Статті в журналах: "Proteasome-like inhibitors"

1

Mitchell, Angela M., and R. Jude Samulski. "Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors." Journal of Virology 87, no. 23 (September 11, 2013): 13035–41. http://dx.doi.org/10.1128/jvi.01826-13.

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Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.

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2

Widjaja, Ivy, Erik de Vries, Donna M. Tscherne, Adolfo García-Sastre, Peter J. M. Rottier, and Cornelis A. M. de Haan. "Inhibition of the Ubiquitin-Proteasome System Affects Influenza A Virus Infection at a Postfusion Step." Journal of Virology 84, no. 18 (July 14, 2010): 9625–31. http://dx.doi.org/10.1128/jvi.01048-10.

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ABSTRACT We have demonstrated that influenza A virus (IAV) RNA synthesis depends on the ubiquitin-proteasome system. IAV replication was reduced both by proteasome inhibitors and in E36ts20 cells, which contain the thermolabile ubiquitin-activating enzyme E1. While virus entry was not affected in E36ts20 cells, the proteasome inhibitor MG132 retained viral particles in the cytoplasm. Addition-removal experiments of MG132 in combination with bafilomycin A1, a well-established inhibitor of IAV entry and fusion, showed that MG132 affected IAV infection at a postfusion step. This was confirmed by the lack of inhibition of IAV entry by proteasome inhibitors in a virus-like particle fusion assay.

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3

Fidor, Anna, Katarzyna Cekała, Ewa Wieczerzak, Marta Cegłowska, Franciszek Kasprzykowski, Christine Edwards, and Hanna Mazur-Marzec. "Nostocyclopeptides as New Inhibitors of 20S Proteasome." Biomolecules 11, no. 10 (October 8, 2021): 1483. http://dx.doi.org/10.3390/biom11101483.

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Nostocyclopeptides (Ncps) are a small class of bioactive nonribosomal peptides produced solely by cyanobacteria of the genus Nostoc. In the current work, six Ncps were isolated from Nostoc edaphicum strain CCNP1411. The bioactivity of these compounds was tested in vitro against 20S proteasome, a proteolytic complex that plays an important role in maintaining cellular proteostasis. Dysfunction of the complex leads to many pathological disorders. The assays indicated selective activity of specific Ncp variants. For two linear peptide aldehydes, Ncp-A2-L and Ncp-E2-L, the inhibitory effects on chymotrypsin-like activity were revealed, while the cyclic variant, Ncp-A2, inactivated the trypsin-like site of this enzymatic complex. The aldehyde group was confirmed to be an important element of the chymotrypsin-like activity inhibitors. The nostocyclopeptides, as novel inhibitors of 20S proteasome, increased the number of natural products that can be considered potential regulators of cellular processes.

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4

Kabore, Albert F., Kristin McCrea, James B. Johnston, and Spencer B. Gibson. "Proteasome Inhibiters Up-Regulate TRAIL/Apo2L and Its Receptors Significantly Contributing to Proteasome Inhibitor-Induced Apoptosis in Primary Chronic Lymphocytic Leukemia (CLL) Cells." Blood 104, no. 11 (November 16, 2004): 2810. http://dx.doi.org/10.1182/blood.v104.11.2810.2810.

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Abstract The proteasome inhibitor, bortezomib has recently emerged as a new therapeutic treatment for refractory multiple myeloma and is presently being evaluated for other hematological malignancies either alone or in combination with other antitumor agents. Proteasome inhibitors cause the accumulation of many proteins but the precise mechanism responsible for their antitumor effect is unclear. In the present study, we have determined that cytotoxic effect the proteasome inhibitor MG-132 in primary chronic lymphocytic leukemia (CLL) cells is through the activation of the TRAIL (tumor necrosis factor-related apoptosis inducing ligand) apoptotic pathway. MG-132 induced apoptosis in approximately 70% of primary CLL cells as measured by annexin V staining. Addition of DR4:Fc that prevents TRAIL ligation with its receptors decreased the amount of MG-132 induced apoptosis by approximately 40% suggesting MG-132 caused activation of the TRAIL apoptotic pathway. MG-132 also up-regulated both the mRNA and protein levels of TRAIL and protein levels of TRAIL receptors DR4 and DR5. This upregulation correlated with activation of caspase 8 and cleavage of pro-apoptotic Bcl-2 family member Bid. Moreover, MG-132 treatment also induced a substantial reduction in the FLICE-like inhibitory protein (c-FLIP) protein levels. In contrast to CLL cells, proteasome inhibitors failed to activate the TRAIL apoptotic pathway in normal B-cells. This indicates that proteasome inhibitors are inducing apoptosis in primary CLL cells through activation of the TRAIL apoptotic signaling pathway through up-regulation of TRAIL and its cognate receptors and reduced FLIP expression. Thus, proteasome inhibitors may have a therapeutic role in CLL, either when used alone or in combination with TRAIL or antibodies against DR4/DR5.

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5

Crawford, Lisa J. A., Huib Ovaa, Brian Walker, Dharminder Chauhan, Kenneth C. Anderson, Treen C. M. Morris, and Alexandra E. Irvine. "Evaluation of the Specificity and Cytotoxicity of Three Proteasome Inhibitors." Blood 106, no. 11 (November 16, 2005): 3366. http://dx.doi.org/10.1182/blood.v106.11.3366.3366.

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Abstract The proteasome is a mutlicatalytic protease with three main catalytic activities - chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolising (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma (MM). The CT-L activity, considered to be the rate-limiting step in protein degradation, is the primary target of many proteasome inhibitors. We have compared the specificity and potency of the novel proteasome inhibitor BzLLLCOCHO to the previously characterised inhibitors PS-341 (Velcade, bortezomib) and MG-132. Specific fluorogenic substrates were used to measure proteasome proteolytic activity in the presence and absence of the inhibitory compounds. An active site directed probe with a dansyl-sulfonamidohexanoyl hapten tag was used in conjuction with immunoblotting to determine the subunit specificity of the proteasome inhibitors (Nature Methods2005;2:357–362). MM cell lines (U266, OPM-2, KMS-11, KMS-18) were incubated with 10 μM BzLLLCOCHO, 5 nM PS-341 or 1 μM MG-132 for 24 hrs and proteasome activity was measured. Addition of BzLLLCOCHO reduced CT-L activity by 83 ± 13 % in the fluorogenic assay, and T-L and PGPH activities were reduced by 93 ± 6 % and 92 ± 2 % respectively. Immunoblot results revealed a similar pattern, the T-L and PGPH subunits were completely inhibited by BzLLLCOCHO and there was only weak labeling of the CT-L subunit with the active site probe. In contrast, treatment with PS-341 completely inhibited the CT-L and PGPH activities and incubation with MG-132 resulted in weak inhibition of the CT-L and PGPH activities, neither inihibitor significantly affected T-L activity. The ability of the different inihibitors to induce apoptosis in MM cell lines was then evaluated. All three inhibitors were demonstrated to act through both the caspase-8 and caspase-9 signalling pathways. Using Mitosensor™ and Hoescht/Propidium Iodide staining we found that MM cells were more sensitive to the induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO (U266 cells treated for 72 hrs with BzLLLCOCHO 51 % apoptosis, PS-341 79 % apoptosis and MG-132 84 % apoptosis). BzLLLCOCHO is a cell permeable and potent inhibitor of all three proteolytic activities of the proteasome. PS-341 and MG-132 inhibited only two of the three proteasome activities but were more efficient than BzLLLCOCHO at inducing apoptosis in MM cell lines. MG-132 is known to inhibit non proteasomal proteases such as Cathepsin B and Calpain 1 which may contribute to its potency. Further investigation on the effects of these inhibitors on gene and protein expression in the cell may lead to the development of more specific and targeted inhibitors.

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6

Parlati, Francesco, Monette Aujay, Siv Lise Bedringaas, Susan Demo, Bjorn Gjertsen, Eileen Goldstein, Jing Jiang, et al. "Anti-Tumor Activity of Immunoproteasome Selective Inhibitors." Blood 110, no. 11 (November 16, 2007): 1599. http://dx.doi.org/10.1182/blood.v110.11.1599.1599.

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Abstract Background: The immunoproteasome is a form of the proteasome that is distinct from the broadly expressed constitutive proteasome. The immunoproteasome, which is predominant in hematopoetic cells, contains the unique active site subunits, LMP7, LMP2 and MECL1, accounting for the chymotrypsin-like, caspase-like and trypsin-like activity respectively. The proteasome inhibitors bortezomib and carfilzomib have validated the proteasome as a therapeutic target in hematologic malignancies; however these inhibitors target both the constitutive and immunoproteasome. We hypothesized that selective inhibition of the immunoproteasome would have anti-tumor activity on hematologic tumors and may avoid toxicities associated with constitutive proteasome inhibition. Methods: The level of immunoproteasome expression in hematologic malignant cells was not known. Hematologic tumor cell lines and primary patient samples were assessed for relative levels of immunoproteasome versus constitutive proteasome using an ELISA that quantitates proteasomal subunits. Epoxyketone-based inhibitors were generated that are >20-fold selective for the immunoproteasome subunit, LMP7, over the corresponding constitutive subunit, Beta5. Anti-tumor activity of LMP7 selective inhibitors was determined by treating tumor cell lines and primary patient cells that are predominately LMP7 expressing. Furthermore, anti-tumor effects were assessed by combining LMP7 and Beta5 inhibition using genetic and chemical methods Results: Immunoproteasome expression was variable in primary patient samples and tumor cell lines. In the majority of acute myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia patient samples, the immunoproteasome accounted for >95% of the total proteasome. In HS-Sultan, Molt4 and RL cell lines, the immunoproteasome accounted for 70%, 50% and 50% of the total proteasome, respectively. In these cell lines, complete inhibition of LMP7 was not sufficient to induce to cell death. LMP7 selective inhibitors were only cytotoxic in these cell lines under conditions where Beta5 was depleted either genetically or chemically. Genetic depletion was accomplished using Beta5-specific shRNA expression in HS-Sultan, Molt4 and RL cells which increased the relative LMP7 levels to 95%, 80% and 70% respectively. These cells were significantly more sensitive to LMP7 inhibition than their parental cells, suggesting that cell kill requires suppression of LMP7 and Beta5. Chemically, combinations of Beta5 and LMP7 selective inhibitors showed similar results, i.e. greater cytotoxicity in combination than either inhibitor alone. Acute lymphocytic leukemia patient samples were screened for LMP7 expression and anti-tumor activity of the LMP7 selective inhibitors correlated with the relative levels of LMP7 expression. Conclusion: This work suggests that tumor cells with high relative levels of LMP7 will be sensitive to LMP7 selective inhibition. These results support the development of immunoproteasome-selective inhibitors for the treatment of hematologic malignancies where high levels of immunoproteasome expression are observed. An LMP7 selective inhibitor has been identified as a clinical candidate for development in leukemia.

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7

Kirk, Christopher J., Monette A. Aujay, Mark Ho, Jing Jiang, Guy J. Laidig, Evan R. Lewis, Yan Lu, et al. "Pharmacologic Evaluation of Orally Bioavailable Inhibitors of the 20S Proteasome." Blood 108, no. 11 (November 16, 2006): 3581. http://dx.doi.org/10.1182/blood.v108.11.3581.3581.

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Abstract Clinical application of proteasome inhibitors in the treatment of hematologic malignancies such as myeloma and lymphoma is restricted in part by the necessity of frequent IV administration and would be improved by oral (PO) administration. Selective inhibitors of the protease subunits of the 20S proteasome can be generated from peptidyl aldehydes, boronates, vinyl sulfones, and epoxyketones. Many of these peptide based proteasome inhibitors are cell permeant and capable of systemic proteasome inhibition upon intravenous (IV) administration to experimental animals such as mice and rats. In the cases of the peptidyl boronate bortezomib (Velcade™) and the epoxyketone PR-171, proteasome inhibition can be achieved in patients with IV administration. However, systemic exposure following PO administration of these inhibitors may be limited by several factors including gastric pH, gastric and intestinal peptidases, efflux pumps, biliary excretion and intestinal and hepatic metabolic activities. We have tested over 80 peptide epoxyketones with potent (IC50 <100 nM) in vitro inhibitory activity against the chymotrypsin-like activity of the 20S proteasome for bioavailability following PO administration in mice. Systemic exposure was monitored by measuring chymotrypsin-like inhibition in a number of tissues and an approximation of the relative bioavailability for selected compounds was measured by comparing the pharmacodynamics of IV and PO administration using a range of doses. These compounds were also tested in vitro for solubility, intestinal cell permeability, intestinal and hepatic metabolism, and sensitivity to the multidrug resistance protein 1 (MDR1) efflux pump in order to determine which properties were associated with oral bioavailability. We have found that oral bioavailability in mice is associated with increased intrinsic solubility and metabolic stability and reduced MDR1 sensitivity. Proteasome inhibition following PO administration is rapid, resulting in maximal proteasome inhibition within 15 minutes. Rapid absorption and clearance of selected compounds was also confirmed in mice and rats by pharmacokinetic analysis. Repeated oral administration was well tolerated at doses that resulted in significant (>80%) proteasome inhibition in most tissues. The anti-tumor efficacy of these orally bioavailable proteasome inhibitors are being assessed in both human tumor xenograft and mouse syngeneic models. The results from these studies will enable further pre-clinical development of potent, orally bioavailable proteasome inhibitors for the treatment of malignant diseases.

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8

Harding, C. V., J. France, R. Song, J. M. Farah, S. Chatterjee, M. Iqbal, and R. Siman. "Novel dipeptide aldehydes are proteasome inhibitors and block the MHC-I antigen-processing pathway." Journal of Immunology 155, no. 4 (August 15, 1995): 1767–75. http://dx.doi.org/10.4049/jimmunol.155.4.1767.

