4 research outputs found
Proteasome Subunit Selective Activity-Based Probes Report on Proteasome Core Particle Composition in a Native Polyacrylamide Gel Electrophoresis Fluorescence-Resonance Energy Transfer Assay
Most
mammalian tissues contain a single proteasome species: constitutive
proteasomes. Tissues able to express, next to the constitutive proteasome
catalytic activities (β1c, β2c, β5c), the three
homologous activities, β1i, β2i and β5i, may contain
numerous distinct proteasome particles: immunoproteasomes (composed
of β1i, β2i and β5i) and mixed proteasomes containing
a mix of these activities. This work describes the development of
new subunit-selective activity-based probes and their use in an activity-based
protein profiling assay that allows the detection of various proteasome
particles. Tissue extracts are treated with subunit-specific probes
bearing distinct fluorophores and subunit-specific inhibitors. The
samples are resolved by native polyacrylamide gel electrophoresis,
after which fluorescence-resonance energy transfer (FRET) reports
on the nature of proteasomes present
Systematic Analyses of Substrate Preferences of 20S Proteasomes Using Peptidic Epoxyketone Inhibitors
Cleavage
analyses of 20S proteasomes with natural or synthetic
substrates allowed to infer the substrate specificities of the active
sites and paved the way for the rational design of high-affinity proteasome
inhibitors. However, details of cleavage preferences remained enigmatic
due to the lack of appropriate structural data. In a unique approach,
we here systematically examined substrate specificities of yeast and
human proteasomes using irreversibly acting α′,β′epoxyketone
(ep) inhibitors. Biochemical and structural analyses provide unique
insights into the substrate preferences of the distinct active sites
and highlight differences between proteasome types that may be considered
in future inhibitor design efforts. (1) For steric reasons, epoxyketones
with Val or Ile at the P1 position are weak inhibitors of all active
sites. (2) Identification of the β2c selective compound Ac-LAE-ep
represents a promising starting point for the development of compounds
that discriminate between β2c and β2i. (3) The compound
Ac-LAA-ep was found to favor subunit β5c over β5i by three
orders of magnitude. (4) Yeast β1 and human β1c subunits
preferentially bind Asp and Leu in their S1 pockets, while Glu and
large hydrophobic residues are not accepted. (5) Exceptional structural
features in the β1/2 substrate binding channel give rise to
the β1 selectivity of compounds featuring Pro at the P3 site.
Altogether, 23 different epoxyketone inhibitors, five proteasome mutants,
and 43 crystal structures served to delineate a detailed picture of
the substrate and ligand specificities of proteasomes and will further
guide drug development efforts toward subunit-specific proteasome
inhibitors for applications as diverse as cancer and autoimmune disorders
Structure-Based Design of β5c Selective Inhibitors of Human Constitutive Proteasomes
This work reports
the development of highly potent and selective
inhibitors of the β5c catalytic activity of human constitutive
proteasomes. The work describes the design principles, large hydrophobic
P3 residue and small hydrophobic P1 residue, that led to the synthesis
of a panel of peptide epoxyketones; their evaluation and the selection
of the most promising compounds for further analyses. Structure–activity
relationships detail how in a logical order the β1c/i, β2c/i,
and β5i activities became resistant to inhibition as compounds
were diversified stepwise. The most effective compounds were obtained
as a mixture of <i>cis</i>- and <i>trans</i>-biscyclohexyl
isomers, and enantioselective synthesis resolved this issue. Studies
on yeast proteasome structures complexed with some of the compounds
provide a rationale for the potency and specificity. Substitution
of the N-terminus in the most potent compound for a more soluble equivalent
led to a cell-permeable molecule that selectively and efficiently
blocks β5c in cells expressing both constitutive proteasomes
and immunoproteasomes
Incorporation of Non-natural Amino Acids Improves Cell Permeability and Potency of Specific Inhibitors of Proteasome Trypsin-like Sites
Proteasomes degrade the majority of proteins in mammalian
cells
by a concerted action of three distinct pairs of active sites. The
chymotrypsin-like sites are targets of antimyeloma agents bortezomib
and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple
myeloma cells to these agents. Here we describe systematic effort
to develop inhibitors with improved potency and cell permeability,
yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone
(<b>4a</b>, LU-102), and a fluorescent activity-based probe
for this site. X-ray structures of <b>4a</b> and related inhibitors
complexed with yeast proteasomes revealed the structural basis for
specificity. Nontoxic to myeloma cells when used as a single agent, <b>4a</b> sensitized them to bortezomib and carfilzomib. This sensitizing
effect was much stronger than the synergistic effects of histone acetylase
inhibitors or additive effects of doxorubicin and dexamethasone, raising
the possibility that combinations of inhibitors of the trypsin-like
site with bortezomib or carfilzomib would have stronger antineoplastic
activity than combinations currently used clinically