To demonstrate the potential for the use of QUBE/SOMD in prospective drug discovery efforts, we analyze the relative alchemical binding free energies of a series of NNRTIs with HIV-1 RT. Crystal structures are available for compounds 1c, a catechol diether that incorporates a 7-cyano-2-naphthyl substituent, and 2a, where the 2-napthyl group is replaced by an indolizine. Figure shows overlays of compounds 1c and 2a from SOMD simulations using the QUBE force field, with the corresponding crystal structures. In both cases, the cyano group projects out below Trp229 into a solvent-exposed channel, while close aryl-aryl contacts with Tyr188 and Trp229 are maintained. The orientation of residue Tyr181 shows sensitivity to the identity of the ligand, which is consistent with previous simulations 54 and crystallography. In complex with compound 1c, Tyr181 switches orientation during MD simulations, from a face-to-face interaction with the catechol diether ring of the ligand, to form closer contact with the 2-napthyl group. Our implementation of the QUBE force field in SOMD now allows us to perform highthroughput alchemical free energy calculations to investigate the effects on binding of small changes to the ligand. A disadvantage of the chosen case study is that enzyme-based activities are unavailable for these compounds. Following previous studies, we provide here EC 50 data measured using human T-cells infected by wild-type HIV-1 (MT-2 cell assays) for comparison with our computational results. Although it has been demonstrated that cell-and enzymebased activities are well-correlated for several NNRTI series, we cannot guarantee that experimental data are not affected by, for example, differences in cell permeability between compounds. We therefore focus here on general observations and convergence studies, and refer the reader to previous work where we benchmark the accuracy of QUBE in proteinligand binding. displays the binding free energies of the 2-napthyl ethers (relative to compound 1e) computed using the QUBE force field, along with experimental assay data. For comparison, we also report relative binding free energies computed with the AMBER force field, using the same free energy protocols (Table ). Compounds 1a-1e incorporate a series of small methyl and halogen transformations at the R 1 and R 2 positions. With R 2 = Me, the Me and Cl substitutions at R 1 are favored over F (1e < 1c < 1b) in both computational free energy studies with QUBE and in experimental assays. Experimentally, the two compounds (1a and 1d) with R 2 = Cl are equipotent and less strongly bound than 1e, while QUBE slightly favors compound 1d.