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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Chem Biol. Author manuscript; available in PMC 2012 May 27.
Published in final edited form as:
PMCID: PMC3134264

Specific cell-permeable inhibitor of proteasome trypsin-like sites selectively sensitizes myeloma cells to bortezomib and carfilzomib


Proteasomes degrade the majority of proteins in mammalian cells, are involved in the regulation of multiple physiological functions, and are established targets of anti-cancer drugs. The proteasome has three types of active sites. Chymotrypsin-like sites are the most important for protein breakdown and have long been considered the only suitable targets for anti-neoplastic drugs; however, our recent work demonstrated that inhibitors of caspase-like sites sensitize malignant cells to inhibitors of the chymotrypsin-like sites. Here we describe the development of specific cell-permeable inhibitors and an activity-based probe of the trypsin-like sites. These compounds selectively sensitize multiple myeloma cells to inhibitors of the chymotrypsin-like sites, including anti-myeloma agents bortezomib and carfilzomib. Thus, trypsin-like sites are co-targets for anti-cancers drugs. Together with inhibitors of chymotrypsin- and caspase-like sites developed earlier we provide the scientific community with a complete set of tools to separately modulate proteasome active sites in living cells.


Proteasomes are proteolytic machines that are responsible for turnover of the majority of proteins in mammalian cells. The proteasome inhibitor bortezomib (Velcade) is being used for treatment of multiple myeloma, and at least five second-generation proteasome inhibitors—carfilzomib (PR-171) (Demo et al., 2007; O’Connor et al., 2009), NPI-0052 (Chauhan et al., 2005), CEP-18770 (Piva et al., 2008), MLN-9708 (Kupperman et al., 2010), and ONX-0912 (PR-047) (Zhou et al., 2009)—are in clinical testing.

Proteasomes have three different types of active sites, chymotrypsin-like (ß5), trypsin-like (ß2), and caspase-like (ß1). Cells of the immune system express γ-interferon inducible immunoproteasomes, which have slightly different catalytic subunits, namely the ß5i (LMP7), ß2i (MECL1), and ß1i (LMP2). Of these, the chymotrypsin-like sites (ß5 and ß5i) have long been considered as the only suitable targets for drug development. Bortezomib and all drugs presently undergoing trials were developed to target these sites (Adams, 2004). However, bortezomib, CEP-18770, and MLN-9708 co-target the caspase-like sites (Altun et al., 2005; Berkers et al., 2005; Kisselev et al., 2006; Kupperman et al., 2010; Piva et al., 2008), whereas NPI-0052 co-targets trypsin-like and caspase-like sites (Chauhan et al., 2005). This raises the question of whether inhibition of these sites is important for these drugs’ anti-neoplastic activity. Recently we have demonstrated that, in most multiple myeloma cell lines, cytotoxicity of inhibitors does not correlate with inhibition of the chymotrypsin-like sites but does correlate with loss of specificity and onset of inhibition of the trypsin-like sites (Britton et al., 2009). These data strongly suggest that the trypsin-like sites are important co-targets for anti-neoplastic agents (Britton et al., 2009). Cell-permeable inhibitors of these sites are needed to test this hypothesis. Efforts to develop specific inhibitors of the trypsin-like site have met with limited success to date. Most proteasome inhibitors are short N-terminally capped peptides with an electrophilic group at the C-terminus. This electrophile interacts, reversibly or irreversibly, with the catalytic N-terminal threonine of the proteasome active site. The peptide moiety of the inhibitor binds to the substrate binding pocket of the active site and is largely responsible for the specificity (Groll and Huber, 2004; Kisselev and Goldberg, 2001), although the specificity may be influenced by the electrophile (Screen et al., 2010). The trypsin-like sites cleave peptide bonds after a basic residue and also prefer basic residues in the P3 position (Groll et al., 2002; Harris et al., 2001; Nazif and Bogyo, 2001). Thus an ideal inhibitor would have basic residues, preferably arginines, in the P1 and P3 positions. This presents a challenge from the synthetic point of view and would, most likely, render the inhibitor cell-impermeable. In fact, the few ß2-specific aldehydes (Loidl et al., 1999) and vinyl sulfones (Groll et al., 2002; Nazif and Bogyo, 2001) are not cell permeable. A cell-permeable peptide vinyl ester (ve) Hmb-VSL-ve, recently reported as specific inhibitor of the trypsin-like sites (Marastoni et al., 2005), did not show any inhibitory activity in our assays (Screen et al., 2010). Thus, at the onset of our work, no cell-permeable, ß2-specific inhibitors or activity-based probes were available.

