Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit

摘要

The mammalian 26S proteasome is a 2500?kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin–proteasome system in cells. We show that these compounds are entirely selective for the β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S β5 site in vitro , and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin–luciferase reporter, activation of NFκB (nuclear factor κB) in response to TNF-α (tumour necrosis factor-α) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the β5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the β5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.Abbreviations: Ac, acetyl; AMC, 7-amino-4-methylcoumarin; Boc, t-butoxycarbonyl; HBTU, O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HEK, human embryonic kidney; LC50, half-maximal lethal concentration; MPD, 2-methyl-2,4-pentanediol; NFκB, nuclear factor κB; IκB, inhibitory protein of NFκB; NFκB-Luc, NFκB–luciferase; PA, proteasomal activator; PDL, poly-D-lysine; RNAi, RNA interference; siRNA, small interfering RNA; Suc, succinyl; TEV, tobacco etch virus; TNF-α, tumour necrosis factor-α; 4xUb-Luc, tetra-ubiquitin–luciferase; UPS, ubiquitin–proteasome system; Z, benzyloxycarbonyl