Patent Application: US-201213623415-A

Abstract:
a method of treating in a person a cancer tumor refractory to treatment with bortezomib or an agent sharing the apoptosis generating activity of bortezomib or any other anti - cancer drug , comprises administering to the person , in a pharmaceutically acceptable carrier , a pharmacologically effective dose of an agent selected from the group consisting of b - ap15 and other proteasome inhibitor abrogating the deubiquitinating activity of the 19s rp dubs .

Description:
in vitro proteasome activity assays were performed in black 96 - well microtitier plates using human 20s proteasome ( boston biochem ) in reaction buffer ( 25 mm hepes , 0 . 5 mm edta , 0 . 03 % sds ) with suc - llvy - amc , z - lle - amc or boc - lrramc used as substrates for proteasome activity . de - ubiquitinase activity assays were performed with human 19s rp ( boston biochem ) with ubiquitin - amc as substrate . for fadu xenograft studies a 100 - μl - cell suspension containing 1 × 10 6 cells was injected subcutaneously into the flank of scid . upon tumor take mice were randomized into control or treatment groups and administered with 5 mg kg − 1 b - ap15 or vehicle . in vivo levels of apoptosis and cell death were determined from the detection of caspase cleaved and total levels of cytokeratin - 18 in plasma using m30 apoptosense ® and m65 elisa ® s assays ( peviva ). the methods are described below in more detail . reagents were obtained from the following sources : 20s proteasome ( e - 360 ), 26s proteasome ( e - 365 ), 19s proteasome ( e - 366 ), suc - llvy - amc ( s - 280 ), z - lle - amc ( s - 230 ), boc - lrr - amc ( s - 300 ), ubiquitin - amc ( u - 550 ), tetra - ubiquitin k63 ( uc - 310 ), tetra - ubiquitin k48 ( uc - 210 ), deconjugating enzyme set ( ke10 ), ha - ubiquitin vinyl sulfone ( u - 212 ) ( boston biochem ); anti - β - actin ( ac - 15 ), odc - 1 ( hpa001536 ) ( sigma aldrich ); anti - lc - 3 ( 2775 ), anti - gapdh ( 2118 ), anti - p44 / 42 mapk ( 4695 ), anti - phospho - p44 / 42 mapk ( 9101 ) ( cell signaling ); n - ethylmaleimide ( 34115 ) ( emd chemicals ); anti - ubiquitin k48 ( apu2 ), anti - ubiquitin ( mab1510 ) ( millipore ); anti - p53 ( d01 ), anti - uchl5 ( h - 110 ), hdm2 ( smp14 ) ( santa cruz ); anti - parp ( c2 - 10 ), anti - p27 ( g173 - 524 ), anti - active caspase 3 ( c92 - 605 ) ( bd biosciences ); anti - usp14 ( a300 - 919a ) ( bethyl laboratories ); anti - ha ( 12ca5 ) ( roche ); b - ap15 ( nsc687852 ) was obtained from the developmental therapeutics program of the us national cancer institute ( http :// www . dtp . nci . nih . gov ) or synthesized by oncotargeting ab ( uppsala , sweden ). bortezomib was obtained from the department of oncology , karolinska hospital , sweden . mcf7 cells were maintained in mem / 10 % fetal calf serum . hct - 116 p53 +/+, p53 −/−, bcl - 2 +/+, puma −/− and bax −/− cells were maintained in mccoy &# 39 ; s 5a modified medium / 10 % fetal calf serum . the hct - 116 p53 +/+, p53 −/−, puma −/− and bax −/− were generated as described ( 36 ). the hct - 116 bcl - 2 +/+ cell line was generated by transfecting parental hct - 116 p53 +/+ cells with pcep4 bcl - 2 ( addgene plasmid 16461 ) ( 37 ) and isolating high expression clones . fadu and llc3 cells were maintained in dmem high glucose medium supplemented with 10 % fetal calf serum , na pyruvate , hepes and non - essential amino acids . 4t1 . 12b carcinoma cells were maintained in rpmi medium supplemented with 10 % fetal calf serum . the proteasome reporter cell line meljuso ub - yfp was generated as described ( 38 ). cells were maintained in dulbecco &# 39 ; s modified eagle &# 39 ; s medium / 10 % fetal calf serum . the retinal epithelial cell line was generated as described ( 39 ). all cells were maintained at 37 ° c . in 5 % co 2 . the microarray based gene expression analysis and the connectivity map ( cmap ) analysis was performed as previously described ( 40 ). briefly , mcf7 cells were exposed to b - ap15 ( 1 μm , 6 h ) or vehicle ( 0 . 