cyclin dependent kinases ( cdks ) and their 
 natural inhibitors ( cdkis ) 
 are central to cell cycle 
 regulation , and their functions are commonly altered in tumor cells .
deregulation of cdk2 and cdk4 through inactivation 
 of cdkis such as p16 , p21 ( p21 ) , p27 , and p57 provides a means for cancer cells 
 to override the g1 checkpoint .
compounds that mimic 
 the ternary complex of cdkis with cdk / cyclins should lead to reinstatement 
 of cdk inhibition and therefore represent an opportunity for pharmacological 
 interference with tumor progression . a particular 
 hypothesis for tumor selective cell death through inhibiting
the phosphorylation 
 of cdk substrates comes from observations that the cdk2/cyclin a ( cdk2a ) 
 complex is a key regulator of e2f1 transcriptional activity .
e2f activity must be terminated in a timely fashion 
 during s - phase , as persistent function results in a powerful apoptotic 
 signal mediated by transcriptional effects .
inhibition of cdk activity with cyclin groove inhibitors ( cgi ) therefore 
 results in tumor selective induction of apoptosis in cells already 
 possessing deregulated e2f .
cdk2 activity appears to 
 be redundant for the proliferation of 
 normal cells and in some cases for cancer cells , leading to doubts 
 as to the validity of cdk2 as a drug target .
studies suggest that 
 its nonessential role is a result of the substitution of one cdk for 
 another .
this can occur , since 
 the various cdk isoforms are in high abundance throughout the cell 
 cycle and are transiently activated by cyclin binding and subsequent 
 phosphorylation .
inhibition of the cell cycle cdks through the cyclin 
 groove rather than the atp binding site offers the possibility to 
 overcome the switch to a different cdk family member when the activity 
 of one particular isoform is downregulated .
as the transient expression 
 of a specific cyclin is obligatory both for activation of the kinase 
 and for substrate recruitment of critical cell cycle regulatory proteins 
 and resulting progression , the cancer cell will be unable to bypass 
 cdk activity directly .
it is believed that a component of the anticancer 
 activity of cdk inhibitors is through the transcriptional inhibition 
 of cdk7 and cdk9 . while it has been suggested that 
 transcriptional cdk inhibition may be beneficial for cancer therapy , 

it is also probable that this will lead to significant toxicities 
 and has led to the failure of cdk2 inhibitors in clinical trials . 

protein interaction involved in cdk2 
 substrate recruitment therefore offers the possibility of generating 
 cell cycle selective cdk inhibitors . with
only cyclins a , d , and e 
 containing a functional cyclin binding groove ( cbg ) , it is possible 
 in principle to inhibit the g1 and s phase cdks ( cdk2 , -4 , and -6 ) 
 selectively while avoiding those involved in transcriptional regulation . 

cyclin groove inhibitors should therefore avoid undesirable side effects 
 of atp competitive cdk inhibitors .
highly potent 
 peptidic inhibitors of cdk activity have been described and in cell 
 permeable form result in antitumor activity therefore providing proof 
 of concept for non - atp competitive targeting . to exploit protein 
protein interactions as drug targets , 
 replace , a unique drug discovery strategy has been validated and applied 
 to discover first generation inhibitors of the cyclin groove that 
 serve as the basis for oncology drug development .
further progress in delineating 
 the structure  activity of such inhibitors is described here 
 providing essential information for the conversion of peptides into 
 nonpeptidic molecules .
modification of cgi compounds was successfully 
 undertaken through the principles of replace resulting in optimized 
 inhibitors with enhanced druglike properties , anticancer activity , 
 and confirmation of on - target mechanism of action through cell cycle 
 analysis .
while structure  activity relationships 
 for the cyclin binding 
 motif ( cbm ) have previously been described , contributions of key residues 
 have not been adequately investigated .
in addition , analysis 
 is required to determine the consequences of these substitutions in 
 the context of smaller peptides that are a starting point for drug 
 discovery .
furthermore , detailed structure  activity relationships 
 have been conducted in both cdk2/cyclin a ( cdk2a ) and cdk4/cyclin 
 d1 ( cdk4d ) contexts which has not been previously carried out . to 
 this end , peptides derived from p21 ( cdk inhibitory protein ) and p107 
 ( rb like substrate ) were modified to study the key determinants of 
 binding .
these include an acidic region of the cyclin binding groove 
 interacting with basic residues of the cbm , and two hydrophobic pockets , 
 the larger of which interacts with the c - terminal lif and lfg motifs . 