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Abstract Class I MHC (MHC-I) molecules present peptides derived from Ag that are processed in the cytosol. The proteasome is a multicatalytic protease complex that is present in the cytosol and has been implicated in cytosolic Ag processing. Novel dipeptide aldehydes were designed, synthesized, and demonstrated to specifically inhibit the chymotrypsin-like protease activity of isolated proteasomes, but produced relatively little inhibition of cathepsin B, a vacuolar cysteine protease. The inhibitors were membrane permeable and inhibited intracellular cleavage of a membrane-permeable fluorogenic substrate of the chymotrypsin-like proteasome activity. When a model Ag, OVA, was introduced into the cytoplasm of M12.B6 murine B cells by electroporation, the proteasome inhibitors blocked its processing for subsequent presentation by MHC-I molecules. The inhibitors had little effect on class II MHC processing of exogenous Ag. The potencies of different inhibitors for blockade of MHC-I Ag processing correlated directly with their potencies for inhibition of the chymotrypsin-like proteasome activity. In contrast, conventional inhibitors of vacuolar cysteine proteases (e.g., leupeptin and benzyloxycarbonyl-Phe-Ala-CHN2) had little effect on MHC-I processing or the chymotryspin-like activity of isolated proteasomes. These results directly demonstrate that inhibition of proteasome activity blocks MHC-I Ag processing, confirming a role for proteasomes in this pathway. Moreover, they suggest that the chymotrypsin-like activity of the proteasome may be of major importance to the cytosolic processing of at least some Ag.

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9

Sha, Zhe, Helena M. Schnell, Kerstin Ruoff, and Alfred Goldberg. "Rapid induction of p62 and GABARAPL1 upon proteasome inhibition promotes survival before autophagy activation." Journal of Cell Biology 217, no. 5 (March 13, 2018): 1757–76. http://dx.doi.org/10.1083/jcb.201708168.

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Proteasome inhibitors are used as research tools and to treat multiple myeloma, and proteasome activity is diminished in several neurodegenerative diseases. We therefore studied how cells compensate for proteasome inhibition. In 4 h, proteasome inhibitor treatment caused dramatic and selective induction of GABARAPL1 (but not other autophagy genes) and p62, which binds ubiquitinated proteins and GABARAPL1 on autophagosomes. Knockdown of p62 or GABARAPL1 reduced cell survival upon proteasome inhibition. p62 induction requires the transcription factor nuclear factor (erythroid-derived 2)-like 1 (Nrf1), which simultaneously induces proteasome genes. After 20-h exposure to proteasome inhibitors, cells activated autophagy and expression of most autophagy genes by an Nrf1-independent mechanism. Although p62 facilitates the association of ubiquitinated proteins with autophagosomes, its knockdown in neuroblastoma cells blocked the buildup of ubiquitin conjugates in perinuclear aggresomes and of sumoylated proteins in nuclear inclusions but did not reduce the degradation of ubiquitinated proteins. Thus, upon proteasome inhibition, cells rapidly induce p62 expression, which enhances survival primarily by sequestering ubiquitinated proteins in inclusions.

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10

Kraus, Marianne, Bobby Florea, Jürgen Bader, Nan Li, Paul Geurink, Wouter van der Linden, Hermen S. Overkleeft та Christoph Driessen. "Selective Inhibition of the proteasome's β2 Catalytic Subunit Alone Does Not Induce Cytotoxicity, but Resensitizes Bortezomib-Refractory Myeloma Cells for Bortezomib Treatment". Blood 118, № 21 (18 листопада 2011): 2915. http://dx.doi.org/10.1182/blood.v118.21.2915.2915.

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Abstract Abstract 2915 Bortezomib is a reversible first-generation proteasome inhibitor that inhibits the β5 and to a lesser extent the β1 catalytic site of the proteasome. However, bortezomib does not inhibit the β2 catalytic proteasomal site at clinically relevant concentrations, and bortezomib-resistance is accompanied by upregulation of the β2 subunit, suggesting that increased β2 activity may compensate for the loss of β1/ β5 activity during bortezomib-treatment. The second generation proteasome inhibitor carfilzomib, due to the chemistry of its epoxyketone warhead, has a higher substrate specificity and functions as an irreversible proteasome inhibitor, but is still a β1/ β5 inhibitor that does not affect the β2 active site. We investigated the effect of β2-specific proteasome inhibition on myeloma and acute myeloid leukemia (AML) cells and tested the hypothesis that β2-selective proteasome inhibition may overcome bortezomib-resistance. To this end we have developed a set of epoxyketone- and vinylsulfone-based, cell permeable proteasome inhibitors of which we selected the compounds PR523A and PR671A for further testing in cell-based assays. PR671A is a peptide-vinylsulfone that selectively inhibits the proteasome's β2/ β2i subunit in an irreversible fashion in human cell lines and primary cells at low micromolar concentrations without inhibition of other protease species. PR523A is a β5-selective peptide-epoxyketone with otherwise similar properties. Treatment of myeloma and AML cell lines (AMO-1, U-266, HL-60, THP-1) with PR523A induced ER-stress mediated apoptosis, very similar to bortezomib. The combination of bortezomib with PR523A led to additive, but not synergistic induction of apoptosis, as expected. Selective β2 inhibition by PR671A resulted in the induction of ER stress and the accumulation of poly-ubiquitinated protein, however, this was not effectively translated into apoptotic cell death. This indicates that selective inhibition of the β2 proteasome subunit alone has only a poor cytotoxic effect on myeloma and AML cell lines, suggesting that the function of β2 is largely redundant and can be compensated when the remaining proteasome catalytic subunits (β1 and β5) remain active. However, when the β2 inhibitor PR671A was combined with agents that target the proteasome's β5 active site (PR523A) or the β5 and the β1 site (bortezomib), the combination of either inhibitor with the β2 inhibitor PR671A was highly synergistic for both activation of ER stress and the induction of apoptotic death. Importantly, the bortezomib-resistance in bortezomib-adapted myeloma and AML cell lines could be overcome by combining PR671A with either bortezomib or PR523A, while β2 inhibition by PR671A alone had no effect on the viability of bortezomib-adapted cells. We conclude that PR671A is a β2 selective proteasome inhibitor. Selective Inhibition of the proteasome's β2 subunit has little effect on viability or ER stress both in normal and bortezomib-resistant myeloma and leukemia cells, suggesting that the function of the β2 catalytic site is largely redundant. However, when β1/ β5 proteasome activity is inhibited by drugs like bortezomib or carfilzomib, proper function of the β2 proteasome active site is crucial for cell survival, also in bortezomib-resistant myeloma cells. The use of specific β2 inhibitors like PR671A in combination with β1/ β5 inhibitors like bortezomib or carfilzomib is therefore a promising strategy to overcome resistance against β1/ β5-selective proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

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11

Tang, Guangqing, and Stephen H. Leppla. "Proteasome Activity Is Required for Anthrax Lethal Toxin To Kill Macrophages." Infection and Immunity 67, no. 6 (June 1, 1999): 3055–60. http://dx.doi.org/10.1128/iai.67.6.3055-3060.1999.

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ABSTRACT Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines such as RAW 264.7. LF is translocated by PA into the cytosol of target cells, where it acts as a metalloprotease to cleave mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins. In this study, we show that proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not. The inhibitor concentrations that block LF cytotoxicity are similar to those that inhibit the proteasome-dependent IκB-α degradation induced by lipopolysaccharide. The inhibitors did not interfere with the proteolytic cleavage of MEK1 in LeTx-treated cells, indicating that they do not directly block the proteolytic activity of LF. However, the proteasome inhibitors did prevent ATP depletion, an early effect of LeTx. No overall activation of the proteasome by LeTx was detected, as shown by the cleavage of fluorogenic substrates of the proteasome. All of these results suggest that the proteasome mediates a toxic process initiated by LF in the cell cytosol. This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis. Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates.

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12

Terpos, Evangelos, Eleni N. Tsakiri, Efstathios Kastritis, Tina Bagratuni, Vassilis G. Gorgoulis, Ioannis P. Trougakos, and Meletios A. Dimopoulos. "The Novel Proteasome Inhibitors Carfilzomib and Oprozomib Induce Milder Degenerative Effects Compared To Bortezomib When Administered Via Oral Feeding In An In Vivo Drosophila Experimental Model: A Biological Platform To Evaluate Safety/Efficacy Of Proteasome Inhibitors." Blood 122, no. 21 (November 15, 2013): 1930. http://dx.doi.org/10.1182/blood.v122.21.1930.1930.

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Abstract The proteasome is involved in the degradation of both normal, short-lived ubiquitinated proteins and mutated or damaged proteins. Carfilzomib is a tetrapeptide epoxyketone–based proteasome inhibitor and oprozomib is an orally bioavailable tripeptide epoxyketone-based proteasome inhibitor. The primary target for both agents is the chymotrypsin-like β5 subunit of the constitutive proteasome and immunoproteasome. Oprozomib is 5-fold less potent than carfilzomib, but displays similar cytotoxic potential with longer exposure times due to its time-dependent proteasome inhibition. In contrast, bortezomib is a slowly reversible proteasome inhibitor with potency of proteasome inhibition similar to carfilzomib. We propose the fruit fly Drosophila melanogaster as an in vivo platform for screening and characterizing proteasome inhibitors at the whole organism level. Drosophilais well-suited to this line of investigation, due to its powerful genetics, its similarities in key metabolic and aging pathways with humans, the fact that it expresses proteasomes that structurally resemble those from mammals, and also because it comprises a soma-germ line demarcation composed of both post-mitotic and mitotic cells. Moreover, flies live for few months and thus, drug screening on large cohorts can be completed in a reasonable time. We validate our model by investigating the effects of orally administered carfilzomib and oprozomib vs. bortezomib. In isolated Drosophila proteasome in vitroassays, carfilzomib showed a pattern of inhibitory activity similar to bortezomib, whereas oprozomib was less effective. After continuous oral administration of the inhibitors (∼50 μM of carfilzomib and ∼300-400 μM of oprozomib) to young flies (by adding the inhibitor in the flies’ culture medium) a proteasome inhibitory effect in somatic tissues roughly similar to 1 μM bortezomib was induced. Similar findings were noted when we analyzed distinct somatic tissue parts (i.e., head, thorax and abdomen), indicating that orally administered proteasome inhibitors are equally distributed to different body parts. As in the case of bortezomib, the effects of the inhibitors were less pronounced in the reproductive tissues. At the molecular level, carfilzomib (as compared to bortezomib) induced a milder disruption of fly somatic tissue proteostasis, lower rates of somatic tissue oxidative stress and less intense activation of genomic antioxidant response elements that correlated with reduced intensities of proteasome genes and protein subunit upregulation. Proteasome subunit induction was found to depend on the activity of the transcription factor Nrf2, a master regulator of cellular anti-oxidant responses. Furthermore, carfilzomib promoted the induction of lysosomal enzymes (e.g. cathepsins) and autophagy-related genes but less intensively compared to bortezomib. At concentrations that induced rates of proteasome inhibition that were similar to bortezomib, there were no significant toxic effects of either carfilzomib or oprozomib to oogenesis or to embryogenesis. Compared to bortezomib, both inhibitors exerted a significantly milder impact on the neuromusculatory system (locomotor performance) of the flies. Finally, we found that sustained oral administration of either carfilzomib or oprozomib exerted significantly milder effects (as compared to bortezomib) on flies’ mortality rate, healthspan and overall longevity. Our in vivo data support that carfilzomib is significantly less toxic compared to bortezomib, including neuromusculatory toxicity. Oprozomib was also less toxic but it is worth noting that it showed reduced activity against fly proteasomes. In support, our preliminary analyses indicated that in comparison to bortezomib and carfilzomib, oprozomib was less potent when tested in human osteosarcoma cancer cell lines. The validity of our in vivo pharmacological model is exemplified by the observed similarities with the reported clinical adverse effects, while the ratio of the different doses used to achieve similar rates of proteasome inhibition in Drosophila somatic tissues (i.e. ∼1 μM bortezomib, ∼50 μM carfilzomib) is reminiscent of the doses used in the clinic (i.e. ∼1.3 mg/m2 bortezomib and ∼25-56 mg/m2 carfilzomib). We conclude that fruit flies represent a valid biological platform for evaluating the efficacy and toxicity of proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

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Jia, Li, Ganga Gopinathan, Johanna T. Sukumar та John G. Gribben. "Potentiation of Bortezomib-Induced Killing of Lymphoma Cells by Inhibition of Autophagy and Prevention of I-κBα Degradation". Blood 116, № 21 (19 листопада 2010): 116. http://dx.doi.org/10.1182/blood.v116.21.116.116.

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Abstract Abstract 116 Previous studies have shown that germinal center B cell-like (GCB) diffuse large B-cell lymphoma (DLBCL) are resistant to proteasome inhibitors such as bortezomib. The mechanism by which GCB-DLBCL cells escape from proteasome inhibitor-induced apoptosis is unclear. To investigate this further, we examined the proteasomal pathway, expression of anti-apoptotic proteins and autophagy. Using bortezomib or MG-262, we show that DLBCL cells have differential sensitivity to proteasome inhibitor-mediated cell death, even though the effects on proteasome inhibition were similar. DLBCL cells that either over-express anti-apoptotic proteins such as Bcl-2, or lack pro-apoptotic proteins including Bax/Bak, are more resistant to proteasome inhibitor-induced reduction of mitochondrial membrane potential and activation of caspase-3. Treatment with bortezomib induced autophagy in both sensitive and resistant DLBCL cells, as demonstrated by an accumulation and aggregation of the autophagy marker protein LC3-II. Bortezomib induced accumulation of ubiquitinated proteins and a decrease in the adaptor protein p62, indicating activation of autophagic degradation. Fluorescent microscopy revealed that bortezomib induced p62 recruits both ubiquitinated proteins and LC3-II, suggesting that p62 leads ubiquitinated protein to autophagosomes. Treatment with bortezomib thereby promotes I-kBα degradation, demonstrating that the route of I-κBα degradation is not via the ubiquitin-proteasome degradation system. Bortezomib-induced I-kBα degradation was detected in both DLBCL cell lines and primary DLBCL and follicular lymphoma samples. Chloroquine (CQ), an inhibitor of autophagy, significantly increased bortezomib-induced accumulation of p62 and ubiquitinated proteins, including I-κBα, Bax and p53. CQ alone induced a dose-dependent increase in I-kBα protein levels, indicating that I-κBα protein can be degraded by autophagy even in the absence of proteasome inhibition. Importantly, the combination of proteasome and autophagy inhibitors shows great potential to kill apoptosis-resistant lymphoma cells. Proteasome inhibitor with or without CQ induced cell death in DLBCL cells cannot be blocked completely by either caspase inhibitors or knockdown of Bax/Bak proteins, suggesting that cell death occurs via a caspase-independent pathway. In summary, proteasome inhibitors induce autophagy and confer DLBCL cells resistance by eliminating I-κBα and possibly other pro-apoptotic proteins. Addition of autophagy inhibitors to bortezomib has the potential to induce increased killing in patients with resistant lymphoma. Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

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14

Ahn, Won-Gyun, Yeejin Jeon, Yeong-In Yang, Jaeseung Kim, Seung-Joo Lee, Uwe Koch, Gunther Zischinsky, et al. "Abstract 416: A novel non-covalent and rapidly reversible proteasome inhibitor for multiple myeloma and various solid cancers." Cancer Research 82, no. 12_Supplement (June 15, 2022): 416. http://dx.doi.org/10.1158/1538-7445.am2022-416.