1 % dmso , 6 h ). rna was isolated ( rneasy miniprep kit , qiagen ) followed by quality control , labelling and hybridization to genome u133 plus 2 . 0 arrays ( affymetrix inc ). raw data was normalized using mass ( affymetrix inc .) and rank ordered . for selection of the 30 most induced ( up tags ) and the 30 most suppressed ( down tags ) transcripts the following criteria were used : up tags , present call and expression over 300 arbitrary in the b - ap15 experiment ; down tags , present call after both b - ap15 and vehicle treatment , and expression over 300 arbitrary units in the vehicle experiment . for cmap compatibility only tags ( i . e . probes ) present on hg u133a were used . raw and normalized expression data have been deposited at gene expression omnibus ( http :// www . ncbi . nlm . nih . gov / geo /) with accession number gse24150 . in vitro proteasome activity assays using 20s cp ( 2 nm ) ( boston biochem ) were performed at 37 ° c . in 100 - μl reaction buffer ( 25 mm hepes , 0 . 5 mm edta , 0 . 03 % sds ). samples were incubated for 10 min with indicated compound followed by addition of 10 μm suc - llvy - amc , z - lle - amc or boc - lrr - amc for the detection of chymotrypsin - like , caspase - like and trypsin - like activity respectively . for dub inhibition assays 19s rp ( 5 nm ), 26s ( 5 nm ) uch - l1 ( 5 nm ), uch - l3 ( 0 . 3 nm ), usp2cd ( 5 nm ) usp7cd ( 5 nm ) usp8cd ( 5 nm ) and bap1 ( 5 nm ) were incubated with b - ap15 followed by addition of ubiquitin - amc ( 1000 nm ). fluorescence was monitored using wallac multilabel counter or tecan infinite m1000 equipped with 360 nm excitation and 460 nm emission filters . substrate overlay assays . native gel electrophoresis was performed as described ( 41 ). in brief 4 μg of purified 26s proteasome ( boston biochem ) was mixed with 10 or 50 μm b - ap15 and incubated at 37 ° c . for 10 min . samples were resolved on 4 % non - denaturing page . gels were submerged in assay buffer ( 20 mm tris - hcl , 5 mm mgcl 2 , 1 mm atp , 0 . 1 mm suc - llvy - amc ) and proteasomes were visualized under uv illumination . the recombinant ub - gfp plasmid pet19b ub - m - gfp was generated as described ( 42 ). in brief recombinant ub - gfp was purified from bl21 e . coli cells by his affinity purification . for cleavage assays 19s rp ( 25 nm ) was incubated with 10 mm nem , 250 μm tpen or 50 μm b - ap15 for 10 min followed by the addition of recombinant ub - gfp ( 200 nm ). ubiquitin chain disassembly reactions were performed essentially as above except k48 - or k63 - linked ubiquitin tetramers ( 50 ng ) were substituted for ub - gfp . the level of ub - gfp cleavage or ubiquitin disassembly was determined by immunoblotting with anti ubiquitin antibodies . the ubiquitinated hdm2 substrate was generated according to the boston biochem protocol ( k - 200 ). for the cleavage assay 19s rp ( 25 nm ) was incubated with 50 μm b - ap15 or dmso for 10 min followed by the addition of ubiquitinated hdm2 substrate ( 100 nm ). the cleavage of ubiquitinated hdm2 substrate and ubiquitinated hdm2 was determined by immunoblotting with anti - hdm2 antibodies . hct - 116 cells were treated with bortezomib ( 100 nm ) or b - ap15 ( 1 μm ) for 3 hours . after stimulation , the cells were lysed in 50 mm hepes ph 7 . 4 , 250 mm sucrose , 10 mm mgcl 2 , 2 mm atp , 1 mm dtt and 0 . 025 % digitonin . samples were sonicated briefly and incubated for 15 min on ice . proteasomes from these samples were isolated according to the manufacturer &# 39 ; s protocol . for labelling of dubs in cell lysates sub confluent cells were harvested by trypsinization , washed three times with pbs , and centrifuged at 1500 rpm for 5 min . cell pellets were lysed with buffer ( 50 mm hepes ph 7 . 