Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit
| Autor: | Edward J. Olhava, Paul Hales, Cynthia Barrett, Jane X. Liu, Matthew Jones, Frank J. Bruzzese, Kenneth M. Gigstad, Khristofer Garcia, Christopher Tsu, Darshan S. Sappal, Teresa A. Soucy, Paul E. Fleming, Christopher Blackburn, Michael D. Sintchak, Jonathan L. Blank, Nancy Bump, Lawrence R. Dick |
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| Rok vydání: | 2010 |
| Předmět: |
Models
Molecular IκB inhibitory protein of NFκB Plasma protein binding PA proteasomal activator Crystallography X-Ray β5-subunit TNF-α tumour necrosis factor-α Biochemistry Bortezomib Ubiquitin Correction Article Luciferases 26S proteasome ubiquitin–proteasome system (UPS) Molecular Structure biology NF-kappa B Boronic Acids Boc t-butoxycarbonyl RNAi RNA interference Pyrazines RNA Interference HT29 Cells Oligopeptides Proteasome Inhibitors Protein Binding Research Article medicine.drug Proteasome Endopeptidase Complex UPS ubiquitin–proteasome system LC50 half-maximal lethal concentration Molecular Sequence Data Cell Line TEV tobacco etch virus immunoproteasome HEK human embryonic kidney PDL poly-D-lysine Cell Line Tumor medicine Humans Protease Inhibitors Amino Acid Sequence AMC 7-amino-4-methylcoumarin Suc succinyl Binding site Z benzyloxycarbonyl Molecular Biology Cell Proliferation Ac acetyl Binding Sites Sequence Homology Amino Acid proteasome inhibitor HBTU O-benzotriazole-N N N′ N′-tetramethyluronium hexafluorophosphate chymotrypsin-like MPD 2-methyl-2 4-pentanediol Cell Biology HCT116 Cells NFKB1 In vitro Protein Structure Tertiary NFκB-Luc NFκB–luciferase Kinetics Protein Subunits NFκB nuclear factor κB 4xUb-Luc tetra-ubiquitin–luciferase Proteasome siRNA small interfering RNA Cancer cell biology.protein Proteasome inhibitor |
| Zdroj: | Biochemical Journal |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20100383 |
| Popis: | 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 based [corrected] 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 beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (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 beta5 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 NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 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 beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells. |
| Databáze: | OpenAIRE |
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