the proprotein convertases ( pcs ) are members 
 of a mammalian family 
 of endoproteases related to the bacterial subtilisin and the yeast 
 kexin .
there are seven pcs that cleave proteins at paired basic 
 amino acid residues , namely furin , pc2 , pc1/3 , pc4 , pace4 , pc5/6 , 
 and pc7 .
the optimal pc recognition sequence 
 is r - x - k / r - r , while the minimal consensus sequence is r - x - x - r. 
 a variety of substrates have been described including precursors of 
 hormones , enzymes , growth factors , receptors , cell membrane proteins , 
 and plasma proteins but also a number of pathogenic proteins such 
 as viral glycoproteins and bacterial toxins .
our previous work showed that pace4 has a role in prostate cancer 
 cellular proliferation .
pace4 has a wide 
 expression pattern and is constitutively secreted into the extracellular 
 media .
it has been suggested from immunohistochemical 
 observations that in addition to its localization within the secretory 
 pathway , pace4 is also localized at the cell surface through interactions 
 between its cysteine - rich domain ( crd ) and heparan sulfate proteoglycan 
 ( hspg ) or tissue inhibitors of metalloproteinases 
 ( timps ) .
recently , two independent studies 
 ( including one from our group ) showed a specific overexpression of 
 pace4 mrna in prostate cancer tissues .
this overexpression 
 is correlated with higher circulating protein levels in some patients . using a molecular inhibition approach ,
the relevance 
 of pace4 in a prostate cancer model has been demonstrated . as the expression levels of other
pcs remains 
 unchanged , it was suggested that a selective pace4 inhibitor , with 
 limited inhibition toward furin , might provide a useful tool against 
 prostate cancer . to our knowledge
, no such inhibitor has been yet 
 reported ( for complete review see ref . ) . designing 
 specific pc inhibitors represent an important challenge . 

the high homology level deep within the catalytic cleft suggests that 
 small - molecule inhibitors acting as competitive inhibitors will be 
 unlikely to produce any specificity . indeed
, structural evidence indicates that the pc 
 active sites are nearly identical in their s1s4 subsites.a however , there are notable 
 differences found at the s5 subsite and beyond .
this suggests that peptide - based inhibitors could 
 be designed to achieve the desired specificity , although they would 
 require a minimum of six residues .
there is some proof for this concept 
 based on discovered endogenous peptide inhibitors , such as the 7b2 
 ct - peptide , which is a highly potent ( nm range ) and specific pc2 inhibitor .
of course , each pc also has an endogenous inhibitor within its structure , 
 namely their prodomains , of which the c - terminal 
 region provides the critical positions for inhibition at the catalytic 
 sites .
pc prodomains first act in the er as intramolecular chaperones 
 and then as activity delayers through interactions in cis with the 
 active site of their cognate pc .
the derived prodomains have been 
 shown to be potent pc inhibitors in trans but display minimal levels 
 of specificity .
last , the screening of peptide combinatorial libraries 
 has led to the identification of polyarginine peptides as furin inhibitors , however , these are also not highly specific .
the present study 
 reports the development of a new pace4 inhibitor , 
 named the multi - leucine ( ml)-peptide .
our focus remains primarily 
 on discriminating between our target pace4 and furin , which is the 
 only ubiquitously pc enzyme in normal tissues .
since it is known that 
 furin inhibition can be lethal ( i.e. , as demonstrated in furin knockout 
 mice ) , it appears logical to design an inhibitor 
 that primarily avoids furin as a target .
we also present some of the 
 characteristics of the ml - peptide as a potent inhibitor of proliferation 
 in prostate cancer cell lines .
pc prodomains 
 act as cis regulatory inhibitors during the maturation process and 
 have been considered as lead compounds for pc inhibition .
our previous 
 work assessed the inhibitory potency of the prodomains as trans competitive 
 inhibitors .
however , their inhibitory 
 potency has never been carried out for pace4 until now ( figure 1a ) .
we observed that most pc prodomains displayed 
 similar inhibition potencies for pace4 and furin , with ki values ranging from low nanomolar ( 12.4 nm ) to subnanomolar 
 levels ( 0.34 nm ) .
the inhibition ratios ( furin / pace4 ) were always 
 in the range of 15 for all prodomains , with the exception 
 of the pc7 prodomain , which displayed a 36-fold inhibition preference 
 for pace4 over furin .
the ki obtained 
 with the pc7 prodomain on pace4 ( 0.34 nm ) is lower than the ki previously reported for inhibition of pc7 
 by its own prodomain .
( a ) pcs prodomains were produced 
 and purified to perform inhibition assays toward pace4 and furin . 
 the ratio between ki for furin and pace4 , 
 namely the specificity ratio , point out the selectivity of pc7 prodomain 
 toward pace4 .
kis in the table 
 are means and standard deviations of three independent experiments . 