4 , 250 mm sucrose , 10 mm mgcl 2 , 2 mm atp , 1 mm dtt ) on ice for 15 min . debris was removed by centrifugation and 25 μg of protein was labelled with 1 μm ha - ubvs for 30 min at 37 ° c . samples were resolved by sds - page and analyzed by immunoblotting with indicated antibodies . for determination of apoptosis parental hct - 116 p53 +/+ cells were treated with the increasing doses of bortezomib or b - ap15 for 24 h . treatment doses were based on the drug concentration that resulted in maximal apoptosis over a 24 h period . hct - 116 cells were seeded in 96 - well microtiter plates at 10 , 000 cells per well and incubated overnight . cells were treated with indicated drug for 24 h . at the end of the incubation period , np40 was added to the tissue culture medium to 0 . 1 % and 25 μl of the content of each well was assayed using the m30 - apoptosense ® elisa as previously described ( 43 ). cell viability was determined by measuring acid phosphatase activity or using the fmca method ( 44 ). for the acid phosphatase activity cells were seeded at 5000 cells per well in 96 - well culture plates and incubated for 12 h at 37 ° c . compounds were added to the cells in growth media and incubated for 72 h at 37 ° c . cells were washed with 200 μl warm pbs . 100 μl of para - nitrophenyl phosphate ( pnpp , 2 mg / ml ) in na acetate buffer ph 5 ( naac 0 . 1 m , 0 . 1 % triton - x - 100 ) was added per well . cells were incubated for 2 h after which reaction was stopped by addition of 1n naoh . absorbance was measured at 405 nm . for the fmca assay cells were seeded in the drug - prepared 384 - well plates using the pipetting robot precision 2000 ( bio - tek instruments inc ., winooski , vt .). the plates were incubated for 72 h and then transferred to an integrated hts saigan core system consisting of an orca robot ( beckman coulter ) with co 2 incubator ( cytomat 2c , kendro , sollentuna , sweden ), dispenser module ( multidrop 384 , titertek , huntsville , ala . ), washer module ( elx 405 , bio - tek instruments inc ), delidding station , plate hotels , barcode reader ( beckman coulter ), liquid handler ( biomek 2000 , beckman coulter ) and a multipurpose reader ( fluostar optima , bmg labtech gmbh , offenburg , germany ) for automated fmca . survival index ( si ) is defined as the fluorescence of test wells in percentage of controls with blank values subtracted . for determination of cell cycle hct - 116 cells were treated with b - ap15 or dmso cells were harvested by trypsinisation , washed and fixed in 70 % ice cold etoh for 12 h . cells were re - suspended in staining solution containing propidium iodide ( 50 μg / ml ) and rnase a ( 0 . 5 μg / ml ) in pbs . samples were run on bd facscalibur . the percentage of cells in each phase of the cell cycle was determined using modfit software . animal experiments were conducted in full accordance with swedish governmental statutory regulations on animal welfare under permission from local ethical committees . animals were housed at a max of five per cage and provided with sterile water and food ad libitum . all mice were monitored and weighed daily . for the head and neck carcinoma model a 100 - μl cell suspension containing 1 × 10 6 fadu cells was injected subcutaneously into the right rear flank of the animals . after injection , tumor growth was measured daily with calipers and the tumor volume calculated by the formula l × w 2 × 0 . 44 . when tumors had grown to a size of approximately 200 mm 3 ( day 0 ) mice were randomized to receive either vehicle ( n = 10 ) or b - ap15 5 mg / kg by subcutaneous injection s . c . ( n = 15 ) daily . for the colon carcinoma model , 2 . 