Therapy resistance remains a challenge for the clinics. Here, dual-active chemicals that simultaneously inhibit independent functions in disease-relevant
proteins are desired though highly challenging. As a model, we here addressed the unique
protease threonine aspartase 1, involved in various
cancers. We hypothesized that targeting basic residues in its bipartite
nuclear localization signal (NLS) by precise bisphosphate
ligands inhibits additional steps required for
protease activity. We report the bisphosphate anionic bivalent inhibitor 11d, selectively binding to the basic NLS cluster (220KKRR223) with high affinity (K D = 300 nM), thereby disrupting its interaction and function with
Importin α (IC50 = 6 μM). Cell-free assays revealed that 11d additionally affected the
protease's catalytic substrate trans-cleavage activity. Importantly, functional assays comprehensively demonstrated that 11d inhibited
threonine aspartase 1 also in living
tumor cells. We demonstrate for the first time that intracellular interference with independent key functions in a disease-relevant
protein by an inhibitor binding to a single site is possible.