In this work, we describe the development of several cell-permeable peptide epoxyketone inhibitors as well as an active-site probe specific to the trypsin-like proteasome sites. We demonstrate that the most potent of these compounds sensitizes multiple myeloma cells to the specific inhibitors of the chymotrypsin-like sites, to bortezomib, and to the second-generation proteasome inhibitor carfilzomib.


Design and initial characterization of inhibitors

We have designed several peptide epoxyketones to target the trypsin-like site (Fig. 1a). Peptide epoxyketones are the most specific of the several structural classes of proteasome inhibitors (Groll and Huber, 2004; Kisselev, 2008; Kisselev and Goldberg, 2001). By forming a stable morpholino adduct with the proteasome catalytic N-terminal threonine, they take specific advantage of the proteasome’s unique mechanism for cleaving peptide bonds (Groll et al., 2000). In fact, in more than a decade of research since the discovery of this class of proteasome inhibitors (Meng et al., 1999), no off-target effects of epoxyketones have been found.

Figure 1
Effect of inhibitors on purified 26 proteasomes

Consistent with the nomenclature used in our previous work (Britton et al., 2009) we refer to inhibitors of the trypsin-like sites as NC-0X2, where “NC” stands for the Norris Cotton Cancer Center, “2” indicates that a compound inhibits ß2 and ß2i sites, and the character in the position marked by “X” changes from compound to compound. The first compound, NC-002 (Ac-LLR-ek), is the epoxyketone derivative of leupeptin. Leupeptin (Ac-Leu-Leu-Arg-al) is a cell-permeable inhibitor of cysteine proteases. In the context of purified proteasome, this peptide aldehyde is a specific inhibitor of the trypsin-like sites (Kisselev et al., 2006; McCormack et al., 1998). Peptide aldehydes inhibit serine, cysteine, and threonine proteases. We reasoned that replacing the aldehyde in leupeptin with a highly proteasome-specific epoxyketone (Groll et al., 2000) to generate Ac-LLR-amc (NC-002) would eliminate reactivity with lysosomal cysteine proteases, retain specificity to the trypsin-like sites, and not alter cell-permeability of the compound.

The design of the second compound, NC-012 (Ac-RLR-ek), is based on the sequence of the best substrate of the trypsin-like site (Ac-RLR-amc) we developed earlier (Kisselev and Goldberg, 2005). The third inhibitor, NC-022 (Hmb-VSR-ek) has the same left-handed peptide fragment as the peptide vinyl-ester inhibitor of the trypsin-like sites reported in the literature (Marastoni et al., 2005) that lacked inhibitory activity in our hands (Screen et al., 2010). We chose this fragment because it was optimized to improve specificity towards these sites.

In order to enable the synthesis of the epoxyketone derivatives of arginine, we have modified the established procedure for the synthesis of leucine epoxyketones (Zhou et al., 2009) to allow for proper protection of the guanidine functional group during the procedure (See Supplementary Methods).

We initially evaluated the proteasome inhibitory potential of our compounds on purified 26S proteasomes from rabbit muscles (Fig. 1b-c). All three are potent and specific inhibitors of the trypsin-like sites. NC-012, as expected for the compound derived from the best substrate, was the most potent and specific in the series.

Next we treated NCI-H929 multiple myeloma (MM) cells with these compounds overnight and determined their proteasome inhibition profile (Fig. 2a–c). NC-002 and NC-022 specifically inhibited trypsin-like activity at sub-micromolar concentrations, but much higher concentrations of NC-012, the most potent inhibitor of the purified enzyme, were required to achieve inhibition in live cells. We attribute this decrease in potency with live cells to poor cell permeability. For cell-permeable compounds, maximal inhibitory effect was achieved within 6–10 h after addition of NC-022 (Fig. 2d) or NC-002 (Fig. S1). Importantly, NC-002, the epoxyketone derivative of the cysteine protease inhibitor leupeptin, does not inhibit lysosomal cysteine proteases (Fig. 2e).