5 × 10 6 hct - 116 colon carcinoma cells stably transfected with bcl - 2 ( hct - 116 bcl - 2 + ) were inoculated subcutaneously into the right flank of nude mice . one day after inoculation mice were treated with 5 mg / kg − 1 by intra peritoneal injection ( i . p .). animals were inspected daily to establish the tumor onset and growth . for the lung carcinoma model a 100 - μl cell suspension containing 2 × 10 5 lewis lung carcinoma ( llc ) cells was injected subcutaneously into the right rear flank of c57 / b6 mice . when tumors had grown to a size of approximately 50 mm 3 ( day 0 ) mice were randomized to receive either vehicle ( n = 4 ) or b - ap15 5 mg / kg − 1 i . p . ( n = 4 ) with a treatment cycle consisting of two days treatment followed by two days no treatment ( 2 days on / 2 days off ) for two weeks . for the breast carcinoma model a 100 - μl cell suspension containing 1 × 10 5 4td cells was injected subcutaneously into the right mammary fat pad of balb / c mice . when tumors had grown to a size approximately 25 mm 3 ( day 0 ), mice were randomized to receive either vehicle ( n = 5 ) or b - ap15 2 . 5 mg / kg − 1 i . p . ( n = 5 ) with a treatment cycle consisting of one days treatment followed by three days no treatment ( 1 day on / 3 days off ) for 3 weeks . in the aml studies female c57bl / 6j mice were injected i . v . in the tail vein with 5 × 10 5 c1498 aml cells . after eight days mice were randomized to receive either b - ap15 5 mg / kg − 1 ( n = 10 ) or vehicle ( n = 10 ) i . p . for 7 days ( day + 8 till + 14 ). nineteen days after malignant cell injection all of the mice were killed and histopathological manifestations of liver , ovary ( target organs for this model of tumor ) were evaluated and compared between groups . for administration of drug b - ap15 was dissolved in cremphor el : peg 400 ( 1 : 1 ) by heating to give a working concentration of 2 mg / ml . working stock was 1 : 10 diluted in 0 . 9 % normal saline immediately prior to injection . for measurement of the apoptosis - related ck18 - asp396 fragment , 12 . 5 ml of plasma was collected 24 h after last treatment and analyzed using the m30 - apoptosense ® assay . each sample was mixed with 0 . 4 ml of heterophilic blocking reagent ( scantibodies laboratory inc ). since the 4t1 cells are resistant to 6 - thioguanine , metastases can be determined by culturing homogenized tissue in the presence of 6 - thioguanine . for determination of metastastic 4t1 cells the protocol was as described ( 45 ). in brief lungs from treated or untreated animals were homogenized and treated with collagenase and elastase . cells were grown in the presence of 60 μm 6 - thioguanine for 2 weeks and the number of metastatic colonies determined by giemsa staining . tumor sections were de - paraffinized with xylene , rehydrated and then incubated over - night with k - 48 ubiquitin or active - caspase 3 ( 1 / 500 ) diluted in 1 % ( wt / vol ) bovine serum albumin and visualized by standard avidin - biotin - peroxidase complex technique ( vector laboratories ). counterstaining was performed with mayer &# 39 ; s haematoxylin . for comparisons of treatment groups , we performed the unpaired t test ( mann - whitney ), repeated measures anova and kaplan - meier survival ( mantel - cox test ). all statistical analyses were performed using graphpad prism software ( version 5 . 0 ). statistical significance was achieved when p was less than 0 . 05 . cmap readout of mcf7 cells treated with b - ap15 ( 1 μm ) for 6 h is shown in table 3 . b - ap15 inhibits degradation of ubiquitin - tagged yfp in a proteasome reporter cell line ( fig1 a ). levels of ubg76v - yfp accumulation were determined by flow cytometry and immunoblotting ; immunoblot of ubiquitin conjugation in hct - 116 cells treated with b - ap15 ( 1 μm ) or bortezomib ( 100 nm ) ( fig1 b ). immunoblot of ubiquitin conjugates , caspase 3 activation parp cleavage , p53 . p21 cip1 and p kip1 in hct - 116 cells following 24 h treatment with the indicated concentrations of b - ap15 ( fig1 c ). immunoblot of odc - 1 levels in hct - 116 cells following treatment with bortezomib ( 100 mm or b - ap15 ( 1 μm ) ( fig1 d ); values represent quantified optical density units of odc - 1 normalized to β - actin . cell cycle profiles of b - ap15 treated hct - 116 cells ( fig1 e ); cells were analyzed by propidium iodide staining and flow cytometry . levels of caspase activity in isogenic hct - 116 cells as determined by elisa for caspase cleaved cytokeratin - 18 ( ck18 - asp398 ) following treatment with bortezomib ( 100 nm ) or b - ap15 ( 1 μm ) (**′ p = 0 . 