PI-1840, a novel non-covalent proteasome inhibitor with anti-tumor activity Discovery of PI-1840, a novel non-covalent and rapidly reversible proteasome inhibitor with anti- tumor activity

The proteasome inhibitor Bortezomib is effective in hematologic malignancies such as multiple myeloma but has little activity against solid tumors, act covalently and is associated with undesired side effects. Therefore, non-covalent inhibitors that are less toxic and more effective against solid tumors are desirable. Structure activity relationship studies led to the discovery of PI-1840, a potent and selective inhibitor for chymotrypsin-like (CT-L) (IC 50 value = 27 ± 0.14 nM) over Trypsin-Like (T-L) and peptidylglutamyl peptide hydrolyzing (PGPH) (IC 50 values >100 μM) activities of the proteasome. Furthermore, PI-1840 is over 100-fold more selective for the constitutive proteasome over the immunoproteasome. Mass-spectrometry (LC-MS/MS) and dialysis studies demonstrate that PI-1840 is a non-covalent and rapidly reversible CT-L inhibitor. In intact cancer cells, PI-1840 inhibits CT-L activity, induces the accumulation of proteasome substrates p27, Bax and IκB-α, inhibits survival pathways and viability, and induces apoptosis. Furthermore, PI1840 sensitizes human cancer cells to the mdm2/p53 disruptor, nutlin, and to the pan Bcl-2 antagonist BH3-M6. Finally, in vivo, PI-1840 but not Bortezomib suppresses the growth in nude mice of human breast tumor xenografts. These results warrant further evaluation of non-covalent and rapidly reversible proteasome inhibitor as potential anticancer agents against solid tumors. Introduction Dysregulation of the catalytic processes mediated by the ubiquitin/proteasome system (UPS) contributes to the pathogenesis of many diseases, including cancer (1, 2). More than 80 % of cellular proteins are degraded by the UPS (3), including proteins that regulate cell cycle progression, DNA repair and apoptosis (4-6). Deregulation of various components of 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M113.533950 The latest version is at JBC Papers in Press. Published on February 25, 2014 as Manuscript M113.533950 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 4, 2017 hp://w w w .jb.org/ D ow nladed from PI-1840, a novel non-covalent proteasome inhibitor with anti-tumor activity the UPS resulting in increased degradation of cell cycle inhibitors or pro-apoptotic proteins (e.g. p21, p27, p53, Bax) contributes to malignant transformation (3, 7). The UPS has two distinct steps: recognition/ubiquitination and degradation (5, 8). The ubiquitin-protein ligase system results in the transfer of multiple ubiquitin molecules to the target protein (9). Degradation of such multi-ubiqitinated proteins occurs on a large 26S proteaome complex (5, 8) that contains three proteolytic enzymes, peptidylglutamyl peptide hydrolyzing (PGPH), trypsin-like (T-L), and chymotrypsin-like (CT-L) activities, residing in the β1, β2, and β5 catalytic subunits, respectively (3, 7). In contrast to normal cells, cancer cells generally have higher levels of proteasome activity (3), and have acquired a series of mutations that render them dependent on strong activation of survival pathways (10). One of these is the phosphorylation-dependent recognition and subsequent degradation of cellular proteins by the UPS. Furthermore, compare to normal cells, cancer cells show higher sensitivity towards the pro-apoptotic effects of proteasome inhibition. Therefore, the UPS has become a promising target for anti-cancer strategies (3, 7, 11, 12). Although two proteasome inhibitors, Bortezomib and Carfilzomib, are FDA approved and others are in clinical trials, they are all covalent inhibitors (13, 14). Covalent inhibitors have highly reactive and unstable chemical groups, and are therefore less specific (15). This is believed to be a major cause for toxicity to patients. Furthermore, Bortezomib is active against liquid but not solid tumors; and its covalent binding which would limit its widespread tissue distribution could be a