title: Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S beta5-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 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.
authors.affiliation: Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Blackburn
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Gigstad
authors.forename: Paul
authors.initials: P
authors.lastname: Hales
authors.forename: Khristofer
authors.initials: K
authors.lastname: Garcia
authors.forename: Matthew
authors.initials: M
authors.lastname: Jones
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Bruzzese
authors.forename: Cynthia
authors.initials: C
authors.lastname: Barrett
authors.forename: Jane X
authors.initials: JX
authors.lastname: Liu
authors.forename: Teresa A
authors.initials: TA
authors.lastname: Soucy
authors.forename: Darshan S
authors.initials: DS
authors.lastname: Sappal
authors.forename: Nancy
authors.initials: N
authors.lastname: Bump
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Olhava
authors.forename: Paul
authors.initials: P
authors.lastname: Fleming
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Dick
authors.forename: Christopher
authors.initials: C
authors.lastname: Tsu
authors.forename: Michael D
authors.initials: MD
authors.lastname: Sintchak
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Blank
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018360:Crystallography, X-Ray; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
chemical_list: D001897:Boronic Acids; D016328:NF-kappa B; D009842:Oligopeptides; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D021122:Protein Subunits; D011719:Pyrazines; D025801:Ubiquitin; D000069286:Bortezomib; D008156:Luciferases; D046988:Proteasome Endopeptidase Complex