protein kinase c ( pkc ) is a family of 
 serine / threonine - specific protein kinases . the pkc isozymes can be 
 classified into three groups : ( i ) the conventional  , i , 
 ii ,  ; ( ii ) the novel  ,  ,  ,  , 
 and ( iii ) the atypical / ( mouse / human ) and . 
 pkc isozymes seem to play important roles in the activation of signal 
 transduction pathways leading to synaptic transmissions , the activation 
 of ion fluxes , secretion , proliferation , cell cycle control , differentiation , 
 and tumorigenesis . because of their role in a complex network of signal 
 transduction pathways , different isoforms have divergent , sometimes 
 opposite roles within a given biological process . as a result , the pkc isozyme has been reported to participate in neoplastic 
 transformation , cardiac hypertrophy , protection from ischemic insult , nociceptor function , macrophage activation , diabetes , and alcohol 
 consumption . a pkc isozyme - specific 
 inhibitor would be a valuable tool for analyzing the function of pkc 
 and is expected to have pharmaceutical potential for cancer , stroke , 
 drug addiction , or pain . typically , kinase inhibitors 
 interact with the atp - binding site , which is well conserved among 
 different kinase families and is even more so within isoforms of a 
 given kinase . this poses a serious hurdle for the development of isozyme - specific 
 inhibitors , as there are about 500 kinases encoded by the human genome . although several selective kinase inhibitors 
 have been reported , it was later found that they also inhibit other 
 targets . for example , the marketed drug imatinib was developed as 
 an inhibitor of the oncoprotein bcr - abl . however , it has turned out 
 to inhibit also other tyrosine kinases such as kit and platelet - derived 
 growth factor receptor , as well as non - kinase targets . similarly , 
 relatively unspecific inhibitors initially intended as pkc-selective , 
 such as ruboxistaurine and enzastaurine , are in clinical trials for diabetic retinopathy 
 and cancer , respectively . rottlerin was described as specific inhibitor 
 of pkc. however , additional modes of action have also been 
 observed more recently for this compound . because of the high degree of conservation among the atp - binding 
 sites in pkc isozymes , it is obviously very difficult to develop monospecific 
 inhibitors . while monospecificity is not a prerequisite for a successful 
 drug , secondary activities create ambiguity in the interpretation 
 of biochemical , pharmacological , and clinical results . we employed 
 a different approach to specifically target pkc signaling by 
 preventing the binding of pkc to its adaptor protein , the pkc-specific 
 receptor of activated c - kinase 2 ( rack2 , cop ) . it has been shown that adaptor proteins bind and translocate activated 
 pkc isozymes to subcellular sites in proximity to their substrates . rack2 binds to activated pkc. the peptide 
 eavslkpt , corresponding to amino acids 1421 of pkc , 
 selectively inhibits the translocation of pkc but not that 
 of other pkc isozymes by binding to rack2 . it 
 has also been shown that the pkc antagonist eavslkpt inhibits 
 protection from hypoxia - induced cell death of cardiac myocytes . protein 
 interactions is aurothiomalate , which interferes with the interaction 
 between pkc and its adaptor molecule par6 . the objective of the current work was 
 to develop a small molecule peptidomimetic of eavslkpt that prevents 
 the pkc/rack2 interaction . it has been shown that small molecule 
 inhibitors of protein  protein interactions are useful for pharmacological 
 purposes . a cell - permeable inhibitor of pkc signal transduction could help to explain the exact function of pkc 
 and could probably also be developed further for pharmaceutical purposes . the lack of any known nonpeptidic inhibitor of the 
 pkc/rack2 interaction and the very limited structural data 
 available render rational drug discovery of an inhibitor of this protein a crystal structure is only available 
 for the n - terminal - c2-like domain of pkc ( pdb entry 1gmi ) , but no structural 
 data are available on rack2 . the pkc protein segment eavslkpt , 
