the complement system 
 is implicated in the onset and progression 
 of a number of autoinflammatory diseases . despite growing efforts to identify new complement - targeted therapeutics , 
 only one ( eculizumab , alexion ) is currently in the clinic .
there is a growing need for new therapeutics to treat chronic inflammatory 
 diseases , which include age - related macular degeneration ( amd ) , systemic 
 lupus erythematosus , and rheumatoid arthritis , among many others . 

most complement therapeutics currently in clinical development are 
 biopharmaceuticals , which are prone to challenges in production , delivery , 
 and bioavailability .
few attempts at developing low - molecular mass 
 complement inhibitors have been successful , largely because of the 
 fact that complement activation cascades are comprised of large protein 
compstatin 
 ( table 1 , parent ) 
 is a cyclic peptide that inhibits complement activation 
 ( reviewed in refs ( 2 , 414 ) ) .
it is one of a small number of low molecular mass complement therapeutics 
 in development .
the peptide binds to complement component c3 ( as well 
 as its derivatives c3(h2o ) , c3b , and c3c ) , the central 
 protein of all complement activation cascades , and prevents its cleavage 
 to c3a and c3b , thus blocking generation of complement effector proteins 
 and complexes . since its discovery
, the 
 sequence of compstatin has been optimized to improve its affinity 
 and complement inhibitory activity .
numerous sequence modifications led to the development of w4a9 ( table 1 ) , the most active compstatin peptide with only 
 natural amino acids .
subsequently , many 
 studies explored incorporation of non - natural amino acids and modifications 
 to the compstatin sequence .
early studies of this type led to development 
 of mew4a9 ( table 1 ) , which is currently being 
 pursued for treatment of amd ( clinicaltrials.gov , identifier numbers 
 nct00473928 and nct01157065 ) .
non - natural amino acid abbreviations : 
 mew = l-1-methyltryptophan ; nal = l-1-naphthylalanine ; 
 rea = r--ethylalanine ; aal = r--allylalanine ; sea = s--ethylalanine ; 
 2nl = l-2-naphthylalanine .
all peptides ( except linear ) are 
 cyclized by a disulfide bond between c2 and c12 .
determination of the structures 
 of free ( parent ) and c3c - bound ( w4a9 ) compstatin 
 peptides paved the way for further structure - based design and optimization 
 of compstatin peptides .
the solution nmr structure and subsequent 
 structure  activity studies by nmr identified two opposite faces 
 in compstatin , a hydrophobic face , proposed to be important for binding 
 and inhibitory activity , and a polar face .
the cocrystal structure revealed that compstatin 
 binding is dominated by hydrophobic interactions , accompanied by several 
 hydrogen bonds .
recent efforts have focused on modifying amino acids at positions previously shown 
 to tolerate mutations , but most designed peptides exhibited no significant 
 improvement in inhibitory activity , and some had poor solubility . 

consequently , molecular dynamics ( md ) simulations were used to explore 
 the possibility of incorporating additional polar amino acids at the 
 compstatin ( w4a9 ) n - terminus and n - terminal extensions .
incorporation of hydrophilic amino acids has 
 been recently reported to increase solubility of stapled peptides .
indeed , incorporation of arg at position 1 and 
 ser - ser and arg - ser extensions at positions 1 and 0 showed 
 similar inhibitory activity and improved solubility compared to w4a9 .
this improvement is attributed to increased 
 polarity at the n - terminus and the potential formation of additional 
 polar contacts ( hydrogen bonds and salt bridges ) with c3c . in this study
, we sought to identify new compstatin peptides 
 with 
 sequence modifications that promote both potent complement inhibition 
 and improved aqueous solubility .
upon the basis of results from our 
 previous study , we designed peptides 
 with varied polar n - terminal extensions .
in addition , we introduced 
 a new computational approach for peptide design with non - natural amino 
 acids .
we incorporated non - natural amino acids ( figure 1 ) at positions 4 and 9 , which were previously shown to be 
 amenable for inhibitory activity optimization by the incorporation 
 of non - natural amino acids .
these peptides , and combinations 
 thereof , were screened in functional in vitro and cell - based assays 
 for complement inhibitory activity and using absorbance spectroscopy 
 and hplc for solubility and hydrophobicity .
we identified several 
 new compstatin peptides with improved pharmacological properties for 
 potential therapeutic development .
building upon our recent studies , we designed 
 new compstatin 
 peptides with the aim of simultaneously improving inhibitory activity 
 and aqueous solubility . here , we incorporated components of de novo 
 design , including a new method for incorporation of non - natural amino 
 acids , rational design based on molecular dynamics simulations and 
 previous experimental data , and structure  activity relations . 

the previously known top compstatin peptides , w4a9 and mew4a9 , include 
 modifications at positions 4 and 9 ( relative to parent ) , which enhance 
 inhibitory activity by at least 10-fold in various functional assays . 

while these modifications enhance peptide inhibitory activity , they 
 contribute to increased peptide hydrophobicity , which has led to aggregation 
 in aqueous solution .
here , we introduced a new computational peptide 
 design method , which allows for the introduction of non - natural amino 
 acids , targeting positions 4 and 9 in order to promote sequence diversity 
 and explore new sets of possible potent and soluble compstatin peptides . 

