We carried out our VS with AutoDock Vina [54 (link)] in the PyRx open-source software package [55 (link)]. In brief, SDF files of our 1604-compound FDA-approved library were downloaded from the ZINC15 database [46 (link),47 (link),48 (link)] and loaded into PyRx with the Open Babel chemical toolbox [64 (link)]. The SDF files were then energy-minimized and appropriately protonated to generate the required PDBQT files. Once all ligands were prepared, FL ε R3 (PDB 6var) [30 (link)] was loaded and prepared as the receptor molecule. The docking grid was prepared in a manner to ensure an unbiased dock (i.e., the grid encompasses the entire receptor molecule), and therefore, dimensions of 64.9 × 57.0 × 39.3 were used. Finally, we enabled nine possible docking poses per ligand. The intention of our VS was to rapidly screen our compound library and rank-order our lead compounds by predicted affinity. We therefore carried out selection criteria on the basis of affinity, commercial availability and drug-like properties, and dock site (Figure 2 a) to identify the lead compounds from our 1604-compound library. For our first selection step, we took the 122 compounds whose top-ranked docking pose had a predicted affinity higher than that of the already known [30 (link)] ε-targeting ligand Raloxifene (−9.5 kcal·mol−1) (Figure 2 b). For our second selection step, we excluded all compounds that were not commercially available and/or had potential adverse effects and proceeded with 66 compounds (Figure 2 c). This step required the manual curation of the compound library. We considered any anticancer drug or any compound with a mode of action that included the inhibition of fundamental cellular processes (e.g., DNA replication) as having a potential adverse effect. Given the functional importance of the PL [22 (link),23 (link),27 (link),29 (link),33 (link),34 (link)] (Figure 1 a) and our previous computational [30 (link),31 (link)] (Figure 1 c) and experimental [30 (link)] (Figure 1 d) data, our final selection step chose compounds that targeted the ε PL. To increase our confidence that we proceeded with authentic ε PL-targeting compounds, we repeated our docking three additional times. Then, we classified confident PL docking on the basis of two criteria: (1) top-rank pose localized to the PL in >50% of the repeated runs and/or (2) >50% of all poses localized to the PL (Figure S1 ). In both instances, PL localization was loosely defined as having more than one contact within 5 Å of a PL nucleotide (i.e., C14–C19, including the adjacent A13, A20, U48, and U49). In total, our VS identified 12 initial lead compounds (Figure 2 e).
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