A Bivalent Supramolecular GCP Ligand Enables Blocking of the Taspase1/Importin α Interaction

Abstract

Taspase1 is a unique protease not only pivotal for embryonic development but also implicated in leukemia as well as solid tumors. As such, it is a promising target in cancer therapy, although only a limited number of Taspase1 inhibitors lacking general applicability are currently available. Here we present a bivalent guanidiniocarbonyl‐pyrrole (GCP)‐containing supramolecular ligand that is capable of disrupting the essential interaction between Taspase1 and its cognate import receptor Importin α in a concentration‐dependent manner in vitro with an IC 50 of 35 μM. Here, size of the bivalent vs the monovalent construct as well as its derivation with an aromatic cbz‐group arose as critical determinants for efficient interference of 2GC . This was also evident when we investigated the effects in different tumor cell lines, resulting in comparable EC 50 values (∼40–70 μM). Of note, in higher concentrations, 2GC also interfered with Taspase1’s proteolytic activity. We thus believe to set the stage for a novel class of Taspase1 inhibitors targeting a pivotal protein‐protein interaction prerequisite for its cancer‐associated proteolytic function.

Recognition of a Flexible Protein Loop in Taspase 1 by Multivalent Supramolecular Tweezers

“…Systematic experiments involving the full-length protease corroborated a tendency toward increased potencies from the mono- to the pentavalent ligand, revealing IC 50 values of less than 2 μM. Earlier approaches using sequence-controlled macromolecules or bivalent guanidiniocarbonyl-pyrrole (GCP)-containing supramolecular ligands were substantially less effective in targeting the Taspase 1 loop and especially in interfering with proteolytic activity.…”

“…A customized biochemical pull-down assay was subsequently employed to evaluate the effects of our multivalent molecular tweezers on the Taspase 1/Importin α interaction (Figures and and Supporting Information Figures S7–S10). , Because Importin binding crucially relies on the basic NLS clusters present in the Taspase 1 loop, the assay serves as a reliable and direct readout for tweezer binding to the responsible lysine and arginine residues. Briefly, recombinant Importin α is expressed with an N-terminal glutathione S -transferase (GST) affinity tag and immobilized on a glutathione sepharose column.…”

“…The observed correlation between valency and inhibitory potential revealed for the flexible protein loop in Taspase 1 is sharply contrasted by the preferred recognition of compactly folded protein regions with defined distances of single lysine residues. In these scenarios, tweezer dimers or small oligomers with rigid linkers are superior to their larger counterparts, as shown for the critical interface on microtubule-binding NDC80 and the segregase p97. − The fact that multivalency of tweezer molecules also allows to gradually increase their inhibitory potential towards the proteolytic activity of full-length Taspase 1 implies that bulky, multivalent ligands resembling a kraken most likely hinder substrate access to the active center where they outperform smaller ligands . In the future, the nature of the flexible arm will be optimized with respect to length and rigidity.…”

“…pET22b-Taspase1 D233A/T234 -His, pET22b-WT_Taspase1-His, pET41-GST-PreScn-Importinα, pET22b-GST-MLL 2700–2850 -GFP-His, and the 15 N-labeled Taspase 1 loop 178–233 for NMR titrations were expressed and purified as previously reported. ,, Purity was validated using SDS polyacrylamide gel electrophoresis (PAGE) and immunoblotting.…”

“…Importantly, Taspase 1 is preferentially expressed during embryonic development, and thus, pharmacological intervention should not affect healthy adult cells. ,,− In contrast, tumor cells have been demonstrated to upregulate the expression of Taspase 1 to promote their proliferation and counteract apoptotic cell death. ,,, This classifies Taspase 1 as a ″non-oncogene addiction″ protease. , However, the full repertoire of physiological and pathological targets and pathways affected by Taspase 1 is still not fully understood. Although Taspase 1 belongs to the family of threonine proteases, it is not affected by general protease inhibitors. , Some more or less specific inhibitors have been suggested for Taspase 1, but their mode of action is often not understood or they need to be used in high concentrations. ,,,, This clearly impedes the molecular dissection of Taspase 1’s (patho)­biologic functions and hence the full exploitation of its therapeutic potential. ,,− Until today, Taspase 1 is not only a promising target for intervention but also a challenging model for rational drug design. , …”