possible reason. In contrast to covalent inhibitors, non-covalent inhibitors have the advantage of rapid binding and dissociation kinetics that would allow broader tissue distribution, reaching both liquid and solid tumors. Only very few non-covalent inhibitors have been identified and none have entered clinical trials (16, 17). It is important to point out that at present it is not known whether non-covalent inhibitors suffer from the same drawbacks as covalent inhibitors. In this manuscript, we describe the development of a novel non-covalent chemical probe, PI1840, and provide data that give further support to the notion that non-covalent inhibitors are more effective against solid tumors. Experimental Procedures: Materials. DMEM, RPMI-1640, DMEM/Ham’s F-12, horse serum, penicillin and streptomycin were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum was from Atlanta Biologicals (Atlanta, GA). Purified 20S proteasome (rabbit), purified 20S immunoproteasome (human), fluorogenic peptide substrates Suc-Leu-Leu-Val-TyrAMC (for the proteasomal CT-L activity), benzyloxy-carbonyl (Z-Leu-Leu-Glu-AMC (for the proteasomal PGPH activity) were purchased from Boston Biochem (Cambridge, MA). Fluorogenic peptide substrate Bz-ValGly-Arg-AMC (for the proteasomal T-L activity) was obtained from Biomol International (Plymouth Meeting, PA). Antibodies were obtained from the following suppliers: p27 (BD Biosciences, San Jose, CA), and β-actin (Sigma-Aldrich, St. Louis, MO); Phospho-Akt (S473), phospho-S6 Ribosomal Protein (Ser240/244), S6 Ribosomal Protein (5610), cleaved PARP (Asp214) (D64E10) XP, cleaved Caspase-3 (Asp175) (5A1E) (Cell Signaling, Danvers, MA); Akt1/2 (N-19), survivin (FL-142), IKBα (C-21), Bax (N20) (Santa Cruz Biotechnology, Santa Cruz, CA), MTT (Calbiochem, ). The pan Bcl-2 antagonist BH3-M6 and the proteasome inhibitors PI1833 and PI-1840 were all synthesized inhouse as reported previously (18, 19). Bortezomib was purchased from Selleckchem, Houston, TX. Nutlin 1 was purchased from Sigma-Aldrich. All other reagents were from Sigma-Aldrich unless otherwise noted. Determination of CT-L, T-L and PGPH proteolytic activities. These assays were performed exactly as described by us previously (20). Briefly, 1 nM of purified 20S rabbit proteasome or 2 by gest on O cber 4, 2017 hp://w w w .jb.org/ D ow nladed from PI-1840, a novel non-covalent proteasome inhibitor with anti-tumor activity immunoproteasome was incubated with 20 μM Suc-Leu-Leu-Val-Tyr-AMC for the CT-L activity, Bz-Val-Gly-Arg-AMC for the T-L activity, and benzyloxycarbonyl Z-Leu-LeuGlu-AMC for the PGPH activity for 1 h at 37oC in 100 μl of assay buffer (50 mM TrisHCl, pH 7.6) with or without compound, and the hydrolyzed 7-amido-4-methyl-coumarin (AMC) was measured using a WALLAC Victor Counter. To determine proteasome activity in whole cell extracts (5μg) from cultured cells, lysates were used instead of 20S rabbit proteasome. Whole cell extracts were prepared by homogenizing the cells in lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40), centrifuging the lysates at 12,000 g, and collecting the supernatants as whole cell extracts as described previously by us (20). Protein Digestion, Peptide Purification and LC-MS/MS Analysis. These procedures were performed exactly as described by us previously (18). Briefly, after purified 20S Proteasome (rabbit) (1 nM) was incubated for 30 min with inhibitors in 50 mM Tris-Hcl, pH 7.6, acetonitrile and trypsin were added (4 hr, 37° C). The digest was concentrated and the peptides were extracted with C18 reversed phase pipette tip columns, and injected into mass spectrometer. To assess LC-MS/MS performance, tryptic peptides from horse apomyoglobin (25 fmol) were spiked in each LC-MS/MS analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide sequencing experiments were performed using a nanoflow liquid chromatograph (U3000, Dionex, Sunnyvale, CA) interfaced with an electrospray ion trap mass spectrometer in order to detect and localize modified peptides from the proteasome exactly as described