Novel inhibitors for the protease Taspase1

Abstract The threonine protease Taspase1 is of high interest as new target for cancer therapeutics, due to its implication in leukemias and solid tumors. Although Taspase is not an oncogene itself, it is crucially required for the proteolytic activation of the MLL oncogene as well as MLL fusion proteins generated by chromosomal translocations. Hence, MLL‐dependent cells can successfully be targeted by inhibition of Taspase activity. However, Taspase is insensitive to inhibition by general serine, cysteine or metalloprotease inhibitors. Furthermore, the limited number of available Taspase inhibitors display either low inhibitory potential or broad bioactivity. Thus, novel Taspase inhibitors are still urgently required. In a first step, detailed characterization revealed for the first time that recombinant Taspase and protein overexpressed in eukaryotic cells are comparable with respect to their catalytic parameters. Moreover, the specific catalytic activity (0.03 U/mg) was determined, as well as a 20‐fold higher efficiency regarding cleavage of the CS2 cleavage site of MLL over CS1. Regarding stability, Taspase rapidly loses its catalytic activity (half‐life < 3 h), unless it is stabilized by high concentrations of NaCl (≥ 450 mM). Additionally, the autocatalytic processing event required for catalytic competence was found to occur on a time scale of 2‐3 days. The flexible loop region between glycine 178 and aspartate 233 generated by this activation event is not resolved in the crystal structure. Hence, NMR and CD spectroscopy were applied and the results suggest a helixturn‐helix structure. Furthermore, analytical gel filtration pinpointed that Taspase is active as a hetero‐tetramer in solution. With these data, a fluorogenic in vitro assay was optimized for initial inhibitor tests. While the controversially discussed bisarsonic inhibitor NSC48300 and compounds derived from virtual docking exhibited no inhibitory effect, both silica nanoparticles and succinimide peptides were discovered as novel Taspase inhibitors. In fact, silica nanoparticles are known to bind proteins, albeit nanoparticle‐mediated inhibition of enzyme activity is scarcely described. Surprisingly, IC50 values in the nanomolar and picomolar range indicate high affinity binding, which was observed in vitro and in cells. In addition, comparison of nanoparticles with different diameters (8‐125 nm) suggests a positive correlation of size and affinity. Subsequent analyses characterized the inhibition type as noncompetitive and pinpointed that Taspase does not change its structure upon binding. Importantly, inhibition by nanoparticles is no general feature, as three other enzymes (lactate dehydrogenase, chymotrypsin, and proteinase K) showed no or only weak inhibition by silica nanoparticles. Another approach for Taspase inhibition exploited the cleavage sequence and the proposed mechanism to design substrate analog peptides with a succinimide moiety at the cleavage site. The most promising compound exhibited a Ki value of 400 ± 88 nM and displays thus a tenfold better inhibitory potential than the best reported Taspase inhibitor. Moreover, the competitive inhibition type and the resistance against Taspase cleavage were verified. However, degradation by other proteases and/or limited cellular uptake as well as competition with chloride ions prevents the in vivo inhibition of Taspase. Altogether, these findings demonstrate the potential of silica nanoparticles as well as succinimide peptides as Taspase inhibitors and tools for subsequent studies.