 which as a peptide is known to disrupt pkc/rack2 interaction , 
 is highly solvent - exposed and expected to undergo conformational changes 
 during the interaction with rack2 . the interface is large ( with respect 
 to the size of small molecule inhibitors ) , only vaguely characterized , 
 and interaction hot spots remain speculative . conformations observed 
 for eavslkpt ( in particular glu14 and lys19 ; numbering according to 
 the crystal structure ) in the crystal structure are likely to be influenced 
 by crystal packing . lacking known ligands and structural data 
 for modeling , several pharmacophore models were derived directly from 
 eavslkpt in its crystal structure conformation ( figure 1a ) . the latter 
 attempts to resemble the hydrophobic area presented to rack2 by val16 , 
 while the hydrogen bonding features characterize the side chain properties 
 of glu14 , ser17 , and lys19 . the rationale for selecting these features 
 was their surface exposure and , consequently , an increased likelihood 
 of being involved in the interaction with rack2 . in order to account 
 for protein flexibility ( as far as possible and without becoming overly 
 unspecific ) , the tolerance spheres of the pharmacophore feature and 
 projection points were set to 1.5 and 2.0  , respectively ( the 
 latter are not indicated in figures 1b and 1d ) . this 
 pharmacophore model was used for virtual screening of the asinex gold 
 and platinum collections ( asinex ltd . , moscow , russia ) , which contain 
 approximately 330  000 compounds in total . ( a ) x - ray 
 structure of the c2 domain of pkc ( pdb code 1gmi ) . the protein backbone 
 is illustrated as cartoon , while the protein segment eavslkpt is highlighted 
 as sticks in the yellow box . ( b ) pharmacophore model derived from 
 eavslkpt ( extracted from the crystal structure ) . hydrogen bonding 
 and hydrophobic features are indicated with red / green and yellow spheres , 
 respectively . ( a ) , 
 ( b ) , and ( d ) were generated using ligandscout 3.0 . the hit list was refined 
 by ranking candidate molecules according to their geometric fit to 
 the pharmacophore model and by visual inspection . nineteen compounds 
 ( supporting information table 1 ) were selected 
 and purchased for in vitro testing . one of them , 3-amino - n-(6-ethoxy-1,3-benzothiazol-2-yl)-7-methoxythieno[2,3-b]quinoline-2-carboxamide ( 1 ) , showed significant 
 activity . it disrupted the interaction of pkc and rack2 with 
 an ic50 of 25.5 m ( figure 1c , figure 1d ) . nineteen further thienoquinolines , 
 structurally related to 1 , were purchased or synthesized 
 for in vitro testing . significant disruption of the pkc/rack2 interaction was 
 detected for several tested thienoquinolines ( table 1 ) , with the most active compound being n-(3-acetylphenyl)-9-amino-2,3-dihydro-1,4-dioxino[2,3-g]thieno[2,3-b]quinoline-8-carboxamide ( 8) ( figure 1c ) . acetylphenyl substituents at r1 appear favorable 
 for activity , with a preference for meta- over para - substitution ( as 
 exemplified by 8 and 13 ) . also the amine 
 group at r4 increases activity ( see 8 and 11 ) . compounds with a hydrogen bond acceptor at r3 tend to exhibit 
 significant activity ( such as 3 , 6 , 8 , 13 ) . a pharmacophore - based alignment 
 of 8 with the peptide itself might allow the formulation 
 of a binding mode hypothesis . compound 8 may cover the 
 pharmacophore features of the solvent - exposed side chains of eavslkpt . however , these considerations remain highly speculative , and hopefully 
 more data on structure  activity relationships will help to 
 shed some light on the potential molecular binding mode of 8 in the future . compound 8 led to a dose - dependent 
 inhibition of the pkc/rack2 interaction ( figure 2a ) and exhibited an ic50 of 5.9 m . seven arginines were added 
 to eavslkpt to increase internalization of the peptide for experiments 
 in intact cells . ic50 for the inhibition of the pkc/rack2 
 interaction by eavslkpt - rrrrrrr in this assay was 1.02 m ( supporting information figure 1 ) . it has been 
 shown previously that pkcii interacts with the adaptor protein 