Figure 2
Effect of compounds on proteasomes in NCI-H929 cells

Multiple myeloma cells express constitutive proteasomes and immunoproteasomes, and substrates used for the measurement of activity (Fig. 2a-c) are cleaved by both. To determine whether there are any differences in inhibition of constitutive proteasomes or immunoproteasomes by NC-002, NC-012, and NC-022 we used the fluorescent activity-based probe MV-151 (Verdoes et al., 2006) in a label-competition experiment. Extracts of RPMI-8226 MM cells (which express more immunoproteasomes than NCI-H929 cells) were treated first with the NC inhibitors and then with the MV-151 probe. This was followed by fractionation on SDS-PAGE to separate proteasome subunits and by imaging to reveal those subunits labeled by the probe (i.e., unmodified by the inhibitors). All three inhibitors blocked modification of ß2 and ß2i sites by the probe to a similar extent (Fig. 2f). Thus, we conclude that NC-002, NC-012, and NC-022 are equipotent inhibitors of the trypsin-like sites of constitutive and immunoproteasomes.

Specific inhibitors of trypsin-like sites sensitize cells to specific inhibitors of chymotrypsin-like sites

Next, we used our compounds to characterize trypsin-likes sites as targets and co-targets of anti-neoplastic agents. For this purpose we used NC-022, the most potent cell-permeable inhibitor. First we tested whether selective inhibition of trypsin-like sites is sufficient to reduce cell viability. We treated NCI-H929 cells with NC-022 for 48 h and assayed cell viability with Alamar Blue mitochondrial conversion dye. No loss of viability was detected even at concentrations that completely inhibited the trypsin-like sites (data not shown). Thus, targeting trypsin-like sites is not sufficient to induce cytotoxicity in multiple-myeloma cells. (It should be noted that NCI-H929 is the most sensitive to proteasome inhibitors among myeloma cell lines (Britton et al., 2009).)

We next tested whether NC-022 sensitizes myeloma cells to inhibitors of the chymotrypsin-like sites. In the past few years, we have developed several peptide epoxyketone inhibitors of the chymotrypsin-like sites (Britton et al., 2009; Geurink et al., 2010); in these experiments we used the most specific of these, a pentafluorophenylalanine-containing compound referred to as here as LU-005 (Figs. (Figs.2g,2g, S2; Geurink et al., 2010). (“LU” stands for Leiden University and “5” indicates that the compound targets ß5/ß5i active sites.) In the first experiment, we determined whether NC-022 sensitizes cells to LU-005, and what concentrations are needed to achieve this sensitization. Consistent with the treatment condition used in our previous work (Britton et al., 2009), where we demonstrated that a specific inhibitor of the caspase-like sites sensitizes myeloma cells to NC-005 (a specific inhibitor of chymotrypsin-like sites), we treated cells with LU-005 for 1 h and then incubated them in the presence of different concentrations of NC-022 for 48 h, whereupon an Alamar Blue assay for cell viability was performed. Dramatic dose-dependent sensitization was observed, with the IC50 of LU-005 increasing up to 8.5-fold. This maximal sensitization was achieved at 3 μM NC-022 (Fig. 2h), which causes 90% inhibition of the trypsin-like sites within 4–6 h after addition of NC-022 (Fig. 2d). NC-002 caused similar sensitization to NC-005 (data not shown). Thus, near-complete inhibition of the trypsin-like sites is needed to achieve maximal sensitization effect.

Development of a ß2-specific activity-based probe

To further confirm that our compounds are specific for the trypsin-like sites and that their biological activity is not due to off-target effects, we have synthesized az-NC-002, an NC-002-derived, activity-based probe (Fig. 3a). We have chosen NC-002 over NC-022 for derivatization because it was easier to introduce an azido group into this molecule. Addition of the azido group does not alter the specificity of the inhibitor (Fig. 3B). Polypeptides modified by this probe were visualized on Western blot after treating extracts of probe-treated NCI-H929 cells with azido-reactive biotinylated phosphane (BioP) in a Staudinger-Bertozzi ligation (Ovaa et al., 2003). One major az-NC-002-specific streptavidin-reactive band was detected (Fig. 3C, lane 5). This matches the size of the band of the ß2 subunit, which harbors the catalytic threonines of the trypsin-like sites. A weaker band of slightly lower mobility, matching the mobility of ß2i band, was also detected. Corroborating that these bands are of proteasomal subunits, az-NC-002 treatment prevented subsequent modification of ß2 and ß2i subunits by another proteasome-specific probe (Ada-K(Bio)-Ahx3L3VS, lane 4). (A number of endogenously biotinylated proteins in the 70-100 kDa region were also detected and can serve as a loading control.)

