High‐Throughput Synthesis and Screening of a Cyanimide Library Identifies Selective Inhibitors of ISG15‐Specific Protease mUSP18

High-throughput screening (HTS) of large compound collections is a critical early step in many drug discovery programs. Its success depends heavily on the quality of the compound libraries used, and as such, the development of targeted libraries has emerged to enhance the effectiveness of HTS efforts. However, the acquisition of such libraries remains costly and labor-intensive, often yielding compound quantities far exceeding required amounts. We present a high-throughput synthesis-to-screening method for the efficient in-plate generation and immediate HTS of a deubiquitinase (DUB)-focused compound library. Central to our approach is the Echo acoustic liquid handler, which transfers nanoliter volumes of DMSO-based solutions, facilitating miniaturized synthesis directly in 1536-well plates. We constructed a library of 7536 compounds featuring a DUB-privileged cyanimide warhead and screened against twelve ubiquitin(-like) proteases. This identified two structurally related molecules with selective inhibitory activity against the interferon-stimulated gene 15 (ISG15) protease mUSP18, which we further developed into a first-in-class mUSP18 inhibitor with 35 nM potency. This compound, BB07CA902, demonstrated exceptional specificity for mUSP18 across 41 DUBs and effectively increased ISGylation levels in cells by inhibiting mUSP18 activity. Our technology enables the efficient preparation of large DUB-targeted cyanimide-based libraries, which will accelerate future DUB inhibitor development.