 rack1 . as 
 shown in figure 2b , compound 8 does not prevent the pkcii / rack1 interaction , indicating 
 specificity of 8 for the pkc/rack2 interaction . ( a ) compound 8 prevents the 
 in vitro interaction of pkc with rack2 in a dose - dependent 
 manner . ( b ) compound 8 does not prevent the in vitro 
 interaction between pkcii and rack1 . in ( a ) and ( b ) , the mean 
 values ( sd ) of four independent experiments in which four samples 
 were taken within each experiment are shown . because the color development 
 varies from experiment to experiment , the data were normalized to 
 the control ( 100% ) . therefore , the standard deviation for the control is 0 . upon activation , pkc 
 associates with rack2 and for these experiments pc-3 prostate adenocarcinoma cells were employed 
 because they express relatively high levels of endogenous pkc 
 ( figure 3 ) . in pc-3 cells , treatment with 12-o - tetradecanoylphorbol 13-acetate ( tpa ) led to an increase 
 of pkc in the membrane fraction . accordingly , the amount of 
 pkc in the cytosol decreased ( figure 3 ) . tpa increased the phosphorylated form of pkc. compound 8 does not inhibit pkc in vitro ( supporting information figure 2 ) . treatment of cells with 8 similarly did not reduce ser729-phosphorylated pkc 
 at the membrane ( figure 3 ) . this result illustrates 
 that pkc is phosphorylated following treatment with tpa and 8 does not prevent this phosphorylation . however , compound 8 partially inhibits the cellular translocation of activated 
 and phosphorylated pkc to the membrane fraction . pkc 
 shows a high degree of homology with pkc. therefore , we investigated 
 the influence of 8 on pkc. as shown in figure 3 , compound 8 did not decrease pkc 
 in the membrane . short - term treatment with tpa led 
 to an increase of pkc in the plasma membrane . compound 8 prevented the tpa - induced pkc translocation to the 
 plasma membrane ( figure 4 ) . igfi-r and gapdh 
 were used as loading controls for the membrane and the cytosolic fraction , 
 respectively . in the densitometric analysis , four independent experiments 
 were scanned and the mean values ( sd ) are shown : (  ) p = 0.02 . depending on the exposure time of the membrane 
 to the film , the bands vary from experiment to experiment . therefore , 
 the data were normalized to the control ( 100% ) and no standard deviation 
 can be reported . compound 8 inhibits this tpa - induced 
 translocation of pkc. experiments 1 , 2 , and 3 are three independent 
 experiments . pkc 
 is situated in the signal transduction cascade upstream of raf-1 . in a hela cell line containing a doxycycline - inducible 
 constitutively active pkc , active pkc leads to phosphorylation 
 of the transcription factor elk-1 ( figure 5a , figure 5b ) . to obtain 
 information on whether 8 is able to prevent the pkc/rack2 
 interaction in intact cells , we tested the compound for inhibition 
 of elk-1 phosphorylation in a pathdetect hela luciferase ( hlr ) trans - reporting 
 hela cell line . in these cells , as shown in figure 5c , compound 8 inhibited the phosphorylation of 
 elk-1 in a dose - dependent manner with an ic50 of 11.2 m 
 in intact cells . ( a ) in hela 
 cells , a doxycycline - inducible constitutively active pkc leads to phosphorylation of elk-1 . the cells 
 were left untreated or induced with doxycycline ( 2 g / ml ) for 
 24 h. additional stimulation with tpa ( 50 nm ) was performed for 10 
 min . phosphorylation of elk-1 was detected with a phospho - specific 
 antibody against the ser383 residue of elk-1 . elk-1 phosphorylation 
 was normalized to the gapdh loading control , and data are expressed 
 relative to untreated cells ( control ) . ( c ) compound 8 inhibits the phosphorylation of elk-1 in pathdetect hela - hlr cells . data shown 
 are the mean values ( sd ) of three independent experiments . because 
 the relative light units vary from experiment to experiment , the data 
 were normalized to the controls . in cells , pkc 
 phosphorylates myristoylated alanine - rich protein kinase c substrate 
 ( marcks ) . as shown in figure 6a , compound 8 inhibited the phosphorylation 
