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

Dual p97 and proteasome inhibition induces high levels of apoptosis and disrupts protein degradation in MMCs.

<p>(<b>A</b>) A panel of human MMC lines were treated with the indicated concentrations of bortezomib (BTZ) or Eer1 for the indicated time. The proportion of live cells relative to DMSO-treated controls was determined by staining with Annexin V-FITC and PI (mean and SEM of 3 independent experiments). (B) MMC lines were treated with the indicated concentrations of BTZ and Eer1 for 48h (OPM-2, RPMI 8226) or 24h (U-266, KMS-11) and the proportion of live cells compared to controls determined as in A (*p<.05, **p<.001, two-sided student’s t-test). (<b>C</b>) BTZ-adapted AMO-1 MMCs co-cultured with human bone marrow MSCs, primary MMCs grown in the presence of IL-6, and healthy donor PBMNCs were subjected to single and dual inhibition with Bortezomib and Eer1 (the median of 3 technical replicates is shown). (<b>D</b>) Immunoblotting for ubiquitinated proteins and tubulin (loading control) carried out on whole cell extracts prepared from MMC lines treated with bortezomib, Eer1, or both inhibitors, for 24h (14h in KMS-11 cells due to their higher apoptotic sensitivity).</p

Combined inhibition of p97 and the proteasome dramatically affects ER configuration.

<p>(<b>A</b>) RPMI 8226 myeloma cells were stained with ER Tracker Blue-White DPX following treatment for 24h with bortezomib (BTZ; 5nM), Eer1 (5µM), or both. Representative confocal microscopic images show minor ER alterations after BTZ treatment, transformation of tubule-lamellar into globular ER structures after treatment with Eer1, and widespread ER vacuolisation after dual treatment. (<b>B</b>) Representative electron microscopic images of OPM-2 cells after treatment with BTZ (5nM), Eer1 (5µM), or BTZ and Eer1, for 24h. Arrows indicate classical ER in control cells, black arrowheads indicate moderately dilated and disrupted ER in Eer1-treated cells, and open arrowheads indicate vacuolised ER with reduced ribosomes on the cytosolic ER surface. Another cell treated with Eer1 and BTZ is shown at lower magnification (right panel). Areas of dilated perinuclear space are indicated by asterisks. Nu, nucleus. (<b>C</b>) BTZ and Eer1 have different effects on ER volume as shown by staining of OPM-2 cells with BFA-BODIPY after treatment for 24h with BTZ (5nM), Eer1 (5µM), or with BTZ and Eer1. A representative histogram (left panel) and the mean and SEM of 6 experiments (right panel) are shown (*p<.05, **p<.001, two-sided student’s t-test). (<b>D</b>) Immunoblotting for lumenal ER chaperones and tubulin (loading control) was carried out on whole cell extracts prepared from OPM-2 cells treated as in (C).</p

Structurally different ERAD inhibitors have comparable effects in MMCs.

<p>(<b>A</b>) Representative microscopic images of parental and BTZ-adapted AMO-1 cells stained with ER Tracker Blue-White DPX after treatment for 18h with Eer1 (5µM), DBeQ (5µM) or Eer1/DBeQ plus Bortezomib (5nM). The images show small to medium-sized globular/vesicular ER after p97 inhibition and ER vacuolisation after dual ERAD inhibition. (<b>B</b>) The proportion of live RPMI8226 cells was determined after treatment with bortezomib (10nM), DBeQ (10µM), or both, for 4h followed by drug wash-out and a further 20h incubation in culture medium (mean and SEM of 3 experiments; *p<.05, **p<.001, two-sided student’s t-test). (<b>C</b>) Primary MMCs were isolated from a bone marrow aspirate, incubated in culture medium containing IL-6 (10ng/ml) for 6h, followed by addition of bortezomib (5nM), DBeQ (5µM), or both for 24h and analysis of apoptosis levels. Live cells (Annexin V-FITC and PI-negative) are shown in green in the left lower quadrant, early apoptotic (Annexin V-FITC positive, right lower quadrant) and dead (Annexin V-FITC and PI-positive, right upper quadrant) cells are shown in orange and red, respectively.</p

Dual p97/proteasome inhibition deregulates key cell survival and protein translation control pathways in MMCs.

<p>(<b>A</b>) Immunoblotting for the indicated survival- and apoptosis-related proteins was carried out on whole cell extracts from MMC lines and primary human fibroblasts prepared after treatment with either BTZ, Eer1, or both inhibitors, for 24h (14h in KMS-11 cells due to their higher apoptotic sensitivity to inhibitors). (<b>B</b>) Cell death induced by dual p97/proteasome inhibition is predominantly caspase-dependent and JNK-independent. The proportion of live cells was determined after treatment with BTZ (5nM) and Eer1 (5µM) with or without the pan-caspase inhibitor zVADfmk (50µM) or the JNK inhibitor SP600125 (10µM) for 24h (36h for OPM-2 cells; all values are the mean and SEM of 3 experiments; *p<.05, **p<.001, two-sided student’s t-test).</p

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