( b ) pcs prodomain sequence alignment was performed for the region 
 p7p1 downstream primary cleavage site .
dark background indicates 
 conserved residues , while light - gray background indicates residues 
 of same type than consensus .
uniprotkb accession numbers are the following : hfurin ( p09958 ) , mpc1/3 
 ( p63239 ) , hpc2 ( p16519 ) , mpc4 ( p2921 ) , hpc5/6 ( q92824 ) , hpc7 ( q16549 ) , 
 and hpace4 ( p29122 ) . to further investigate the selectivity of pc7 prodomain 
 inhibition 
 toward pace4
, a sequence alignment was performed , focusing on the c - terminal regions ( figure 1b ) .
although 
 prodomains are approximately 100 amino acids in length , our analysis 
 focused on the dissimilarities in the p1p7 region of the primary 
 cleavage site . because those residues are directly implicated in molecular 
 recognition by pcs , we hypothesized that the 36-fold difference might 
 be related to this c - terminal region .
the sequence 
 alignment ( figure 1b ) reveals that the pc7 
 prodomain differs from the consensus in position p6 , as it does not 
 exhibit a basic amino acid at this position .
apart from the pc2 prodomain , 
 a poor inhibitor of both furin and pace4 , only pc7 is dissimilar from 
 consensus in this position .
it is well - known that the presence of 
 a basic residue in position p6 promotes increased inhibition potency 
 for furin .
however , the observation of leu residues 
 at p6 and p7 positions of the pc7 prodomain suggests that pace4 may 
 have a preference for hydrophobic residues .
the use 
 of combinatorial libraries has proven its relevance for the rational 
 design of inhibitors .
this method has also been used for pc recognition 
 pattern studies . an 8-mer positional scanning - synthetic peptide combinatorial libraries 
 ( ps - spcl ) approach was used to further study differences in furin 
 and pace4 inhibition . because it is hypothesized that specific pc 
 inhibition will be reached by varying the p5p8 positions , 
 this library was synthesized by fixing the p1p4 positions 
 with the core consensus motif rvkr while using the combinatorial method 
 for the p5p8 positions .
this pattern ensures that each peptide 
 in the library has the ability to be recognized and bind to the pc s 
 active site .
the inhibition profiles for pace4 and furin at the p6 
 position are shown in ( figure 2 ) .
as expected , 
 peptides with arg and lys at the p6 position were the most potent 
 pace4 inhibitors ( ki = 40 and 37 nm , respectively ) . 

it is interesting to note that peptides with hydrophobic leu residue 
 displayed equally strong pace4 inhibitory potency ( ki = 49 nm ) ( figure 2 ) .
in contrast , 
 when testing furin inhibition , peptides containing leu in p6 had a 
 much higher inhibition constant ( 900 nm ) whereas peptides with lys 
 and arg at this position maintained mid - nm range inhibition constants 
 ( 230 and 300 nm , respectively ) . for this purpose ,
the specificity 
 ratios were most important and led to the observation that peptides 
 containing a leu residue showed a 18-fold ratio ( furin / pace4 ) .
the 
 second highest ratio was obtained with peptide containing his residue 
 with a 11-fold specificity ratio .
it is relevant to mention that ph 
 differences in the enzymatic assays might influence this result ( ph 
 6.5 for pace4 and ph 7.5 for furin ) given that the histidine side - chain 
 pka is 6.08 .
use of sp - spcl to profile 
 pc - inhibitor recognition . to better understand 
 the recognition patterns of pace4 and furin , sp - spcl was used toward 
 both enzymes .
for each sample in this table , the general recognition 
 pattern rvkr is present in positions p1p4 and p6 position 
 are occupied by a unique amino acid .
the other positions of those 
 peptides are occupied by an equimolar mixture ( x ) of the 19 natural 
 amino acids , cysteine excluded .
ki was 
 calculated from ic50 using the cheng and prussof equation 
 for competitive inhibitors .
thus , this partial combinatorial library approach 
 confirmed that 
 pace4 tolerates the presence of a hydrophobic amino acid in the p6 
 position whereas furin did not and presents a clear preference for 
 basic amino acids in this position . on the basis of these results , 
 peptides containing leu at the p6 position
as 
 a leu containing peptide could offer a selective inhibition toward 
 pace4 , the effects of leu n - terminal extensions of 
 the core sequence ac - rvkr - nh2 , a poor micromolar inhibitor of pcs , was investigated in vitro 
 ( figure 3 ) .
the peptide ac - llrvkr - nh2 was a midnanomolar inhibitor of pace4 , but the progressive 
 addition of n - terminal leu decreased the inhibition 
 constant to the low nanomolar range ( 1822 nm ) for both ac - lllrvkr - nh2 and ac - llllrvkr - nh2 .
however , the subsequent 
 addition of leucine residues increased ki values , reaching higher nanomolar values ( 300 nm ) for the decapeptide 
 ac - llllllrvkr - nh2 .
peptides 
 containing three to four leu residues were the most potent inhibitors 
 of pace4 evaluated in this study and were significantly more effective 
 on pace4 than furin ( 2022-fold ) .
the peptide ac - llllrvkr - nh2 and now designated as the 
 ml - peptide was chosen as lead inhibitor for further characterization 
 on pace4 inhibition .
the inhibitory potency of the ml - peptide was 
 also assayed with other members of the pc family and also showed high 
 levels of specificity ( supporting information 
 table s1 ) . multi - leucine peptides . to stabilize pc 
inhibitor 
 interaction , n - terminal leucine extensions were added 
 to the core rvkr 
 sequence .
( a , b ) each peptide was assayed with pace4 and furin in an 
 inhibition assay .
( c ) kis in this table 
 are means and standard deviations of three independent experiments . 