 of marcks in hela cells . these results show that 8 exhibits 
 the features of an inhibitor of pkc signaling in vitro and 
 also in intact cells . compound 8 inhibits marcks phosphorylation , 
 cell proliferation , and invasion following treatment with 8 . ( a ) hela cells were starved for 16 h in medium without fetal calf 
 serum . subsequently , they were treated with 50 m solution of 
 compound 8 for 30 min and 100 nm tpa for the last 10 
 min . an amount of 150 g of protein from lysed cells was loaded 
 onto sds 816% gels . below 
 a representative blot , a bar graph corresponding to quantitative scans 
 of three independent experiments ( sd ) is shown . ( b ) cell proliferation 
 following treatment of hela and pc-3 cells with 8 . the 
 mean values ( sd ) of three independent experiments , in which 
 three samples were taken within each experiment , are shown . cell invasion was determined as described 
 in experimental section . in hela cells with 
 a doxycycline - inducible pkc the mean of three experiments 
 ( sd ) is shown : (  ) p = 0.03 . a major question is whether such an inhibitor is toxic or in other 
 words whether the observed effects are due to inhibition of cell proliferation 
 rather than of elk-1 mediated signaling . therefore , we tested the 
 compound for inhibition of cell proliferation in hela - hlr and human 
 pc-3 prostate adenocarcinoma cells . these cell lines were used because 
 hela - hlr cells were employed for elk-1 phosphorylation and pc-3 cells 
 for the pkc translocation experiments described above . as shown 
 in figure 6b , in both of the cell lines even 
 50 m 8 did not show any inhibition of cell proliferation . it has been shown previously that pkc does not increase cell 
 proliferation . however , it increases cell migration and 
 is associated with metastatic spread and invasiveness of human cancer 
 cells . invasion of cells through an extracellular matrix was indeed inhibited 
 by 8 ( figure 6c ) . as shown in 
 figure 7 , untreated hela cells containing a 
 constitutively active doxycycline - inducible pkc showed only 
 low migration into a scratch made with a pipet tip on a tissue culture 
 dish . if the scratch was made into the monolayer cells and the expression 
 of constitutively active pkc was induced by doxycycline , after 
 24 h a significant part of the scratch was covered with cells . pkc 
 increased migration of cells into a gap , and 8 indeed 
 inhibited this pkc-induced migration ( figure 7 ) . another 
 question in the cancer area is whether the compound inhibits angiogenesis . 
 as shown in figure 8 , in a chicken egg assay 
 the induction of angiogenesis by vascular endothelial growth factor the expression of constitutively active pkc was induced by 
 doxycycline for 24 h after a scratch was made into monolayer cells 
 with a pipet tip . angiogenesis was observed in a chicken egg assay 
 with and without treatment with 8 . earlier , we found that the barbituric acid derivative bas 02104951 
 prevented pkc/rack2 interaction ( ic50 = 28.5 m ) . as shown in supporting information figure 2 , 50 m 
 compound inhibited pkcs  , i , and  to approximately 
 80% . if compared to the inhibition of the pkc/rack2 interaction 
 ( ic50 = 5.90 m ) , this is not a major effect . however , 
 these are significant inhibitions , in particular for pkc. this 
 has to be considered in further investigations into this compound . a screen of 109 kinases showed that many kinases are not or only 
 slightly affected by 8 ( table 2 ) . among the kinases inhibited by 8 are erk1 , nuak1 
 pim3 , btk , and rsk2 . as shown in figure 2a , 25 m solution of 8 inhibited 
 the pkc/rack2 interaction to approximately 10% of untreated 
 controls , whereas 25 m solution of 8 inhibited 
 the most affected kinase rsk2 to 27% ( table 2 ) . screening was performed by the 
 national centre for protein kinase profiling , division of signal transduction 
 therapy , university of dundee . the data are portrayed as mean % activity 
 and standard deviation of assay duplicates . the 
 exact functions of the different pkcs in the cells are still being 
 actively investigated . the corresponding sequences , in particular in pkc 
