the PDE10A dataset, and rivals the FEP+ method in some tests with a speed improvement of several orders of magnitude. We applied LumiNet in the scaffold hopping process, which accurately guided the discovery of the optimal ligands. Furthermore, we provide a web service for the research community to test LumiNet. The visualization of predicted inter-molecular energy contributions is expected to provide practical value in drug discovery projects.

Relative binding free energy (RBFE) calculations, widely used to predict the potencies of congeneric small molecules binding to a protein receptor, can greatly increase the efficiency of the hit-to-lead and lead optimization stages of the drug discovery process. Traditional RBFE methods, however, cannot be easily applied to small molecules lacking a common core or binding mode, precluding their use in a challenging but crucial component of many drug discovery campaigns. In principle, an absolute binding free energy (ABFE) method can be applied to such molecules, but ABFE often suffers from high computational cost and poor statistical convergence due to the large amount of additional sampling required when compared to RBFE. Here, we introduce core-hopping binding free energy (CBFE) calculations, a computationally efficient framework for the accurate determination of relative binding free energies between small molecules with different cores, leveraging several recently developed techniques such as Alchemical Enhanced Sampling (ACES) with optimized transformation pathways and flexible λ-spacing, as well as λ-dependent Boresch restraints. We benchmark the performance of CBFE across 4 protein systems consisting of 56 small molecules, and find that the results are consistent with RBFE for a congeneric series of ligands and offer considerable improvement in computational cost and precision relative to ABFE results for a series of small molecules with diverse cores and binding modes. All CBFE-related developments are fully implemented in the GPU-accelerated AMBER free energy module (pmemd.cuda) and are available as part of the latest official AMBER release.

The recent advances in relative protein-ligand binding free energy calculations have shown the value of alchemical methods in drug discovery. Accurately assessing absolute binding free energies, although highly desired, remains a challenging endeavour, mostly limited to small model cases. Here, we demonstrate accurate first principles based absolute binding free energy estimates for 128 pharmaceutically relevant targets. We use a novel rigorous method to generate protein-ligand ensembles for the ligand in its decoupled state. Not only do the calculations deliver accurate protein-ligand binding affinity estimates, but they also provide detailed physical insight into the structural determinants of binding. We identify subtle rotamer rearrangements between apo and holo states of a protein that are crucial for binding. When compared to relative binding free energy calculations, obtaining absolute binding free energies is considerably more challenging in large part due to the need to explicitly account for the protein in its apo state. In this work we present several approaches to obtain apo state ensembles for accurate absolute ΔG calculations, thus outlining protocols for prospective application of the methods for drug discovery.

Alchemical absolute binding free energy (ABFE) calculations have substantial potential in drug discovery, but are often prohibitively computationally expensive. To unlock their potential, efficient automated ABFE workflows are required to reduce both computational cost and human intervention. We present a fully automated ABFE workflow based on the automated selection of λ windows, the ensemble-based detection of equilibration, and the adaptive allocation of sampling time based on inter-replicate statistics. We find that the automated selection of intermediate states with consistent overlap is rapid, robust, and simple to implement. Robust detection of equilibration is achieved with a paired t-test between the free energy estimates at initial and final portions of a an ensemble of runs. We determine reasonable default parameters for all algorithms and show that the full workflow produces equivalent results to a nonadaptive scheme over a variety of test systems, while often accelerating equilibration. Our complete workflow is implemented in the open-source package A3FE (https://github.com/michellab/a3fe).

Absolute binding free energy (ABFE) calculations can be an important part of the drug discovery process by identifying molecules that have the potential to be strong binders for a biomolecular target. Recent work has used free energy perturbation (FEP) theory for these calculations, focusing on a set of 16 inhibitors of the severe acute respiratory syndrome coronavirus 2 main protease (Mpro). Herein, the same data set is evaluated by metadynamics (MetaD), four different docking programs, and molecular mechanics with generalized Born and surface area solvation. MetaD yields a Kendall τ distance of 0.28 and Pearson r2 of 0.49, which reflect somewhat less accuracy than that from the ABFE FEP results. Notably, it is demonstrated that an ensemble docking protocol by which each ligand is docked into the 13 crystal structures in this data set provides improved performance, particularly when docking is carried out with Glide XP (Kendall τ distance = 0.20, Pearson r2 = 0.71), Glide SP (Kendall τ distance = 0.19, Pearson r2 = 0.66), or AutoDock 4 (Kendall τ distance = 0.21, Pearson r2 = 0.55). The best results are obtained with "superconsensus" docking by averaging the 52 results for each compound using the 4 docking protocols and all 13 crystal structures (Kendall τ distance = 0.18, Pearson r2 = 0.73).