peptides ac - lllrvkr - nh2 and ac - llllrvkr - nh2 were 
 the most potent and the most selective inhibitors of pace4 of this 
 library .
the peptide ac - llllrvkr - nh2 , named multi - leu peptide ( ml ) , was selected as lead compound 
 for further studies . in a previous study ,
pace4 was proposed as a new therapeutic target 
 in prostate cancer based on a molecular 
 inhibition approach using the prostate cancer cell line du145 . because 
 the molecular inhibition of pace4 in du145 cells had dramatic effects 
 on cell proliferation in vitro and in vivo
, we decided to test the 
 ml - peptide as a pharmacological inhibitor to achieve identical results . 
 in the present study , two additional prostate cancer cell lines
thus , pace4 expression level 
 was first evaluated in lncap and pc3 cell lines in comparison to du145 
 cells using a rt - qpcr approach ( figure 4a ) . 

pace4 was most highly expressed in lncap cells , with nearly 6-fold 
 higher levels than du145 cells , but was almost absent in pc3 cells . 

du145 and lncap cells also exhibited higher levels of furin mrna than 
 pc3 cells .
similar expression levels were observed for pc5/6 and pc7 
 within all cell lines investigated and pc1/3 and pc2 were undetectable . 

the effect of the multi - leu peptide on cellular proliferation of each 
 cell line was evaluated using mtt assays ( figure 4b ) .
the ml - peptide showed a very poor inhibition of pc3 cells , 
 whereas the half - maximal inhibitory concentrations ( ic50 ) were in the micromolar range for du145 and lncap cells ( 100  
 10 and 180  60 m , respectively ) .
thus , the ml - peptide 
 inhibited the proliferation of du145 and lncap cells , but not pc3 
 cells , showing a strong correlation with cellular pace4 expression .
in previous work , our research team proposed pace4 as a therapeutic 
 target against prostate cancer progression .
( a ) du145 , lncap , and pc3 prostate - cancer derived cell lines were 
 first screened to compare their pcs expression levels using rt - qpcr . 

( b ) to assess the efficiency of our new pace4 inhibitor in such 
 context , mtt assays were performed on those cell lines .
ml is efficient 
 to inhibit proliferation and metabolic activity in du145 and lncap , 
 two pace4-expressing cell lines , indicating a possible role for ml 
 as a prostate cancer therapeutic .
( c ) to prove that the inhibition 
 observed is a pcs dependent mechanism , the peptide ac - llllrvk[darg]-nh2 was used as negative control . 
 because the p1 position is a key residue of the recognition pattern , 
 the replacement of p1 arg by darg significantly affected the ki for this peptide .
( d , e ) as expected from inhibition 
 constant values , the peptide ac - llllrvk[darg]-nh2 is a poor proliferation inhibitor in a mtt assays with 
 du145 and lncap .
an additional control experiment was performed 
 to test the pc - specific 
 interaction of the ml - peptide resulting in cell proliferation inhibition 
 by designing a ml - peptide substituted at the p1 position with a darg . 
 as the p1 arg position is critical for pc recognition
, this modification 
 should strongly abrogate the observed effects unless they are not 
 pc - mediated .
as expected , the peptide ac - llllrvk-[darg]-nh2 showed a substantial loss of affinity 
 in vitro going from a nm to a m inhibitor ( figure 4c ) ( kis = 1380 and 2600 nm for 
 pace4 and furin , respectively ) .
consistent with this affinity loss , 
 this peptide also showed a significant loss of potency in both du145 
 ( figure 4d ) and lncap ( figure 4e ) cell - based assays ( ic50 440  80 and 390 
  10 m , respectively ) .
these data make a strong case that 
 decreased cell proliferation is mediated by pc inhibition .
pace4 is localized both at the cell surface 
 and within the intracellular secretory pathway .
thus , it was relevant to determine whether 
 the inhibitory effect of the ml - peptide is mediated by cell surface 
 and/or intracellular pace4 . to elucidate this question , ml - peptide 
 analogues bearing n - terminal modifications with strikingly 
 different cell penetration properties
was coupled with an n - terminal amino - poly(ethylene 
 glycol)-8 ( -amine--propionic acid octaethylene glycol , 
 known as peg8 ) group that should render the peptide much less hydrophobic , 
 whereas a n - terminal hydrophobic 8-amino - octanoyl 
 ( c8 ) chain was used in order to increase its hydrophobicity . in vitro 
 enzymatic activity assays demonstrated that pegylation and alkylation 
 of the ml - peptide did not alter its potency or selectivity for pace4 
 inhibition ( figure 5a ) .
the uptake of fitc - labeled 
 versions of these ml - peptide analogues were tested on du145 cells 
 and analyzed using a fluorescence - activated cell sorter ( facs ) ( figure 5b ) .
the fitc--ala - ml - peptide had excellent 
 cell penetration properties , considering it is relatively unmodified , 
 suggesting that the hydrophobic content of this peptide is sufficient 
 to penetrate cell membranes ( see also confocal microscopy in supporting information figure s1 ) .
however , cells 
 treated with fitc-[peg8]ml - peptide showed a geometric mean of the 
 distribution ( geomean ) of about 1 log unit lower than the one measured 
 for the fitc--ala - ml - peptide ( 5.437.84 vs 48.9052.82 ) . 

