Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S [beta]5-subunit

Journal Article · Mon Apr 30 04:00:00 EDT 2012 · Biochem. J.

DOI:https://doi.org/10.1042/BJ20100383· OSTI ID:1037488

Blackburn, Christopher; Gigstad, Kenneth M; Hales, Paul; Garcia, Khristofer; Jones, Matthew; Bruzzese, Frank J; Barrett, Cynthia; Liu, Jane X; Soucy, Teresa A; Sappal, Darshan S; Bump, Nancy; Olhava, Edward J; Fleming, Paul; Dick, Lawrence R; Tsu, Christopher; Sintchak, Michael D; Blank, Jonathan L ^[1]

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1. Millennium

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 {beta}5 (chymotrypsin-like) site over the {beta}1 (caspase-like) and {beta}2 (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 IC{sub 50} values for the human 20S {beta}5 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 NF{Kappa}B (nuclear factor {Kappa}B) in response to TNF-{alpha} (tumor necrosis factor-{alpha}) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the {beta}5 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 {beta}5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

• Research Organization: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
• Sponsoring Organization: INDUSTRY
• OSTI ID: 1037488
• Journal Information: Biochem. J., Journal Name: Biochem. J. Journal Issue: 09, 2010 Vol. 430; ISSN 0264-6021
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
CRYSTALLOGRAPHY
IN VITRO
LYMPHOMAS
NECROSIS
NEOPLASMS
OPTIMIZATION
PROTEINS
SYNTHESIS