peptides were evaluated based on predicted physical interactions ( contacts , 
 clashes , hydrogen bonds ) with c3c and screened according to computationally 
 predicted binding affinity ( k * ) , relative to control 
 peptides w4a9 and mew4a9 ( table s1 in supporting 
 information ) .
we identified three peptides with higher predicted 
 binding affinity than both w4a9 and mew4a9 ( peptides 1 , 3 , and 4 ) , 
 which were selected for experimental evaluation .
in addition , peptides 
 with similar non - natural modifications ( peptides 2 and 5 ) were included 
 in experimental evaluation as well ( table 1 , set 1 ) .
our most recent study showed that the n - terminal 
 sequence arg - ser - ile 
 forms additional polar and nonpolar contacts with c3c , based on results 
 from molecular dynamics simulations .
, 2013 , sequence ac - rsicv{mew}qdwgahrct - nh2 , cyclized by disulfide bridge ) exhibited potent complement 
 inhibition ( in in vitro and cell 
 based assays ) and improved solubility ( in uv absorption and rp - hplc 
 experiments ) . therefore , we screened 
 combinations of polar amino acids in positions 1 , 0 , and 1 
 and evaluated predicted binding to c3c relative to control peptides 
 ( table s2 ) .
six peptides were identified 
 with predicted c3c binding similar to or better than w4a9/mew4a9 ( table s3 ) , and three of these were selected based 
 on polarity ( table 1 , set 2 ) . in addition
, 
 the arg - ser - ile extension was tested without mew at position 4 ( table 1 , set 2 ) .
additionally , we selected components 
 of both sets of peptides into 
 two new sets , n - terminal extensions with nal9 and n - terminal extensions 
 with nal4/9 ( table 1 , sets 3 and 4 ) . set 3 
 peptides were selected in order to balance the potential interaction 
 enhancement of nal at position 9 with enhanced solubility from polar 
 n - terminal extensions . set 4 additionally included nal at position 
 4 , which was thought to improve hydrophobic contacts with c3 . in light 
 of our results from the first four sets of peptides and previous studies 
 regarding compstatin modifications , we 
 combined selected features to design peptides with optimal binding 
 and solubility ( table 1 , set 5 ) , and included 
 tyr at position 4 to improve solubility ( tyr4 was previously shown 
 to enhance inhibitory activity like other aromatic amino acids at 
 position 4 ) .
we evaluated 20 
 newly designed compstatin peptides ( plus four controls ) 
 for complement inhibition in vitro .
for each peptide , we measured 
 the ability to inhibit formation of both c3b and c5b-9 in elisa , as 
 well as the ability to inhibit complement - mediated lysis of rabbit 
 erythrocytes in hemolytic assays ( figure 2 and table s4 ) .
we observe that for each peptide , 
 inhibition of the different complement effectors occurs at similar 
 concentration .
this is in line with the ability of compstatin peptides 
 to block all complement pathways at the c3 level and subsequent downstream 
 activation .
the newly designed peptides exhibit similar or slightly 
 reduced inhibition ( ic50 values equal or higher ) compared 
 to mew4a9/w4a9 but were approximately an order of magnitude more potent 
 than parent .
notably , peptides 35 from set 1 ( figure 2a  c ) and peptides 79 from set 2 ( figure 2d  f ) show similar inhibitory activity compared 
 to mew4a9/w4a9 .
-modified alanine analogs at position 9 
 ( in peptides 35 ) are conservative substitutions , in terms 
 of both c3 interaction and solubility , and thus have similar inhibitory 
 activity to mew4a9/w4a9 .
furthermore , polar n - terminal extensions 
 seem to maintain inhibitory activity ( as shown previously ) , with the exception of peptide 6 , in which the 
 negatively charged glu residue may destabilize intermolecular salt 
 bridging interactions .
all peptides containing nal at position 9 ( peptides 
 1 , 2 , and 1017 ) required slightly higher concentrations for 
 complement inhibition , likely because of higher aggregation propensity . 

however , some of the n - terminal extensions ( i.e. , asn - arg - leu , peptides 
 12 and 16 ) improved inhibition of the nal9 peptides ( figure 2g  l ) .
the final set of peptides ( set 5 ) showed 
 improved complement inhibition ( figure 2m  o ) 
 but remained less potent compared to the aforementioned peptides from 
 sets 1 and 2 .
indeed , previous work has shown that incorporation of 
 tyr , nal , or 2nl at position 4 increased inhibitory activity relative 
 to parent but had slightly less activity compared to w4a9 .
we hypothesized that addition of the polar rsi 
 n - terminal extension may compensate for the aforementioned activity 
 loss while simultaneously improving solubility .
it seems that the 
 extension neither improves nor diminishes inhibition , and therefore , 
 the slightly reduced activity of set 5 peptides is most likely attributed 
 to the substitutions at position 4 .
w4a9 has an ic50 value 
 approximately 1 order of magnitude better than parent in all three 
 assays ( figure 2p  r ) , which is in agreement 
 with our previous data .
interestingly , we observe 
 that mew4a9 has only slightly better inhibitory activity than w4a9 
 ( within a 2-fold difference ) , which differs from previous studies 
 that report nearly an order of magnitude difference in activity .
we expect this discrepancy is due to differences in biochemical 
 and functional assays used , as well as the aggregation propensity 
 of mew4a9 .
concentration - dependent inhibition curves of compstatin peptides 
 in c3b and c5b-9 elisas and hemolytic assays .
data show c3b , c5b-9 , 
 and hemolysis inhibition ( from left to right ) for set 1 ( a  c ) , 
 set 2 ( d  f ) , set 3 ( g  i ) , set 4 ( j  l ) , set 5 
 ( m  o ) , and control ( p  r ) peptides .
data points indicate 
 mean percent inhibition  sem ( standard error of the mean ) .
the 
 intersection of dashed lines shows the ic50 value for mew4a9 
 in all plots .
curves to the right and left of the intersection point 
 represent peptides with higher ic50 values and lower ic50 values compared to mew4a9 , respectively .
bar plots 
 show 
 ic50 values for compstatin peptides 120 and positive 
 control peptides w4a9 , mew4a9 , and parent in c3b elisa ( a ) , c5b-9 
 elisa ( b ) , and hemolytic assay ( c ) .
bars show mean ic50 from three independent runs of each experiment ( 95% confidence 
 interval ) .
the dashed horizontal line shows the ic50 value 
 for the mew4a9 control peptide , for ease of comparison .
a subset of peptides ( peptides 5 , 8 , 
 12 , 18 , and 19 ) was selected 
 for evaluation of complement inhibition in a retinal pigmented epithelial 
 cell model , as described previously .
selected 
 peptides represent top - performing peptides from each set ( 15 ) , 
 as determined through elisas and hemolytic assays .
all tested peptides 
 significantly inhibited complement activation ( c5b-9/apoe fluorescence ) 
 at 1 m concentration , compared to linear and the positive control 
 ( serum without inhibitor ) , and displayed similar complement inhibition 
 compared to w4a9 ( figure 4 , tables s5 and s6 ) .
notably , there was no significant difference 
 in the levels of complement activation between parent and the positive 
 control or the linear peptide at 1 m .
this is likely due to 
 the fact that typical ic50 values for parent range between 
 4 and 13 m in biochemical and functional assays ( figures 2 and 3 , table s4 ) , and 1 m concentration is not sufficient 
 to reduce complement activation in rpe cells . to address this point , 
 we tested higher concentrations of parent in the in vitro rpe cell 
 assay .
while no complement inhibitory effects were noted for parent 
 at 1 or 10 m , a significant ( p - value of < 0.001 ) 
 inhibitory effect , similar to that of the w4a9 peptide at 1 m , 
 was observed for parent at 50 m ( figure 5 ) .
these results are consistent with the results of c3b and c5b-9 
 elisa analyses of complement inhibition ( figure 2 ) .
effects of compstatin peptides on complement activation in the 
 rpe cell in vitro assay .
the ratio of c5b-9/apoe fluorescence ( sem , n = 10 ) is plotted as a percentage of the positive control 
 ( pos ) for two hfrpe cell lines , 072810 ( gray ) and 081309 ( black ) . 