these results indicate that the fitc-[peg8]-ml - peptide penetrates 
 the cell membrane very poorly . as expected , the fitc - c8-ml - peptide 
 displayed greater cell penetration properties with 5-fold greater 
 geomean compared to the control fitc--ala - ml - peptide ( 260.08261.95 
 vs 48.9052.82 , respectively ) .
when all three peptides were 
 tested on du145 cells , the [ c8]-ml - peptide gave very similar results 
 to the ml - peptide by maintaining antiproliferative effects ( ic50s 80100 m ) while the [ peg8]-ml - peptide could 
 no longer reduce cell proliferation ( ic50 was estimated 
 to be > 500 m ) ( figure 5c , d ) .
this 
 result 
 provides strong evidence that the antiproliferative actions of the 
 ml - peptide were largely mediated by intracellular pace4 .
a second 
 conclusion is that the cell penetration properties of the unmodified 
 ml - peptide are sufficient to achieve a near maximal effect .
ml - peptide 
 targets intracellular pace4 to inhibit prostate cancer 
 cell lines proliferation .
( a ) to determine whether the cell proliferation 
 effects are mediated by cell surface or intracellular pace4 , ml n - terminal extensions were design to modify cell penetration 
 properties of our inhibitor . neither pegylation ( peg8 ) nor alkylation 
 ( c8 ) modified the affinity of multi - leucine inhibitor toward pace4 
 and furin , as determined in a kinetic assay .
( b ) the uptake of fitc - labeled 
 peptides was tested on du145 cells and analyzed by facs .
comparison of geomeans 
 ( numbers adjacent the peaks ) obtained with these three peptides indicates 
 that pegylation prevents cell entry , whereas alkylation increases 
 the uptake of the inhibitor .
( c ) using a mtt metabolic assay , it was 
 clearly demonstrated that alkylation increases inhibitory potency 
 of ml peptide , whereas pegylation leads to a poor proliferation inhibitor 
 in du145 cell line .
( d ) ic50 in the table are means of 
 five independent experiments . n.c .
means the curve did not converged 
 to 50% with doses up to 500 m .
this assay demonstrates that 
 ml peptide must enter the cell in order to inhibit du145 proliferation . to obtain 
 further support for the antiproliferation effects observed , a dna 
 content analysis on lncap treated cells
was performed with 100 or 
 200 m of ml - peptide ( figure 6 ) . as these 
 assays are performed over a 96 h period , which could result in peptide 
 degradation , a modified analogue ac-[dleu]lllrvkr - nh2 was also used .
the substitution of 
 the n - terminal leu by a dleu provided improved stability 
 of the peptide to amino - peptidases ( unpublished data ) . a dose - dependent 
 g0/g1 accumulation and s phase decrease were 
 observed following exposure to ml - peptide and ml - peptide analogue . 
 following a 200 m treatment with the ac-[dleu]lllrvkr - nh2 , a 10% increase in the g0/g1 population
was observed along with an increase 
 in cells with hypodiploid dna content ( sub - g1 ) proportions , 
 which represent apoptotic cells .
the effect of pace4 inhibition on cell cycle distribution was observed 
 by flow cytometry .
lncap cells were treated for 96 h with 100 or 200 
 m of peptide .
( a ) ac - llllrvkr - nh2 or ( b ) ac-[dleu]lllrvkr - nh2 .
cell cycle distribution was assessed from 
 cellular dna content analysis of cells treated with propidium iodine . 

the significance of the results were established from 
 an unpaired two - tailed t test .
( * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 ) .
( c ) data in the table are mean and standard deviation 
 of a representative experiment .
furin was the first discovered 
 pc , and ever since the discovery 
 of additional pcs , issues of distinct and redundant functions have 
 been debated for this family of enzymes .
while this remains 
 a fundamental interrogation , it can also be envisaged that an answer 
 to this question could have repercussions on the druggability of pcs 
 in various pathologies .
structural data have been very clear that 
 within the deep subsites of the catalytic cleft ( s1s4 ) , the 
 pcs appear to be virtually identical .
this has an immediate consequence 
 from a medicinal chemistry point of view , that small - molecule inhibitors 
 ( i.e , small organic compounds ) will not be specific if they are competitive 
 inhibitors .
it can not be excluded that small molecules could specifically 
 inhibit pcs if they have an allosteric mechanism of action , but to 
 date no such inhibitors have been reported nor have druggable allosteric 
 pc sites been established . therefore , the most probable option presently 
 is to use peptides as lead compounds to design specific pc inhibitors . 