Figure 3
Az-NC-002 is a ß2/ß2i specific active site probe

To further confirm that the probe covalently modifies ß2 and ß2i subunits, we denatured the proteasome after BioP modification, isolated biotinylated polypeptides on streptavidin beads, and identified bound polypeptides by mass-spectrometry after on-beads trypsin digestion. Peptides derived from ß2 and ß2i subunits were present in the samples isolated from extracts of the probe-treated cells but not from extracts of the control cells (Fig. 3d, Table S1). No peptides derived from other catalytic subunits were detected. Thus, we conclude that az-NC-002 is a trypsin-like site-specific activity-based probe.

Surprisingly, several other polypeptides were also reproducibly identified as specific az-NC-002 targets. These include the aspartic protease cathepsin D (29 kDa), molecular chaperone hsc71 (71 kDa), and thioredoxin domain-containing protein TXNDC5 (48 kDa, Fig. 3d). Of these, lysosomal aspartic protease cathepsin D (Benes et al., 2008) was of greatest concern to us. It has the same molecular weight as the ß2 subunit, so some of the streptavidin-reactive material in the ß2-band (Fig. 3c) may be cathepsin D. To determine the significance of this potential off-target effect, we measured inhibition of cathepsin D by az-NC-002 but could not detect any significant inhibition (Fig. 3f,g). We conclude that this probe either reacts with cathepsin D outside of the active site or inhibits a small fraction of the enzyme, detectable in the mass-spectrometry experiment but not in the activity assay. Similarly, NC-022 did not inhibit cathepsin D even at concentrations as high as 27 μM (Fig. 3g). Thus, chemical modification of cathepsin D is unlikely to contribute to the biological effects of the NC compounds.

There are no major streptavidin-reactive az-NC-002-specific bands in the 45–50 kDa and ~70 kDa region of the gel, where two other targets of az-NC-002, TXNDC5 and hsc71, migrate (Fig. 3c). Probe modification of these proteins is responsible for one of the background bands in lane 5 on Fig. 3c. We used Western blot to determine which fraction of cellular hsc71 binds to streptavidin beads in extracts of az-NC-002-treated cells. Under conditions when most of ß2-antibody reactive material was detected in streptavidin-bound fraction, the majority of hsc71-antibody reactive material was detected in the streptavidin-unbound fractions (Fig. 3e). Thus, az-NC-022 modifies a small fraction of hsc71 and is therefore very unlikely to affect the overall protein-folding capacity of the cell.

Trypsin-like sites are better co-targets of anti-myeloma agents than caspase-like sites

In the next set of experiments (Fig. 4), we tested whether NC-022 sensitizes other MM cells to LU-005 and whether it is a more potent sensitizer than a specific inhibitor of caspase-like sites NC-001(Britton et al., 2009). We have chosen four additional myeloma cell lines—MM1.R, RPMI-8226, KMS-18, and KMS-12-BM—for these experiments. These cell lines vary up to 40-fold in their sensitivity to bortezomib and NC-005 (Britton et al., 2009). In all experiments, NC-022 was used at a concentration that inhibited trypsin-like activity by more than 90% after 6 h incubation.

Figure 4
Comparative analysis of sensitization of different myeloma cells to specific inhibitor of the chymotrypsin-like sites (LU-005) by NC-022 and specific inhibitor of caspase-like sites (NC-001)

In all MM cell lines, NC-022 reduced the IC50 for LU-005 by 4–10-fold. In three (MM1.R, RPMI-8226, KMS-18), NC-022 caused similar sensitization as NC-001 (Fig. 4c–e). In two others (NCI-H929 and KMS-12-BM, Fig. 4a, b), NC-022 was a more potent sensitizer than NC-001. Thus, the trypsin-like sites are important co-targets of anti-neoplastic drugs in multiple myeloma cells; in fact, they are better co-targets than the caspase-like sites.

