Comparative Selectivity and Specificity of the Proteasome Inhibitors BzLLLCOCHO, PS-341, and MG-132

Abstract

The 26S proteasome is a multicatalytic protease responsible for regulated intracellular protein degradation. Its function is mediated by three main catalytic activities: (a) chymotrypsinlike (CT-L), (b) trypsin-like, and (c) peptidylglutamyl peptide hydrolysing (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma. Here, we profile the contributions of the three catalytic activities in multiple myeloma cell lines and compare the specificity and cytotoxicity of the novel proteasome inhibitor BzLLLCOCHO and inhibitors PS-341 (Velcade, bortezomib) and MG-132. Using fluorogenic substrates and an active site-directed probe specific for proteasome catalytic subunits, we show differential subunit specificity for each of the inhibitors. Addition of BzLLLCOCHO strongly inhibited all three catalytic activities, treatment with PS-341 completely inhibited CT-L and PGPH activities, and treatment with MG-132 resulted in weak inhibition of the CT-L and PGPH activities. Multiple myeloma cells were more sensitive to induction of apoptosis by PS-341 and MG-132 than BzLLLCO-CHO. This study emphasizes the need for further investigation of the effects of these compounds on gene and protein expression in the cell to allow for the development of more specific and targeted inhibitors. (Cancer Res 2006; 66(12): 6379-86)

A novel proteasome inhibitor NPI-0052 as an anticancer therapy

“…Higher doses of Bortezomib are required to inhibit CT-L and C-L activity, whereas little, if any, inhibition of T-L activity was noted. Importantly, our study showed that MM cells exhibit higher constitutive levels of T-L proteasome activity than either CT-L or C-L activities (Crawford et al, 2006). These data, coupled with the results that NPI-0052, but not Bortezomib, efficiently inhibits CT-L þ T-L activities (Chauhan et al, 2005a), suggest that NPI-0052 may trigger more proteolysis than Bortezomib in MM cells.…”

“…It is likely that blockade of one or more of these proteasomal activities along with the kinetics of inhibition confer therapeutic advantage. Moreover, previous reports showed that different organs/cell types express different ratios of constitutive-, immuno-, and hybrid-proteasomes (Stohwasser et al, 1997;Dahlmann et al, 2000;Crawford et al, 2006). Our ongoing studies using MM patient cells and a panel of MM cell lines are addressing these questions by designing more sensitive and specific proteasome probes against each proteasome subunits.…”

Identification of an HLA‐A24‐restricted cytotoxic T lymphocyte epitope from human papillomavirus type‐16 E6: The combined effects of bortezomib and interferon‐γ on the presentation of a cryptic epitope

“…It has been shown that 3 HLA-A*0201 restricted epitopes (E7 [11][12][13][14][15][16][17][18][19][20] , E7 82-90 and E7 [86][87][88][89][90][91][92][93] ) induce CTL responses in HLA-A*0201 transgenic mice, and the generated CTLs lyse HLA-A*0201 1 HPV-16-positive CaSki cells. 34 As reported by Muderspach et al, 35 increase of E7 epitope-specific reactivity in cytokine release and cytotoxicity assays was demonstrated in 10 of 16 HPV-16-positive HLA-A*0201 1 patients with high-grade cervical or vulvar CIN after vaccination with either E7 [11][12][13][14][15][16][17][18][19][20] or E7 86-93 epitopes, and a proportion of these patients achieved partial clearance of virus infections and regression of lesions. Therefore, we sought to identify HLA-A*2402-restricted CTL epitopes from HPV-16 E6 and E7 oncoproteins by reverse immunology to facilitate immunotherapy for cervical cancer patients.…”

“…39 Although IFN-g treatment upregulates immunoproteasomes and TAP proteins in CaSki cells, forced expression of E6 by a recombinant vaccinia virus encoding HPV-16 E6/E7 fusion protein was required to render them susceptible to CTL lysis. 39 In contrast, E7 has been shown to be more abundantly expressed than E6 in HPV-16-positive and HPV-18 1 cervical cancer cell lines 40,41 may explain why CTLs specific for 3 HLA-A*0201-restricted E7 epitopes (E7 [11][12][13][14][15][16][17][18][19][20] , E7 82-90 and E7 86-93 ) 36 could kill CaSki cells (HLA-A*0201 1 ). 34 Second, IFN-g gave a marginal effect on E6 mRNA expression, but its favorable effect on increased cell surface expression of HLA class I molecules was evident in most cell lines tested, which is critical for efficient recognition by CD8 1 T cells.…”

“…14 Bortezomib is a selective but reversible inhibitor of both standard proteasomes and immunoproteasomes. [15][16][17][18] In the present study, using a reverse immunological approach, we identified an HLA-A*2402-restricted HPV-16 E6-derived nonameric epitope, E6 [49][50][51][52][53][54][55][56][57] . A CD8 1 T cell clone, specific for this epitope, initially failed to recognize untreated HPV-16 1 cervical cancer cell lines; however, recognition was evident on bortezomib treatment, particularly with simultaneous IFN-g treatment in combination.…”