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Abstract The existing proteasome inhibitors, such as bortezomib and ixazomib, are effective in multiple myeloma, but have little activity against solid tumors. These are covalent boronic acid-based compounds and are associated with undesired side effects, mainly hematologic toxicity and peripheral neuropathy. A variety of improved covalent proteasome inhibitors have been developed, but lack of oral availability and low distribution in tumors make them ineffective in solid tumors. In addition, they still have a narrow therapeutic window due to unexpected adverse effects such as cardiac and pulmonary toxicity. We report a non-covalent and rapidly reversible proteasome inhibitor as a potential anti-cancer agent against solid tumors as well as multiple myeloma. These novel inhibitors, called QL compounds, are non-covalent chymotrypsin-like selective proteasome inhibitors that can be taken orally. They effectively inhibited tumor growth in multiple myeloma xenograft model without hematologic toxicity through improved PK properties, especially partition of compound between plasma and RBC. Moreover, this improved PK properties of the QL compounds allowed for sufficient distribution outside the blood compartment and induced tumor growth inhibition in some types of solid cancer xenograft models that were sensitive to proteasome inhibition. These results indicate that non-covalent and rapidly reversible proteasome inhibitors are an ideal strategy for multiple myeloma and potential agents for solid tumors. Citation Format: Won-Gyun Ahn, Yeejin Jeon, Yeong-In Yang, Jaeseung Kim, Seung-Joo Lee, Uwe Koch, Gunther Zischinsky, Axel Choidas, Ayesha Pasha, Bert Klebl, Robert Huber, Michael Hamacher, Kiyean Nam. A novel non-covalent and rapidly reversible proteasome inhibitor for multiple myeloma and various solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 416.

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15

Shirazi, Fazal, Richard J. Jones, Ram K. Singh, Jianxuan Zou, Isere Kuiatse, Zuzana Berkova, Hua Wang, et al. "ActivatingKRAS,NRAS, andBRAFmutants enhance proteasome capacity and reduce endoplasmic reticulum stress in multiple myeloma." Proceedings of the National Academy of Sciences 117, no. 33 (August 3, 2020): 20004–14. http://dx.doi.org/10.1073/pnas.2005052117.

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KRAS,NRAS, andBRAFmutations which activate p44/42 mitogen-activated protein kinase (MAPK) signaling are found in half of myeloma patients and contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms are not fully understood. We established myeloma cell lines expressing wild-type (WT), constitutively active (CA) (G12V/G13D/Q61H), or dominant-negative (DN) (S17N)-KRASand -NRAS, orBRAF-V600E. Cells expressing CA mutants showed increased proteasome maturation protein (POMP) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) expression. This correlated with an increase in catalytically active proteasome subunit β (PSMB)-8, PSMB9, and PSMB10, which occurred in an ETS transcription factor-dependent manner. Proteasome chymotrypsin-like, trypsin-like, and caspase-like activities were increased, and this enhanced capacity reduced PI sensitivity, while DN-KRASand DN-NRASdid the opposite. Pharmacologic RAF or MAPK kinase (MEK) inhibitors decreased proteasome activity, and sensitized myeloma cells to PIs. CA-KRAS, CA-NRAS, and CA-BRAFdown-regulated expression of endoplasmic reticulum (ER) stress proteins, and reduced unfolded protein response activation, while DN mutations increased both. Finally, a bortezomib (BTZ)/MEK inhibitor combination showed enhanced activity in vivo specifically in CA-NRASmodels. Taken together, the data support the hypothesis that activatingMAPKpathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue these mutations promote myeloma survival by reducing cellular stress, thereby distancing plasma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma.

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16

Ponduru, Sridevi, Raymond Moellering, Edward Greenberg, John Paul Ying-Ching Shen, Benjamin Z. Stanton, and James E. Bradner. "Discovery and Characterization of Macrocyclic Thiopeptide Proteasome Inhibitors for Hematologic Malignancies." Blood 112, no. 11 (November 16, 2008): 3669. http://dx.doi.org/10.1182/blood.v112.11.3669.3669.

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Abstract The ubiquitin proteasome pathway comprises a coordinated, dynamic cellular system critical to cellular metabolism, signaling and proliferation. The expanding clinical utility of the peptide boronate, bortezomib, in the treatment of patients with multiple myeloma and other hematologic malignancies has established the human 26S proteasome as a validated target in cancer. Still, only one FDA-approved proteasome inhibitor presently exists. Restricted activity against one enzymatic function of the proteasome and dose-limiting toxicities associated with bortezomib warrant further discovery efforts aimed at the identification of structurally and functionally distinct protein degradation inhibitors (PDIs). Here, we report a novel family of natural product proteasome inhibitors discovered by high-throughput, high-content screening at the National Cancer Institute Initiative for Chemical Genetics. A primary screen of 14,000 small molecules was performed in 384-well plate format using a cell line stably transfected with a destabilized fluorescent protein chimera. Assay positives were retested in the primary screen in dose-response format. Thiostrepton was selected for further characterization due to its unique macrocyclic chemical structure, the recent publication of its total synthesis, reports of anticancer properties and the lack of prior annotation as a PDI. First, thiostrepton was linked to previously characterized molecules acting on the protein degradation pathways by transcriptional small molecule connectivity mapping (CMAP). Subsequent cell-state analyses confirmed strong induction of functional and annotated gene sets associated with misfolded protein stress and proteasome inhibition. Mechanism of action was confirmed by biochemical profiling of human 20S proteasome active site inhibition and specificity using homogeneous assays and selective substrates for each of three catalytic active sites. Importantly, inhibitory activity of thiostrepton differs from bortezomib by blocking both the chymotryptic-like and PGPH active sites with sub-micromolar potencies. Dose-dependent inhibition of multiple myeloma cell growth was observed, with a concomitant increase in polyubiquitinated protein stress and induction of apoptosis. Inhibition of conferred proliferation by bone marrow stroma was confirmed using a novel miniaturized high-content assay modeling the bone marrow stroma-multiple myeloma microenvironment. Structurally related compounds to thiostrepton, nosiheptide and siomycin, were confirmed also as proteasome inhibitors as above. Our discovery of this class of natural products as proteasome inhibitors and a recent report of siomycin inhibition of Sonic Hedgehog (Shh) signaling begged the question whether established proteasome inhibitors would inhibit Shh signaling in human cancer. This hypothesis was confirmed in a set of reporter-gene assays. In sum, these studies identify thiopeptide macrocycles as a class of naturally-occurring proteasome inhibitors poised for clinical development in hematologic malignancies, establish novel high-throughput assays for modeling MM-stroma microenvironment interactions and pave the way for the development of proteasome inhibitors in disease states where Shh signaling is central to pathogenesis.

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17

Sterz, Jan, Christian Jakob, Ulrike Kuckelkorn, Hannes A. Braun, Martin Kaiser, Claudia Fleissner, Lorenz Kleeberg, et al. "BSc2118, a Novel Proteasome Inhibitor, Shows Anti-Tumor Activity in Multiple Myeloma and Mantle Cell Lymphoma." Blood 108, no. 11 (November 16, 2006): 3475. http://dx.doi.org/10.1182/blood.v108.11.3475.3475.

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Abstract The ubiquitin-proteasome pathway was recently identified as a promising new therapeutic target in cancer treatment. An increased proteasome activity was described in certain cancer types, especially in multiple myeloma (MM) and also in mantle cell lymphoma (MCL). The proteasome inhibitor bortezomib has been approved for the treatment of refractory MM and has also shown reproducible activity in MCL. Recently we described a novel tripeptide compound, BSc2118, with inhibitory activity against all three proteolytic activities (post-glutamyl peptide hydrolase-like, trypsin-like and chymotrypsin-like activities) of the 20S proteasome (Cancer Res2006;66:7598–605). We investigated the in vitro effects of BSc2118 in MM and MCL cell lines by MTT cell viability and AnnexinV apoptosis-assays. Furthermore, the intracellular chymotrypsin-like proteasome activity and NF-κB activity were detected in MM and MCL cells by detection of proteasome-degraded peptides or NF-κB p65 subunit. In MM cell lines OPM-2 and U266 we could show a significant time and dose-dependent reduction of cell viability by BSc2118 with an IC50 at 48hrs of 52nM and 65nM, respectively, whereas the MM cell line RPMI-S was less sensitive with an IC50 of 287nM. Using AnnexinV assay, a dose-dependent induction of apoptosis by BSc2118 was shown after 48hrs incubation. Comparably, in MCL we also found a time and dose-dependent reduction of cell viability in the cell lines HBL-2, JeKo-1 and Granta-519 with an IC50 of 82nM, 130nM and 262nM, respectively. Furthermore, BSc2118 induced apoptosis in all three MCL cell lines. Additionally, we detected a significant dose-dependent inhibition of intracellular chymotrypsin-like proteasome activity in MM and MCL cells, and a dose-dependent inhibition of TNFα-induced NF-κB activation in the MM cell lines OPM-2 and RPMI-S. This is the first report of anti-tumor effects of the novel proteasome inhibitor BSc2118 in MM and MCL cells. The compound effectively reduces the cell survival and shows a high pro-apoptotic activity in the MM cell lines OPM-2 and U266 and a significant activity in MCL cell lines HBL-2 and JeKo-1. Mechanisms of action are the inhibition of proteasome and NF-κB activity. Since previous clinical trials have shown an activity of the proteasome inhibitor bortezomib in MM and MCL, and adverse effects of other proteasome inhibitors may differ, our preclinical data support the idea to consider BSc2118 as a promising new agent in anti-tumor drug development.

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Parlati, Francesco, Monette A. Aujay, Susan D. Demo, Eileen D. Goldstein, Jing Jiang, Christopher J. Kirk, Guy J. Laidig, et al. "Immunoproteasome-Selective Keto-Epoxide Inhibitors: Potential Therapeutic Agents for the Treatment of Hematological Disorders." Blood 108, no. 11 (November 16, 2006): 4392. http://dx.doi.org/10.1182/blood.v108.11.4392.4392.

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Abstract The clinical development of the dipeptide boronate bortezomib (Velcade™) has validated proteasome inhibition as a treatment for multiple myeloma and non-Hodgkin’s lymphoma. In hematopoetically derived normal and malignant cells, the form of the proteasome expressed, termed the immunoproteasome, differs from that found in most tissues, referred to as the constitutive proteasome. Bortezomib inhibits the chymotrypsin-like activity of the immunoproteasome and the constitutive proteasome with equal potency and induces side effects such as peripheral neuropathy and gastrointestinal toxicity that may be related to proteasome inhibition in cells expressing the constitutive proteasome. This finding suggests that development of immunoproteasome-specific inhibitors would selectively target hematological malignances without affecting non-hematological cells. We have undertaken an effort to profile hematological tumor cell lines for immunoproteasome expression to determine which hematological cancers could be effectively targeted using an immunoproteasome selective inhibitor. We have observed that several cell lines derived from hematologic malignancies express predominantly the immunoproteasome while others express a mixture of both proteasome types. To develop selective inhibitors of the immunoproteasome, we generated analogs of PR-171, a peptide keto-epoxide that is currently being investigated in clinical trials for the treatment of myeloma and lymphoma. Purified 20S preparations were used to screen for compounds that selectively inhibit the chymotrypsin-like activity of the immunoproteasome (subunit LMP7) over the constitutive proteasome (subunit beta5). While the parental molecule PR-171 targets both forms of the proteasome with similar potency, several analogs have been identified with an IC50 below 50 nM on LMP7 and greater than 10-fold selectivity compared to beta5. To evaluate the immunoproteasome selectivity of our compounds in cells, an active site-specific ELISA was utilized. Cells expressing both constitutive and immunoproteasome were treated with the analogs and the occupancy of the LMP7 and beta5 active sites was determined. Several compounds retained their immunoproteasome selectivity in cells with an IC50 below 300 nM for LMP7 inhibition. In cell viability studies, some of these compounds demonstrated enhanced cytotoxicity in hematological tumor cell lines expressing the immunoproteasome as compared to solid tumor cell lines which express the constitutive proteasome. Finally, when pharmacodynamics was evaluated following intravenous administration to mice, LMP7 selectivity was retained in tissues expressing both the immunoproteasome and constitutive proteasome. The in vivo anti-tumor properties and toxicity profiles of the immunoproteasome-selective inhibitors are currently being evaluated.

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Lai, Chao-Yang, Da-Wei Yeh, Chih-Hao Lu, Yi-Ling Liu, Li-Rung Huang, Cheng-Yuan Kao, Huan-Yuan Chen, et al. "Thiostrepton inhibits psoriasis-like inflammation induced by TLR7, TLR8, and TLR9." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 124.41. http://dx.doi.org/10.4049/jimmunol.196.supp.124.41.

