The top 500 compounds from each of four active learning runs (two optimising predicted pK, one optimising protein-ligand interactions, and one optimising the combined scoring function) were checked for availability from the Enamine store. Interestingly, very few of the top scored by predicted pK were available (four in total). This is likely due to an important unavailable building block(s), and could be mitigated in future by increasing diversity and/or including direct store queries in the search process. In any case, we focussed here on outputs from the remaining two runs, and submitted the top 10 protein-ligand interaction and top 25 combination scoring compounds for costing. Finally, a total of 19 designed compounds were purchased (of which 15 had been optimised used the combination score) based on quoted price and excluding similar compounds (based on visual inspection). Two control compounds were also included; one known binder from a crystallographic fragment screen (Enamine ID: Z44592329; PDB: 5R83) and one elaborated compound from the COVID Moonshot study (Enamine ID: Z4943052515 (literature IC 50 0.288 µM)). The twenty one purchased compounds (Figure ) were evaluated in a fluorescence-based Mpro activity assay at 1000, 500, 10 µM (Figure ). Compounds 5 and 6 were excluded from the study due to solubility issues at 1000 µM in the assay conditions. Five compounds (8, 10, 12, 14 and 21 (the positive control 4 )) showed reduction of Mpro activity ≤ 50% at 1000 µM. The IC 50 values of these compounds, except 8 which displayed background autofluorescence, were further determined (Figure ). Compounds 10, 12 and 14 showed a concentration-dependent inhibition of Mpro activity (measured pIC 50 2.10, 3.01, 2.80 respectively). Nirmatrelvir, an orally bioavailable antiviral drug targeting Mpro, showed inhibition (pIC 50 6.01), which was slightly higher than the reported IC 50 (0.022 µM 55 ), likely due to the limit of the assay (the enzyme concentration was at 0.2 µM). Figure shows the predicted structures of compounds 12 and 14 from the active learning design runs. Both compounds form hydrogen bonding interactions with the backbone of Glu166, as well as hydrophobic interactions in the S1' pocket.