since detailed sar information is a key component of replace , two 
 peptide sequences were chosen as the basis for these modifications 
 and included sakrrlfg ( p107 derived ) and hakrrlif ( optimized p21 octapeptide ) . 

for these octapeptides , substitutions and deletions of the c - terminal 
 motif were undertaken to study the binding requirements for cdk2a 
 and cdk4d and to probe structural differences of the major hydrophobic 
 pocket in each cyclin context .
peptide activity ( table 1 ) was quantified with a competitive binding assay previously 
 described employing fluorescence polarization ( fp ) of a fluorescein 
 labeled octapeptide as the readout . in the first instance , the contributions of the c - terminal 
 residues 
 and functional groups were explored .
sakrrlfg - nh2 ( 1 ) and sakrrlfg - oh ( 2 ) were synthesized and shown 
 to have comparable binding activity for cdk2a ( 0.07 vs 0.05 m ) 
 and cdk4d ( 0.88 vs 0.66 m ) , suggesting that the amide and carboxylic 
 acid groups interact similarly in both contexts . 3-thienylalanine 
 ( 3ta , 3 ) was incorporated as a substitution for phe8 
 and found to be a successful non - natural replacement . in the fp assay , 

the activity of this compound was determined to be 0.18 m for 
 cdk2a and 1.67 m for cdk4d , suggesting a 2-fold decrease in 
 binding to both contexts .
-homoleucine ( -leu ) 
 was hypothesized as an efficient 
 way of mimicking the spacing functionality of the ile residue in the 
 lif motif of the p21 sequence and improving binding activity of lfg 
 containing peptides .
exploring the -leu 
 replacement in the p107 context ( sakrr[leu]fg , 4 ) showed a 2- to 3-fold potency increase relative to the native leu 
 residue ( 2 ) in both cyclin contexts .
further to this , 
 the -leu substitution was examined in the 3ta containing peptide . 

a dramatic enhancement in binding affinity for cdk2a was also determined 
 for sakrr { leu}{3ta}g ( 5 ) with an 18-fold increase 
 in potency observed compared to 3 .
replacement of arg5 
 with asn ( 6 ) resulted in a 3- to 4-fold decrease in binding 
 in both contexts .
cyclohexylalanine was found to be another effective 
 replacement for phenylalanine ( 7 ) as part of the p21 
 derived peptide hakrrlif .
compound 7 had an ic50 value of 0.02 m for cdk2a and 0.18 m for cdk4d , indicating 
 very potent binding affinity to both cdk protein complexes and comparable 
 contribution to the native residue in hakrrlif ( 8) . in order to further validate key leads and control compounds , their 
 activity was examined in a direct binding format .
recently a protocol 
 has been developed for soluble expression of monomeric cyclin a2 ( 174432 
 fragment ) and its activity confirmed using control compounds in an 
 intrinsic tryptophan assay ( manuscript in preparation ) . in this case 
 binding to the cyclin groove is directly quantified by measuring fluorescence 
 of trp217 , the only such residue in cyclin a. by use of this method , 
 compound 8 was found to have a kd value of 0.019  0.004 m and therefore was very 
 similar to the result obtained in the fp competitive binding assay 
 validating both sets of observed activities .
further to the studies exploring sar in the 
 p21 and p107 octapeptide sequences , more detailed investigation into 
 the positional requirements of smaller peptides was completed ( table 2 ) .
the truncated p21 
 pentapeptide rrlif was selected as representing a realistic size and 
 molecular weight for conversion to more druglike compounds through 
 the replace strategy ( figure 1 ) .
three series 
 of peptide analogs derived from rrlif ( 9 , ic50 = 1.01 m and ic50 = 25.12 m for cdk2a and 
 cdk4d , respectively ) were designed to introduce replacements for key 
 binding determinants .
both natural and non - natural amino acid alternatives 
 for arg4 , arg5 , leu6 , and phe8 were incorporated to examine the sensitivity 
 and requirement for binding to the cbg .
complex of rrlif and 
 cdk2/cyclin a. ( a ) tertiary structure of cdk2 
 ( red ribbon ) , cyclin a ( blue ribbon ) , and rrlif ( 9 ) bound 
 to the cyclin groove .
( b ) close - up view of the interactions of rrlif 
 with the cyclin groove where 9 ( yellow carbons ) and the 
 key interacting side chains of cyclin a ( green carbons ) are shown 
 as a stick representation .
residues of the peptide are indicated by 
 three - letter amino acid code , whereas those of the protein are labeled 
 with one - letter codes . to examine the sensitivity of the critical arg4 position , 