To confirm that LU-005 functions as a specific inhibitor of the chymotrypsin-like sites and to determine whether sensitization occurs upon clinically achievable inhibition of the chymotrypsin-like sites, we measured inhibition of all sites at the end of 1 h treatment with LU-005 (Table 1). In patients treated with bortezomib, inhibition of the chymotrypsin-like sites that can be achieved at maximal tolerated doses does not exceed 70% (Hamilton et al., 2005); in patients treated with carfilzomib, it approaches 90% (O’Connor et al., 2009). As can be seen from Table 1, in all but the KMS-18 cell line, sensitization by NC-022 is observed upon clinically achievable 50–80% inhibition of the chymotrypsin-like sites. Thus, sensitization of myeloma cells to specific inhibitors of the chymotrypsin-like sites by NC-022 is of potential clinical significance.

Table 1
NC-022 and, more dramatically, a mixture of NC-022 and NC-001 sensitize multiple myeloma cells to LU-005 upon clinically achievable proteasome inhibition

Effects of combined inhibition of caspase-like and trypsin-like sites on myeloma cells

Due to the lack of effective, selective, and cell-permeable inhibitors of the trypsin-like sites, the effects of combined inhibition of the trypsin-like and caspase-like sites (in the absence of inhibition of the chymotrypsin-like sites) on growth and viability of mammalian cells could not be studied hitherto. We observed that continuous exposure to a mixture of NC-022 and NC-001 (at concentrations at which caspase-like and trypsin-like sites are both blocked by more than 90%) reduced cell viability by 20–50% (Fig. 4f). Proteasome inhibitors block cell proliferation and induce apoptosis. This moderate decrease could be a consequence of inhibition of cell proliferation without cell death. To determine whether apoptosis occurs, we measured caspase activation in the NCI-H929 and MM1.R cell lines. We found that treatment with a combination of NC-001 and NC-002, in contrast to LU-005 treatment, did not cause any significant increase in caspase activity (Fig. 4g). Therefore, we conclude that the moderate decrease in viability in cells co-treated with NC-001 and NC-002 is not due to apoptosis and most likely reflects inhibition of cell proliferation. We would like to emphasize that this is the first example of a biological effect on mammalian cells due to inhibition of the caspase-like and trypsin-like sites in the absence of inhibition of the chymotrypsin-like sites.

We next studied the effects of the mixture of NC-001 and NC-022 on MM cells sensitivity to LU-005. As in the previous experiments, cell were treated with LU-005 for 1 h and then cultured with a NC-001/NC-022 mixture after removal of LU-005. The mixture of NC-001 and NC-022 appeared to be a much stronger sensitizer than NC-022 alone (Fig. 4a–e). Notably, there was always a concentration of LU-005 at which a mixture of NC-001 and NC-002 caused a dramatic loss of cell viability as compared to the effect of LU-005 as a single agent (i.e., from 80–100% to 10–20%, Fig. 4A–E). At this concentration, LU-005 inhibited chymotrypsin-like sites by a clinically achievable 50–85% (Table 1). A mixture also sensitized cells at much lower concentrations of LU-005 (i.e., upon much smaller inhibition of chymotrypsin-like sites, Table 1) than either NC-001 or NC-002 alone.

NC-022 specifically sensitizes myeloma cells to bortezomib and carfilzomib

To further strengthen the clinical relevance of our observations, we tested whether NC-022 sensitizes MM cells to the FDA-approved proteasome inhibitor bortezomib and to carfilzomib, a second-generation peptide epoxyketone proteasome inhibitor undergoing phase II–III clinical trials (Demo et al., 2007; O’Connor et al., 2009). We used two cell lines in these experiments, one of the most bortezomib-sensitive (NCI-H929) and one of the most bortezomib-resistant (KMS-12-BM) (Britton et al., 2009). Both cell lines were sensitized to the two agents (Fig. 5). In NCI-H929 cells, sensitization to both compounds occurred upon clinically achievable proteasome inhibition. In KMS-12-BM cells, sensitization to bortezomib, although more dramatic than in NC-H929 cells, was observed above clinically achievable inhibition of the chymotrypsin-like sites. Sensitization to carfilzomib was observed at clinically achievable levels.

