Kooij, R.; Pol, V.; Gan, J.; van Doodewaerd, B.; Sapmaz, A.; Fritz, G.; Knobeloch, K.-P.; Geurink, P. P.

High-throughput screening (HTS) of (large) compound collections is a critical early step in many drug discovery programs, enabling the rapid identification of lead molecules with desirable biological activity. The success of HTS depends heavily on the quality of the compound libraries used, and as such, the development of targeted libraries has emerged as a promising approach to enhance the effectiveness of HTS efforts. However, the acquisition and synthesis of such libraries remain costly and labor-intensive, often yielding compound quantities far exceeding the small amounts required for screening. To address these challenges, 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 use of an Echo acoustic liquid handler, which enables precise nanoliter-scale transfers of DMSO-based solutions, facilitating efficient and miniaturized synthesis directly in 1,536-well plates. Using this platform, we constructed a library of 7,536 compounds featuring a DUB-privileged cyanimide warhead and screened it against a panel of twelve DUBs and ubiquitin-like proteases. This identified two structurally related molecules with selective inhibitory activity against the interferon-stimulated gene 15 (ISG15) protease USP18, which we further developed into a first-in-class USP18 inhibitor with 35 nM potency. This compound, BB07CA902, demonstrated exceptional specificity for USP18 across a panel of 41 DUBs and effectively increased ISGylation levels in cells by inhibiting USP18 activity. With our technology, we enabled the efficient preparation of large DUB-targeted cyanimide-based libraries, which will accelerate future DUB inhibitor development.