untreated cells that were not incubated with complement - competent 
 human serum served as negative control ( neg ) . at 1 m
, the parent 
 compound is not significantly different from the positive or linear 
 peptide controls .
all test peptides ( w4a9 , pep 5 , pep 8 , pep 12 , pep 
 18 , and pep 19 ) displayed significant complement inhibition relative 
 to their corresponding positive control ( see tables 
 s5 and s6 ) .
effects of varying concentrations 
 of parent on complement activation 
 in the rpe cell in vitro assay .
the ratio of c5b-9/apoe fluorescence 
 ( sem , n = 10 ) is plotted as a percentage of 
 the positive control .
parent was tested at concentrations of 1 , 10 , 
 and 50 m ( par1 , par10 , and par50 ) .
the concentration of w4a9 
 was 1 m . all values are expressed relative to the positive 
 control .
parent shows no significant difference from the positive 
 control at 1 m or 10 m concentrations . at 50 m 

the effect of parent is equivalent to that of 1 m w4a9 . both 
 parent at 50 m and w4a9 at 1 m are significantly different 
 than the positive control ( p - value of < 0.001 , 
 two - tailed mann 
newly designed compstatin 
 peptides were tested for solubility via absorbance measurements at 
 280 nm .
the peptides showed a wide range of solubility , ranging from 
 0.1 to > 5 mg / ml ( table s7 ) .
control 
 peptide 
 mew4a9 showed moderate solubility in this assay ( 1.9 mg / ml ) , significantly 
 lower than w4a9 and parent , which exhibited apparent solubilities 
 of 3.2 and 4.5 mg / ml , respectively .
this result is consistent with 
 the propensity of mew4a9 to aggregate in aqueous environments .
peptides 1 and 2 , which contain 
 nal at position 9 , exhibited the poorest solubility ( 0.1 mg / ml ) , 
 much lower than all control peptides .
addition of polar n - terminal 
 extensions ( peptides 1017 ) improved solubility only slightly 
 ( < 0.4 mg / ml ) .
peptides with -modified alanine analogs at 
 position 9 ( peptides 35 ) showed much improved solubility , 
 with values near the detection limit in this assay ( and similar to 
 w4a9 and parent ) .
 peptides 35 > peptides 12 
  
 peptides 1017 and , in turn , his > ala  rea  
 aal  sea > nal at position 9 . thus , increased hydrophobicity 
 of residues at position 9 strongly influences the solubility of compstatin 
 peptides .
as in the case of complement inhibition , set 5 peptides 
 showed intermediate solubility .
there is likely a balancing effect 
 between the polar arg - ser - ile n - terminal extension and the hydrophobic 
 residue ( tyr , nal , or 2nl ) at position 4 .
the importance of position 
 4 is also evidenced by decreased solubility of peptides containing 
 mew at position 4 ( peptide 2 and mew4a9 ) .
interestingly , while peptide 
 18 was initially highly soluble ( 5 mg / ml ) , we observed a time - dependent 
 decrease in concentration when stored in a polypropylene tube ( figure 6 , table s7 ) .
thus , it 
 is unclear whether the change in solubility of peptide 18 is due to 
 time - dependent aggregation or simply interaction with the container . 

it should be noted that while complement inhibitory activity of compstatin 
 peptides has been associated with increased hydrophobicity in past 
 studies , we observed 
 that solubility is in fact associated with inhibitory activity ( figure 6 ) . this is a direct result of our peptide design , 
 as we engineered polar residues in regions of compstatin that should 
 not affect binding .
a clear exception to the aforementioned trend 
 is parent , in which solubility - enhancing substitutions are at positions 
 that strongly influence binding .
ic50 values for compstatin peptides in c3b elisa ( a ) , c5b-9 
 elisa ( b ) , and hemolytic assay ( c ) are plotted against solubility . 

horizontal and vertical error bars represent the standard deviation 
 and 95% confidence intervals of solubility and ic50 , respectively . 

points inside the ellipse ( lower right corner ) have a favorable balance 
 between activity and solubility .
note the peptide 8 solubility was 
 measured using less starting material compared to the rest , and thus , 
 its solubility is not directly comparable to that of other peptides .
modifications 
 of peptide amino acid content can have a significant effect on peptide 
 inhibitory activity , solubility , and lipophilicity .
the inhibitory activity of the compstatin peptides depends 
 in part on hydrophobic interactions with c3 , which can be mimicked 
 by the interactions of hydrophobic amino acid side chains of the peptides 
 with the c18 hplc stationary phase .
the retention time of each peptide 
 on the hplc column can be used to calculate logarithm of the retention 
 factor ( log(k ) ) , which is related to its lipophilicity 
 ( and hydrophobicity ) .
figure 7 shows log(k ) values for compstatin peptides . a full summary of rp - hplc 
 retention times , retention factors ( k ) , and log(k ) values are summarized in table s8 , with the peptides organized according to substitution patterns . 

the peptides are listed in order of increasing relative lipophilicity 
 in table s9 . as expected , peptide lipophilicity 
 is inversely related to solubility with an r = 0.72 ( figure s1 ) .
bar plots show 
 log(k ) values for compstatin peptides 120 
 and positive control peptides w4a9 , mew4a9 , and parent in c3b elisa . 

the dashed horizontal line shows the log(k ) value 
 for the mew4a9 control peptide , for ease of comparison .
modifications of peptide sequence can have a large 
 effect on their 
 rp - hplc retention behavior .
comparison of the hplc results for peptides 
 2 and 1013 demonstrates that addition of the charged and polar 
 amino acids at positions 1 , 0 , and 1 reduces the retention 
 time while substitution of the hydrophobic l-1-naphthylalanine 
 ( nal ) for trp increases the interaction with the stationary phase . 