01 , ***′ p = 0 . 001 ) ( fig1 f ). inhibition of ub - amc cleavage by 19s rp or 26s proteasomes following treatment with b - ap15 ; ubiquitin aldehyde ( ubal ), a general dub inhibitor , was included as a control ( fig2 a ). immunoblot of 19s rp mediated cleavage of ub - gfp ( fig2 b ); 19s rp were pre - treated with dmso or indicated concentrations of b - ap15 followed by addition of recombinant ub - gfp as a dub substrate . kinetics of 19s rp ub - gfp cleavage following b - ap15 ( 50 μm ) treatment ( fig2 c ). b - ap15 inhibits de - ubiquination of hdm2 ( fig2 d ); ubiquinated hdm2 was added to dmso or b - ap15 ( 50 μm ) treated 19s rp followed by immunoblotting . ubiquitin chain disassembly reactions of k63 / k48 linked ubiquitin tetramers by 19s rp following treatment with dmso or b - ap15 ( 50 μm ) ( fig2 e ). 19s rp were pre - treated with dmso , nem ( 10 mm ) b - ap15 ( 50 μm ) ( fig3 a ) or tpen ( 250 μm ) ( fig3 b ) followed by addition of ub - gfp and immunoblotting with anti - gfp antibodies . active site directed labelling of proteasomal dubs ( fig3 c ); purified 19s or 26s proteasomes were pre - treated with dmso , nem or bap15 followed by labeling with ha - ubvs and immunoblotting . immunoblot of hct116 cells treated with b - ap15 ( 1 μm ) for 3 h ( fig3 d ); dubs from whole cell lysates were labelled with ha - ubvs followed by sds - page and immunoblotting with indicated antibodies . scid mice bearing fadu human tumor xenografts were randomized at tumor take ( 200 mm 3 ) and treated by daily subcutaneous injection with either vehicle ( n = 10 ) or 5 mg kg − 1 b - ap15 ( n = 15 ) for 10 days . mean tumor volume ± sem shown (*** p =& lt ; 0 . 001 ) ( fig4 a ). total levels of tumor derived ck18 and caspase cleaved ( ck 18 - asp396 ) in circulation following b - ap15 treatment (** p = 0 . 01 ) ( fig4 b ). disease free survival of nude mice challenged with hct - 116 bel - 2 + cells ( fig4 c ). mice were treated with vehicle ( n = 6 ) or 5 mg kg − 1 b - ap15 ( n = 6 ) 4 - 5 times weekly for 3 weeks and monitored for tumor onset ( log - rank , p = 0 . 0136 , hazard ratio = 7 . 9 ). c57bl / 6j mice bearing syngenic lung carcinoma ( llc ) tumors were treated with either vehicle ( n = 4 ) of 5 mg kg − 1 b - ap15 ( n = 4 ) in a one day on / two days off cycle ( fig4 d ); mean tumor volume ± sem shown ( p =& lt ; 0 . 01 ). balb / c mice bearing orthotopic breast carcinomas ( 4t1 ) were treated with either vehicle ( n = 5 ) or 2 . 5 mg kg − 1 b - ap15 ( n = 5 ) in a one day on / three days off cycle ( fig4 e ); mean tumor volume ± sem shown (** p =& lt ; 0 . 01 ). box and whisker plots of pulmonary metastatic colonies from vehicle or b - ap15 treated 4t1 breast carcinomas ( fig4 f ); boxes represent upper and lower quartiles and median , whiskers show maximum and minimum values . representative immunohistochemical staining for k48 - linked ubiquitin accumulation and cleaved caspase - 3 in vehicle and b - ap15 treated 4t1 tumors , original magnification × 20 ( fig4 g ). aml infiltration in liver and ovary of vehicle and b - ap15 treated mice ( fig4 h ). liver of vehicle treated mice showed invasion of leukemic blasts along with glycogen depletion and non - specific hemorrage . ovary section of vehicle treated mice showed massive invasion of leukemic blasts and interstitial bleeding . in contrast , liver and ovary from b - ap15 treated mice showed few infiltrated blasts and normal morphology ( original magnification × 20 ). hct - 116 cells were treated with b - ap15 or doxorubicin ( 100 nm , as a positive control for genotoxic stress for 18 h ) ( fig5 ). cell lysates were immuno - blotted with antibodies for phosphorylated p53 and