The original plan was to keep the amine in GRL-0617 in all the analogues, however the amine group was not compatible with most of the reactions carried out in this study, added synthetic challenges, and got oxidized easily. Therefore, different groups have been used to replace the amine group including nitro, hydroxy, methyl, Cl and H. This was done to avoid the reactivity of the amine, and at the same time increase the reactivity for the R1 group to perform coupling reactions. These compounds were tested against PL pro at 50 μM using a peptide (Z-RLRGG-AMC) as a fluorescent substrate. Focusing on protease activity, all the compounds lost affinity when the amine group was absent (Table ). SP-1f, although not as active as GRL-0617 (SP-1a), achieved 60% inhibition at 50 μM concentration with peptide as a substrate. The most potent compound, SP-5a, the designed hybrid analogue of VIR-251 and GRL-0617, displayed 80% inhibition at 50 μM concentration with peptide as a substrate. From the dose-response curve, the IC50 of this compound is 13.3 ± 9 μM, reaching low micromolar potency, similar to GRL0617 (Figure ). Virtual Screening. As the synthesis of the hybrid structures turned out to be a lot more difficult than anticipated (see Supporting Information), a virtual screening campaign was carried out concomitantly. High throughput docking and large computational resources may be envisioned (e.g., screening millions or even billions of compounds). This approach has been successful, illustrated by the screening of 40B molecules against 3CL pro by Cherkasov and co-workers using a combined machine learning (ML)/docking approach. Challenges with this approach include: analyzing the large resulting data (visual inspection is always recommended) can be time consuming, many of these docked molecules may be intrinsically not good candidates (e.g., too large, featuring toxicophores, not drug-like) and some post-processing analysis removing undesired molecules is often required. In the case of covalent drugs, an additional step is to remove compounds not featuring a reactive warhead susceptible to make a covalent bond with the catalytic cysteine. In addition, it requires ultrafast docking programs, which are often less accurate as a trade-off.