The goal of the second round was to build confidence in advanced hits by experimentally verifying that their chemical analogs were also binding to the target. Compounds associated with experimental orange flags, such as signs of aggregation or poor solubility, were advanced to Round 2 to avoid false negatives and unfairly discounting computational methods. Seventeen teams selected up to 50 analogs of their Round 1 compounds of interest (compounds showing a binding signal by SPR), leading to 618 Round 2 molecules that were screened at 50 µM in an SPR binding assay, followed by dose-response and measurements of aggregation and solubility, as in Round 1 (Tables ). Compounds were also tested in an ATPase assay (Tables and respectively), and no correlation was observed with SPR data, as in Round 1. F-NMR was used as an orthogonal binding assay for fluorinated molecules.