histone h2 ax marker for dna damage of for total levels of p53 and β - actin as loading controls . b - ap15 induces apoptosis and inhibits cell survival of hct - 116 cells whereas pbmc ( peripheral blood mononuclear cells ) and immortalized htert - rpe1 are less sensitive hct - 116 cells were treated with increasing concentrations of b - ap15 for 24 h and the levels of apoptosis were determined by measuring the levels of caspase cleaved cytokeratin - 18 ( ck18 ) by elisa assay ( fig6 a ). hct - 116 cells were treated with increasing concentrations of b - ap15 for 48 h ). cell viability was determined by acid - phosphatase activity assay . mean values ± s . d . shown ( fig6 b ). hct - 116 or htert - rep1 cells were treated with increasing concentrations of b - ap15 for 72 h followed by analysis of cytotoxicity using the fmca method ( 44 ) ( fig6 c ). hct - 116 or htert - rep1 cells were treated with increasing concentrations of bortezomib for 72 h followed by analysis of cytotoxicity using the fmca method ( fig6 d ). htert - rpe1 is an immortalized human retinal pigment epithelial cell line ( 29 ). ic50 was determined from log concentration / effect curves in graph pad prism ( graphpad software inc . ca , usa ) using non - linear regression analysis ( four parameter model with variable hill slope ) ( fig6 e , 6 f ). concentration / response curves were generated in two - fold dilutions at eight concentrations of b - ap15 and bortezomib in triplicate using the fmca assay . the results are expressed as log ic50 + sd from four or five independent experiments ( hct - 116 , n = 5 ; pbmc , n = 4 ; htert - rpe1 , n = 5 ). dose response curves of apoptosis induction in isogenic clones of hct - 116 cells hct - 116 cells were treated with increasing concentrations of bortezomib or b - ap15 for 24 h and the levels of apoptosis were determined by measuring the levels of caspase cleaved cytokeratin - 18 ( ck - 18 ) by elisa assay ( mean fold change ± s . d , n = 4 ) ( fig7 ). 20s cp ( 2 nm ) was pretreated with dmso , b - ap15 ( 50 μm ) or bortezomib ( 100 nm ) for 5 min in assay buffer ( 25 mm hepes , 0 . 5 mm edta , 0 . 03 % sds ) followed by the addition of 100 μm of the fluorogenic substrates suc - llvy - amc , z - lle - amc or boc - lrr - amc for analysis of proteasome chymotrypsin - like , caspase - like and trypsin - like activities , respectively ( fig8 a ). 26s proteasomes ( 2 nm ) in assay buffer ( 25 mm hepes , 50 mm nacl , 1 mm mgcl 2 , 2 mm atp , 1 mm dtt ) were treated as in the experiment illustrated in fig8 a ( fig8 b ). values represent fold cleavage in relative fluorescent units . b - ap15 does not cause dissociation of 19s and 20s particles or alter ubiquitin binding substrate overlay assay of b - ap15 treated proteasomes ( fig9 a ). purified 26 s proteasome was treated with b - ap15 ( 10 μm or 50 μm ) separated by native gel electrophoresis and assayed for proteolytic activity using suc - llvy - amc as a fluorogenic substrate for peptidase activity . analysis of the gels showed the presence of doubly ( rp 2 cp ) and singly ( rp 1 cp ) capped proteasomes in both control and b - ap15 lanes . the addition of 0 . 03 % sds did not reveal an increase in the presence of uncapped 20s core particles . b - ap15 does not alter proteasome - ubiquitin binding activity ( fig9 b ). hct - 116 cells were treated with bortezomib ( 100 nnm ) or b - ap15 ( 1 μm ), and the proteasomes were affinity purified . levels of associated polyubiquitin were determined by immunoblotting . htc - 116 cells were treated for 3 h with b - ap15 ( 1 μm ) ( fig1 ). lysates treated with 10 mm n - ethylmaleimide ( nem ) were included as a control for total dub inhibition . dub activity was determined from cell lysates by measuring cleavage of the fluorogenic substrate ubiquitin - 7 - amido - 4 - methylcoumarin ( ub - amc ). dose response of b - ap15 ( fig1 a ): purified 10s proteasomes ( 5 nm ) were treated with indicated concentrations of b - ap15 , and dub activity was determined by detection of ub - amc cleavage . the ic50 value ( 2 . 