 isozymes , exhibit strong structural similarities . therefore , kinase 
 inhibitors frequently are not selective for a particular kinase . in 
 order to identify a selective inhibitor of pkc signaling , we 
 sought to prevent the pkc/rack2 interaction . a pharmacophore - based 
 approach utilizing the crystal structure of the c2 domain of pkc was employed to identify a candidate molecule ( 1 , figure 1c ) , which is based on a thienoquinoline core . further 
 thienoquinolines related to the structure of 1 were explored , 
 leading to the discovery of 8 , which prevents this interaction 
 with a half - maximal inhibitory concentration of 5.9 m . although 8 inhibits other kinases to a certain extent ( figure 2a , table 2 ) , this level of 
 selectivity is acceptable for an inhibitor lead . in addition , detergent - sensitive 
 aggregation effects may also be involved at such high concentrations 
 of inhibitors . medicinal chemistry - driven 
 optimization of this lead structure is expected to lead to a substantial 
 improvement of inhibitor specificity . the aim of developing 
 an inhibitor of signal transduction is ultimately to use it for therapeutic 
 purposes . however , because of their particular physicochemical 
 properties ( extensive , hydrophobic , surface - exposed interfaces ) , protein  protein 
 interfaces are more difficult to target than classical ligand - binding 
 sites . protein interactions have 
 been reported in recent years , but experience is still quite limited 
 and experimental aspects are challenging as well . the eavslkpt peptide , derived from the binding site of 
 pkc to rack2 and used for molecular modeling , inhibits this 
 interaction with an ic50 of 1.02 m . therefore , a 
 peptidomimetic of eavslkpt with an ic50 of 5.9 m 
 may be approaching the maximum achievable inhibitory activity , considering 
 the size of this molecule . also aurothiomalate , which prevents the 
 interaction of pkc and its adaptor protein par6 , is active 
 in the range of 10 m . for clinical 
 application a compound with an ic50 in the nanomolar range 
 would be preferable . however , this is not essential . for example , 
 the ribonucleotide reductase inhibitor hydroxyurea is used in the 
 clinic as an anticancer agent . furthermore , many clinically relevant kinase inhibitors that are 
 active at nanomolar concentrations in vitro have cellular ic50 values closer to the micromolar range because of the higher physiological 
 concentrations of atp relative to those typically used for in vitro 
 assays . in particular for compounds with high ic50 values 
 solubility might be a problem . therefore , 
 an inhibitor of the pkc signal transduction might exhibit antitumor 
 activity . however , as shown in figure 6b , the 
 proliferation of hela and pc-3 tumor cells was not inhibited by 8 , which illustrates that the compound seems not to exhibit 
 general antitumor activity , although mechanism - based anticancer applications 
 can not be ruled out in tumors in which pkc activation is associated 
 with the driving mutation . the data also indicate that the compound 
 does not exhibit toxicity , a particularly favorable characteristic 
 for a potential application as an inhibitor of pkc signaling 
 to treat diseases such as myocardial hypertrophy , diabetes , stroke , or pain . our results are in agreement with reports showing that pkc does not increase cell proliferation ( figure 6b ) but is involved in cell migration and invasion 
 ( figures 6c and 7 ) . the potential use of the compound for pharmaceutical purposes has 
 to be examined in additional projects . sequence 
 similarities within the 10 members of the pkc family render the development 
 of isozyme - selective small molecule inhibitors of pkc a highly 
 challenging task . in this study , our aim was to develop a molecule 
 blocking the activity of pkc by preventing it from binding 
 to its adaptor protein , rack2 . we successfully identified a cluster 
 of active molecules based on a thienoquinoline ligand core . the most 
 active molecule , compound 8 , interferes with the pkc/rack2 
 interaction with an activity comparable to that of a known peptidic 
