2.1. Identification of hit molecules from virtual screening. We compiled a small peptidic library from the collection of TüKIC, which contains over 10,000 members that target a nucleophilic cysteine residue in a variety of kinases. Initially, compounds were screened at 10 μM against CatL, and three hit compounds that inhibited CatL by ≥50% were chosen to examine concentration-dependent inhibition. The Michael acceptor I (Figure ) inhibited CatL with a Ki value of 0.152 μM, while the nitroaniline derivative II and the proteasome inhibitor carfilzomib (III) showed moderate activity with Ki values of 1.62 and 4.29 μM, respectively. Compound II was previously reported as a covalent reversible inhibitor of SARS-CoV-1 M pro with an IC50 value of 30 nM. With the structural information from these hits and by particularly using compound I as a lead CatL inhibitor, we designed and synthesized new representatives of this chemotype by systematically incorporating various warhead groups and extending the lead structure at the N-terminus. The biochemical data of the generated series of peptidomimetics were analyzed, and structure-activity relationships (SARs) were deduced.