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Abstract Toll-like receptors 7, 8 and 9 (TLR7-9) comprise a subfamily of TLR. Activation of these TLRs has been linked to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and psoriasis. Thus antagonists of these TLRs are being investigated for their therapeutic applications on these diseases. Bortezomib is a proteasome inhibitor known to suppress activation of these TLRs. This drug is approved for the treatment of multiple myeloma, and its inhibitory effects on autoimmune disorders such as psoriasis, RA, and SLE have also been investigated in animal models. In an attempt to develop novel TLR7-9 inhibitors, we searched the Gene Expression Omnibus database for gene expression profiles in cells treated with bortezomib. These profiles were then used as an input to screen the Connectivity Map database for chemical compounds with similar functions as bortezomib. Here we report that the antibiotic thiostrepton is a novel TLR7–9 inhibitor. Like bortezomib, thiostrepton effectively inhibits TLR7–9 activation in cell-based assays and dendritic cells. In contrast to bortezomib, thiostrepton is less cytotoxic to dendritic cells, and its inhibitory activity is more specific to TLR7–9. Thiostrepton inhibits TLR9 localization in endosomes for activation via two mechanisms. One mechanism is similar to the proteasome inhibitory function of bortezomib, and the other is through inhibition of endosomal acidification. In different animal models, thiostrepton attenuated LL37- and imiquimod-induced psoriasis-like inflammation. These results indicated that thiostrepton is a novel and specific inhibitor to TLR7–9.

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Ma, Wanlong, Hagop Kantarjian, Susan O’Brien, Iman Jilani, Xi Zhang, Zeev Zestrov, Elihu Estey, et al. "Measurement and Clinical Relevance of Proteasome Enzymatic Activity in Plasma of Patients with Acute Lymphoblastic Leukemia." Blood 108, no. 11 (November 16, 2006): 4475. http://dx.doi.org/10.1182/blood.v108.11.4475.4475.

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Abstract The ubiquitin-proteasome pathway plays a major role in regulating proteins involved in cell cycle regulation and apoptosis. Three different types of enzymatic activity have been reported for proteasomes: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L) (postglutamyl peptide hydrolytic-like). Various proteasome inhibitors affect each of the three activities differently and at different concentrations. For example, NPI-0052 inhibits Ch-L and Tr-L activities at lower concentrations than does bortezomib, while bortezomib inhibits Cas-L at lower concentrations than does NPI-0052. These enzymatic activities are usually measured in normal or tumor cells to monitor therapy with proteasome inhibitors. We developed fluorogenic kinetic assays using peptide-AMC (7-amino 4-methylcoumoran) substrates to measure Ch-L, Tr-L, and Cas-L activities in cell-free plasma. We evaluated plasma activities in 57 patients with acute lymphoblastic leukemia (ALL) and assessed their correlations with clinical behavior. Significantly (P < 0.001) higher Ch-L, Tr-L, and Cas-L activities were detected in patients with ALL (medians: 1.40, 2.06, and 2.04 pmol AMC/sec/mL, respectively) than in healthy volunteers (n = 42) (medians: 0.80, 0.74, and 0.81 pmol AMC/sec/mL, respectively). While there was no significant difference between Ch-L and Cas-L activities in healthy controls, there was a significant difference between the 2 activities in patients with ALL. Cas-L, Tr-L, and Ch-L all correlated positively with lactic dehydrogenase (P <0.01). However, ALL patients with Cas-L activity ≥2.7 pmol AMC/sec/mL had significantly longer survival (P = 0.01) than did patients with lower activity. The increased cell-free circulating proteasome activities most likely reflect the leukemic cells and may represent a marker not only for the disease, but also for monitoring therapy. These data also suggest that patients with ALL may benefit differentially from proteasome inhibitors depending on the specific therapeutic properties of the inhibitor.

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21

Hu, Zongyi, Zhensheng Zhang, Edward Doo, Olivier Coux, Alfred L. Goldberg, and T. Jake Liang. "Hepatitis B Virus X Protein Is both a Substrate and a Potential Inhibitor of the Proteasome Complex." Journal of Virology 73, no. 9 (September 1, 1999): 7231–40. http://dx.doi.org/10.1128/jvi.73.9.7231-7240.1999.

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ABSTRACT The hepatitis B virus X protein (HBX) is essential for the establishment of HBV infection in vivo and exerts a pleiotropic effect on diverse cellular functions. The yeast two-hybrid system had indicated that HBX could interact with two subunits of the 26S proteasome. Here we demonstrate an association in vivo of HBX with the 26S proteasome complex by coimmunoprecipitation and colocalization upon sucrose gradient centrifugation. Expression of HBX in HepG2 cells caused a modest decrease in the proteasome’s chymotrypsin- and trypsin-like activities and in hydrolysis of ubiquitinated lysozyme, suggesting that HBX functions as an inhibitor of proteasome. In these cells, HBX is degraded with a half-life of 30 min. Proteasome inhibitors retarded this rapid degradation and caused a marked increase in the level of HBX and an accumulation of HBX in polyubiquitinated form. Thus, the low intracellular level of HBX is due to rapid proteolysis by the ubiquitin-proteasome pathway. Surprisingly, the proteasome inhibitors blocked the transactivation by HBX, and this effect was not a result of a squelching phenomenon due to HBX accumulation. Therefore, proteasome function is possibly required for the transactivation function of HBX. The inhibition of protein breakdown by proteasomes may account for the multiple actions of HBX and may be an important feature of HBV infection, possibly in helping stabilize viral gene products and suppressing antigen presentation.

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Blackburn, Christopher, Kenneth M. Gigstad, Paul Hales, Khristofer Garcia, Matthew Jones, Frank J. Bruzzese, Cynthia Barrett та ін. "Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit". Biochemical Journal 430, № 3 (27 серпня 2010): 461–76. http://dx.doi.org/10.1042/bj20100383.

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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 used 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 β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (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 β5 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 NFκB (nuclear factor κB) in response to TNF-α (tumour necrosis factor-α) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the β5 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 β5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

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Demo, Susan D., Monette A. Aujay, Eileen Goldstein, Guy J. Laidig, Francesco Parlati, Yan Lu, Kevin D. Shenk, et al. "Identification of Peptide Epoxy-Ketone Proteasome Inhibitors with Differential Active Site Selectivity." Blood 108, no. 11 (November 16, 2006): 4398. http://dx.doi.org/10.1182/blood.v108.11.4398.4398.

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Abstract The proteasome is a multi-subunit structure responsible for degrading the majority of proteins in the cell. The three catalytic subunits in the 20S core of the proteasome are defined by their preference for amino acid side chains at the P1 position: the beta5 subunit (chymotrypsin-like), the beta2 subunit (trypsin-like), and the beta1 subunit (caspase-like) hydrolyze substrates after hydrophobic, basic, and acidic residues, respectively. Bortezomib (Velcade®), currently an approved drug for the treatment of multiple myeloma, is a potent inhibitor of the beta5 subunit but also has activity against the beta1 subunit (9-fold less active) and the beta2 subunit (250-fold less active). PR-171, a peptide epoxy-ketone proteasome inhibitor currently in Phase I clinical trials in multiple myeloma and NHL patients, has a distinct profile of inhibition. Similar to bortezomib, PR-171 is most potent against the beta5 subunit, but differs in its activity against the beta2 and beta1 subunits (45-fold less active and 140-fold less active, respectively). Although both bortezomib and PR-171 are cytotoxic to a variety of tumor cell types, the connection between inhibition of specific proteasomal subunits and cellular consequences, including induction of apoptosis, is currently unclear. To investigate this relationship, we synthesized a series of PR-171 analogs and profiled them in our proteasome-specific active site ELISA binding assay to identify compounds with differential binding profiles for the three catalytic subunits. Using this approach, we have identified compounds that bind all three catalytic sites with approximately equivalent activity as well as compounds that have increased specificity for the beta5 subunit. We have extended this approach to search for compounds with differential binding profiles for the immunoproteasome catalytic subunits. The immunoproteasome is the predominant form of the proteasome expressed in cells of hematopoetic origin and in other cell types after interferon-g stimulation. In the 20S core of the immunoproteasome the beta5, beta2 and beta1 subunits of the “constitutive” proteasome are replaced by the homologous subunits LMP7, MECL1, and LMP2. We have successfully identified inhibitors with distinct active site profiles for the immunoproteasome as well as the constitutive proteasome. The impact of differential active site inhibition on tumor cell viability is under investigation in a variety of solid and hematological tumor cell lines expressing the constitutive proteasome or the immunoproteasome.

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Dunys, Julie, Toshitaka Kawarai, Sherwin Wilk, Peter St. George-Hyslop, Cristine Alves Da Costa, and Frédéric Checler. "Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins." Biochemical Journal 394, no. 2 (February 10, 2006): 501–9. http://dx.doi.org/10.1042/bj20051197.

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PS (presenilin)-dependent γ-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the γ-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S proteasome participates to PEN2 proteolysis in deficient fibroblasts remains to be established.

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Baldisserotto, Anna, Mauro Marastoni, Claudio Trapella, Riccardo Gavioli, Valeria Ferretti, Loretta Pretto та Roberto Tomatis. "Glutamine vinyl ester proteasome inhibitors selective for trypsin-like (β2) subunit". European Journal of Medicinal Chemistry 42, № 5 (травень 2007): 586–92. http://dx.doi.org/10.1016/j.ejmech.2006.12.008.

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Demo, Susan D., Tonia J. Buchholz, Guy J. Laidig, Francesco Parlati, Kevin D. Shenk, Mark S. Smyth, Carol M. Sullivan, et al. "Biochemical and Cellular Characterization of the Novel Proteasome Inhibitor PR-171." Blood 106, no. 11 (November 16, 2005): 1588. http://dx.doi.org/10.1182/blood.v106.11.1588.1588.

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Анотація:

Abstract Recent clinical studies have identified the proteasome as an important therapeutic target for hematologic malignances. The proteasome inhibitor, bortezomib, has been approved for the treatment of relapsed or refractory multiple myeloma and ongoing clinical trials suggests a potential benefit for the treatment of Non-Hodgkin’s lymphoma. PR-171 is a novel epoxomicin derivative that is a potent and irreversible inhibitor of the human proteasome. It inhibits the chymotrypsin-like activity of purified human 20S proteasome with a kinact/Ki of 34,000 M−1s−1 and is >300-fold selective over the other proteasome catalytic activities. In addition, PR-171 has been found to exhibit minimal activity in a broad diversity panel of biochemical assays that includes 67 receptor/ligand and 37 enzyme assays. PR-171 retains its potency for inhibition of the proteasome chymotrypsin-like activity in mammalian cells displaying IC50 values <10 nM in multiple tumor cell lines. Although PR-171 is a covalent irreversible inhibitor, proteasome activity in cells recovers with a t1/2 of approximately 24 hr after removal of the compound. This recovery is likely due to induction of de novo synthesis since other proteasome inhibitors have been shown to promote transcription of multiple proteasome subunit genes. The cellular consequences of proteasome inhibition by PR-171 include accumulation of polyubiquitinated proteins, cell cycle arrest, and induction of apoptosis. Potent cytotoxic activity of PR-171 is observed across a broad panel of human tumor cell lines (IC50 range: 2–40 nM). Pulsatile exposure studies designed to mimic the anticipated drug exposure in vivo have demonstrated that cytotoxicity is dependent upon the magnitude and duration of proteasome inhibition. These studies have also shown that hematological tumor lines are most sensitive to brief PR-171 exposure, with solid tumor lines exhibiting intermediate sensitivity and non-transformed cells being the least sensitive to such treatment. Finally, PR-171 has been found to retain its cytotoxic potential on cells made resistant to bortezomib in vitro. These studies motivate the clinical investigation of PR-171 in hematological malignances.

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Kuhn, Deborah J., Peter M. Voorhees, John S. Strader, Kevin D. Shenk, Congcong M. Sun, Susan D. Demo, Mark K. Bennett, and Robert Z. Orlowski. "Potent Activity of a Novel, Irreversible Inhibitor of the Ubiquitin-Proteasome Pathway Against Pre-Clinical Models of Multiple Myeloma." Blood 106, no. 11 (November 16, 2005): 1576. http://dx.doi.org/10.1182/blood.v106.11.1576.1576.

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Abstract The proteasome is a multi-catalytic proteinase complex that is integral to intracellular proteolysis, and plays a key role in many cell functions. Targeting the proteasome with small molecule inhibitors has been validated as a rational therapeutic strategy for patients with relapsed/refractory myeloma with the approval of the first proteasome inhibitor, bortezomib (VELCADE®), for this indication. Additional studies are ongoing to better define the role of this agent in myeloma and other diseases, including non-Hodgkin’s lymphoma. Since bortezomib is a reversible proteasome inhibitor, we considered the possibility that an irreversible agent might have novel, potentially attractive properties. To begin to evaluate this hypothesis, we have studied the efficacy of a novel epoxomicin-related proteasome inhibitor, PR-171, which binds irreversibly and with a high degree of specificity in vitro to the chymotrypsin-like subunit of the proteasome. PR-171 was able to inhibit proliferation of both interleukin (IL)-6-dependent ANBL-6 and KAS-6 cell lines, as well as IL-6-independent models, including RPMI 8226 and U266 cells, in a concentration- and time-dependent fashion. IL-6-dependent cells generally displayed a greater sensitivity to PR-171-mediated effects than IL-6-independent cells. Experiments modeling the in vivo pharmacokinetics of proteasome inhibitors, with a one-hour pulse of drug followed by a washout, showed that PR-171 indeed inhibited the chymotrypsin-like activity of the proteasome without effects on other proteasome proteases. Inhibition of cell proliferation was associated with an induction of programmed cell death, as judged by the appearance of apoptotic oligonucleosome DNA fragments, as well as by the activation of caspase-3. This common effector caspase was activated by both the extrinsic and intrinsic pathways, in that both caspase-8 and caspase-9 were potently induced. Additionally, pulse treatment of PR-171 induced activation of c-Jun-N-terminal kinase, a key signaling molecule in stress-induced and proteasome inhibitor-induced apoptosis. Other members of the stress response-signaling pathway, including heat shock protein-70 and mitogen activated protein kinase phosphatase-1, were induced as well. Finally, both continuous and pulse treatment with PR-171 was also able to inhibit proliferation in freshly purified patient-derived multiple myeloma plasma cells, including isolates from patients with both newly diagnosed, previously untreated disease, as well as isolates from patients who had progressed on other standard therapies, including bortezomib. Importantly, PR-171 was active in both myeloma cell line models and patient-derived samples with chromosome 13 abnormalities. Taken together, these data indicate that PR-171 is a promising, novel proteasome inhibitor with activity against models of multiple myeloma, providing a rational basis for its translation into the clinic.