 inhibitor of this interface ( eavslkpt ) . compound 8 inhibits 
 the phosphorylation of the pkc-downstream target elk-1 , the 
 phosphorylation of marcks , and the tpa - induced translocation of pkc 
 ( but not that of its nearest relative pkc ) from the cytosol 
 to the membrane . it reduces cell migration into a gap and invasion 
 through a reconstituted basement membrane matrix and inhibits angiogenesis 
 in a chicken egg assay . we believe that the herein reported disruptor 
 will be useful for investigating the function of pkc and may 
 serve as an interesting starting point for the development of drug 
 molecules targeting this protein with adequate specificity . for 
 in vitro screening purposes , all 19 initially selected compounds ( including 
 compound 1 , reported in supporting 
 information table 1 ) were purchased from asinex ltd . , this stock was incubated at 56 c 
 for 1 h to dissolve the compound completely . for biological characterization of compound 8 a fully characterized batch synthesized in house and checked 
 for identity and purity was used . mp > 250 c ( dec ) ( crystallized 
 from dmf  meoh ) . h nmr ( dmso - d6 ) :  2.58 ( s,3h ) , 4.42 ( d , j = 6 hz , 
 4h ) , 7.457.49 ( m,3h ) , 7.55 ( s,2h ) , 7.67 ( d , j = 6 hz , 1h ) , 8.01 ( d , j = 6 hz , 1h ) , 8.34 ( s , 1h ) , 
 8.89 ( s , 1h ) , 9.58 ( s , 1h ) . hrms - esi calcd ( c22h17n3o4s + h ) , 420.101  82 ; found , 420.1043 
 ( m + h ) ; 442.0866 ( m + na ) ; 861.1839 ( 2 m + 
 na ) . hplc , 95.05% ( h2o + h3po4 , 0.01 m , ph 2.6/mecn ) . identity and purity ( > 95% ) of 8 was determined using thin - layer chromatography and liquid 
 chromatography  mass spectrometry . h nmr , esi - ms , 
 and hplc analyses of 8 are shown in the supporting information figures 36 . pdb entry 1gmi was used as a basis for the definition 
 of structure - based pharmacophores of the pkc protein fragment 
 eavslkpt . , san diego , ca , usa ) 
 was employed for pharmacophore modeling and screening . , 
 moscow , russia ) were generated applying the fast conformational search 
 algorithm with a maximum of 250 conformers created per molecule . hela cells containing a tetracycline / doxycycline - inducible 
 constitutively active pkc were described previously . a pathdetect hela hlr cell line was obtained 
 from agilent , technologies , wokingham , u.k . hela hlr and 
 pc-3 cells were seeded at 10  000 cells per well in 
 96-well plates . after 4 h various concentrations of 8 were added and left for 72 h. cell proliferation was determined 
 by the srb assay . recombinant rack2 tagged with maltose - binding 
 protein ( rack2-mbp ) was purified on columns with amylose resin ( new 
 england biolabs , ipswich , ma ) as described by the manufacturer . the 
 recombinant protein was analyzed by coomassie blue staining and western 
 blotting after sds  page . the interaction between pkc and rack2 was measured using an 
 elisa - based assay . the 96-well eia / ria high binding plates ( costar ) 
 were coated with 100 ng of recombinant pkc ( proqinase , freiburg , 
 germany ) in buffer a ( 20 mm tris - hcl/100 mm nacl , ph 7.5 ) at 4 c 
 on a shaker with gentle agitation overnight . the plate was washed 
 twice with 225 l / well buffer a. after blocking of unspecific 
 binding sites with 225 l of sterile - filtered 3% bovine serum 
 albumin ( bsa ; sigma - aldrich , st . louis , mo ) in buffer a at room temperature 
 for 3 h , the plate was washed twice with 225 l of this buffer . pkc was left untreated or activated by addition of 60 g / ml 
 phosphatidylserine ( sigma - aldrich ) and 100 nm tpa ( sigma - aldrich ) 
 in a volume of 50 l of buffer a for 10 min at 30 c . dr . daria mochly - rosen , stanford 
 university , usa , as described in ref ( 15 ) ) was either left untreated or incubated with 
 eavslkpt - r7 or 8 at room temperature for 30 min in a 
 final volume of 50 l of buffer a. then 500 ng rack2-mbp was 
 added to untreated or activated pkc for 1 h at room temperature 
 for binding . the plate was washed twice with 225 l of buffer 
 a. then 100 l of rack2-specific rabbit anti - rack2 polyclonal 