substitution 
 of this residue with citrulline in cit - rlif ( 10 ) was 
 undertaken to probe the replacement of the salt bridge interaction 
 of the highly basic guanidinium side chain with the noncharged urea 
 isostere .
the resulting activity data showed a substantial potency 
 decrease of 40-fold with cdk2a ( 43.17 m ) but a lesser impact 
 for the cdk4d context ( 3 fold decrease , 40.46 m ) . further 
 to this , a number of exploratory substitutions were incorporated 
 at the arg5 position which has previously been shown to be less sensitive 
 to substitution than arg4 .
the citrulline replacement 
 at this position ( 11 ) confirmed this with activity values 
 of 5.19 m for cdk2a and 15.76 m for cdk4d , suggesting 
 a 5-fold decrease in affinity compared to rrlif and an increase in 
 binding to cdk4d .
substitution with isosteric non - natural amino acids 
 including lysine with dimethylation of the -amino group ( 12 ; r(n , n--dimethyllys)lif , 
 ic50 = 20.62 m ) resulted in approximately 20-fold 
 potency loss against cdk2a .
incorporation of n - me 
 arg ( 13 , r(nmearg)lif , 1.70 m ) suggests that backbone 
 methylation of this residue does not affect activity and with cdk4d 
 improves potency by 2-fold ( table 2 ) . in further studies of the tolerance of arg5 substitution for binding 
 to the cyclin groove
proline substitution ( 14 , rplif ) was shown to be detrimental to activity with ic50 values greater than 100 m for both cdk / cyclin complexes . 

the alanine surrogate ( 16 , ralif ) retained some activity 
 with a competitive binding ic50 of just over 20 m 
 in both contexts , a respectable value considering the loss of ion - pairing 
 capability . in order to examine the structural basis for the potency 
 variations upon modification of the peptide sequences ,
a computational 
 approach was used to model the interactions of each peptide in this 
 study .
the complex of rrlif bound to cdk2a has been previously determined 
 ( 1okv ) and was 
 therefore used for binding mode analysis and interaction energy calculations . 

the competitive binding efficiency for each peptide substitution as 
 determined by fp assay was compared with the interaction energy obtained 
 for the peptide complexes with cdk2a , and a good correlation between 
 these two parameters was observed with an r value of 0.855 as shown ( figure 2 ) .
these 
 results demonstrate that the loss of ion pairing interactions occurring 
 in the citrulline and other nonconservative replacement derivatives 
 was accurately reflected in the obtained interaction energy values . 

these also suggest that prospective modeling of additional mutations 
 and capping group substitutions will be possible by computational 
 design .
correlation of the interaction energies calculated for the peptide 
 analogs tested in table 2 with the experimental 
 affinity values as measured by inhibition constants determined using 
 the fp binding assay .
the correlation observed ( r = 0.857 ) suggests that the computational method is predictive 
 of actual binding .
further to the exchanges 
 of natural and non - natural amino acids 
 for the second arginine ( arg5 ) , peptoid substitutions were utilized 
 in order to stabilize the backbone of the cgi compound and to probe 
 the consequence of shifting the side chain one atom toward the n - terminus . 