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Kuhn, Deborah J., Qing Chen, Peter M. Voorhees, John S. Strader, Kevin D. Shenk, Congcong M. Sun, Susan D. Demo, Mark K. Bennett, Fred W. van Leeuwen, and Robert Z. Orlowski. "The Novel, Irreversible Proteasome Inhibitor PR-171 Demonstrates Potent Anti-Tumor Activity in Pre-Clinical Models of Multiple Myeloma, and Overcomes Bortezomib Resistance." Blood 108, no. 11 (November 16, 2006): 3461. http://dx.doi.org/10.1182/blood.v108.11.3461.3461.

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Abstract Introduction: The ubiquitin-proteasome pathway has been validated as a therapeutic target with the approval of the small molecule proteasome inhibitor, bortezomib (VELCADE®), in multiple myeloma and non-Hodgkin lymphoma. However, the overall response rate of patients with multiple myeloma in phase III clinical trials was 43%, underscoring the need for a next generation of inhibitors with the potential for greater efficacy. Methods: PR-171 is a novel, tetrapeptide epoxomicin-related inhibitor that binds the proteasome irreversibly, and our objectives were to evaluate its activity and mechanism of action in pre-clinical models of multiple myeloma. Results: PR-171 potently bound and inhibited the chymotrypsin-like subunit of the proteasome in vitro, in cellulo, and in vivo at low concentrations. At higher concentrations, however, unlike bortezomib, which targeted the chymotrypsin-like and peptidyl-glutamyl peptide hydrolyzing activities in vivo, PR-171 also displayed significant inhibition of the trypsin-like and the peptidyl-glutamyl peptide hydrolyzing activities. PR-171-induced proteasome inhibition was associated with accumulation of polyubiquitinated substrates and pro-apoptotic Bax. Brief pulse PR-171 exposure, which simulates the in vivo pharmacokinetics of bortezomib, led to PR-171-mediated inhibition of cellular proliferation linked to induction of caspase-3-dependent apoptosis through both intrinsic (caspase-9) and extrinsic (caspase-8-dependent) pathways. Pretreatment with caspase-3, -8, and -9 inhibitors rescued the anti-proliferative effect of PR-171. Furthermore, pulse PR-171 treatment activated c-Jun-N-terminal kinase, a key-signaling molecule in proteasome inhibitor-induced apoptosis, and cleavage of poly-ADP-ribose polymerase, while abrogation of c-Jun-N-terminal kinase signaling with a dominant-negative c-Jun inhibited PR-171-induced effects. PR-171 displayed enhanced anti-proliferative activity compared to bortezomib in multiple myeloma cell lines and freshly isolated patient-derived CD138+ plasma cells, associated with enhanced phosphorylation of c-Jun-N-terminal kinase and capase-3, -8, and -9 activation. Lastly, PR-171 was a potent inhibitor of proliferation in a multiple myeloma cell line model resistant to bortezomib and in isolates from two patients, one with primary and the other with acquired bortezomib-resistance. Conclusions: These data indicate that PR-171 has enhanced activity against preclinical models of multiple myeloma, perhaps owing to its irreversible binding and subunit specificity, and provide a rationale for its translation into the clinic.

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Kuhn, Deborah J., Peter M. Voorhees, Qing Chen, John S. Strader, Frank Bruzzese, Jeffrey P. Ciavarri, Zhigen Hu, and Robert Z. Orlowski. "Development of Immunoproteasome-Specific Inhibitors as Novel Anti-Cancer Drugs." Blood 108, no. 11 (November 16, 2006): 3458. http://dx.doi.org/10.1182/blood.v108.11.3458.3458.

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Abstract Introduction: The proteasome is a large (~2.5 MDa), ATP-dependent, intracellular protease responsible for degrading ubiquitinated proteins as part of the ubiquitin-proteasome pathway. The immunoproteasome is a unique proteasomal variant with distinct catalytic subunits termed low molecular mass proteins that functions predominately in cells derived from hematopoietic precursors, and differs from the constitutive proteasome found in most other cells. Bortezomib (VELCADE®; Millennium Pharmaceuticals, Inc.) is a first-in-class proteasome inhibitor, which is approved for treatment of multiple myeloma patients who have received at least one prior therapy. While the overall safety profile of bortezomib is manageable and predictable, some toxicities, such as peripheral neuropathy, associated with bortezomib treatment can be dose-limiting. These toxicities may be due to the inhibition of all isoforms of the proteasome. Development of immunoproteasome-specific inhibitors (IPSIs) would allow for targeted therapy against cancers arising from hematologic origins, thereby sparing normal tissues, such as gastrointestinal and neurological tissues. Methods: We have identified several novel IPSIs, most notably IPSI-001, with selective activity against the immunoproteasome, which we therefore sought to characterize. Results: Expression of proteins associated with the immunoproteasome (low molecular mass protein-2; 11S Reg-α) was found primarily in cell lines of hematopoietic origin, while solid tumor cell lines exhibited expression of constitutive proteasome proteins (β5; 19S S6′). IPSI-001 exposure induced preferential inhibition of the chymotrypsin-like activity, the rate-limiting step of proteolysis, in hematologic cell lines over solid tumors. This inhibition was associated with an increase in ubiquitinated substrates, activation of c-Jun N-terminal kinase, and accumulation of Bax. IPSI-001 treatment led to preferential induction of apoptosis as evidenced by DNA fragmentation assays and cleavage of β-actin by caspase-3 into an apoptotic marker, fractin. Furthermore, IPSI-001 had potent chymotrypsin-like inhibitory activity in patient samples of chronic lymphocytic leukemia and acute myeloid leukemia. A dose-dependent decrease in proliferation was observed in additional patient samples of acute myeloid leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma treated with IPSI-001. Also, IPSI-001 exposure induced apoptosis in multiple myeloma and chronic lymphocytic leukemia patient samples. In an effort to increase the efficacy of IPSIs, a series of boronic acid analogs were made of several IPSIs. Conversion into the boronate analogs increased the potency by up to 1000-fold against the chymotrypsin-like activity of the immunoproteasome in vitro and in cellulo. Dose-dependent inhibition of proliferation was observed in ANBL-6, KAS-6/1, and Ramos cell lines, which was associated with induction of apoptosis. Conclusions: Studies are ongoing to characterize the specificity and molecular effects associated with IPSI-boronic acid derivatives exposure in immunoproteasome- and constitutive proteasome-containing cell types.

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Powell, Jason A., Melissa R. Pitman, Julia R. Zebol, Paul A. B. Moretti, Heidi A. Neubauer, Lorena T. Davies, Alexander C. Lewis, et al. "Kelch-like protein 5-mediated ubiquitination of lysine 183 promotes proteasomal degradation of sphingosine kinase 1." Biochemical Journal 476, no. 21 (November 11, 2019): 3211–26. http://dx.doi.org/10.1042/bcj20190245.

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Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.

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Chauhan, Dharminder, Ta-Hsiang Chao, Laurence Catley, Benjamin Nicholson, Mugdha Velanker, Teru Hideshima, Celia Berkers, et al. "In Vitro and In Vivo Proteasome Activity Profiles of Bortezomib and a Novel Proteasome Inhibitor NPI-0052." Blood 106, no. 11 (November 16, 2005): 3363. http://dx.doi.org/10.1182/blood.v106.11.3363.3363.

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Abstract Proteasome inhibition is an effective anti-cancer therapy. Proteasome function is mediated by three catalytic activities: chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L). Kinetics of inhibition of catalytic activities may define the pharmacologic utility of proteasome inhibitors. Here we utilized two structurally distinct proteasome inhibitors Bortezomib, a dipeptide boronic acid; and a non-peptide proteasome inhibitor NPI-0052 to determine their effect on proteasome activities in vitro and in animal model. Examination of the proteasome activity using human erythrocyte 20S proteasomes and fluorogenic substrates shows that NPI-0052 and Bortezomib inhibit all three proteasome activities, albeit at different concentrations: NPI-0052 inhibits CT-L and T-L activities at lower concentrations than Bortezomib (NPI-0052: EC50 = 3.5 ± 0.3 nM versus Bortezomib: 7.9 ± 0.5 nM for CT-L activity; and NPI-0052: EC50 = 28 ± 2 nM versus Bortezomib: EC50 = 590 ± 67 nM for T-L activity); in contrast, higher concentrations of NPI-0052 than Bortezomib are required to inhibit C-L activity (NPI-0052 EC50 = 430 ± 34 nM versus Bortezomib: EC50 = 53 ± 10 nM for C-L activity). We next compared the effects of NPI-0052 and Bortezomib on all three proteasome activities in vivo. Mice were treated with a single MTD dose of NPI-0052 (0.15 mg/kg i.v) or Bortezomib (1 mg/kg i.v); blood samples were collected at 90 mins, 24h, 48h, 72h, or 168h; and whole blood cells were then analyzed for proteasome activity. NPI-0052 completely inhibited CT-L activity by 90 mins, which was recoverable by 168h; whereas Bortezomib-inhibited CT-L activity is recoverable at 24h. T-L activity is significantly inhibited by NPI-0052 at 90 mins, 24h, 48h, and 72h; and is recoverable by 168h; in contrast, Bortezomib enhances T-L activity. Finally, NPI-0052 inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and this activity recovered at 168h, whereas Bortezomib significantly inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and is similarly recoverable at 168h. We next utilized a novel methodology to measure proteasome activity by immunoblotting using dansylAhx3L3VS as a probe (Berkers et al., Nature Methods, 2005), which also allow for determining subunit specificity of a proteasome inhibitor. Multiple myeloma (MM) cells were cultured in the presence or absence of various concentrations of either NPI-0052 (2 nM; 7 nM: IC50; or 20 nM) or Bortezomib (2 nM; 5 nM: IC50; or 20 nM). Competition experiments between either NPI-0052 or Bortezomib and dansylAhx3L3VS revealed that NPI-0052 (7 nM) markedly inhibits the CT-L activity represented by beta-5 subunit of the proteasome and decreased the dansylAhx3L3VS-labeling of the beta-1 (C-L activity) and -2 (T-L activity) subunits. Slightly higher concentrations of Bortezomib are necessary to markedly inhibit beta-5 and -1 subunits, whereas beta-2 subunits are not inhibited. Importantly, both agents trigger apoptosis in MM cells; however, NPI-0052 is remarkably less toxic to normal lymphocytes than Bortezomib. Our data show that NPI-0052, like Bortezomib, targets the proteasome, but triggers a proteasome activity profile distinct from Bortezomib. The mechanistic insights gained from these studies will allow for improved drug design based on targeting specific proteasome subunits.

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Parlati, Francesco, Susan J. Lee, Monette Aujay, Erika Suzuki, Konstantin Levitsky, James B. Lorens, David R. Micklem, et al. "Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like activity of the proteasome." Blood 114, no. 16 (October 15, 2009): 3439–47. http://dx.doi.org/10.1182/blood-2009-05-223677.

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Abstract Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits β5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138+ tumor cells. Although specific inhibition of either LMP7 or β5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both β5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2α suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors.

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Prösch, Susanna, Christina Priemer, Conny Höflich, Christa Liebenthal, Nina Babel, Detlev H. Krüger, and Hans-Dieter Volk. "Proteasome Inhibitors: A Novel Tool to Suppress Human Cytomegalovirus Replication and Virus-Induced Immune Modulation." Antiviral Therapy 8, no. 6 (August 1, 2002): 555–67. http://dx.doi.org/10.1177/135965350300800608.

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Recently, we like others, demonstrated that systemic inflammation is the most important mechanism involved in (re)activation of human cytomegalovirus (HCMV) in both immunocompromised and immunocompetent patients. By in vitro studies the eukaryotic transcription factor NF-κB could be identified as the key mediator of TNF-α- and IE1-dependent stimulation of the HCMV IE1/2 enhancer/promoter activity, which is crucial for initiation of viral gene expression during reactivation from latency as well as productive infection. The enzymatic proteasome complex plays a central role in regulating intracellular processes, including the activation of NF-κB. As present antiviral strategies target mainly late events in HCMV replication (DNA replication, virus assembly) that do not completely prevent virus mediated immunopathogenesis, we wondered whether proteasome inhibitors might be a novel tool for targeting the interaction between inflammation and HCMV (re)activation. Here, proteasome inhibitors like MG132, PSI, II and III (MG262) have been shown to block both TNF-α-associated up-regulation of the HCMV IE1/2 enhancer/promoter in monocytic cells in an in vitro transient transfection system and HCMV replication in permissive human embryonal lung fibroblasts. Importantly, ganciclovir-resistant HCMV strains are sensitive to proteasome inhibitors. The effect of proteasome inhibitors on HCMV replication was found to be specific as replication of other herpes viruses, like HSV-1 and HSV-2, under identical experimental conditions was not influenced. Inhibition of HCMV replication correlated with a delayed and significantly reduced expression of IE proteins, particularly of the IE2 protein, suggesting that MG132 blocks HCMV replication at an immediate early stage of infection. Early and late protein synthesis as shown exemplary for the pp52 (DNA-binding protein) and p68 (structural protein) protein production and viral DNA synthesis were also inhibited. Suppression of HCMV replication could be correlated with an increased cytosolic accumulation of IκB as well as a reduced NF-κB binding activity in nuclear extracts of MG132-treated cells, which mainly regards NF-κB p50. MG132 also reduced the immune modulatory activity of the virus by abrogating virus-induced up-regulation of cellular ICAM-1. These data suggest that short-term therapy with proteasome inhibitors might be an alternative strategy to prevent (re)activation, replication and immune modulatory activity of HCMV in patients with systemic inflammation.

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ROBERTSON, John D., Kaushik DATTA, Shyam S. BISWAL, and James P. KEHRER. "Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis." Biochemical Journal 344, no. 2 (November 24, 1999): 477–85. http://dx.doi.org/10.1042/bj3440477.

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Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-xL-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be ≈ 30% lower in bcl-xL cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome β-subunit, as determined by Western blot analyses, were similar in control and bcl-xL cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-xL cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-7-amino-4-methylcoumarin (Ac-DEVD-AMC), was twice that observed in bcl-xL cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-xLcells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-xL cells by 12 h, although the maximal increases found in bcl-xL cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-xL provide some protection against proteasome inhibitor-induced apoptosis.