this is plausible based on the observed structural differences within 
 the s4s7 subsites , as deduced by homology modeling of the 
 furin structure .
peptide - based inhibitors afford a number of advantages , 
 the principal one being a great variety of structures that could provide 
 an optimal fit , providing the sought specificity .
the disadvantages 
 of peptide - based inhibitors are in their further use as in vivo drug 
 compounds because they can be readily degraded , may be excreted rapidly , 
 and are said not to be very cell penetrable .
there is mounting 
 evidence of the potential benefits of inhibiting pace4 in prostate 
 cancer cells .
should a therapeutic application be possible , it would 
 have to be through the development of a relatively specific pace4 
 inhibitor .
thus , we have focused our studies on a peptide - based approach 
 to target pace4 , with the full knowledge of the obstacles that may 
 arise . as the starting point to design a specific pace4 inhibitor , 
 the 
 potency and the selectivity of purified pc prodomains toward pace4 
 and furin were evaluated ( figure 1a ) .
as furin 
 is the only basic amino acids cleaving pc ubiquitously expressed ( i.e. , 
 defined as expressed in every single normal cell ) and furin full knockouts 
 in mice have proven to be lethal , it 
 seemed appropriate to establish an inhibitor that favors pace4 as 
 much as possible over furin .
this objective may appear almost insurmountable 
 due to the stated structural similarities of the two enzymes and multiple 
 data suggesting very similar substrates . nonetheless , the peptide 
 designs focus on obtaining the best specificity ratio of furin / pace4 
 inhibitory potency ( furin ki /pace4 ki ) . as previously reported in various studies , including 
 our own previous 
 work , pcs prodomains are highly potent inhibitors of pcs 
 ( nm and sub - nm range ) , except for pc2 prodomain , which displays no 
 inhibition at concentrations up to 1 m
. however , one observation 
 stands out , namely the specificity ratio of the pc7 prodomain , which 
 was 36-fold ( furin ki / pace4 ki ) .
the alignment of pc prodomains was performed for region 
 p7 to p1 , as this region contains key residues for substrate - pcs recognition 
 ( figure 1b ) and highlights the higher density 
 of hydrophobic residues in this region .
the pc7 prodomain clearly 
 stands out as it differs from the consensus in position p6 .
this suggests 
 that basic residue in p6 position could be crucial for potent furin 
 inhibition , whereas pace4 may tolerate a broad range of residues for 
 this position .
the combinatorial peptide library provided additional 
 support for 
 this hypothesis ( figure 2 ) .
this library was 
 anchored at the c - terminal with the consensus motif 
 rvkr to allow the study of p5p8 positions .
it was first observed 
 that kis were higher for furin than pace4 
 for every peptide mixture . as positions p8 , p7 , and p5 of those samples 
 are occupied by an equimolar mix of the 19 natural amino acids ( cysteine 
 excluded ) , this could indicate that pace4 can accommodate a more diversified 
 selection of amino acids .
thus , when looking at kis for position p6 for furin , it can be concluded that 
 furin has a marked preference for basic residues , as lys and arg peptides 
 are the only midnanomolar inhibitors of this enzyme in this library . 
 on the other hand , although p6 lys and p6 arg peptides are mid - nm 
 inhibitors of pace4 , similar levels of inhibition can be reached with 
 p6 leu peptides .
the highest specificity ratios for p6 position were 
 obtained from leu , his , val , ile , and ala containing peptides .
on 
 the basis of the hydrophobic nature of leu , val , ile , and ala , this 
 suggests that pace4 tolerates the presence of hydrophobic residues 
 in position p6 and this opens possibilities to design specific inhibitors .
the synthesized series of ml - peptides validated the differences 
 between furin and pace4 in the p5p8 region . to date , the ml - peptide 
 is the best pace4 specific inhibitor described .
although furin and 
 pace4 share very identical catalytic domains and an almost identical 
 catalytic cleft , our design of the ml - peptide selective 
 pace4 inhibitor shows that even with these similarities , both enzymes 
 have different binding affinities .
the results highlight the fact 
 that furin does not provide nonpolar peptide stabilization in its 
 catalytic cleft while pace4 has good binding affinity with both cationic 
 and nonpolar peptides . to hypothesize on the origin of partial selectivity 
 between differently charged substrates , a homology model of pace4 
 containing peptide ac - rvkr - cmk in the binding cleft 
 was built using modeler 9v8 from crystalline furin 1p8j(31 ) ( figure 7 ) .
stereoscopic views of ( a ) mouse furin 
 crystal catalytic cleft ( 1p8j ) and ( b ) pace4 homology model .
asp and glu negative 
 charges are shown in red , whereas arg and lys positive charges are 
 shown in blue .
green spheres represent dicationic calcium ions and 
 decanoyl - rvkr - cmk inhibitor has been modified to 
 ac - rvkr - cmk for clarity .
a close examination of the whole catalytic domain 
 of pcs shows 
 various degrees of charge excess , as previously reported for other 
 subtilisins . by looking at the number 
 of positively charged arg and lys ( i.e. , his being mostly neutral 
 at cytoplasmic ph
is left out ) and negatively charged asp and glu , 
 the overall total charge calculated for both convertase catalytic 
 domains are 7 for human furin and 0 for human pace4 ( i.e. , 
 the catalytic domain of furin goes from n - terminal 
 prodomain primary cleavage site asp108 to val444 and from gln150 to 
 ala496 in pace4 ) .
the active site of both furin and pace4 also reveals 
 that the s1 , s2 , and s4 sites are formed by residues with similar 
 properties , e.g. , glu257 and glu230 in furin replace asp309 and asp282 
 in pace4 , respectively ( figure7 ) . on the other 
 hand ,
the differences in affinity observed with multiple leu extension 
 in the ml - peptide propose the existence of notable differences in 
 subsite s5 and beyond . to gain a deeper insight of the phenomenon , 
 it is necessary to dissect the binding cleft of both pcs .
one common 
 feature among all the subtilases is the presence of a groove running 
 from the catalytic site to helixes 3 and 4 that stabilizes 
 a peptidic substrate in an antiparallel -sheet conformation 
 through an induced fit process .
this interaction can also be seen 
 in the noncatalytic pcsk9 structure ( 2pmw ) between the prodomain and the region 
 homologous to the catalytic cleft of the other pcs ( ser147 to gln152 ) . despite the fact that other residues might be 
