Alternatively, we replaced this post-processing step with a pre-processing step with the goal of using our docking program FITTED which, sacrifices some runtime for higher accuracy. . We started from libraries of sizes similar to those used in high throughput docking (several millions of molecules) and we removed compounds that are unlikely to bind and/or are not drug-like before docking. From the crystal structures of PL pro co-crystallized with ligands, it was clear that hydrogen bond donors and acceptors were required. Finally, based on known inhibitors and on the structure of PLpro catalytic/binding site, we selected molecules with a net charge of 0 or +1 for docking to PL pro . With all these filters, we generated a library of over 85,000 compounds featuring a covalent warhead from the ZINC database and of over 25,000 from the ChemSpace database. These compounds were docked using FITTED which allows for automated covalent docking. More specifically, FITTED automatically recognizes any potential warheads and docks the molecules considering both covalent and non-covalent binding modes in a single run. Analysis. When the docking was complete, the 200 best scoring compounds were extracted, and their binding modes were investigated by our team of medicinal chemists, biochemists, and computational chemists. Through a voting system, 25 were selected for PL pro and 21 of them purchased (Figure ). From virtual screening, a few molecules showed satisfactory interactions with PL pro in their docking. For example, the catalytic Cys111 is predicted to react with the Michael acceptor warhead on ZINC3276278 to form a covalent bond (Figure ). The N-H on the backbone of Cys111 and Gly271 form H-bonds with the methoxy oxygen and the amide carbonyl, while the backbone of Gly163 acts both as a H-bond donor and acceptor. The side chain of Thr273 forms an H-bond with the carbonyl of the benzophenone moiety on the molecule. On the other hand, the catalytic Cys111 is predicted to react with the ketone group of CSSS58908458. The resulting hydroxyl is stabilized by the backbone of both Cys111 and Asn109. The carbonyl on the backbone of Gly163 forms H-bonds with both the amide N-H and the positively charged N-H on the molecule, while the amide carbonyl forms an H-bond with the backbone of Gly271. Overall, these two virtual hits demonstrate complementary interactions with PL pro in docking. Inhibitory potency. Fluorescence assays were performed on the 21 purchased compounds. These compounds were tested against PL pro at 25 μM using ubiquitin-AMC (Ub-AMC) and a peptide (Z-RLRGG-AMC) as fluorescent substrates. These two substrates allowed us to evaluate two activities of PL pro as discussed above. GRL0617 was used as a positive control. Four classes of inhibitors were identified with potencies in the mid-micromolar range which, for hit molecules, is close to GRL0617, one of the best inhibitors reported to date (IC50 = 5.8 ± 1 µM) (Figure ). We decided to resynthesize and optimize two of the molecules (ZINC3276278 and CSSS58908458) and carry out a preliminary structure-activity relationship of the other two hit molecules (CSSS28078573 and CSSS28008898) using commercially available analogues to increase their potencies (Figure ). Interestingly, a closer look at the docked binding modes of the first two hits identified by virtual screening (see for example ZINC3276278 in Figure ) suggested that these compounds bind covalently and fill up the tunnel as does VIR251 and feature aromatic functionalities in the allosteric site as does GRL0617. As a result, these are very similar to the hybrid molecule we designed and discussed above.