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Huang, Xiangao, Kathryn Bailey, Maurizio Di Liberto, Francesco Parlati, Ruben Niesvizky, Susan Demo, and Selina Chen-Kiang. "Induction of Sustained Early G1 Arrest by Selective Inhibition of CDK4 and CDK6 Primes Myeloma Cells for Synergistic Killing by Proteasome Inhibitors Carfilzomib and PR-047." Blood 112, no. 11 (November 16, 2008): 3670. http://dx.doi.org/10.1182/blood.v112.11.3670.3670.

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Abstract Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Dysregulation of the cyclin-dependent kinases CDK4 and CDK6 precedes uncontrolled proliferation of myeloma cells in vivo, in particular during relapse and drug resistance. This finding reinforces the critical importance of targeting CDK4/6 in myeloma, but success with broad-spectrum CDK inhibitors has been modest. Using the only known selective inhibitor of CDK4/6, PD 0332991, we have developed a novel approach to prime chemoresistant myeloma cells for synergistic killing by diverse cytotoxic agents. We show that selective inhibition of CDK4/6 by PD 0332991 leads to sustained cell cycle arrest in early G1 in the absence of apoptosis. However, it markedly augments cytotoxic killing by PR-171 (carfilzomib), a selective inhibitor of the chymotrypsin-like activity of the proteasome, or PR-047, an orally bioavailable analog of carfilzomib. Synergistic killing of myeloma cells arrested in early G1 by carfilzomib (or PR-047) is caspase-dependent, and requires only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 60 nM. This effect is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9 within 6 hours of removal of carfilzomib or PR-047. As PD 0332991 acts as an ATP-competitive inhibitor of the CDK4/6 kinase domain, inhibition of CDK4/6 and the cell cycle by PD 0332991 is reversible. Importantly, targeting CDK4/6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/6 in combination with carfilzomib (or PR-047), therefore, not only halts cell proliferation but also potently induces synergistic killing that is likely to eliminate cell cycle reentry and generation of resistant cells. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as shown by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase I trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfizomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies.

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Trougakos, Ioannis P., Eleni-Dimitra Papanagnou, Tina Bagratuni, Efstathios Kastritis, Issidora S. Papassideri, Evangelos Terpos, and Meletios A. Dimopoulos. "Translating Findings of Proteasome Inhibitors Effects from the in VivoDrosophila Experimental Model to Humans: The Paradigm of the Molecular-Cellular Responses to Bortezomib and Carfilzomib." Blood 124, no. 21 (December 6, 2014): 4814. http://dx.doi.org/10.1182/blood.v124.21.4814.4814.

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Abstract Cellular proteostasis (homeostasis of the proteome) is ensured by the efficient maintenance of proteome quality and functionality. Central to the proteostasis process is the proteasome, which is involved in the degradation of both normal short-lived ubiquitinated proteins and mutated or damaged proteins. Recent findings indicate that the over-activation of the proteostasis ensuring mechanisms represents a hallmark of advanced tumors, and thus their inhibition provides a strategy for the development of novel anti-tumor therapies. In line with this concept, proteasome inhibitors (i.e. bortezomib and carfilzomib) have demonstrated clinical efficacy in the treatment of multiple myeloma (MM) and mantle cell lymphoma and are under evaluation for the treatment of other malignancies. By using the fruit fly Drosophila melanogaster as an in vivo platform for screening the effects of proteasome inhibitors at the whole organism level we recently reported that proteasome functionality in flies’ somatic tissues is sex- and tissue- dependent and that it declines with aging. Administration of bortezomib to young flies caused dose-dependent decrease of proteasome activities in the somatic tissues; induction of the proteasome genes expression, disruption of proteostasis, reduced motor function (a phenotype that recapitulates peripheral neuropathy of bortezomib treatment in the clinic) and a marked reduction of flies’ lifespan. Our in vivo data also showed that carfilzomib was less toxic compared to bortezomib, including neuromusculatory toxicity and effects on flies’ longevity. To address the question whether these findings can be translated to humans we started characterizing proteasome regulation and functionality in both healthy donors, as well as in MM patients treated with either bortezomib or carfilzomib. Initially, we screened isolated red blood cells (RBCs) and peripheral blood mononucleated cells (PBMCs) from male and female healthy donors of different ages; these two cell types represent either an anucleate relatively “long-lived” proteome (RBCs) or cell lineages with the capacity to mobilize genome responses after proteasome inhibition (PBMCs). Our analyses revealed significant variability of basal proteasome peptidase activities among different donors in both RBCs and PBMCs. PBMCs expressed (independently of sex) higher basal proteasome activities as compared to RBCs. Moreover, RBCs isolated from female donors had elevated (as compared to males’ RBCs) basal chymotrypsin-like activity; whereas, males’ PBMCs exhibited higher trypsin-like and caspase-like enzymatic activities as compared to PBMCs from females of similar age. In line with our observations in flies’ somatic tissues, we also found that the proteasome peptidase activities decrease significantly during aging (in a sex-independent manner) in both RBCs and PBMCs. Our studies in isolated RBCs and PBMCs from MM patients treated with either bortezomib or carfilzomib revealed drug-, donor- and cell type-specific readouts. Specifically, in most cases proteasome activities were suppressed in both RBCs and PBMCs after drug administration. Also, we noted that RBCs were particularly sensitive to both inhibitors and their proteasome activities remained low during the entire course of treatment. On the other hand, PBMCs were characterized by phases of relapsed proteasome activities during the periods of no drug administration. Finally, as in the case of the in vivo Drosophila experimental model, proteasome dysfunction in PBMCs triggered in most patients a significant upregulation of the proteasome 20S and 19S genes expression. Moreover, we noted an induction of genes involved in cellular antioxidant responses; this finding is in line with our observations in flies showing that administration of proteasome inhibitors results in increased cellular oxidative stress that mobilizes genomic antioxidant responses. Data on the clinical outcomes of the treated patients in correlation with the recorded molecular responses will be presented at the meeting. Overall, our findings indicate that the molecular-cellular responses to proteasome inhibitors observed at the in vivo Drosophila Experimental model are largely translatable to humans. Moreover, we anticipate that our employed methodologies will set the basis towards a more personalized clinical therapeutic approach for multiple myeloma patients. Disclosures No relevant conflicts of interest to declare.

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Chang, Tsui-Ling, and Chi-Hsien Wang. "Combination of quercetin and tannic acid in inhibiting 26S proteasome affects S5a and 20S expression, and accumulation of ubiquitin resulted in apoptosis in cancer chemoprevention." Biological Chemistry 394, no. 4 (April 1, 2013): 561–75. http://dx.doi.org/10.1515/hsz-2012-0277.

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Abstract To look for oral proteasome inhibitors, daily injested food is the best source for cancer chemoprevention. A combination of active components from vegetables, coffee, tea, and fruit could be more efficient to inhibit 26S proteasome activities for preventing cancer diseases. Tannic acid and quercetin have been shown to strongly inhibit 26S proteasome activity, but the molecular target involved remains unknown. Overlay assay, peptide assay, Western blot, and 2-D gels were used to assess the combination of quercetin and tannic acid as a potential inhibitor. Here, we demonstrated that the combination of quercetin and tannic acid (1) synergistically suppresses chymotrypsin-, caspase-, and trypsin-like proteolytic activities, (2) are tightly binding substrates, (3) do not perturb the proteasome structure, (4) inhibit the 26S proteasome affected by ubiquitin, ATP, or β-casein, and (5) inhibit β-casein degradation by the 26S proteasome in vitro. Finally, the inhibition of the proteasome by a combination of quercetin plus tannic acid in Hep-2 cells resulted in the induction of S5a at low dose, accumulation of ubiquitin, and the cleavage of pro-caspase-3, followed by the induction of apoptotic cell death. Evaluating the combination of quercetin and tannic acid as an oral drug to prevent cancer may provide a pharmacological rationale to pursue preclinical trials of this combination.

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Kraus, Marianne, Jürgen Bader, Thomas Mehrling, and Christoph Driessen. "Edo- S101, a New Alkylating Histone-Deacetylase Inhibitor (HDACi) Fusion Molecule, Has Superior Activity Against Myeloma and B Cell Lymphoma and Strong Synergy with Proteasome Inhibitors in Vitro." Blood 124, no. 21 (December 6, 2014): 2249. http://dx.doi.org/10.1182/blood.v124.21.2249.2249.

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Abstract Background: EDO-S101 is a first-in-class alkylating histone-deacetylase inhibitor (HDACi) fusion molecule that combines the strong DNA damaging effect of bendamustine, with a fully functional pan-HDAC inhibitor, vorinostat. Bendamustine has substantial clinical activity against B-cell malignancies, lacks cross resistance with many other anticancer drugs, has superior antimyeloma activity compared to melphalan, and can safely be combined with proteasome inhibitors. The Histone deacetylase inhibitor (HDACi) vorinostat has a broad spectrum of epigenetic activities and sensitizes lymphoma and myeloma cells for a variety of cytotoxic drugs. Vorinostat in particular has a strong synergy with proteasome inhibitors, presumably due to the inhibition of HDAC6. HDAC6 allows polyubiquitinated substrate protein to bypass the inhibited proteasome towards degradation via the autophagy pathway. The EDO-S101 molecule was designed to create a very potent cytotoxic agent for systemic use upon exploiting the synergies of a bi-functional mode of action. Methods: The aim of the in vitro study was to compare the cytotoxicity of EDO-S101 against Multiple Myeloma (MM), leukemia and lymphoma cells with established alkylating agents and to investigate its cellular and molecular effects in combination with proteasome inhibitors. Results: The IC50 of EDO-S101 ranged between 5-13 μM in 8 myeloma cell lines and thus one order of magnitude lower than the IC50 for bendamustine (70 - > 200 μM). Myeloma cell lines with adaptive resistance against bortezomib or carfilzomib did not differ from non-adapted cells in their IC50 for EDO-S101. Likewise, the IC50 for 3 ABC type DLBCL cell lines ranged between 3-8 μM for EDO-S101, compared to bendamustine > 50 μM. EDO-S101 had significant synergistic cytotoxicity with the proteasome inhibitors bortezomib and carfilzomib across all cell types tested, in contrast to melphalan and bendamustine. In a panel of 6 MM cell lines, the combination of EDO-S101 yielded a mean combination index for synergistic cytotoxicity of 0.12 (± 0.06) and 0.08 (± 0.06) for bortezomib or carfilzomib combinations, respectively (with values < 0.8 indicating significant synergism), in contrast to bendamustine 1.35 (± 0.87) and 1.29 (± 0.86), and melphalan 1.09 (± 0.66) and 1.20 (± 1.44). Likewise, EDO-S101 showed synergistic cytotoxicity with bortezomib and carfilzomib against mantle cell lymphoma cells (mean CIs 0.6 and 0.2), in contrast to bendamustine (CIs 1.72, 1.22) and melphalan (CIs 1.16 and 1.17), as well as ABC type DLBCL (CIs 0.32 and 0.28 for EDO S-101, compared to 15 and 34 for bendamustine and 0.87 and 0.78 for melphalan). To dissect the molecular mechanism for the unique synergistic cytotoxicity of EDO-S101 with proteasome inhibition, which contrasted to the established alkylating drugs, we analysed proteasome activity, protein acetylation status, accumulation of polyubiquitinated proteins as well as regulatory and effector proteins of the unfolded protein response (UPR) in RPMI8226 myeloma cells by western blot. EDO-S101 induced strong protein and histone acetylation, confirming its HDACi-like activity. Interestingly, and in contrast to bendamustine, melphalan and vorinostat, EDO-S101 was a strong inducer of pIRE-1, the key activator protein of the UPR in MM cells. IRE1 activation and induction of the UPR have recently been shown to be the major determinants of proteasome inhibitor sensitivity in human MM. Conclusions: We conclude that EDO-S101, an alkylating HDAC inhibitor fusion molecule, displays bi-functional activity. Compared to bendamustine and melphalan, it has superior monoactivity in vitro against hematologic malignancies including MM, mantle cell lymphoma and ABC type DLBCL. Of particular interest is the strong synergy of EDO-S101 with proteasome inhibitors which also stands out in comparison to the established alkylating agents. The latter is associated with induction of pIRE1, the key regulator of the UPR by EDO- S101. Both, the superior monoactivity of EDO-S101 and its mechanism-based synergy with proteasome inhibitors warrant further development of the compound towards clinical testing. Disclosures Driessen: Mundipharma: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Chauhan, Dharminder, Ajita Singh, Mohan Brahmandam, Klaus Podar, Teru Hideshima, Robert Schlossman, Paul Richardson, Nikhil Munshi, Michael Palladino, and Kenneth C. Anderson. "Combination of Proteasome Inhibitors Bortezomib and NPI-0052 Trigger In Vivo Synergistic Cytotoxicity in Multiple Myeloma." Blood 110, no. 11 (November 16, 2007): 2524. http://dx.doi.org/10.1182/blood.v110.11.2524.2524.

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Abstract Bortezomib (Velcade™), the first in class proteasome inhibitor is FDA approved drug for the treatment of relapsed and relapsed/refractory multiple myeloma (MM). However, as with other agents, dose-limiting toxicities and the development of resistance limits its long-term utility. Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in MM cells; and importantly, is distinct from bortezomib in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 + bortezomib-induced synergistic apoptosis is associated with: activation of caspase-8, caspase-9, caspase-3, and PARP; induction of ER-stress response and JNK; inhibition of migration of MM cells and angiogenesis; suppression of chymotrypsin-like (CT-L), caspase-like (C-L) and trypsin-like (T-L) proteolytic activities; and blockade of NF-kappa B signaling. Studies in a xenograft MM model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth. Importantly, analysis of resected xenografted tumors show that 30–40% proteasome inhibition of all three (CT-L, C-L and T-L) proteasomal activities is sufficient to trigger significant MM cell apoptosis, confirming both the sensitivity of MM cells to proteasome inhibition and the importance of inhibiting all three proteolytic activities to obtain maximum response. Immunohistochemical analysis of MM tumors excised from NPI-0052 + bortezomib-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. The clinical observation that bortezomib therapy can be associated with toxicity and drug-resistance, coupled with our present preclinical findings demonstrating that low doses of bortezomib together with NPI-0052 trigger a potent anti-MM effect in vitro and in vivo, suggests the promise of combination treatment strategies to enhance anti-MM activity, reduce toxicity, overcome drug resistance, and improve outcome in MM patients. In addition to the above studies, data related to combination of NPI-0052 with lenalidomide (Revlimid™) and with histone deacetylase inhibitors such as MS-275, Tubacin or LBH589, will be presented.