peptides 1417 are more retained than peptides 1013 , 
 which differ in structure only by a nal substitution at position 4 . 

peptides 1 and 2 also contain nal at position 9 in place of ala in 
 structurally similar peptides mew4a9 and w4a9 , respectively , and are 
 more highly retained than these analogs .
a similar observation can 
 be made when comparing peptides 9 and 19 , which differ at position 
 4 by the substitution of nal .
substitution of tyr for trp in peptide 
 18 reduces its interaction with the stationary phase as reflected 
 by its smaller value of log(k ) .
structural 
 modifications of amino acid residues can also affect 
 the interactions of peptides with the hydrophobic stationary phase . 

for example , the addition of a methyl group to a trp residue at position 
 4 increases the retention compared with the trp analogs .
peptides 
 1 and mew4a9 are more highly retained than peptides 2 and w4a9 , respectively . 

the position of the naphthyl addition on alanine also affects the 
 interaction of the peptide with the stationary phase .
peptide 19 , 
 which contains l-1-naphthylalanine , is less retained than 
 peptide 20 , which contains l-2-naphthylalanine at the same 
 position .
similarly , peptides 3 and 5 , which differ only by substitution 
 of r- and s--ethylalanine , 
 respectively , can be resolved by hplc with peptide 3 being more highly 
 retained than peptide 5 .
although continual efforts 
 to improve compstatin have yielded peptides 
 with potent complement inhibition , many are characterized by poor 
 solubility in aqueous environments containing physiological ionic 
 strength .
the inhibitor mew4a9 ( al-78898a , alcon ) has recently completed phase ii clinical trials 
 ( clinicaltrials.gov , identifier numbers nct00473928 and nct01157065 ) 
 for amd ; however , the results showed that this peptide did not yield 
 reduced retinal thickness in amd patients in comparison to ranibizumab 
 ( genentech / novartis ) , a current amd therapeutic . 

it is likely that the low aqueous solubility of mew4a9 contributes 
 to its poor performance .
thus , recent studies have aimed at improving solubility of compstatin 
 peptides while simultaneously maintaining or improving complement 
 inhibitory activity . in this study , we designed and tested new 
 compstatin peptides with 
 diverse polar n - terminal substitutions and extensions .
we also employed 
 a new computational design framework for non - natural amino acids to 
 identify non - natural amino acids at positions 4 and 9 predicted to 
 improve c3 binding .
furthermore , we tested peptides containing combinations 
 of these features , selected using rational design arguments .
we note 
 that all of our designed peptides have inhibitory activities similar 
 to controls mew4a9 and w4a9 and significantly better than parent ( figures 25 ) .
most importantly , 
 we found that peptides containing either -modified non - natural 
 alanine analogs at position 9 ( peptides 3 , 4 , and 5 ) or n - terminal 
 natural amino acid extensions ( peptides 7 , 8 , and 9 ) have inhibitory 
 activity similar to mew4a9 while exhibiting greatly improved aqueous 
 solubility . given the clinical interest in mew4a9 for treatment of 
 a variety of complement - mediated disorders , in conjunction with problems 
 recently encountered in clinical trials , we believe that our newly 
 designed compstatin peptides ( i.e. , peptides 3 , 4 , 5 , 7 , 8 , and 9 ) 
 represent more promising therapeutic alternatives .
indeed , some compstatin 
 peptides bind to c3 with affinities of < 1 nm , with correspondingly 
 low ic50 values in complement assays . while improved affinities are attractive for therapeutic 
 design when taken at face value , we must remain aware that human plasma 
 c3 concentrations are at least 5 m , and thus high compstatin 
 concentrations are required for therapeutic efficacy despite high 
 binding affinity .
consequently , we conclude that ( a ) it is unnecessary 
 to further improve compstatin binding affinity and inhibitory activity 
 and ( b ) compstatin must be highly soluble to inhibit c3 cleavage in 
 vivo .
recent studies have addressed several of these 
 properties , including solubility , half - life , and degradation .
we have identified several highly 
 soluble compstatin peptides with potent inhibitory activity . in the 
 past
, it was thought that a high degree of hydrophobicity was required 
 for potent compstatin activity .
we illustrate here that the incorporation 
 of n - terminal extensions can help to circumvent this activity  hydrophobicity / solubility 
 paradigm .
our inhibitory activity versus solubility data ( figure 6 ) denote peptides with similar inhibitory activity 
 compared to highly active controls ( i.e. , mew4a9 ) but with improved 
 solubility characteristics .
furthermore , several peptides ( peptides 
 69 and 18 ) are notably less lipophilic than both mew4a9 and 
 w4a9 ( figure 7 and table 
 s9 ) .
these peptides , as well as additional peptides containing 
 alternative combinations of top - performing modifications , represent 
 promising candidates for therapeutic development , for treatment of 
 a wide variety of complement - mediated autoinflammatory diseases .
sequence 
 selection , fold specificity , and approximate binding affinity calculations 
 for natural amino acid design were performed following the procedure 
 described previously in protein wisdom and are described below .
the purpose of the design was to produce novel 
 natural amino acid sequence extensions using the previously designed 
 sequences ac - rsicvwqdwgahrct - nh2 ( sequence of peptide 
 9 in table 1 , called rsi hereafter ) and ac - ssicvwqdwgahrct - nh2 ( called ssi hereafter ) as templates .
both 
 sequences are identical to the w4a9 variant of compstatin with two 
 positions added to the n - terminus , termed positions 1 and 
 0 .
these sequences were used as input to further design these positions 
 for c3c binding .
rsi was chosen as a design template , as it previously 
 yielded improved ic50 values relative to w4a9 in c3b elisa 
 and hemolytic assays , as well as significant complement inhibition 
 according to a human retinal pigmented epithelial cell - based model 
 mimicking drusen biogenesis .
ssi had 
 near w4a9 calculated affinity for human c3 and simultaneously high 
 calculated affinity for non - primate c3 . because of the previous designs of both of these sequences , md simulations 
 were previously performed and therefore were utilized as design templates 
 in this next generation of design .
the peptide structure template for 
 sequence selection was constructed based on the structure of the w4a9 
 analog ( pdb code 2qki ) .
molecular dynamics simulations of 
 the template compstatin extensions were performed previously and were 
 used as a flexible template for this design . in all cases ,
both extended positions , 1 and 0 , as well 
 as the adjacent position 1 , were allowed to mutate . because of the 
 relatively small sequence space in this study ( three mutatable positions 
 only ) , the three positions were allowed to mutate to all possible 
 amino acids except for proline and cysteine , which were excluded because 
 of their unique chemical and geometric properties .
centroid 
 potential energy function was used as the input force field , which serves as a look - up table of energies 
 of all combinations of amino acid pairs at a fixed set of distance 
 bins .
the flexible distance  bin sequence selection method was used to solve for a rank - ordered list of 
 low energy sequences according to the following model.1asubject to1b1c1d1e1fthe set i = 1 , ... , n defines each 
 residue position in the design template . 
 at each position
i , mutations are represented by j{i } = 1 , ... , mi , where mi = 20 if the sequence position is allowed to mutate to any 
 of the 20 natural amino acids . alias sets
i and l  j , with k > i , are used to represent the pairwise 
 interactions between all residue positions and amino acid types .
the 
 decision variables yij and ykl are solved for in order to 
 determine the amino acid types in each position
. the yij variable will assume the value of 1 if the 
 model assigns amino acid j to position i and assume the value of zero otherwise ( similarly for ykl ) .
the objective function represents the 
 sum of all pairwise energy interactions in the design template .
the model incorporates the distance information 
 from the template structures by introducing a binary variable bikd , which equals 1 if the 
 distance between residues i and k falls within distance bin d and equals zero otherwise . 

