Hence, we set up the ligands initially in pose 1, but employed the replica exchange with solute tempering (REST) enhanced sampling method with the goal of reducing the dependence of the computed binding free energies on the starting conditions. Importantly, in MCPRO, the REST algorithm may be employed alongside the 'flip' protocol, in which selected dihedral angles (here, φ 1 and φ 2 ) undergo Monte Carlo moves that are much larger than typical. shows a single peak at φ 2 = 250 • , indicating a strong preference for pose 2. This agrees with previous observations using the OPLS force field, 29 and x-ray crystal structures of similar ligands 33 (Figure ). Of note, in that former study, MC simulations were required starting from both poses 1 and 2 since interconversion between the two is not expected during these simulations using either standard MC or molecular dynamics. In contrast, the use of the REST/flip algorithm facilitates binding mode determination and free energy prediction from a single MC run. Despite being asymmetric, 12 shows similar behavior to 1, with peaks around φ 2 = 30 • and φ 2 = 210 • (Figure ). This is perhaps reasonable, since 12 is similar in chemistry to 17, but the bulkier Br atom may hinder full inclusion into the pose 2 binding pocket. Overall, we conclude that using the QUBE force field and REST enhanced sampling algorithm described here, the asymmetric ligands 4-11 and 13-15 bind in pose 1, ligands 16-18 bind in pose 2, and ligand 12 is intermediate between the two. Having elucidated the preferred binding poses of the 17 inhibitors, we turn now to the prediction of protein-ligand relative binding free energies. Figure compares the errors in the relative binding free energies computed using the QUBE protein/ligand force field with experiment. For comparison, the corresponding quantities are also displayed for the OPLS force field from previous work. Full details of the transformations and computed free energies are given in the Supporting Information. Overall, the mean unsigned error (MUE) is 0.98 kcal/mol, which is competitive with the generally accepted accuracy of standard biological force fields for transformations of this type 2 and, in particular, with previous calculations using the OPLS force field on this system (0.88 kcal/mol). The largest errors, using the QUBE force field are for ligands 12-15, which all include bulky and/or polar substituents at the R 1 position (Figure ), as well as ligand 7. The torsional profiles of these ligands are all reasonable, and so it seems likely that non-bonded interactions and/or sampling errors are to blame. We have found previously that QUBE can underestimate hydration free energies of some molecules containing bulky hydrophobic and hydroxyl functional groups by up to around 2 kcal/mol. Other possibilities, highlighted by Luccarelli et al., are that changes in solvent distribution in the binding pocket and/or protein side chain conformational changes are not properly sampled during alchemical perturbation. To investigate the adequacy of the REST method for sampling the complex binding mode of 12, we re-ran the 12→18 and 18→1 transformations starting with the ligand in pose 2. However, the error in the relative binding free energy of 12 fell only from 1.8 kcal/mol to 1.5 kcal/mol, indicating that the binding mode is sufficiently sampled during our simulations.