 involved ,
the same trend remains for furin and pace4 : a way of hosting 
 a peptidic substrate or inhibitor adopting an antiparallel -sheet . on the basis of these data
, we propose that 
 the linear conformation of the peptide brings the p6p8 residues 
 of the inhibitor in the vicinity of the residues located at the tip 
 of helix 4 , and this region of the enzyme would represent the 
 subsites s6 , s7 , and s8 .
we also focused our analysis on the disparities 
 in this region to better understand the specificity observed for ml - peptide 
 because the solvent accessible residues of helix 4 differ in 
 pace4 and furin . from the homology model of pace4
, it appears that 
 the most critical dissimilarity in the helix 4 comes from the 
 replacement of acidic residues in furin by either basic or neutral 
 residues in pace4 ( glu271 and glu272 in furin exchanged for lys323 
 and gln324 in pace4 ) .
consequently , the n - terminal 
 end of helix 4 has a charge of 1 in furin and + 2 in 
 pace4 .
finally , an intramolecular quench of charges in pace4 may explain 
 its potential to complex neutral ligands .
such self - quenching is unlikely 
 for furin in the n - terminal region of the helix 4 . 
 in sum , the fact that furin catalytic clef is more negatively charged 
 than pace4 might very well account for much of the selectivity showed 
 by the two pcs .
the cellular effects of the ml - peptide are remarkable , 
 as they 
 phenocopy the antiproliferation effects observed in our previous molecular 
 studies knocking down pace4 in du145 cells .
this study also provided additional evidence for the pace4 targeted 
 antiproliferation using lncap cells .
however , the effects of the ml - peptide 
 are largely ineffective in the pc3 cell lines , as it expresses very 
 little if any pace4 . as a control ,
the polybasic peptide ac - rarrrkkrt - nh2 , which is a potent furin 
 inhibitor of similar length , was tested 
 in the mtt cell - based assays ( with lncap and du145 cells ) and no antiproliferative 
 effects could be observed with this peptide ( supporting 
 information figure s2 ) .
we are confident that 
 the furin target was reached by the peptide , as in a previous study 
 we showed that this potent furin inhibitor has cell penetration properties .
additional control peptides provide evidence 
 that the ml - peptide indeed targets pace4 because the ac - llllrvk[darg]-nh2 shows a substantial loss 
 of affinity in vitro and is also a poor inhibitor of du145 and lncap 
 cell proliferation ( figures 4d , e ) .
pace4 
 is localized at the cell surface and in the extracellular 
 matrix due to interactions between its cycteine - rich domain ( crd ) , 
 heparin sulfate proteoglycans ( hspgs ) , and tissue 
 inhibitors of metalloproteinases ( timps ) . to assess the importance of cell surface pace4 in tumor progression , 
 the ml - peptide was modified with n - terminal pegylation , 
 significantly reducing its cell penetration property while not affecting 
 its inhibitory potency ( i.e , pegylated ml - peptide has a similar ki to the ml - peptide ) .
because the pegylated 
 ml - peptide significantly losses its ability to inhibit du145 proliferation , 
 it leads to the conclusion that intracellular pace4 is required for 
 this function .
a reduction of cellular proliferation could occur 
 by a number of 
 mechanisms downstream of pace4 inhibition , but most likely a number 
 of important growth factors which are substrates of pace4 have lost 
 their activity due to lack of processing .
this general lack of growth 
 factor activity would eventually have effects on the cell cycle .
therefore , 
 a cell cycle analysis was performed to evaluate if cell cycle arrest 
 or slow - down was possible .
a dose - dependent accumulation of cells 
 in g0/g1 phase was observed , thus preventing 
 cells entry into s phase .
the transition between g1 and 
 s phase is a finely regulated mechanism controlled by a combination 
 of environmental considerations mostly influenced by the presence 
 of growth signals and the discontinuation of extracellular inhibitory 
 signals .
this could suggest that the ml - peptide inhibits the processing 
 of growth factors required to go beyond the restriction point and 
 therefore triggers a cell cycle arrest at this point .
however , while 
 outside of the scope of the present manuscript , further studies identifying 
 these substrates and signaling pathways involved will be required . 
 nonetheless , various potential substrates have been suggested in various 
 other studies . of interest was the observation 
 of apoptosis following an exposure to 200 m ac-[dleu]-lllrvkr - nh2 in lncap cells .
this phenomenon 
 may be easily explained by the fact that cell cycle arrest is usually 
 poorly tolerated and prolonged cytostasis must be escaped by cell 
 death .
if this is true , then more potent 
 and stable versions of the ml - peptide inhibitor could result in exceptional 
 drugs that reduce prostate cancer cell proliferation as well as inducing 
 specific cell death . in the context of the metastasis phase of prostate 
 cancer
synthesis of sp - spcl peptide library 
 was performed as described previously ( torrey pines institute for 
 molecular studies ) .
all other peptides were obtained 
 by solid - phase peptide synthesis ( 50 mol scale ) on a polystyrene 
 resin , tentagel s ram ( rapp polymere , capacity 0.23 mmol / g ) , with 
 a pioneer peptide synthesizer ( applied biosystems ) , according to standard 
 coupling procedures and fmoc strategy .
the protected amino acids were coupled at 3-fold excess using hatu 
 as coupling agent in the presence of dipea in dmf .
after the final 
 fmoc deprotection , with the exception of fitc - labeled peptides , n - terminal acetylation was carried out in dmf with acetic 
 anhydride ( 0.5% ) and 2.6-lutidine ( 0.6% ) .
the fitc - peptides were labeled 
 with fluorescein isothiocyanate isomer i ( fitc ) through their n - terminus ( -ala was used as a spacer for the peptide : 
 fitc-[-ala]-llllrvkr - nh2 ) .
dcm ( 1:4 ) was added 
 to the resin and allowed to couple overnight .
after completion of 
 the synthesis , the protected peptidyl resins were treated with tfa  h2o 
tis ( 95:2.5:2.5 ) and stirred for 3 h. the solutions 
 with the released peptides were filtered and evaporated in vacuo to 
 a volume of about 1 ml . then the peptides were precipitated with diethyl 
 ether to afford crude products .
the crude compounds were purified 
 by semipreparative hplc ( agilent technologies , 1100 series hplc equipped 
 with a diode array detector ( dad ) ) on reversed - phase support agilent 
 c18 column ( 15 m , 100  , 7.8 mm  300 mm ) . 