1 ± 0 . 411 μm ) was determined from log concentration curves in graph pad prism using non - linear regression analysis ( mean values ± sd , n = 3 ). it should be noted that ic50 observed in cell - free assays is somewhat higher than that observed in cells , probably due to the hydrophobicity of b - ap15 ( x log p = 3 . 3 ) resulting in enrichment of the compound in cells ( 11 ). reversibility of b - ap15 inhibition ( fig1 b ): the reversibility of inhibition was determined by measuring recovery of dub activity after rapid dilution of the enzyme / b - ap15 complex . a reaction mix containing 50 times the 19s concentration normally used in reactions ( 250 mm ) and 10 times the calculated ic50 value for b - ap15 ( 25 μm ) was incubated on ice for 15 min followed by a 50 - fold dilution in reaction buffer to give a final concentration of 5 nm for 19 ′ s and 0 . 5 μm for b - ap15 . the linear reaction curves of ub - amc cleavage show that b - ap15 is a reversible inhibitor . determination of whether b - ap15 reacts non - specifically with cysteine residues ( fig1 c ). 19s ( 5 nm ) was treated with b - ap15 ( 10 μm ) or b - ap15 ( 10 μm ) mixed with reduced glutathione ( gsh ( 2 mm ). the presence of glutathione did not reduce b - ap15 mediated inhibition of 19s dub activity . hct - 116 cells were treated for 3 h with b - ap15 ( 1 μm ) and the proteasomes were affinity purified ( fig1 a ). proteasome dub activity is expressed as cleavage of ub - amc / suc - llvy - amc to normalize for proteasome recovery ( p = 0 . 012 , unpaired t - test , two tailed ). b - ap15 does not inhibit non - proteasomal dubs ( fig1 b ). recombinant non - proteasomal dubs were treated with b - ap15 and % activity determined . cell lysates from 293t and hela cells were treated with b - ap15 ( 50 μm ) followed by active labelling with ha - ubvs ( fig1 c ). all samples were run on sd - page followed by immunoblotting with α - ha antibodies . b - ap15 treatment does not significantly alter animal weight ( fig1 ) the difference in weight at the start and the endpoint between control and treated animals for the xenografts shown in fig4 was : fadu , − 1 . 3 %; llc , + 2 . 1 %; 4t1 + 5 . 8 %. boxes represent the upper and lower quartiles and median , whiskers show maximum and minimum values . sensitivity of cell lines in the nc160 cell line to b - ap15 and bortezomib ( fig1 ) shown are ic50 values for individual cell lines ( left hand graphs ) and median ic50 for each tumor type ( right hand graphs ). data have been taken from www . dtp . nci . nih . gov . arrows indicate the two most sensitive tumor cell types for each drug . expression of chaperone genes observed in bap15 - treated cells ( table 1 ) is indicative of induction of a proteotoxic response . further analysis by quantitative pcr showed that b - ap15 induces a stronger hspa6 ( hsp70b ′), hspa1b and dnajb1 ( hsp40 ) expression than bortezomib ( table 2 ). hspa6 , which is known to be induced in response to accumulation of damaged proteins ( 35 ), was induced & gt ; 1000 - fold by b - ap15 . these findings indicate that high molecular weight ubiquitin substrate complexes accumulating as a result of dub inhibition can generate strong cytotoxicity that is insensitive to bcl - 2 over - expression . # hct116 cells were treated with ic90 concentrations of b - ap15 or bortezomib and mrna levels were determined after reverse transcription and real time pcr , fold induction is expressed as fold untreated control . the experiment was repeated with similar results . the cellular response to b - ap15 is not only distinct from that of bortezomib in regard of involvement of apoptosis regulators but also in regard of the sensitivity of tumor cell lines in the nci - 60 cell line panel ( http :// dtp . nci . nih . gov ). inhibitors of 19s rp dub activity should display a therapeutic spectrum different from that of inhibitors of 20s enzymatic activity , and therefore expand the arsenal of therapy options in oncology . 