Bortezomib-treated multiple myeloma cells undergo apoptosis at different rates.

<p>Caspase-3 activity (A) and cleavage of PARP (B) were measured in extracts of cells treated with bortezomib for 1 h, and then cultured in drug-free media. B. Cleavage of PARP was assessed by western blotting. C. The % of Annexin V-positive cells was determined by flow cytometry. Cells on the first 3 graphs were analyzed 24 h after the start of 1-h bortezomib treatment. Values are averages ± S.E.M of 2–4 independent experiments.</p

Effect of inhibitors on degradation of short-lived proteins in multiple myeloma cells.

<p>Degradation of short-lived proteins was analyzed after 1-h pulse labeling with [³H]Leu followed by 1-h chase. Total degradation was calculated as % of [³H]Leu incorporated during released in the TCA-soluble fraction during 1 h chase. Treatment with inhibitors was performed during pulse labeling.</p><p>% inhibition of degradation (at 1 h) was calculated as [1–(%TCA soluble radioactivity in the presence of inhibitor)/(% TCA soluble in the absence of inhibitor)]×100%. Inhibition of proteasomal degradation was determined by dividing inhibition by bortezomib by inhibition by ZL3ek.</p><p>Values are averages ± SEM of <i>n</i> independent experiments for inhibition of protein degradation. Data on viable cells are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056132#pone-0056132-g001" target="_blank">Fig. 1B</a>, where errors are shown.</p

Comparison of 48-h continuous and a 1-h treatment of multiple myeloma cells with bortezomib.

<p>A. Cells were treated with bortezomib for 48 h, and then assayed for viable cells with the Alamar Blue mitochondrial dye conversion assay. Mock-treated cells served as control. Values are means±S.E.M of two experiments. B. Cells were treated with bortezomib for 1 h, then cultured in a drug-free media for an additional 47 h and finally assayed for viable cells with Alamar blue. Values are mean ± S.E.M of 4–13 measurements. IC₅₀ values are presented in the legend.</p

Contribution of long-leaved proteins to proteasome load.

1<p>Calculated as % of [³H]Leu incorporated into proteins (i.e., TCA-precipitable) after 16-h labeling/16 h.</p>2<p>% Of [³H]Leu released into TCA-soluble fraction/h, relative to the total radioactivity incorporated in proteins after 16-h labeling.</p>3<p>Long-lived protein degradation (as % of total [³H]Leu used for labeling/h) = Long-lived protein degradation (as % of [³H]Leu incorporated/h, column 3)×[³H]Leu incorporation (%/h, column 2)/100%.</p>4<p>Proteasome load from long-lived protein (LLP) = % of total [³H]Leu used for labeling/h (column 4)×% inhibition by ZL3ek (column 5)/100%.</p>5<p>Proteasome load total = LLP load on proteasome+short-lived proteins load on proteasome (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056132#pone-0056132-t002" target="_blank">Table 2</a>, column 6).</p>6<p>LLP contribution to total load = LLP load on proteasome/total load×100%.</p

One-hour treatment with bortezomib causes similar inhibition of proteasome inside multiple myeloma cells.

<p>A. Inhibition of active sites was measured in cells immediately after 1-h of treatment with bortezomib. Mock-treated cells served as control. An aliquot of cells was cultured in fresh drug-free media for an additional 48 h, followed by Alamar Blue assay for cell viability. Values are averages±S.E.M of 2 or 3 experiments. The % of viable cells differ from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056132#pone-0056132-g001" target="_blank">Fig. 1B</a> where they are averages of more repeats. B. Recovery of activity in cells treated for 1 h with 100 nM bortezomib. The first measurement was immediately after removal of bortezomib. Values are averages ± S.E.M of 2 independent experiments. Mock-treated cells served as controls. The activity is normalized to cell count at time zero. In NCI-H929, MM1.R and MM1.S cells, no data is presented at time points beyond 6 h because the number of viable cells decreases rapidly due to cell death (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056132#pone-0056132-g002" target="_blank">Fig. 2</a>). Western blots analyzing proteasome amounts are shown on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056132#pone.0056132.s004" target="_blank">Fig. S1</a>.</p

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