the parameter disbin(xi , xk , d ) equals 1 if the distance between residue positions i and k in any of the input template structures falls 
 into distance bin d and equals zero otherwise , which 
 forces only one distance bin per amino acid pair to contribute to 
 the total energy .
since we started 
 with template structures with sequences of both 
 rsi and ssi , three separate runs were performed for the design .
run 2 used only 
 ssi structures as the input template , and run 3 used a combined rsi 
 and ssi structure set as the input template . for each run
, 500 low - energy 
 sequences were generated using the flexible distance  bin sequence 
 selection method . from runs 13 ,
this resulted 
 in a total of 1019 sequences , which were then validated using the 
 fold specificity metric .
the aim of the fold specificity calculation 
 is to determine the stability of a designed sequence in the target 
 template structure as compared to the stability of the native sequence . 

the flexible template structure defined bounds on the minimum and 
 maximum cc distances and  and  
 angles experienced .
ensembles are next generated using a torsion angle 
 dynamics simulated annealing containing 500 conformers using cyana 
 2.1 .
a local energy minimization is performed 
 on every conformer in tinker 3.6 using 
 the amber force field , and the final 
 potential energy of each conformer is tabulated .
the energetics of 
 the native sequence is similarly tabulated . the fold specificity calculation 
 is next performed as2with 
 more positive values being favorable .
three separate runs of fold specificity 
 were performed , based on which template structures were used in the 
 calculation : run 1 using rsi structures only , run 2 using the ssi 
 structures only , and run 3 using the combined rsi and ssi structure 
 set .
the sequences were then rank - ordered by fold specificity for 
 each set , and a consensus set of sequences was determined for validation 
 using approximate binding affinity ( table s2 ) .
sequences were chosen for validation to maximize fold specificity 
 across runs , as well as to cover a large chemical space .
this resulted 
 in 15 sequences with mutations in positions 1 , 0 , and 1 and 
 in 7 sequences with mutations in positions 1 and 0 only .
the approximate binding 
 affinity calculation in protein wisdom consists of several steps , 
 which include diverse ligand ensemble generation , clustering , docking , 
 and final ensemble generation .
the goal is to generate a diverse ensemble 
 of docked ligand poses to the receptor to assess the relative energetics 
 of the complex , receptor , and ligand through calculation of approximations 
 of their partition functions .
the approximate binding affinity is defined as k * = qpl/(qpql ) , where qpl = bb  e , qp = ff  e , and ql = lle are the partition functions of the protein  ligand complex , 
 the free protein , and ligand , respectively .
the sets b , f , and l , contain rotamerically based conformations 
 of the bound complex , free protein , and free ligand .
two - thousand structures for each candidate compstatin extension 
 sequence are generated using the rosetta abrelax function as part 
 of the rosetta 3.4 package .
monte carlo sampling is used 
 to replace local protein structures with structural fragments derived 
 from homologous sequences .
the structures are clustered based on their 
  and  angles using oreo . the average 
 structure from the 10 largest clusters and the lowest energy structure 

the 10 lowest energy docked poses for each of 
 the 11 ligands are passed to rosettadesign to generate 200 rotamer conformations per starting complex structure 
 ( 22  000 total states representing the bound set ) .
the ligand 
 ensemble is constructed by taking the 10 lowest energy structures 
 from each of the 10 largest clusters plus the 10 lowest overall energy 
 peptide conformations and generating 200 rotamer conformations ( 22  000 
 free ligand structures ) for each .
the protein ensemble is generated 
 directly by assembling 2000 rotamer structures from the single starting 
 c3c structure . with these ensembles and their corresponding rosetta 
 energies ,
the 22 sequences 
 from fold specificity were run through the approximate 
 binding affinity protocol in order to 
 determine which designed peptides were predicted to bind more strongly 
 than the w4a9 peptide .
of the 22 tested peptides , six peptides were 
 predicted to have higher binding affinity than w4a9 and peptide 9 . 

recently , efforts have expanded to create methods for 
 designing non - natural 
 amino acids into proteins .
we have created new force fields based on quantum chemical calculations 
 for post - translational modifications and unnatural amino acids for 
 amber .
thus , we developed a novel framework combining 
 our force fields , integer linear optimization , and molecular dynamics 
 simulations to aid us in generating lead compounds for experimental 
 exploration .
the first step in the procedure is to derive candidate 
 sequences containing post - translational modifications and non - natural 
 amino acids .
the overall idea is that we want to introduce modified 
 amino acids to enhance the number of contacts and hydrogen bonds relative 
 to the starting peptide while minimizing the clashes introduced by 
 the modified amino acid .
in addition , we want to preserve the local 
 electrostatic landscape and any salt - bridges that are formed between 
 the original or designed sequence scaffold and the target receptor . 