4 - nitrobenzaldehyde ( 12 . 39 g , 82 mmol ) and 4 - piperidone . hcl ( 6 . 14 g , 40 mmol ) were suspended in acetic acid ( 27 . 5 ml ). hydrochloric acid gas generated by dropwise addition of sulfuric acid ( 400 mmol ) on sodium chloride ( 400 mmol ) was bubbled through the reaction mixture , followed by conc . sulfuric acid ( 0 . 5 ml ) and stirring overnight ( 16 h ) at room temperature . lcms analysis showed almost complete conversion to product . the reaction mixture was filtered and the collected solid suspended in sat k 2 co 3 ( 80 ml ) acetone ( 80 ml ). the ph was adjusted to 12 with saturated aqueous na 2 co 3 and the mixture stirred for 30 min at room temperature . the slurry was filtered and washed with water ( 160 ml in small portions ) to give a fine yellow powder . drying overnight under vacuum in a desiccator gave 7 . 81 g ( 3e , 5e )- 3 , 5 - bis [( 4 - nitrophenyl ) methylidene ] piperidin - 4 - one ( 53 %), & gt ; 98 % pure according to lcms ( ace c8 , 3 . 0 μm , 50 × 3 . 0 mm , 10 % to 97 % acetonitrile in 3 min , 1 ml / min , detection at 305 ± 90 nm ). k 2 co 3 ( 4 . 0 g , 29 mmol ) was dissolved in water ( 10 ml ) and mixed with acetone ( 10 ml ). to the clear 2 - phasic liquid system cooled on an ice - bath was added ( 3e , 5e )- 3 , 5 - bis [( 4 - nitrophenyl ) methylidene ] piperidin - 4 - one ( 2 . 1 g , 5 . 75 mmol ). over a few minutes acrylic acid chloride ( 0 . 80 g , 0 . 715 ml , 8 . 84 mmol ) was added dropwise to the 2 - phasic system . the reaction mixture was stirred at 0 ° c . for 10 min , then for 1 h at room temperature . the reaction vessel was cooled in an ice - bath , and more acrylic acid chloride ( 0 . 40 g , 0 . 358 ml , 4 . 42 mmol ) was added dropwise . the reaction mixture was allowed to warm to room temperature and was stirred for 3 h . workup by adding 20 ml water , filtering and washing with 20 ml water rendered a solid residue , which was purified on silica with a gradient of 0 - 5 % acetone in dichloromethane . solvent was evaporated from the combined pure fractions to 1 . 12 g ( 46 %) yellow solid b - ap15 . basic ( xbridge c18 , 3 . 5 μm , 50 × 3 . 0 mm , 10 % to 97 % mecn in 10 mm nh 4 hco 3 ( ph10 ) over 3 min , 1 ml / min , detection at 305 ± 90 nm ) and acidic ( ace c8 , 3 . 0 μm , 50 × 3 . 0 mm , 10 % to 97 % acetonitrile in 0 . 1 % aqueous tfa over 3 min , 1 ml / min , detection at 305 ± 90 nm ) lcms methods showed & gt ; 98 % purity . melting point 170 - 172 ° c . b - ap15 ( 25 . 2 mg ) is dissolved in 1 ml of dimethyl sulfoxide . the solution is added dropwise to 10 ml of vigorously stirred saline . the formed suspension , which can be stabilized by adding 1 % by weight of pvp , can be used for intramuscular , intravenous or subcutaneous administration . tablets for oral administration are produced by blending 2 . 0 g of b - ap15 ( powder , & lt ; 10 mu , 90 %) with microcrystalline cellulose ( 1 . 30 g ), corn starch ( 0 . 50 ) g , silica ( 0 . 20 ) g , mg stearate ( 0 . 12 mg ). the mixture is dry compressed to 400 mg tablets , which are sugar coated . b - ap15 ( 14 mg ) was dissolved in 0 . 5 ml of cremophor el ( basf corp .) and absolute ethanol was added to 1 . 0 ml . the clear solution was filled into glass vials for injection . 1 . masdehors , p et al ., increased sensitivity of cll - derived lymphocytes to apoptotic death activation by the proteasome - specific inhibitor lactacystin . br j haematol 105 , 752 - 757 , doi : bjh1388 [ pii ] ( 1999 ). 2 . demartino , g n et al ., pa 700 , an atp - dependent activator of the 20 s proteasome , is an atpase containing multiple members of a nucleotide binding protein family . j biol chem 69 , 20878 - 20884 , http :// www . ncbi . nlm . nih . gov / entrez / query . fcgi ? cmd = retrieve & amp ; 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