the purity of the peptides was controlled using analytical hplc . a 
 seldi - tof mass spectrometer ( bio - rad laboratories )
was used to confirm 
 the identity of the pure products ( molecular ion ) . according to both 
 hplc and mass spectrometry ,
the peptide content analyses were performed 
 in vacuo with a beckman 120c autoanalyzer following 24 h hydrolysis 
 in 5.7 n hcl at 110 c and analyzed using a varian ds604 system 
 integrator .
recombinant 
 soluble human pcs were produced from s2 insect cells and purified 
 as previously described .
briefly , s2 
 conditioned medium was purified using ultrafiltration , anion exchange 
 chromatography , hydrophobic interaction chromatography , and gel filtration . 

the prodomains of pcs were produced in one shot top 10 cells ( invitrogen ) 
 transformed with cdna construct of full - length prosegment .
the recombinant 
 prodomains were purified from bacterial lysate by nickel chromatography 
 and reverse - phase hplc as previously described .
enzyme inhibition assays for 
 furin were performed in 100 mm hepes ph 7.5 , 1 mm cacl2 , 1 mm -mercaptoethanol , and 1.8 mg / ml bsa , while assays for 
 pace4 were performed in 20 mm bis - tis ph 6.5 , 1 mm cacl2 , and 1.8 mg / ml bsa . all assays were performed with the substrate 
 pyroglu - arg - val - lys - arg - methyl - coumaryl-7-amide ( bachem , ca ) at 100 
 m .
assays were carried out at 37 c for 60 min , and real - time 
 fluorescence was measured using a gemini em 96-well spectrofluorometer 
 ( molecular devices , ca ) ( em , 370 nm ; ex .
inhibitory peptides and prodomains 
 were added to the assays at decreasing concentrations to perform a 
 competitive inhibition assays .
kinetics assays were analyzed using 
 softmaxpro5 , and ki values were determined 
 from ic50 using cheng and prusoff s equation with km values of 
 4.61 m for furin and 3.5 m for pace4 .
the quality of the total rna sample 
 was assessed using an agilent bioanalyzer with the rna nano chip ( agilent 
 technologies ) .
briefly , 1 g rna was reverse transcribed and 
 the obtained cdna was used to carry out qpcr analysis .
relative expression were calculated using -actin 
 as a reference gene and the formula ( 1 + amplification efficiency ) 
 
