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

Abstract

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.

Structural and Functional Characterization of the Immunoproteasome

“…Hence, the β1i active site preferentially cleaves proteins C-terminally of small hydrophobic and branched amino acids such as Leu, Ile or Val and significantly enhances the production of high-affinity MHC I ligands. The structural features of subunit β1i are in full agreement with the reported β1i-selective fluorogenic substrate Ac-Pro-Ala-Leu-AMC [110] . In addition to the changes in the S1 pocket, the S3 pocket is characterized by the amino acid substitutions T22A (β1i) and A27V (β1i) as well as Y114H in the neighbouring subunit β2i.…”

“…pockets, the β5i subunit prefers more spacious ones than subunit β5c [110] (see also chapter 6.3.3).…”

Brief treatment with a highly selective immunoproteasome inhibitor promotes long-term cardiac allograft acceptance in mice

“…The synthetic route and methods of compound characterization were similar to those reported for N,C-capped dipeptidomimetics (15,28), with modifications as given in the supplemental online material. All compounds were >95% pure.…”

“…We cultured Karpas1106P B lymphoma cell line (catalog no. 06072607; Aldrich) (15), mouse bone marrow-derived macrophages (18), PBMCs (17,30), and HepG2 human hepatoma cells (31). Karpas cells were cultured in complete medium and the other cells in complete medium with 10% (vol/vol) FBS instead of 20% (vol/vol), all at 37°C in a humidified air/5% (vol/vol) CO 2 .…”

Why the Structure but Not the Activity of the Immunoproteasome Subunit Low Molecular Mass Polypeptide 2 Rescues Antigen Presentation

“…Previous studies have reported that in proteasomes derived from livers of LCMV-infected mice, the three catalytically active constitutive subunits, b1, b2, and b5, are almost completely replaced by the immunoproteasome subunits LMP2, MECL-1, and LMP7 (36). LMP2-deficient and WT immunoproteasomes (from the livers of LCMV-WE-infected mice) were incubated with different concentrations of ML604440 and assayed with a fluorogenic substrate specific for LMP2 activity (Ac-PAL-AMC) (37) (Fig. 2B).…”