 antibody ( obtained from prof . f. wieland , university of heidelberg , 
 germany ) diluted 1:20  000 in 3% bsa / buffer a was added for 
 1 h at room temperature . the plate was subsequently washed three times 
 with 225 l of buffer a , and a goat anti - rabbit hrp - conjugated 
 igg ( santa cruz biotechnology , santa cruz , ca ) diluted 1:20  000 
 in 100 l of 3% bsa / buffer a was added for 1 h at room temperature . after three washes with 225 l of buffer a , 100 l of 
 abts substrate ( 0.5 mg / ml ) diluted in abts buffer ( roche , vienna , 
 austria ) was added and the plate was incubated in the dark for 30180 
 min . color development was measured on a plate reader at a wavelength 
 of 420 nm . 6-his - tagged rack1 was cloned into a pet-30a(+ ) vector ( novagen , 
 merck kgaa , darmstadt , germany ) and purified with ni - nta agarose ( qiagen , 
 hilden , germany ) . final elution was performed with 500 nm imidazole 
 in elution buffer ( 20 mm tris - hcl/300 mm nacl , 20% glycerol , ph 7.5 ) . the integrity of purified recombinant protein was analyzed by coomassie 
 blue staining and western blotting . then 200 ng rack1 was coated onto 
 96-well eia / ria high binding plates ( costar , corning , new york , ny ) 
 at 4 c on a shaker with gentle agitation overnight . then 500 ng of 
 recombinant gst - tagged pkcii ( proqinase ) was activated with 
 cacl2 , phosphatidylserine , and tpa for 10 min . the rack1-coated 
 plates with and without 8 were incubated for 1 h with 
 activated pkcii . the interaction was determined with a primary 
 rabbit anti - gst antibody ( santa cruz ) and corresponding hrp - conjugated 
 goat anti - rabbit hrp conjugated secondary antibody ( santa cruz ) as 
 described above for the pkc/rack2 interaction . elk-1 phosphorylation was determined with the pathdetect system 
 ( agilent , santa clara , ca ) . pathdetect hela hlr - elk-1 cells contain 
 a luciferase reporter cassette and express a unique , stably integrated , 
 trans - acting fusion protein . the fusion protein consists of the activation 
 domain of the elk-1 transcriptional activator that is fused 
 to the yeast gal4 dbd ( residues1147 ) . an amount of 200  000 
 pathdetect hela hlr - elk-1 cells per well was seeded in a six - well 
 plate and grown for 24 h. cells were washed with phosphate buffered 
 saline and starved for 16 h in starvation medium ( dmem - containing 
 0.5% fetal bovine serum and 1% glutamine ) . compounds were added in 
 dmem for 30 min . following treatment with 50 nm tpa for 5 min the 
 cells were washed twice with phosphate buffered saline and incubated 
 for 4 h in starvation medium and compound 8 . lysates 
 were collected and centrifuged at 11000 g at 4 c 
 for 2 min and used immediately for luciferase activity measurement . protein concentration was determined according to bradford , and 20 g of each sample was transferred 
 to a white , opaque 96-well plate . an amount of 150 l of luciferase 
 assay buffer as described by the manufacturer was injected , and light 
 emission from the reaction was measured for 3 s after a delay time 
 of 2 s. relative light units were measured with a 1450 microbeta wallac 
 jet luminometer ( perkin - elmer , waltham , ma ) . for pkc translocation pc-3 cells 
 were starved for 16 h , treated with 8 for 30 min , stimulated 
 with 50 nm tpa for 5 min , lysed , and fractionated with a cnmcs / cnm 
 compartmental protein extraction kit ( biochain institute , newark , 
 ca ) . western blotting was performed by a standard procedure as described 
 by garczarczyk et al . cytosolic and membrane fractions 
 were loaded onto sds gels and transferred to an immobilon membrane 
 ( millipore , billerica , ma ) . the membranes were incubated with rabbit 
 polyclonal igg antibodies for detection of pkc ( santa cruz 
 biotechnology ; dilution 1:2000 ) , for pkc phosphoser ( millipore ; dilution 1:1000 ) , for pkc ( santa cruz biotechnology ; 
 dilution 1:1000 ) , and for marcks phosphoser152/156 ( cell signaling 
 technology , danvers , ma ; dilution 1:1000 ) . for the loading control 
 tubulin ( santa cruz biotechnology ; dilution 1:1000 ) , and as marker 
 for the membrane fraction , an igfi-r rabbit polyclonal igg 