this is modeled as the following integer linear optimization problem:3asubject to3b3c3d3e3f3g3hwith 
 the following sets , parameters , and binary 
 decision variables .
( 1 , 2 , ... , npositions ) ; this is the 
 set of positions in the amino acid sequence .
np  ( ala , 
 arg , asn , ... , tyr ) ; this is the native amino acid in each position p. m  ( np , ... , ptms , ... , non - naturalaminoacids ) ; 
 this is the universe 
 of modification types under consideration for which we have derived 
 parameters for .
this universe comes from a limited set of modifications 
 parametrized early on for forcefield_ptm and forcefield_ncaa .
c  ( 1 , 2 , 3 , ... , ncomplexes ) ; this is the set of complex configurations .
 is the 
 set of complex positions that correspond to the 
 lowest energy configurations from a molecular dynamics simulation , 
 an nmr structural ensemble , or a single crystal structure .
note that 
 the set m contains all the post - translational modifications 
 and unnatural amino acids for which we have parameters for , as well 
 as the native amino acid in position p being designed . 

cmp , charge 
 of modification m at position p. d , charge of original 
 amino acid on template sequence at position p. hmp , hydrophobicity of 
 modification m at position p. h , hydrophobicity 
 of original amino acid on template sequence at position p. goodcontacts(c , m , p ) , number of contacts between modification m at 
 position p and the receptor with van der waals radii 
 overlap of atoms ( i , j )  
 2 in configuration c. clashes(c , m , p ) , number of contacts 
 between modification m at 
 position p and adjacent residues in the peptide or 
 the receptor with a van der waals radii overlap of atoms ( i , j )  0 or  0.4 if atoms
( i , j ) can potentially 
 hydrogen bond in configuration c. hbonds(c , m , p ) , number of valid hydrogen bonds ( distance and angle orientation ) 
 between modification m at position p and the receptor in configuration c. ie(c , m , p ) , energy 
 of binding between protein and peptide in configuration c as a result of modifying position p to modification 
 type m , where this energy is defined assp(p ) = 
 1 if position p is allowed to be modified , 0 otherwise .
s(c , m , p ) = 0.1  goodcontacts(c , m , p )  5  clashes(c , m , p ) + 1  hbonds(c ,
m , p ) , weighted contact score of 
 modification m at position p in 
 configuration c. this score is not an energy that 
 is physically derived but a tabulation of quantities of interactions .
natural amino acids are classified as hydrophobic or hydrophilic 
 if their relative side chain solvent - accessible surface areas ( sasas ) 
 calculated with the program naccess in 
 their position in three - dimensional space within the context of the 
 complex are 20 or 50 , in accordance with work by bellows 
 et al . introducing hydrophobicity constraints 
 in previous designs using only natural amino acids
next , we define mp  m , which is the subset of modifications meeting the charge 
 and hydrophobicity constraints below at position p ,  positions p. this set , derived from the 
 following constraints , reduces the search space . local charge / salt - bridges 
 must be conserved in each position p4local hydrophobicity 
 must be conserved at 
 each p5 we further want to limit the modifications 
 to those that are of the same type as the native amino acid in position p. for example , if the native amino acid in position 5 were 
 a tyrosine , we would allow only for modifications of tyrosine as design 
 choices and not modifications of alanine .
therefore , we define mpnp  mp as the 
 subset of modifications in position p that are allowed 
 given the native amino acid in position p , np . utilizing this subset dramatically 

there are geometric conditions required 
 for a contact , clash , and 
 hydrogen bond to occur .
these conditions reflect the fact that two 
 atoms that hydrogen bond may come closer to each other than their 
 van der waals radii would normally allow .
contacts , clashes , and hydrogen 
 bonds were evaluated between the modified position of the compstatin 
 analog and c3c using custom scripts interfacing with ucsf chimera .
intermolecular contacts were defined with an 
 overlap cutoff of 2 and a hydrogen bond allowance of 0 . both 
 inter- and intramolecular
clashes were calculated with an overlap 
 cutoff of 0.4 and a hydrogen bond allowance of 0.4 .
intermolecular 
 hydrogen bonds were calculated between compstatin analogs and c3c 
 with a 0.4  distance relaxation and a 20 angle relaxation 
 from the default hydrogen bond criteria .
ymp = 1 
 if modification m is assigned to position p , 0 otherwise .
the objective function , eq 3a , aims to maximize a boltzmann - weighted contact 
 score .
equation 3b defines the weights of the 
 contact score as a weighted sum of goodcontacts , clashes , and hbonds . 

the goal of the contact score is to improve affinity / specificity through 
 the optimization of shape - complementarity at the individual amino 
 acid level .
this quantity becomes the 
 denominator for the boltzmann - weighted contact score so that for each 
 position , a probability can be derived based on the sum over the allowed 
 modification types m  mpnp and complex configurations c. equation 3d defines the boltzmann 
 factor that is the ratio of the exponent of the weighted contact score , 
 eq 3b divided by the position - specific partition 
 function , eq 3c .
the constraint 3e states that the average energy of binding across all complex 
 configurations must remain favorable for the modification m to be chosen in position p. this is important 
 to preserve the local electrostatic interactions of the template sequence 
 if any are present .
equation 3f states that 
 only one amino acid is allowed to be selected in each position .
equation 3 g is the integer cut constraints that generate a 
 rank - ordered list of optimal solutions .
a computational pipeline to design 
 proteins and peptides by introducing 
 non - natural amino acids was created .
the pipeline takes as input design 
 positions , as well as a set of complex configurations corresponding 
 to a peptide with natural amino acids bound to a receptor protein . 