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40

Yerlikaya, Azmi, Scot R. Kimball та Bruce A. Stanley. "Phosphorylation of eIF2α in response to 26S proteasome inhibition is mediated by the haem-regulated inhibitor (HRI) kinase". Biochemical Journal 412, № 3 (28 травня 2008): 579–88. http://dx.doi.org/10.1042/bj20080324.

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The present study demonstrates that even brief inhibition of degradation by the 26S proteasome inhibits global protein synthesis, mediated through increased phosphorylation of eIF2α (eukaryotic translational initiation factor 2α) by the HRI (haem-regulated inhibitor) kinase. Exposure of COS-7 cells to the proteasome inhibitor MG-132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal) for 4 h resulted in a 55–60% decrease in protein synthesis rate compared with control cells. This repression of protein synthesis after treatment with MG-132 is not due to induction of apoptosis, which is known to occur after longer periods of 26S inhibition. Instead, we observed a significantly increased phosphorylation of eIF2α, which is known to repress global protein synthesis. In three MEF (mouse embryonic fibroblast) knockout cell lines lacking one of the four kinases known to phosphorylate eIF2α, increased phosphorylation of eIF2α still occurred after inhibition of the 26S proteasome. These three cell lines included a deletion of the PKR (double-stranded-RNA-dependent protein kinase); a deletion of the PERK (PKR-like endoplasmic reticulum resident kinase); or a deletion of the GCN2 (positive general control of transcription-2) kinase, indicating that none of these kinases was primarily responsible for the observed phosphorylation of eIF2α. In contrast, in a fourth MEF knockout cell line, HRI−/− cells lacking the HRI kinase failed to increase eIF2α phosphorylation upon proteasome inhibitor treatment (MG-132 or various doses of Bortezomib), indicating that the HRI kinase is the primary kinase activated by brief treatment of MEFs with 26S proteasome inhibitors.

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Spencer, Andrew, Michael Millward, Paul Mainwaring, Simon Harrison, Laurence Catley, Amanda Townsend, Shawgi Sukumaran, et al. "Phase 1 Clinical Trial of the Novel Structure Proteasome Inhibitor NPI-0052." Blood 114, no. 22 (November 20, 2009): 2693. http://dx.doi.org/10.1182/blood.v114.22.2693.2693.

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Abstract Abstract 2693 Poster Board II-669 Background: NPI-0052 is a proteasome inhibitor with a novel bicyclic structure (other proteasome inhibitors in clinical use are peptide based). Preclinical studies indicate rapid, broad and prolonged inhibition of all 3 catalytic sites of the proteasome, and subsequently unique proteasome inhibition, signal transduction, toxicology and efficacy profiles. Taken together these suggest the potential for improvements in therapeutic ratio and activity in hematologic and solid tumor malignancies. Materials and Methods: Patients with solid tumor, lymphoma, leukemia or myeloma diagnoses without standard treatment options have been treated with IV NPI-0052 on one of two arms (weekly or twice weekly) in this 3+3 design dose escalation study. This is followed by 10 patient Recommended Phase 2 dose Cohorts of patients with lymphomas, CLL and myeloma respectively. Proteasome inhibition (pharmacodynamics) and pharmacokinetics are also assayed in whole blood, and proteasome inhibition in peripheral blood mononuclear cells (PBMC). Results: 44 patients have been treated with NPI-0052 at doses ranging from 0.075 mg/m2 to 0.9 mg/m2. Common adverse events include fatigue, parosmia/dysgeusia, transient peri-infusion site pain, lymphopenia, headaches, dizziness / unsteady gait, closed-eye visuals, cognitive changes. Incidence and grade of these events correlate with dose, being quite tolerable at the MTD of 0.7 mg/m2 on the weekly dosing arm. An MTD has not yet been determined for the twice weekly dosing arm. Pharmacokinetic data has demonstrated a rapid elimination half-life (<20 minutes) and relatively large volume of distribution. Assessment of proteasome inhibition has demonstrated increasing inhibition of chymotrypsin-like activity of up to 88% Day 1 and 100% Day 15. Inhibition of caspase-like and trypsin-like activity of up to 52% and 71% respectively has also been seen. Inhibition remains between doses in whole blood (principally RBC), but recovers between doses in PBMC. Clinical benefit, including stable disease, regression or response, was reported in patients with mantle cell lymphoma, myeloma, Hodgkin's lymphoma, cutaneous marginal zone lymphoma, follicular lymphoma, sarcoma, prostate carcinoma and melanoma. Conclusions: NPI-0052 produces dose-dependent pharmacologic effects through the predicted efficacious range, while producing a toxicity profile that is dissimilar to what is reported with other proteasome inhibitors (notably deficient in peripheral neuropathy, neutropenia and thrombocytopenia) in spite of producing equal or greater proteasome inhibition. These data indicate a broad range of potential uses, and led to additional studies in hematologic malignancies and solid tumors alone and in combination. Disclosures: Longenecker: Nereus Pharmaceuticals: Employment. Palladino:Nereus Pharmaceuticals: Employment, Equity Ownership. Lloyd:Nereus Pharmaceuticals: Employment, Equity Ownership. Neuteboom:Nereus Pharmaceuticals: Employment, Equity Ownership. Spear:Nereus Pharmaceuticals: Employment, Equity Ownership.

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Arastu-Kapur, Shirin, Kevin Shenk, Francesco Parlati, and Mark K. Bennett. "Non-Proteasomal Targets of Proteasome Inhibitors Bortezomib and Carfilzomib." Blood 112, no. 11 (November 16, 2008): 2657. http://dx.doi.org/10.1182/blood.v112.11.2657.2657.

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Abstract The proteasome is a multicatalytic protease complex that has been validated as a therapeutic target in oncology with the approval of bortezomib for the treatment of multiple myeloma and mantle cell lymphoma. Carfilzomib is a next generation proteasome inhibitor that is structurally and mechanistically distinct from bortezomib and has entered clinical development in oncology. Both inhibitors target the chymotrypsin-like activity of the proteasome, but their mechanism of action differs due to their unique chemical pharmacores (or “warheads”): bortezomib is a boronate while carfilzomib is an epoxyketone. Phase 1 studies with carfilzomib suggest a clinical safety profile that has both commonalities and distinctions from bortezomib. Transient thrombocytopenia is observed with both molecules suggesting that this event is a proteasome inhibitor class effect. In contrast, the painful peripheral neuropathy that is commonly observed with bortezomib appears to be less severe and possibly less frequent with carfilzomib, raising the possibility that non-proteasome mechanisms may underlie this toxicity. To gain potential insight into the common and unique clinical toxicities of bortezomib and carfilzomib, we evaluated their propensity to act as inhibitors of non-proteasomal enzymes. Bortezomib and carfilzomib were initially screened in a panel of candidate cysteine, aspartyl, metallo-, and serine proteases. Bortezomib significantly inhibited the serine proteases cathepsin G (IC50= 0.3μM) and chymase (IC50= 1.1μM), while carfilzomib did not inhibit these enzymes (IC50>10μM). These effects were further validated in cell extracts prepared from the Thp1 monocyte cell line and peripheral blood mononuclear cells (PBMC), where the inhibition of cathepsin G and chymase by bortezomib was detected using FP-biotin, a serine hydrolase-specific fluorophosphonate activity-based probe. These results suggest that the boronic acid warhead of bortezomib has greater off-target activity than the epoxyketone warhead of carfilzomib. To further investigate this hypothesis, off-target binding of proteasome inhibitors with either the boronate (Cbz-LLL-boronate) or epoxyketone (Cbz-LLL-epoxyketone) warheads were tested in cell extracts and intact cells using FP-biotin detection. Several serine hydrolases in liver (HepG2), lung (A549), kidney (786-O), and leukemia (Thp1) tumor cell lines were found to bind the boronate inhibitor but not the epoxyketone inhibitor. This result suggests that the boronate warhead is more “promiscuous” than the expoxyketone warhead. To obtain a global inhibition profile of the serine hydrolases that are targeted by bortezomib, multidimensional protein identification technology (MudPIT) analysis was performed on FP-biotin-reactive proteins from HepG2 cell extracts. Bortezomib was found to bind several serine hydrolases, including cathepsin A and dipeptidyl peptidase IV (DPP IV) and we have further validated these targets using specific antibodies. Taken together, these data demonstrate that non-proteasomal enzymes can be targeted by bortezomib (cathepsin G, cathepsin A, DPP IV and chymase), whereas carfilzomib does not appear to inhibit non-proteasomal targets in our assays. Work in progress includes biochemical and cell-based characterization for non-proteasomal targets, further global proteomic inhibition profiling using MudPIT analyses, and assessing the potential contribution of non-proteasomal targets to the differential toxicity profiles of the two proteasome inhibitor classes.

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Miller, Claudia P., Kechen Ban, Melanie E. Dujka, David J. McConkey, Mark Munsell, Michael Palladino, and Joya Chandra. "NPI-0052, a novel proteasome inhibitor, induces caspase-8 and ROS-dependent apoptosis alone and in combination with HDAC inhibitors in leukemia cells." Blood 110, no. 1 (July 1, 2007): 267–77. http://dx.doi.org/10.1182/blood-2006-03-013128.

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The proteasome has been successfully targeted for the treatment of multiple myeloma and mantle cell lymphoma; however, in other hematologic malignancies, bortezomib has been less effective as a single agent. Here, we describe effects of NPI-0052, a novel proteasome inhibitor, in leukemia model systems. In cell lines, NPI-0052 inhibits all 3 proteolytic activities associated with the proteasome: chymotrypsin-, trypsin-, and caspase-like. NPI-0052 also induces DNA fragmentation in leukemia lines and in mononuclear cells from a Ph + acute lymphoblastic leukemia (ALL) patient. Caspase-3 activation by NPI-0052 was seen in wild-type Jurkat cells, but was significantly lessened in Fas-associated death domain (FADD)–deficient or caspase-8–deficient counterparts. NPI-0052–induced apoptosis was further probed using caspase-8 inhibitors, which were more protective than caspase-9 inhibitors. N-acetyl cysteine (NAC) also conferred protection against NPI-0052–induced apoptosis, indicating a role for oxidative stress by NPI-0052. In support of the drug's in vitro activities, biweekly treatment with NPI-0052 lessened total white blood cell (WBC) burden over 35 days in leukemic mice. Interestingly, combining NPI-0052 with either MS-275 or valproic acid (VPA) induced greater levels of cell death than the combination of bortezomib with these histone deacetylase inhibitors (HDACi). These effects of NPI-0052, alone and in combination with HDACi, warrant further testing to determine the compound's clinical efficacy in leukemia.

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Tanaka, Yuko, Seiichi Okabe, Tetsuzo Tauchi, Yoshikazu Ito, and Kazuma Ohyashiki. "Targeting Insulin-like Growth Factor in Multiple Myeloma: Novel Strategies in the Treatment of Proteasome Inhibitor Resistant Cells." Blood 132, Supplement 1 (November 29, 2018): 5155. http://dx.doi.org/10.1182/blood-2018-99-114750.

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Abstract Introduction: Multiple myeloma (MM) is one of the hematological malignancy that affects the plasma cells in bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs. However, most patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Insulin-like growth factor (IGF) cause intracellular signaling that ultimately results in cellular growth and proliferation. Because IGF signaling pathways have crucial functions in hematological malignancies and solid tumors, IGF pathways might regulate myeloma cell survival. Materials and Methods: In this study, we investigated whether IGF1R inhibitor could suppress myeloma cells. We also investigated whether IGF1 was involved in proteasome sensitivity against myeloma cells and cytokines were induced by IGF1 in human umbilical vein endothelial cells (HUVEC). Results: We first investigated the IGF signaling pathways by microarray gene expression data from the online Gene Expression Omnibus (GEO). IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). Gene expression of IGF1, IGF1R, IGFBP2, IGFBP5, IGFBP7 and IGF2 are involved in myeloma and normal control samples from the public microarray datasets of GSE39754. We next investigated the proteasome inhibitor, bortezomib or carfilzomib activity. Proteasome inhibitors exhibit cell growth inhibition against myeloma cells. However, in the presence of IGF1, proteasome inhibitors activity was reduced. Linsitinib is an orally bioavailable small molecule inhibitor of the IGF1R. We investigated whether linsitinib could inhibit the proliferation of myeloma cells. We found linsitinib treatment inhibited the growth of myeloma cells in a dose dependent manner. Activity of Caspase 3/7 and cytotoxicity were also increased. Combined treatment of myeloma cells with carfilzomib and linsitinib caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We examined the intracellular signaling. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased after carfilzomib and linsitinib treatment. We next blocked IGF1R function by small interfering RNA (siRNA). We found drug sensitivity of the cells to the carfilzomib was increased compared to mock-transfected cells. Caspase 3/7 activity and cytotoxicity were increased after carfilzomib treatment in siRNA transfected cells. We next investigated the in vitro effects of IGF1 on cultured HUVEC. Because the entire bone marrow cavity is hypoxic condition, we investigated the IGF signaling pathways in the hypoxic condition by microarray gene expression data from GEO. Gene expression of IGF1R and IGF2 are increased in the hypoxia from the public microarray datasets of GSE36837. Gene amplification was confirmed by RT-PCR analysis. In the immunoblot analysis, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation was increased by IGF1 stimulation in the time dependent manner. We also found that gene expression of monocyte chemotactic protein-1 (MCP-1) was enhanced by IGF1 stimulation. Conclusion: The IGF1 signaling pathway is involved in proteasome inhibitor sensitivity and plays a key role in chemokine production of the HUVEC. Our data also suggested that administration of IGF1R inhibitor, linsitinib, might be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in those residual MM cells. Disclosures Ohyashiki: MSD,: Honoraria, Research Funding; Bristol Meyer Squibb KK,: Honoraria, Research Funding; Kyowakko Kirin KK,: Research Funding; Celegene KK,: Honoraria, Research Funding; Pfizer KK,: Honoraria, Research Funding; Novartis KK,: Honoraria, Research Funding; Taiho Pharmaceutical KK: Honoraria, Research Funding; Takeda Pharmaceutical KK,: Honoraria, Research Funding; Jansen Pharma KK,: Research Funding; Chugai KK,: Honoraria, Research Funding; Nihon-Seiyaku,: Research Funding; Ono Pharmaceutical KK,: Honoraria, Research Funding; Eizai,: Research Funding; Nippon-shinyaku,: Honoraria, Research Funding; Asahikase: Research Funding; Asteras KK,: Research Funding; Dainippon Sumitomo KK,: Honoraria, Research Funding.