Figure 5
NC-022 specifically sensitizes multiple myeloma cells to bortezomib and carfilzomib

To assess whether co-inhibition of trypsin-like sites increases toxicity to normal cells, we tested whether NC-022 increases toxicity of bortezomib and carfilzomib to peripheral blood mononuclear cells (PBMNCs). NC-022 did not sensitize cells from any of the three donors to either of two agents (Figs. (Figs.55 and S3). This lack of sensitization is surprising because NC-001 sensitizes PBMNCs to inhibitors of the chymotrypsin-likes sites (Britton et al., 2009). Thus, NC-022 selectively sensitizes malignant MM cells to bortezomib and carfilzomib.


Site-specific, cell-permeable inhibitors of the proteasome’s trypsin-like sites have long been missing from the otherwise impressive palette of reagents available to study the role of the proteasome and its active sites in different aspects of cellular function. The compounds described herein fill this void.

The significance of this work is two-fold. First, it describes the development of (1) cell-permeable specific inhibitors of the trypsin-like sites of the proteasome and (2) an active site probe derived from these inhibitors. Second, we use one of these compounds, NC-022, to demonstrate that these sites are co-targets of anti-neoplastic-drugs in multiple myeloma. Trypsin-like sites appear to be better co-targets than caspase-like sites for two reasons. First, in two out of five cell lines tested, NC-002 caused better sensitization to chymotrypsin-site-specific inhibitor than NC-001, while in three others sensitization was similar (Fig. 4). Second, NC-022 selectively sensitized MM cells to carfilzomib and bortezomib (Fig. 5); sensitization by NC-001 was not selective (Britton et al., 2009). It remains to be determined whether NC-022 could be developed into a drug to be used in combination with bortezomib and carfilzomib or whether development of newer agents that inhibit chymotrypsin- and trypsin-like sites with equal potency would be a better approach to translate the results of this work into novel treatments for patients.

The importance of this work goes beyond oncology. Several years ago, we found that the ability of leupeptin (used as specific inhibitor of the trypsin-like sites of purified proteasomes) to block degradation of model substrates depends on the content of basic residues in a substrate (Kisselev et al., 2006). Using inhibitors developed in this work as well as proteomic approaches, we can now ask whether basic proteins will be selectively stabilized upon treatment of cells with ß2-specific proteasome inhibitors.

Proteasomes are involved in a variety of biological processes (e.g., inflammation and immune response). One immediate application of these compounds would be to study the role of trypsin-like sites in the generation of MHC class I epitopes. Although it is well established that these peptides or their precursors are generated by proteasomes, the role of individual active sites in the excision of specific epitopes is not known (Groettrup and Schmidtke, 1999; Rock et al., 2002). Specific activity of the trypsin-like sites of immunoproteasomes (ß2i) is several-fold higher than that of their counterparts in the constitutive particles (ß2) (Cascio et al., 2001). Some MHC class I ligands have basic residues at the C-terminus (Rammensee et al., 1995). The C-termini of these specific peptides may be generated by cleavages at the trypsin-like sites. Because of a lack of specific inhibitors of these sites, this hypothesis could not previously be tested; it can be tested now using the reagents developed in this work.


The cell-permeable inhibitors of trypsin-like sites reported here fill the largest remaining void in the impressive palette of proteasome inhibitors available to biologists. This study completes the development of site-specific inhibitors and activity based probes of proteasome different active sites, at least for the constitutive proteasome. These active sites can now be down-regulated individually to the desired extent in living cells. Inhibitors developed in this study will find wide use to study the role of trypsin-like sites in protein degradation, MHC class I antigen presentation and other biological processes, and, as demonstrated in this study for multiple myeloma, to determine whether these sites can be targeted for the treatment of other cancers or different diseases.

Experimental Procedures

Synthesis of inhibitors

Synthesis of NC-002, NC-012, NC-022, and az-NC-002 and analytical data for compounds are described in the Supplementary Materials section. Bortezomib was purchased from LC laboratories. Carfilzomib was synthesized as described (Britton et al., 2009; Demo et al., 2007; Zhou et al., 2009). BioP was synthesized as described (Verdoes et al., 2008).