these configurations were the single state of the c3c : w4a9 crystal 
 structure ( pdb code 2qki ) .
next , the initial energy of binding , charge , and hydrophobicity 
 constraints are populated using amber  11 and chimera .
then for each complex 
 configuration and each allowed modification in each design position , 
 the complex is modified at the specified design position , a local 
 energy minimization is performed , and the contacts , clashes , hydrogen 
 bonds , and binding energies are populated .
the amber program tleap 
 was used to construct the modified side chains according to their 
 orientations defined in forcefield_ptm and forcefield_ncaa . in position 4 , 

tryptophan , 5-hydroxytryptophan , 5-methyltryptophan , n - methyltryptophan , 1-methyltryptophan , and 7-hydroxytrptophan were 
 evaluated . in position 9 ,
alanine , 1-naphthylalanine , n - methylalanine , pyrenylalanine , ( r)--ethylalanine , 
 ( s)--ethylalanine , and r(+)--allylalanine 
 were evaluated .
these were chosen as they met the criteria to be included 
 in each of their respective sets mpnp , and their parameters were included 
 in forcefield_ptm or forcefield_ncaa .
for each modification upon construction , 
 1500 steps of steepest descent followed by 500 steps of conjugate 
 gradient minimization were performed to relax the new non - natural 
 side chain placement in the context of compstatin and the c3c receptor . 

the implicit solvent model by onufriev ( igb = 5 ) 
 was utilized in all amber calculations .
after population of this information 
 for all complex configurations , the integer linear optimization model 
 presented above is solved to global optimality , generating a rank - ordered 
 list of modified amino acid substitutions .
the sequence solutions 
 were next simulated in amber  11 to 
 assess the approximate binding affinity ( k * ) with 
 a procedure described previously .
specifically , 
 three independent simulations are carried out of the complex , protein , 
 and peptide .
structures were minimized with 600 steps 
 of steepest descent with 400 steps of conjugate gradient minimization 
 to relax the new side chains .
the structures were heated in six stages 
 over 30 ps from 0 to 300 k. shake constraints were used for all simulation 
 steps on the heavy - atom to hydrogen bonds , and a nonbonded interaction 
 cutoff of 16  was used .
each c3c : compstatin peptide candidate 
 sequence underwent short 0.5 ns production simulations using a 1 fs 
 time step at 300 k. the theoretical foundation for this calculation 
 was previously described in khoury et al . and
those sequences with 
 the highest k * values relative to the control peptide 
 w4a9 were proposed for experimental testing ( table 
 s1 ) .
we note that the approximate binding affinity is used 
 as a metric to select which peptides may be promising candidates for testing based on physics - based potentials , 
 and we do not attempt to compare the calculated values to the exact 
 experimental values . instead , we aim to utilize metrics that increase 
 our probability that top ranked peptides are indeed inhibitors of 
 c3 .
compstatin peptides 19 , 1420 , 
 w4a9 , mew4a9 , and parent were synthesized by wuxi apptec ( shanghai , 
 china ) .
( piscataway , nj , usa ) . linear ( negative control ) was obtained from 
 either tocris bioscience ( bristol , u.k . ) or wuxi apptec .
all peptides 
 were of > 95% purity , as determined by hplc and ms .
non - natural 
 amino 
 acids used in this study included l-1-methyltryptophan , l-1-naphthylalanine , l-2-naphthylalanine , r--ethylalanine , r--allylalanine , and s--ethylalanine ( figure 1 ) . 

peptides containing l-1-methyltryptophan were synthesized 
 using fmoc-1-methyl - dl - tryptophan ( commercially available 
 preparation ) and subsequently purified by hplc .
approximately 1 mg of each 
 tested peptide was dissolved in 200 l of pbs , ph 7.4 .
it should 
 be noted that the resulting concentration yields saturated solutions 
 for only some peptides , and the maximum solubility reported here is 
 thus 5 mg / ml .
samples were vortexed for 30 s each , then centrifuged 
 at 13000 g for 5 min .
supernatant was collected and 
 measured spectrophotometrically at 280 nm , using a nanodrop lite spectrophotometer . 

molar extinction coefficients were calculated 
 for each peptide based on the number of trp and tyr residues in their 
 sequences . because of their aromatic properties , l-1-methyltryptophan , l-1-naphthylalanine , and l-2-naphthylalanine also contribute 
 to absorbance at 280 nm .
molar extinction coefficients used for these 
 amino acids were 5470 m cm , 3936 m cm , and 3936 m cm , respectively ( the coefficient for l-2-naphthylalanine was assumed to be equal to that of l-1-naphthylalanine ) . 
 dissolved samples were stored at 4 c , and measurements of the 
 same samples were repeated at 24 and 48 h time points .
measurements 
 represent the mean and standard deviation of three measurements performed 
 on the each sample .
nunc maxisorp 
 96-well microtiter 
 plates were coated with 20 g / ml lipopolysaccharides ( lps ) from salmonella enterica serotype enteritidis ( sigma - aldrich , 
 st .
plates were washed 
 three times with pbs ( containing 0.05% tween-20 ; pbs - t ) between each 
 incubation step .
plates were blocked with 4% bsa in pbs - t for 1 h 
 at 37 c . lyophilized compstatin peptides were initially dissolved 
 in pbs , ph 7.4 , and concentration was measured spectrophotometrically 
 at 280 nm .
serial dilutions of each peptide were prepared in veronal - buffered 
 saline containing 0.1% gelatin , 5 mm mgcl2 , and 10 mm egta 
 ( gvbs - mgegta ) .
tyler , 
 tx , usa ) was dissolved in gvbs - mgegta and was preincubated with peptide 
 dilutions ( or buffer ) for 15 min at ambient temperature .
after 
 incubation , plates were incubated with either horseradish peroxidase 
 ( hrp ) conjugated anti - c3 ( mp biomedicals , solon , oh , usa ) or anti - c5b-9 
 ae11 ( abcam , cambridge , ma , usa ) for 1 h at 37 c .
c5b-9 detection 
 additionally involved incubation with an hrp - conjugated secondary 
 antibody , also for 1 h at 37 c
. levels of c3b and c5b-9 deposition 
 were measured using a 3,3,5,5-tetramethylbenzidine 
 substrate solution containing urea hydrogen peroxide in 0.11 m sodium 
 acetate buffer , followed by addition of 1 n h2so4 .
tyler , tx , usa ) were washed and resuspended in veronal - buffered 
 saline containing 5 mm mgcl2 and 10 mm egta ( vbs - mgegta ) . 

normal human serum and compstatin peptides ( serial dilutions ) were 
 diluted in vbs - mgegta and preincubated together in round - bottom 96-well 
 plates for 15 min at ambient temperature .
5  10 rabbit 
 erythrocytes were added to each well , and plates were incubated for 
 20 min at 37 c .
erythrocytes in either deionized water or normal 
 human serum ( dissolved in vbs - mgegta ) were used as positive controls 
 for lysis , and erythrocytes in either vbs - mgegta or normal human serum 
 ( dissolved in vbs - edta ) were used as negative controls for lysis . 

