Functional Linkers Support Targeting of Multivalent Tweezers to Taspase1

Abstract Taspase 1 is a unique protease not only pivotal for embryonic development but also implicated in leukemias and solid tumors. As such, this enzyme is a promising while still challenging therapeutic target, and with its protein structure featuring a flexible loop preceding the active site a versatile model system for drug development. Supramolecular ligands provide a promising complementary approach to traditional small‐molecule inhibitors. Recently, the multivalent arrangement of molecular tweezers allowed the successful targeting of Taspase 1’s surface loop. With this study we now want to take the next logic step und utilize functional linker systems that not only allow the implementation of novel properties but also engage in protein surface binding. Consequently, we chose two different linker types differing from the original divalent assembly: a backbone with aggregation‐induced emission (AIE) properties to enable monitoring of binding and a calix4arene scaffold initially pre‐positioning the supramolecular binding units. With a series of four AIE‐equipped ligands with stepwise increased valency we demonstrated that the functionalized AIE linkers approach ligand binding affinities in the nanomolar range and allow efficient proteolytic inhibition of Taspase 1. Moreover, implementation of the calix4arene backbone further enhanced the ligands’ inhibitory potential, pointing to a specific linker contribution.