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Tong, Jian-Bo, Yuan-Yuan Li, Guo-Yan Jiang, and Kang-Nan Li. "Application of an R-group search technique in the molecular design of dipeptidyl boronic acid proteasome inhibitors." Journal of the Serbian Chemical Society 82, no. 9 (2017): 1025–37. http://dx.doi.org/10.2298/jsc161227047t.

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In this work, a 3D-QSAR model involving for 40 dipeptidyl boronic acid proteasome inhibitors was built based on Topomer CoMFA. The multiple correlation coefficient of fitting, cross-validation and external validation were 0.908, 0.647 and 0.703, respectively. The results indicated that the obtained Topomer CoMFA model has not only the favourable estimation stability but also the good prediction capability. Topomer Search was employed as a tool for virtual screening in lead-like compounds of ZINC database. Finally, 1 R1 group, 7 R2 groups and 6 R3 groups with higher contribution values were employed to alternately substitute the R1, R2 and R3 of the templete compound 23 with highest bioactivity. As a consequence, 33 new molecules with higher activity than that of the model molecule were designed successfully. The results showed that the Topomer Search technology could be effectively apply to screen and design new dipeptidyl boronic acid proteasome inhibitors and has good predictive capability to design new dipeptidyl boronic acid proteasome inhibitors drugs as guidance.

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Ros, Carlos, Christoph J. Burckhardt, and Christoph Kempf. "Cytoplasmic Trafficking of Minute Virus of Mice: Low-pH Requirement, Routing to Late Endosomes, and Proteasome Interaction." Journal of Virology 76, no. 24 (December 15, 2002): 12634–45. http://dx.doi.org/10.1128/jvi.76.24.12634-12645.2002.

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ABSTRACT The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A 1 and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nα-p-tosyl-l-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation.

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Ma, Wanlong, Francis Giles, Susan O’Brien, Iman Jilani, Xi Zhang, Zeev Estrov, Elihu Estey, Alessandra Ferrajoli, Michael Keating, and Maher Albitar. "Variations in Proteasome Enzymatic Activities in Plasma of Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome and Their Value in Predicting Clinical Behavior." Blood 108, no. 11 (November 16, 2006): 4497. http://dx.doi.org/10.1182/blood.v108.11.4497.4497.

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Abstract The ubiquitin-proteasome pathway is responsible for multiple pathways in cancer cells; proteasome inhibition causes rapid apoptosis of tumor cells. Three different types of peptidase activities have been reported for proteasomes: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L) (postglutamyl peptide hydrolytic-like). Various proteasome inhibitors affect each of the 3 activities differently and at different concentrations. For example, NPI-0052 inhibits Ch-L and Tr-L activities at lower concentrations than does bortezomib, while bortezomib inhibits Cas-L at lower concentrations than does NPI-0052. These enzymatic activities are usually measured in normal or tumor cells to monitor therapy with proteasome inhibitors. Because rapidly proliferating leukemic cells pour their proteins, DNA, and RNA into the circulation, we developed fluorogenic kinetic assays using peripheral blood plasma. The assays used peptide-AMC (7-amino 4-methylcoumoran) substrates to measure Ch-L, Tr-L, and Cas-L activities. We measured proteasome activities in plasma from 188 patients with acute myeloid leukemia (AML) and 58 patients with myelodysplastic syndrome (MDS) and assessed their correlations with clinical behavior. Significantly (P < 0.001) higher Ch-L, Tr-L, and Cas-L activities were seen in AML patients (medians: 1.39, 1.51, and 2.40 pmol AMC/sec/mL, respectively) and MDS patients (medians: 1.16, 1.40, and 1.67 pmol AMC/sec/mL, respectively) than in healthy volunteers (n=42) (medians: 0.80, 0.74, and 0.81 pmol AMC/sec/mL, respectively). The difference in Cas-L activity between AML and MDS was significant (P <0.001). While there was no significant difference between Ch-L and Cas-L activities in healthy controls, there was a significant difference between the 2 activities in both AML and MDS. Cas-L and Ch-L, but not Tr-L, correlated with WBC count and lactic dehydrogenase in AML and MDS patients. In AML patients, higher levels of Ch-L and Cas-L were associated with poor response to a variety of therapies (P = 0.004 and P = 0.001, respectively). Cas-L correlated strongly with survival in AML patients when used as an activity-dependent variable (P <0.001) or when the median was used as a cut-off (P = 0.004). This was independent of cytogenetic abnormalities, age, and performance status. Patients with intermediate-risk cytogenetic abnormalities and Cas-L activity >3 pmol AMC/sec/mL had significantly shorter survival (P = 0.04). Ch-L activity was also predictive of survival in AML independent of age and cytogenetic and performance status, but not independent of Cas-L. In MDS, higher levels of Cas-L, but not Ch-L, correlated with shorter survival and this was independent of cytogenetic abnormalities. The increased cell-free circulating proteasome activities most likely reflect the leukemic cells and may be a marker not only for disease, but also potentially for monitoring therapy. These data also suggest that patients with AML may benefit differentially from proteasome inhibitors depending on the specific therapeutic effect of the inhibitor.

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Masdehors, Peggy, Hélène Merle-Béral, Karim Maloum, Satoshi Ömura, Henri Magdelénat, and Jozo Delic. "Deregulation of the ubiquitin system and p53 proteolysis modify the apoptotic response in B-CLL lymphocytes." Blood 96, no. 1 (July 1, 2000): 269–74. http://dx.doi.org/10.1182/blood.v96.1.269.

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Abstract We recently reported increased sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Here, we show that only specific—not nonspecific—proteasomal inhibitors can discriminate between malignant and normal lymphocytes in inducing the apoptotic death response. Indeed, lactacystin and its active metaboliteclasto-lactacystin β-lactone induced apoptotic death in CLL but not in normal lymphocytes. This difference was completely abolished when tripeptide aldehydes such as MG132 or LLnL (which can also inhibit calpains) were used as less specific proteasomal inhibitors. Moreover, B-CLL cells exhibited a constitutive altered ubiquitin-proteasome system, including a threefold higher chymotrypsin-like proteasomal activity and high levels of nuclear ubiquitin-conjugated proteins compared with normal lymphocytes. Interestingly, B-CLL cells also displayed altered proteolytic regulation of wild-type p53, an apoptotic factor reported to be a substrate for the ubiquitin-proteasome system. Nuclear wild-type p53 accumulated after lactacystin treatment used at the discriminating concentration in malignant, but not in normal, lymphocytes. In contrast, p53 was stabilized by MG132 or LLnL in malignant and normal cells undergoing apoptosis, indicating that in normal lymphocytes p53 is regulated mainly by calpains and not by the ubiquitin-proteasome system. This work raises the possibility that two different proteolytic pathways controlling p53 stability may be pathologically imbalanced. This could result in modification of apoptosis control, since in CLL-lymphocytes a highly upregulated ubiquitin-proteasome system, which controls p53 stability among other apoptotic factors, was correlated with an increased propensity of these cells to apoptosis triggered by lactacystin.

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Masdehors, Peggy, Hélène Merle-Béral, Karim Maloum, Satoshi Ömura, Henri Magdelénat, and Jozo Delic. "Deregulation of the ubiquitin system and p53 proteolysis modify the apoptotic response in B-CLL lymphocytes." Blood 96, no. 1 (July 1, 2000): 269–74. http://dx.doi.org/10.1182/blood.v96.1.269.013k10_269_274.

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We recently reported increased sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Here, we show that only specific—not nonspecific—proteasomal inhibitors can discriminate between malignant and normal lymphocytes in inducing the apoptotic death response. Indeed, lactacystin and its active metaboliteclasto-lactacystin β-lactone induced apoptotic death in CLL but not in normal lymphocytes. This difference was completely abolished when tripeptide aldehydes such as MG132 or LLnL (which can also inhibit calpains) were used as less specific proteasomal inhibitors. Moreover, B-CLL cells exhibited a constitutive altered ubiquitin-proteasome system, including a threefold higher chymotrypsin-like proteasomal activity and high levels of nuclear ubiquitin-conjugated proteins compared with normal lymphocytes. Interestingly, B-CLL cells also displayed altered proteolytic regulation of wild-type p53, an apoptotic factor reported to be a substrate for the ubiquitin-proteasome system. Nuclear wild-type p53 accumulated after lactacystin treatment used at the discriminating concentration in malignant, but not in normal, lymphocytes. In contrast, p53 was stabilized by MG132 or LLnL in malignant and normal cells undergoing apoptosis, indicating that in normal lymphocytes p53 is regulated mainly by calpains and not by the ubiquitin-proteasome system. This work raises the possibility that two different proteolytic pathways controlling p53 stability may be pathologically imbalanced. This could result in modification of apoptosis control, since in CLL-lymphocytes a highly upregulated ubiquitin-proteasome system, which controls p53 stability among other apoptotic factors, was correlated with an increased propensity of these cells to apoptosis triggered by lactacystin.

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Suzuki, Erika, Susan Demo, Shirin Arastu-Kapur, Christopher J. Kirk, and Mark K. Bennett. "Bortezomib-Resistant Cell Lines Have Increased Proteasome Levels but Remain Sensitive to Carfilzomib." Blood 114, no. 22 (November 20, 2009): 2852. http://dx.doi.org/10.1182/blood.v114.22.2852.2852.

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Abstract Abstract 2852 Poster Board II-828 Introduction: Carfilzomib (CFZ) is a selective and irreversible proteasome inhibitor that induces anti-tumor activity through inhibition of the proteasome. In vitro, CFZ induces cell death in CD138+ myeloma cells from patients refractory to the dipeptide boronate proteasome inhibitor bortezomib (BTZ) and, in Phase 2 trials, CFZ has demonstrated objective and durable responses in BTZ relapsed and refractory patients. To understand the mechanism underlying the ability of carfilzomib to overcome BTZ resistance, we compared the activity of the two inhibitors in vitro using tumor cells conditioned to be resistant to BTZ. Methods: HT-29, human colorectal adenocarcinoma cells, were cultured in the continuous presence of BTZ by stepwise increases in concentration (20nM to 200nM) over 7 months. Clonal isolates were derived by limiting dilution analysis and cultured in BTZ at 100nM and 200nM for an additional 4 months. Cytotoxic responses were measured as 50% inhibitory concentrations (IC50) following 72 hr compound exposure. Proteasome activity was measured using fluorogenic substrates for chymotrypsin-like, caspase-like and trypsin-like activities and by an active site ELISA (ProCISE) that quantitates the catalytic subunits of the constitutive proteasome (beta5, beta1, beta2) and immunoproteasome (LMP7, LMP2, MECL1). To assess recovery of proteasome activity, cells were exposed to a brief (1hr) “pulse” of either BTZ or CFZ, at concentrations resulting in 80% inhibition, and the chymotrypsin-like proteasome activity was measured at 4 and 24hrs after compound washout. We sequenced the cDNA encoding chymotrypsin-like proteasome subunits, beta5 and LMP7, along with a structural subunit, beta6, to identify potential mutations in the clonal BTZ resistant cell lines. Results: Compared to parental cells, clones grown in 100nM (BR100) or 200nM (BR200) BTZ were 15 – 25-fold less sensitive to BTZ-induced cytotoxicity and remained resistant after long-term culture without BTZ exposure. The cytotoxic response to CFZ was largely unchanged in these cells with <5-fold differences in IC50 values. Basal proteasome catalytic activity was increased by 7-11 fold in both BR100 and BR200 cells. This corresponded to an increase (5-8 fold) in expression of all catalytic subunits except for LMP2. No difference in the initial inhibition of proteasome activity by either compound was seen in resistant cells. However, 4 hr post BTZ exposure, resistant cells showed a more rapid recovery of proteasome activity relative to parental cells. Compared to BTZ, CFZ induced a prolonged proteasome inhibition that was not altered in resistant cells and may therefore relate to the preserved cytotoxic activity of CFZ in these cells. Interestingly, after 24 hrs of recovery from a 1hr exposure to either BTZ or CFZ, resistant cells did not recover their proteasome activity to levels seen parental cells, suggesting alterations in proteasome assembly. Sequence analysis of clonal BR100 and BR200 cell lines identified a mutation in the propeptide of beta5 (Arg24Cys). This mutation, which is in a region required for proper proteasome assembly, has previously been identified at a high frequency in myeloma patients (Wang L., et al Clin Cancer Res 2008;14:3503)). Mutations in beta5 near the interface with the alpha ring (Cys63Phe) and in the propeptide portion of LMP7 (Phe50Ile) were also found in BR200 cells but were absent in BR100 cells. We did not detect mutations in beta6 or a mutation in the active site of beta5 described by other groups. Conclusions: BTZ resistance in vitro is associated with increased proteasome subunit levels and a rapid recovery of proteasome activity at early (4hr) timepoints following BTZ exposure. Our data, which includes the first report of a mutation in LMP7, support a hypothesis that resistance is mediated by a more rapid and efficient proteasome assembly process. Since one of the mutations is prevalent at a high frequency in myeloma patients, this process may be involved in the etiology of disease and/or clinical resistance to BTZ treatment. CFZ overcomes BTZ resistance in these cells, due in part to prolonged inhibition of the proteasome activity, providing a possible mechanism for the activity of CFZ in myeloma patients that are refractory to BTZ therapy. Disclosures: Suzuki: Proteolix, Inc: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Arastu-Kapur:Proteolix, Inc: Employment, Equity Ownership. Kirk:Proteolix, Inc: Employment, Equity Ownership. Bennett:Proteolix: Employment.

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