Accurately predicting protein-ligand binding affinities and binding modes is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes. In this technique, the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically-relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.

Molecular simulations are a valuable tool for studying biomolecular motions and thermodynamics. However, such motions can be slow compared to simulation timescales, yet critical. Specifically, adequate sampling of sidechain motions in protein binding pockets is crucial for obtaining accurate estimates of ligand binding free energies from molecular simulations. The timescale of sidechain rotamer flips can range from a few ps to several hundred ns or longer, particularly in crowded environments like the interior of proteins. Here, we apply a mixed non-equilibrium candidate Monte Carlo (NCMC)/molecular dynamics (MD) method to enhance sampling of sidechain rotamers. The NCMC portion of our method applies a switching protocol wherein the steric and electrostatic interactions between target sidechain atoms and the surrounding environment are cycled off and then back on during the course of a move proposal. Between NCMC move proposals, simulation of the system continues via traditional molecular dynamics. Here, we first validate this approach on a simple, solvated valine-alanine dipeptide system and then apply it to a well-studied model ligand binding site in T4 lysozyme L99A. We compute the rate of rotamer transitions for a valine sidechain using our approach and compare it to that of traditional molecular dynamics simulations. Here, we show that our NCMC/MD method substantially enhances sidechain sampling, especially in systems where the torsional barrier to rotation is high (≥ 10 kcal/mol). These barriers can be intrinsic torsional barriers or steric barriers imposed by the environment. Overall, this may provide a promising strategy to selectively improve sidechain sampling in molecular simulations.

A correct estimate of ligand binding modes and a ratio of their occupancies is crucial for calculations of binding free energies. The newly developed method BLUES combines molecular dynamics with nonequilibrium candidate Monte Carlo. Nonequilibrium candidate Monte Carlo generates a plethora of possible binding modes and molecular dynamics enables the system to relax. We used BLUES to investigate binding modes of caffeine in the active site of its metabolizing enzyme Cytochrome P450 1A2 with the aim of elucidating metabolite-formation profiles at different concentrations. Because the activation energies of all sites of metabolism do not show a clear preference for one metabolite over the others, the orientations in the active site must play a key role. In simulations with caffeine located in a spacious pocket above the I-helix, it points N3 and N1 to the heme iron, whereas in simulations where caffeine is in close proximity to the heme N7 and C8 are preferably oriented toward the heme iron. We propose a mechanism where at low caffeine concentrations caffeine binds to the upper part of the active site, leading to formation of the main metabolite paraxanthine. On the other hand, at high concentrations two molecules are located in the active site, forcing one molecule into close proximity to the heme and yielding metabolites theophylline and trimethyluretic acid. Our results offer an explanation of previously published experimental results.

Flexible ligands often have multiple binding modes or bound conformations that differ by rotation of a portion of the molecule around internal rotatable bonds. Knowledge of these binding modes is important for understanding the interactions stabilizing the ligand in the binding pocket, and also for calculating accurate binding affinities. In this work, we use a hybrid molecular dynamics (MD)/nonequilibrium candidate Monte Carlo (NCMC) method to sample the different binding modes of several flexible ligands and also to estimate the population distribution of the modes. The NCMC move proposal is divided into three parts. The flexible part of the ligand is alchemically turned off by decreasing the electrostatics and steric interactions gradually, followed by rotating the rotatable bond by a random angle and then slowly turning the ligand back on to its fully interacting state. The alchemical steps prior to and after the move proposal help the surrounding protein and water atoms in the binding pocket relax around the proposed ligand conformation and increase move acceptance rates. The protein-ligand system is propagated using classical MD in between the NCMC proposals. Using this MD/NCMC method, we were able to correctly reproduce the different binding modes of inhibitors binding to two kinase targets -c-Jun N-terminal kinase-1 and cyclin-dependent kinase 2 -at a much lower computational cost compared to conventional MD and umbrella sampling. This method is available as a part of the BLUES software package.

Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called Molecular Darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves.We apply this technique to a simple dipeptide system, a ligand binding to T4 Lysozyme L99A, and ligand binding to HIV integrase in order to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal, and rotational/translational degrees of freedom in these systems. File list (2)download file view on ChemRxiv BLUES_moldart_4.pdf (5.25 MiB) download file view on ChemRxiv si_moldart.zip (140.19 MiB)