Click chemistry enables rapid development of potent sEH PROTACs using a direct-to-biology approach

The direct-to-biology (D2B) approach enables biological screening of crude reaction mixtures, eliminating the need for purification steps and thereby accelerating drug discovery. In this study, we developed a miniaturized D2B platform for the rapid synthesis of proteolysis targeting chimera (PROTAC) degraders of soluble epoxide hydrolase (sEH). We used copper-catalyzed azide-alkyne cycloaddition and optimized the conditions for 384-well PCR plate applications with 10 μL reaction volumes on a 300 nmol scale. This approach enabled the D2B synthesis of 92 crude PROTACs from azide-functionalized CRBN-ligands and alkyne-linked sEH inhibitors. Biological screening using a HiBiT lytic degradation assay identified two hits that were resynthesized and exhibited subnanomolar DC₅₀ values and degradation efficacy (D_max). Thus, we established a scalable, cost-effective and time-saving D2B platform for the discovery of PROTACs in very small quantities. This methodology is particularly suitable for early-stage screening and hit validation assessing the degradability of a target.