Proteasome purification and assays

26S proteasomes were purified from rabbit muscle as described (Screen et al., 2010). To determine inhibition of purified proteasomes, they were incubated with inhibitors for 30 minutes at 37 °C followed by assay of activity with fluorogenic substrates Suc-LLVY-amc (chymotrypsin-like site), Ac-RLR-amc (trypsin-like site), and Ac-nLPnLD-amc (caspase-like site). See (Geurink et al., 2010) for detailed description of the procedure. 12% Novex Bis-Tris gels (Invitrogen) with MOPS running buffer were used for electrophoretic separation of catalytic subunits modified by active-site probes. Inhibition of active sites inside cells was assayed using luminescent ProteasomeGlo assay (Promega) (Moravec et al., 2009) as described in our previous study (Britton et al., 2009). Results of the cell-based ProteasomeGlo assay are undistinguishable from the activity measurement with fluorogenic substrates in extracts, in which background cleavage by non-proteasomal proteases is accounted for by subtracting activity left in extract after treatment with high concentrations of highly specific proteasome inhibitor epoxomicin (see (Britton et al., 2009): Fig. S4B for data and Supplementary Experimental Procedures for details of the assay with fluorogenic substrates). Cathepsin B, H, L, S activity was measured with pan-cathepsin substrate Z-FR-amc (Kirschke and Wiederanders, 1994) in extracts of cytosol-less cells at pH 6.0 as described in the previous study (Screen et al., 2010). Cathepsin D activity was measured in cytosol-less extracts using SensoLyte®520 Cathepsin D Fluorometric Assay Kit (AnaSpec). Combined cathepsin D and E activity was measured using the same kit, in which cathepsin D substrate provided with the kit was replaced with 7-Methoxycoumarin-GKPILFFRLK(Dnp)-r-NH2 (where “r” stand for D-Arg) internally quenched fluorogenic substrate of cathepsin D and E. In this case, we used pH 3.0 assay buffer provided with the kit was used for cell extraction. All activity observed using both procedures was inhibited by more than 98% by specific inhibitor of aspartic proteases pepstatin A.

Cell culture

All cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum. Viability of multiple myeloma cells was measured with Alamar Blue mitochondrial dye conversion assay. Viability of PBMNC was measured using Cell Titer-Glo luminescent cell viability assay (Promega), which is based on quantification of ATP present in the cells. Caspase-3/7 activity was measured using ApoONE 3/7 homogeneous assay (Promega). (This assay uses Ac-DEVD-Rhodamine110 cell permeable fluorogenic substrate.)

Isolation and identification of polypeptides modified by az-NC-002

Cells were treated with the activity-based site probe overnight and lyzed with 50 mM Tris-HCl, 10% glycerol, 5 mM MgCl2, 0.5 mM EDTA, 0.5% CHAPS, 1 mM ATP. After 1 h treatment with 100 μM BioP proteins were denatured with 1% SDS, followed by affinity purification of biotinylated polypeptides on Streptavidin coated magnetic beads. After on-beads trypsin digestion, samples were analyzed by LC-MS/MS. See (Florea et al., 2010) for the detailed description of the procedure. IRDye™ 800 CW-conjugated Streptavidin was purchased from Rockland, hsc71 antibodies from Abcam (Cat # 19136), and ß2 antibodies from Abgent (Cat #AP2914b).

Table 2
NC-022 sensitizes multiple myeloma cells to bortezomib and carfilzomib upon clinically achievable proteasome inhibition


  • Cell-permeable inhibitors and activity-based probe of proteasome trypsin-like sites developed
  • Trypsin-like sites are better co-targets (together with chymotrypsin-like sites) of anti-neoplastic drugs than caspase-like sites
  • Co-inhibition of trypsin-like and caspase-like sites does not cause apoptosis in myeloma cells but sensitizes them better to inhibitors of chymotrypsin-like sites than inhibition of individual sites

Supplementary Material


These studies were supported by an R01 grant from the NCI, by the American Recovery and Reinvestment Act Supplement to this Grant to AFK, and by grants from the Netherlands Organization for Scientific Research (NWO) and the Netherlands Genomics Initiative (NGI) to HSO. We thank Hans van der Elst (Leiden Institute of Chemistry) for performing high-resolution mass-spectrometry analysis of inhibitors, Wayne Casey (Dept. of Chemistry, Dartmouth College) for assistance with NMR instruments, and John DeLeong and Jackie Chanon-Smith for providing PBMNCs.


The authors declare no conflicts of interest.


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