two different peptoids incorporating a dimethylamino methyl group 
 ( table 2 , 17 , x1 = ( 2-(dimethylamino)methyl)glycine ) 
 and ( 18 , x2 = ( 2-(dimethylamino)methyl)alanine ) were 
 synthesized .
neither of these peptide  peptoid hybrids displayed 
 measurable binding activity for cdk2a or cdk4d , suggesting nonoptimal 
 positioning of the basic group for ion - pairing with the cyclin .
subsequent to the sar investigation of the n - terminal arginines , 
 residues of the c - terminal region were replaced by non - natural amino 
 acids and furthermore were deleted and/or truncated in the p21 and 
 p107 contexts ( table 3 ) .
previous comparisons 
 of the p21 and p27 c - termini indicate that placement of a spacer residue 
 between the leucine and phenylalanine results in a considerable potency 
 increase due to more optimal complementarity of the lipophilic side 
 chains with the primary hydrophobic pocket .
rrlfg ( 19 , ic50 = 18.88 m ) was synthesized and tested to 
 further examine impact on binding to cyclin a and d and was found 
 to undergo greater than 10-fold decrease in potency .
truncation of 
 the c - terminal glycine resulted in even weaker cdk2a binding ( 20 , ic50 = 35.65 m ) .
these results suggest 
 a significant impact of deletion of the spacer residues in both scenarios . 
 to further underscore the importance of spacing for optimal positioning 
 of the two side chains , incorporation of -leu as a leucine 
 replacement resulted in recapitulation of activity for both cdk2a 
 ( rrleuf , 21 , ic50 = 2.81 m ) 
 and cdk4d ( 21.17 m ) .
readdition of the glycine residue resulted 
 in a slight further potency improvement ( rrleufg , 22 ; , ic50 = 1.73 m cdk2/cyclin a , 20.24 m 
 cdk4d ) .
interchange of the native residue with 4-fluorophenylalanine 
 generated a 2-fold binding increase to both cdk / cyclin complexes ( rrlnpff , 23 , ic50 = 0.70 m cdk2/cyclin a , 9.26 m 
 cdk4d ) . in order to improve the druglikeness and metabolic stability 
 of the peptidic compounds ,
n - methylphenylalanine 
 was used as a replacement in the context of the leu while also 
 exploring the role of the c - terminal functional group .
rr{leu}nmef - oh 
 ( 24 , ic50 = 1.35 m ) and r{leu}nmef - nh2 ( 25 , ic50 = 5.82 m ) showed 
 that n - methylation of the phe backbone resulted in greater inhibition 
 of cdk2a . in previous work , it was shown that capping groups based upon a 
 1-(phenyl)-5-methyl-1h-1,2,4-triazole-3-carboxylic 
 acid core structure represent effective replacements for the n - terminal 
 tetrapapetide of hakrrlif and in the context of the capped tetramer 
 ( rlif ) result in similar activity to the p21 pentapeptide . with this knowledge in hand
, n - capped tripeptides 
 were synthesized incorporating sar information described in the previous 
 section , and their binding to cdk2a was measured ( figure 3 , table 3 ) .
these included 
 the 1-(3,5-dichlorophenyl)-5-methyl-1h-1,2,4-triazole-3-carboxamide 
 ( 35dcpt ) n - capped peptides , 35dcpt - cit-leu - nmephe ( 26 , ic50 > 100 m ) , 35dcpt - arg-leu - nmephe 
 ( 27 , ic50 = 8.95 m ) , and 35dcpt - arg-leu-3ta 
 ( 28 , ic50 = 5.70 m ) .
results indicate 
 that comparable levels of potency to the parent peptide 9 were obtained with n - capped molecules ( see 25 ) .
incorporation 
 of citrulline in this context however results in a substantial decrease 
 in binding for cdk2a relative to the peptides ( see 11 ) .
binding of these cgi compounds to cdk4d was much lower with little 
 activity being observed at relatively high concentrations of inhibitor . 

because of their overall improved druglikeness through inclusion of 
 nonpeptidic , non - natural amino acid components , the three n - capped 
 tripeptides were tested for preliminary antitumor effects in a cellular 
 viability assay .
growth inhibition values obtained in the mtt assay 
 demonstrated that significant antiproliferative activity was observed 
 in two tumor cell lines .
ic50 values in u2os osteosarcoma 
 and du145 prostate tumor cells ( table 3 ) were 
 found for 27 ( 30.7 and 36.5 m , respectively ) and 28 ( 18.3 and 21.8 m , respectively ) with the 3ta containing 
 molecule being the most potent .
not surprisingly the decreased on - target 
 activity and different physicochemical properties of the citrulline 
 containing compound ( 26 ) led to no observable cellular 
 activity .
( a ) 27 ( yellow carbon atoms ) showing the n - methylated phenylalanine 
 residues and ( b ) 28 ( cyan carbon atoms ) illustrating 
 the 3-thienylalanine residue .
both compounds are displayed interacting 
 with the solvent accessible surface of cyclin a and where the west 
 part of the molecule binds to the minor pocket and the second arginine 
 binding site whereas the east side interacts with the major lipophilic 
 site . as an extension of the preliminary 
 data indicating the successful 
 conversion of the p21 peptide rrlif into a more druglike inhibitor , 
 further modification and optimization of structure
it has been reported that incorporation 
 of modified arginine derivatives into peptides results in enhancements 
 both in ion - pairing ability and in cell permeability .
a series of non - natural isosteric analogs were included 
 based on the concept that steric inhibition of aqueous ion solvation 
 through guanidine alkylation promotes ion - pairing interactions . in 
 addition , increased lipophilicity of the non - natural residues may 
 improve pharmacokinetic parameters .
as shown ( table 3 ) , the modified arg5 residues were included as derivatives 
 of 28 containing the 3-thienylalanine , where the guanidine 
 group is monomethylated ( g10 , 29 , ic50 = 46.75 
 m ) , dimethylated ( g11 , 30 , ic50 = 3.69 
 m ) , monoethylated ( g12 , 31 , ic50 = 
 16.39 m ) , and cyclized ( g19 , 32 , ic50 = 4.50 m ) .
evaluation of these compounds in the fp assay 
 revealed that the dimethyl and cyclic guanidines had an approximately 
 2-fold enhancement in potency confirming that the greater magnitude 
 of the charge 
charge interaction improved binding . testing 
 of these compounds in the mtt assay however did not result in a corresponding 
 increase in activity ( table 3 ) relative to 28 and conversely seemed to diminish the antiproliferative 
 effect , thereby suggesting that in this context , the alkylated guanidines 
 did not lead to improvements in cellular permeability .
previous 
 attempts at studying cellular effects and consequences 
 of cyclin groove inhibition centered on the use of chimeric peptides 
 derived from p27 with penetration enhancing sequences appended to 
 the n - terminus .
minimal mechanistic data resulted from the use of 
 these peptides ; however , significant antiproliferative activity was 
 observed . as described above
, the cell permeable compounds 
 developed in this study are the first true mechanistic probes of the 
 consequences of cdk inhibition through the cbg and are therefore highly 
 useful tools in validating cell cycle cdks as antitumor therapeutics . 
 in order to determine these effects in detail , cell cycle analysis 
 through fluorescence activated cell sorting
was carried out using 
 the cell active n - capped tripeptides ( figure 4 ) .
the data obtained for 28 demonstrated an effect upon 
 cell cycle distribution that is consistent with inhibition of cdks 
 that play a key role in regulating the checkpoints .
relative to the 
 dmso control , a dose dependent g1 arrest was observed in asynchronous 
 u2os cells after treatment with 28 in contrast to the 
 effect observed for flavopiridol which resulted in a g2/m accumulation . 