(ct ) for each cell line , as described previously . to perform mtt assays , 
 both du145 and pc3 cell lines were seeded at a density of 1500 cells 
 per well in 96-wells plates .
lncap cells were seeded at a density 
 of 2500 cells per well in a poly lysine coated 96-well plate .
after 
 24 h , media was changed and inhibitory peptides were added to the 
 cells . the peptides were incubated with the cells for 72 h prior to 
 addition of mtt reagent at a final concentration of 1 mg / ml . mtt reagent 
 was incubated 4 h with du145 and lncap and 6 h with pc3 cells , and 
 then media was removed and formazan was solubilized with 100 l 
 of 2-propanol  hcl ( 24:1n ) .
the total metabolic activity was 
 normalized relatively to vehicle treated cells ( sterile bidistilled 
 water 0.1% dmso ) .
each step of mtt , as well as maintenance of du145 , 
 lncap , and pc3 cells , were carried in rpmi 1640 5% fbs for du145 and 
 10% fbs for lncap and pc3 .
the uptake of fitc peptides 
 was tested on du145 cells and analyzed with a facscan cytometer ( becton 
 dickinson , mountain view , ca ) .
first , 4  10 cells 
 were incubated 1 h in serum - free rpmi media with 10 m of fitc--ala - ml , 
 fitc-[peg8]-ml , or fitc-[c8]-ml and collected by centrifugation .
then 
 the cell pellets were washed twice with trypsin ( 0.05% v / v ) during 
 5 min at 37 c to remove nonspecific interactions with the membrane . 

cells were incubated 2 min with propidium iodine ( 10 g / ml ) 
 in order to exclude cells with altered membrane .
a minimum of 10000 
 events per sample was acquired , excluding cell clumps and debris . 

geomeans were determined using cellquest software ( becton dickinson , 
 mountain view , ca ) . to perform cell cycle analyses 
 on lncap , 4 
10 cells were seeded in 10 cm culture 
 dishes and grown for 24 h without treatment .
cells were then treated 
 with vehicle ( 0.1% dmso ) or with 100 m or 200 m of peptides 
 ac - llllrvkr - nh2 or ac-[dleu]lllrvkr - nh2 .
treatments were carried 
 out in complete medium ( 10% fbs ) for a period of 96 h , and cell media 
 were changed every 24 h to offset peptide degradation .
cells were 
 harvested using trypsin , washed once with pbs , resuspended in 0.5 
 ml of pbs , and fixed by dropwise addition of 1.5 ml of ice - cold ethanol . 

after a 30 min incubation at room temperature , cells were washed with 
 pbs and dna staining was performed in 20 mm hepes ph 7.2 , 0.16 m nacl , 
 and 1 mm egta buffer containing 10 g / ml of rnasea and 10 g / ml 
 of propidium iodine .
flow cytometry was performed using a facscan 
 cytometer ( becton dickinson , mountain view , ca ) equipped with a 15 
 mw argon ion laser tuned at 488 nm .
forward and side scatter signals were used 
 to establish the live gate to exclude debris and cell clumps and a 
 second live gate was set using the fl3-a and fl3-w parameters of the 
 doublet discrimination module ( ddm ) , allowing single cell measurements . 

the percentages of cells in different phases of cell cycle were calculated 
 by modfit software ( verity software house , topsham , me ) .
 the proprotein convertases ( pcs ) play an important role 
 in protein precursor activation 
 through processing at paired basic residues . 
 however , significant 
 substrate cleavage redundancy has been reported between pcs . 
 the question 
 remains whether specific pc inhibitors can be designed . 
 this study 
 describes the identification of the sequence llllrvkr , named multi - leu 
 ( ml)-peptide , that displayed a 20-fold selectivity on pace4 over furin , 
 two enzymes with similar structural characteristics . 
 we have previously 
 demonstrated that pace4 plays an important role in prostate cancer 
 and could be a druggable target . 
 the present study demonstrates that 
 the ml - peptide significantly reduced the proliferation of du145 and 
 lncap prostate cancer - derived cell lines and induced g0/g1 cell cycle arrest . 
 however , the ml - peptide must enter 
 the cell to inhibit proliferation . 
 it is concluded that peptide - based 
 inhibitors can yield specific pc inhibitors and that the ml - peptide 
 is an important lead compound that could potentially have applications 
 in prostate cancer . 
Introduction
Results
Discussion and Conclusions
Experimental Section