 antibody ( santa cruz ; dilution 1:1000 ) and , as secondary antibodies , 
 peroxidase - conjugated affinipure goat anti - rabbit igg ( jackson immunoresearch 
 laboratories , west grove , pa ; dilution 1:20000 ) were used . gapdh ( chemicon , 
 millipore , billerica , ma ; dilution 1:10000 ) was used as loading control 
 for the cytosolic fraction . a peroxidase - conjugated secondary antibody 
 ( affinipure goat anti - mouse igg ; jackson immunoresearch laboratories ; 
 dilution 1:20000 ) was employed for detection . pc-3 cells were grown on glass coverslips coated with poly - l - lysine ( sigma - aldrich ) . after treatment with 8 for 
 30 min and with 100 nm tpa for 5 min , the cells were rinsed twice 
 with phosphate buffered saline ( pbs ) and fixed with filter sterilized 
 4% ( w / v ) paraformaldehyde/4% sucrose ( w / v ) ( both from sigma - aldrich ) 
 in pbs at room temperature for 10 min . subsequently the cells were 
 washed three times with pbs and permeabilized with 0.2% triton x-100/0.2% 
 igg - free bsa in pbs at room temperature for 10 min . after blocking 
 with 5% normal goat serum diluted in pbs as described above for 30 
 min , cells were incubated with the primary antibodies for pkc 
 ( santa cruz ; diluted 1:500 ) in 0.2% subsequently , the cells were washed 
 three times with the same buffer and incubated with the labeled secondary 
 antibody ( alexa fluor , invitrogen , life technologies , carlsbad , ca ; 
 1:4000 ) at room temperature for 1 h. after three more washes with 
 0.2% triton x-100/0.2% igg - free bsa in pbs , cells were mounted with 
 mowiol ( sigma - aldrich ) and images were taken with an olympus bx 50 
 optical microscope ( olympus corporation , tokyo , japan ) . for cell migration and motility , a scratch migration 
 assay described by cha et al . was employed . 
 in a tissue culture dish , with logarithmically growing hela cells 
 containing a doxycycline - inducible constitutively active pkc , a migration gap of approximately 1 mm was created by introducing a 
  scratch  to the adherent layer of cultured cells using 
 a sterile gilson 200 l pipet tip . the scratch was administered 
 by hand with sufficient pressure to remove adherent cells from the 
 polystyrene substrate but without causing physical damage to the polystyrene 
 surface . the dish was washed with pbs to remove the cells and further 
 incubated with 2 g / ml doxycycline and 25 m 8 for 24 h. controls were left either untreated or were incubated 
 with 2 g / ml doxycycline and dmso . invasion of hela cells through 
 a reconstituted basement membrane matrix was determined with a cell 
 invasion assay kit from chemicon ( no . eggs from hen 
 were incubated at 37 c for 3 days , opened , and incubated for 
 further 7 days . on day 10 , the growth factor vegf and 8 were added as indicated in figure 8 . ic50 values were determined with calcusyn ( biosoft , cambridge , u.k . ) .
ten 
 protein kinase c ( pkc ) isozymes play divergent roles in signal transduction . 
 because of sequence similarities , it is particularly difficult to 
 generate isozyme - selective small molecule inhibitors . in order to 
 identify such a selective binder , we derived a pharmacophore model 
 from the peptide eavslkpt , a fragment of pkc that inhibits 
 the interaction of pkc and receptor for activated c - kinase 
 2 ( rack2 ) . 
 a database of 330  000 molecules was screened in 
 silico , leading to the discovery of a series of thienoquinolines that 
 disrupt the interaction of pkc with rack2 in vitro . 
 the most 
 active molecule , n-(3-acetylphenyl)-9-amino-2,3-dihydro-1,4-dioxino[2,3-g]thieno[2,3-b]quinoline-8-carboxamide 
 ( 8) , inhibited this interaction with a measured ic50 of 5.9 m and the phosphorylation of downstream target 
 elk-1 in hela cells with an ic50 of 11.2 m . 
 compound 8 interfered with marcks phosphorylation and tpa - induced translocation 
 of pkc ( but not that of pkc ) from the cytosol to the 
 membrane . 
 the compound reduced the migration of hela cells into a 
 gap , reduced invasion through a reconstituted basement membrane matrix , 
 and inhibited angiogenesis in a chicken egg assay .