reactions were quenched by adding ice cold vbs ( containing 50 mm edta ) 
 to each well .
plates were centrifuged at 1000 g for 
 5 min , and supernatant was diluted 1:1 in deionized water in new plates . 

the rpe cell - based in vitro model 
 of drusen formation was employed as previously 
 described .
human fetal rpe cells ( advanced 
 bioscience resources , alameda , ca ) were cultured on millipore ha porous 
 supports ( millipore , catalog no .
piha 01250 ) in miller medium supplemented 
 with 5% fetal calf serum ( fcs ) .
rpe cell cultures derived from two 
 different donor eyes ( line no . 081309 and line no .
samples were rinsed in pbs and then individually exposed to an experimental 
 peptide in serum - free miller medium containing 10% human complement 
 serum ( innovative research , catalog no .
the 1 m peptide concentration employed was previously shown 
 to be in the linear range of inhibitory concentrations during titrations .
one experiment employed the parent compound 
 at 1 , 10 , and 50 m .
negative control cells were exposed to 
 miller medium + 5% fcs ; positive control cells were exposed to miller 
 medium + 10% human complement serum .
experimental and control solutions 
 ( 1 ml ) were mixed on a rocker at room temperature for 30 min , then 
 warmed to 37 c , before sample exposure and overnight incubation 
 at 37 c in a 7.0% co2 incubator . following this period , 

the samples were rinsed with warm , sterile pbs , fixed in cold 4% paraformaldehyde 
 ( pfa ) in pbs for 20 min , and stored in 0.4% pfa until use in immunohistochemical 
 assays .
after rinsing the fixed samples 
 in pbs , the inset membrane was excised with a scalpel and cut into 
 4 mm squares .
duplicate samples from each condition 
 were embedded in 10% agarose ( type xi , sigma - aldrich , catalog no
sections were 
 blocked with normal donkey serum ( 1/20 in pbt / pbs containing 0.5% 
 bovine serum albumin and 0.1% triton x-100 ) overnight at 4 c . 

the sections were co - incubated with two primary antibodies ( polyclonal 
 goat anti - apoe , millipore catalog no .
ab947 , 1/1000 in pbt , and mouse 
 monoclonal anti - c5b-9 , dako catalog no .
mo777 , 1/200 in pbt ) overnight 
 at 4 c , then rinsed in pbt , and then co - incubated in secondary 
 antibodies ( alexa fluor 546-conjugated donkey anti - goat igg and alexa 
 fluor 488-conjugated donkey anti - mouse igg , both 1/200 in pbt , life 
 technologies catalog no .
the immunolabeled sections were then rinsed in pbt , stained with hoechst 
 33342 ( life technologies catalog no .
h3570 ) , and mounted on slides 
 with prolong gold ( life technologies catalog no .
ten single - plane 
 images , captured at a resolution of 1024  768 pixels and saved 
 as 24-bit tiff files , were acquired for each sample .
digital image 
 files were analyzed using metamorph software ( molecular devices , sunnyvale , 
 ca ) . for image quantification ,
the area of c5b-9 specific fluorescence 
 was normalized to the area of apoe specific fluorescence and expressed 
 as the c5b-9/apoe ratio .
this method 
 has been used to evaluate the retention factor , k , of peptides between the hydrophobic stationary phase and hydrophilic 
 mobile phase .
the logarithm of the retention factor was determined 
 based on the peptide retention time , tr , and column dead time , t0 .
these peptides are structurally similar , some 
 differing by only one site or position of substitution .
the isocratic 
 rp - hplc method used in this work allows for comparison of the interaction 
 of the structurally similar peptides with the hydrophobic stationary 
 phase .
the separations were performed using an agilent 1100 
 hplc with uv detection at 280 nm and a waters ( milford , ma , usa ) 4.6 
 mm  150 mm xterra ms c18 column with 5 m particles and 
 a 125  pore size .
the peptides were eluted using a mobile phase 
 a , consisting of 10 mm phosphate buffer at ph of 7.4 with 1% tfa , 
 and acetonitrile as mobile phase b. the peptides were first evaluated 
 using method 1 , running 32% b isocratically at a flow rate of 1.0 
 ml / min at 25 c .
the less hydrophobic peptides 39 and 
 1820 were better resolved using isocratic method 2 which used 
 28% b. triplicate 10 l injections were performed for each peptide 
 at concentrations ranging from 9 to 16 m .
the column dead time 
 was determined to be 1.418  0.004 ( method 1 ) and 1.425  
 0.006 ( method 2 ) using a 5 mg / ml solution of aspartic acid as the 
 unretained analyte .
dibasic sodium 
 phosphate , aspartic acid , and acetonitrile were 
 purchased from fisher scientific ( pittsburgh , pa , usa ) .
trifluoroacetic 
 acid ( tfa ) was purchased from acros ( geel , belgium ) .
hplc - grade water 
 was obtained from burdick and jackson ( muskegon , mi , usa ) .
 compstatin peptides are complement 
 inhibitors that bind and inhibit 
 cleavage of complement c3 . 
 peptide binding is enhanced by hydrophobic 
 interactions ; however , poor solubility promotes aggregation in aqueous 
 environments . 
 we have designed new compstatin peptides derived from 
 the w4a9 sequence ( ac - icvwqdwgahrct - nh2 , cyclized 
 between c2 and c12 ) , based on structural , computational , and experimental 
 studies . 
 furthermore , we developed and utilized a computational framework 
 for the design of peptides containing non - natural amino acids . 
 these 
 new compstatin peptides contain polar n - terminal extensions and non - natural 
 amino acid substitutions at positions 4 and 9 . 
 peptides with -modified 
 non - natural alanine analogs at position 9 , as well as peptides containing 
 only n - terminal polar extensions , exhibited similar activity compared 
 to w4a9 , as quantified via elisa , hemolytic , and cell - based assays , 
 and showed improved solubility , as measured by uv absorbance and reverse - phase 
 hplc experiments . because of their potency and solubility , these peptides 
 are promising candidates for therapeutic development in numerous complement - mediated 
 diseases . 
Introduction
Results
Discussion
Experimental Section