Inhibition of the purified 20S proteasome by non-heme iron complexes

Abstract

Polypyridyl pentadentate ligands N4Py (1) and Bn-TPEN (2), along with their respective iron complexes, have been investigated for their ability to inhibit the purified 20S proteasome. Results demonstrated that the iron complexes of both ligands are potent inhibitors of the 20S proteasome (IC(50) = 9.2 μM for [Fe(II)(OH(2))(N4Py)](2+) (3) and 4.0 μM for [Fe(II)(OH(2))(Bn-TPEN)](2+) (4)). Control experiments showed that ligand 1 or Fe(II) alone showed no inhibition, whereas 2 was moderately active (IC(50) = 96 μM), suggesting that iron, when bound to these ligands, plays a key role in proteasome inhibition. Results from time-dependent inactivation studies suggest different modes of action for the iron complexes. Time-dependent decay of proteasome activity was observed upon incubation in the presence of 4, which accelerated in the presence of DTT, suggesting reductive activation of O(2) and oxidation of the 20S proteasome as a mode of action. In contrast, loss of 20S proteasome activity was not observed with 3 over time, suggesting inhibition through direct binding of the iron complex to the enzyme. Inhibition of the 20S proteasome by 4 was not blocked by reactive oxygen species scavengers, consistent with a unique oxidant being responsible for the time-dependent inhibition observed.

Fig. 1

Polypyridyl ligands.

Fig. 2

Time-dependent inactivation of the purified 20S proteasome. The reaction was performed by incubating the proteasome (0.5 nM) with 10 μM of ferrous complexes 3 (a), 4 (b and d (blue) without DTT, d (red) with DTT, 250 μM) and [Fe^II(TPEN)]²⁺ (c) in 100 mM MOPS buffer at pH = 7.4. The fluorescence intensity was converted to %CT-like activity, with 100% activity equal to fluorescence of the blank reaction containing proteasome, in the absence of the inhibitor at t = 0.

Fig. 3

Binding and oxidation mode of action of iron complexes for 20S proteasome inactivation.

Fig. 4

Model illustrating the coordination sphere around iron centers of the ferrous complexes derived from (a) Bn-TPEN and (b) TPEN. The sixth coordination site in model a is vacant for redox chemistry.

Fig. 5

Inactivation of the purified 20S proteasome by ferrous complex 4 in the presence of ROS scavengers (azide and d-mannitol). The reactions were performed by incubating proteasome with 4 (10 μM) in the presence/absence of DTT (250 μM) for 80 min at rt in 100 mM MOPS buffer at pH = 7.4. The fluorescence intensity was converted to %CT-like proteasome activity, with 100% activity equal to fluorescence of blank reaction containing proteasome in the absence of inhibitor and additive. Data points are averages from three independent experiments, errors equal to the standard deviation of the data set.