Automation of absolute protein-ligand binding free energy calculations for docking refinement and compound evaluation

Abstract

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.

Figure 1

Workflow of the BAT.py software. See text for details.

Figure 2

(Top panel) Attachment of restraints, first to the protein and then the ligand. The protein conformational restraints are denoted by the green squiggle. (Middle panel) Transfer ligand from binding site to bulk solvent, using the double decoupling method. (Bottom panel) Release of the ligand and protein restraints in the unbound state. P1, P2 and P3 indicate protein anchor atoms; N1, N2 and N3 indicate artificial dummy atoms whose locations are fixed in the lab frame; and L1, L2 and L3 indicate ligand anchor atoms.

Figure 3

Thermodynamic cycle showing the calculation of $\mathrm{\Delta }{G}_{\mathit{trans}}$ using the DD method, in which the restrained ligand is brought to the gas phase, both in the bound and unbound states.

Figure 4

(Top) Second BRD4 bromodomain with the restrained backbone section (part of the ZA-loop) in yellow, the rest of the protein in blue, and the ligand colored by element. The structure is from the 5uf0 cocrystal structure. (Bottom) Protein backbone showing Ramachandran torsion angles. The optional backbone restraints in BAT.py are applied to only $\varphi$ and $\psi$ angles.

Figure 5

Example of ligand dihedral restraints, for PDB ID 5uf0. (Left) Section of AMBER parameter/topology file listing all the ligand dihedrals that do not include a hydrogen atom. Each row lists two torsions in terms of five indices; the first four map to specific atoms and the fifth maps to the associated force field parameters. Dihedrals restrained in the BAT procedure are highlighted in purple font, with redundant ones in black and improper dihedrals in red. (Right) Ligand from 5uf0 with restrained torsions highlighted with purple bonds. Cyan: carbon. White: hydrogen. Red: oxygen. Blue: nitrogen.

Figure 6

Definition of ligand anchor atoms and dummy atoms for the co-crystal structure 5uf0. (Left) Strike zone (orange square), ligand anchor atom L1 (yellow), and protein anchor atoms (green). (Right) Same system rotated relative to the left-hand panel, illustrating the definition of the L2 and L3 ligand anchor atoms (yellow). The N1–L1–L2 and L1–L2–L3 angles are shown with green and purple lines, respectively, and dummy atoms are shown in red.

Figure 7

Chemical structures of ligands. (Left) BRD4 ligand 2-methyl-5-(methylamino)-6-phenylpyridazin-3(2H)-one (89J), for which binding free energies were computed. (Middle) Structure of the ligand, 5-methoxy-2-methyl-6-(2-phenoxyphenyl)pyridazin-3(2H)-one ligand, from the cocrystal structure 5uez, which provided the receptor structure for the 89J docking calculations. (Right) MCL-1 6-chloro-3-[3-(4-chloro-3,5- dimethylphenoxy) propyl]-1H-indole-2-carboxylic acid ligand (19H), evaluated in the second free energy calculations.

Figure 8

Crystal structures of proteins considered here. (Left) BRD4 crystal structure 5uf0, showing clear access of the ligand to the solvent. (Right) MCL-1 structure 4hw2, showing a site with possible steric barriers if using a physical path from the binding site to the solvent. The receptors are shown in gray, and the ligands are colored by element, with the chlorine atoms colored green.