by us previously (18). Database Searching and Data Analysis. The data searching and analysis was performed exactly as described by us previously (18). Briefly, the 22 rabbit proteasome protein sequences from the UniProt (http://www.uniprot.org) were searched using Sequest, (21) and the search results summarized in Scaffold 3.0 (www.proteomesoftware.com). For peptide quantification, the integrated peak areas were calculated from ion chromatograms using QuanBrowser from Xcalibur 2.0 (Restriction: m/z (+/0.02); retention time (120 seconds)). To insure proper sequence assignment, manual inspection of the accuracy of the m/z values and the fragmentation patterns of the target peptides was performed exactly as described by us (18). Dialysis using purified rabbit 20S proteasome. We used the same dialysis method that we used in our previous study (18) to determine the effect of dialysis on CT-L activity. Briefly, compounds PI-1840 (1 μM) and lactacystin (2.5μM) or vehicle (DMSO) were added to 20S proteasome (rabbit) at a final concentration of 1 nM in proteasome assay buffer (50 mM Tris-HCl, pH = 7.6) and incubated at room temperature for 30 min. Then, the proteasome-compound mixtures were added to mini dialysis units (3500 MWCO Thermo Scientific Slide-A-Lyzer) (Rockford, IL) and dialyzed against proteasome assay buffer. Immediately (t = 0) and at different time points (20 , 60 , 120 , 240 , 480 and 1080 min) of dialysis at 4 oC, samples were taken from the dialysis cassette and the CT-L activity of 20S proteasome was determined as described by us previously (18). CT-L activity was normalized against CT-L activity of DMSO control. Cells, Cell culture and extract preparation. MDA-MB-468 and MDA-MB-231 (human breast cancer cells), HCT-116, HCT-116-p53/-, HCT-116-HKH2 (human colon cancer cells), PC-3 (human prostate cancer cells) were cultured in DMEM medium. DU145, LNCaP (human prostate cancer cells), RPMI8226, U266 (human multiple myeloma cells), Colo357 (human pancreatic adenocarcinoma cells), RXF-397 (human renal carcinoma cells) were cultured in RPMI-1640 medium. All media were supplemented with 10% fetal bovine serum (FBS), and 1% PenStrep antibiotics. Normal immortalized MCF-10A 3 by gest on O cber 4, 2017 hp://w w w .jb.org/ D ow nladed from PI-1840, a novel non-covalent proteasome inhibitor with anti-tumor activity breast cells were cultured in DMEM/Ham’s F12 containing 5% horse serum, 20 ng/ml epidermal growth factor (EGF), 100 ng/ml cholera toxin, 500 ng/ml hydrocortisone and 0.01 mg/ml insulin. Cells were maintained at 37oC in a humidified incubator in an atmosphere of 5% CO2. Western blot analysis. To prepare whole cell lysates, cells were washed with PBS twice, and lysed in 30 mM Hepes, pH 7.5, 10 mM NaCl, 5 mM MgCl2, 25 mM NaF, 1 mM EGTA, 1% Triton-X-100, 10% glycerol, protease inhibitor cocktail, 2 mM PMSF, 2 mM Na3VO4, and 6.4 mg/ml pnitrophenylphosphate. Lysates were cleared by centrifugation at 12,000 x g for 15 min, and the supernatants were collected as whole cell extracts. The protein concentration was determined by the Bradford assay. Cell lysates (50 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane, probed with specific antibodies, and signals were visualized by enhanced chemoluminescence (ECL, Amersham, Piscataway, NJ) according to the manufacturer's protocol. MTT (3-(4,5-dimethythiazol-2-yl)-2,5diphenyltetrazolium bromide) assay. Cancer cells or normal immortalized MCF10A breast cells were plated in 96-well plates in 100 μl of above specified medium and allowed to attach overnight. Cells were then incubated for different time points with varying concentrations of drugs or appropriate controls. After that, media was aspirated and replaced with100 μl complete media containing 1 mg/ml MTT and incubated for three hours at 37°C in 5% CO2 humidified incubator. After incubation, media was aspirated and DMSO was added. Cells were then incubated for 10 min at room temperature while shaking, and the absorbance was determined at 540 nm using a μQuant spectrophotometric plate reader (Bio-TEK,