u2os cells were treated for 48 h , with dmso only 
 as a control , and adherent cells were fixed with 70% ethanol and stained 
 with dapi ( 4,6-diamidino-2-phenylindole ) .
samples were analyzed 
 by facs and were performed in triplicate with a one - way anova statistical 
 analysis .
the 
 overarching objective of this study is to convert peptidic 
 molecules based on the cyclin binding motif into more druglike compounds . 

as described above , replace is an iterative approach that requires 
 in the first instance a detailed structure  activity relationship 
 for binding of a peptide to its target .
in addition , an in depth understanding 
 of the structural basis for peptide affinity to its receptor is important . 
 this information is crucial to decide which determinants can be truncated 
 and to inform the discovery of fragment alternatives ( capping groups ) 
 to replace the essential residues .
therefore , in order to lay the 
 groundwork for further conversion of p21 and p107 peptides into compounds 
 with more appropriate pharmaceutical properties , the structure 
natural 
 and non - natural residues were incorporated , and both conservative 
 ( isosteric ) and nonconservative mutations were explored to precisely 
 define the roles of the parent amino acid in binding .
this was done 
 to expand the sar information and especially for the pentapeptide 
 series that requires transformation into more druglike compounds . 

furthermore , limited data are available regarding the inhibition of 
 cdk4/6 through the cyclin groove .
this study addresses these issues 
 and allows development of druglike inhibitors based on cyclin groove 
 inhibition .
the cbg is primarily a hydrophobic groove consisting 
 of a major 
 and a minor site interacting with lipophilic side chains through the 
 hydrophobic effect and van der waals interactions ( figure 1 ) . in addition , an acidic component of the binding 
 interface ( glu220 , glu224 , and asp283 ; cyclin a2 numbering ) surrounds 
 the minor hydrophobic pocket and interacts with basic residues ( his , 
 lys , and arg ) on cdk substrates through ion pairing contacts .
hydrogen 
 bonding interactions of the peptide backbone amide groups are formed 
 with various side chains of the cyclin ( including w217 and q254 ) and 
 make significant contributions to affinity of the peptide inhibitor . in the cdk2a context ,
replacement of the guanidinium group with 
 the noncharged isosteric residue citrulline ( table 2 ) resulted in a 40-fold potency decrease for arg4 and only 
 a 4-fold decrease for arg5 .
this is consistent with previous data 
 suggesting a large contribution of arg4 to binding by way of its multiple 
 ion pairing interactions with the cyclin .
in contrast , only a 2-fold decrease in potency was observed for 
 replacement of arg4 and even a slight increase in binding for arg5 
 in terms of cdk4d inhibition ( table 2 ) .
this 
 indicates a diminished role of the critical arginine in the binding 
 to cyclin d1 and is consistent with the absence of one of the critical 
 ion pairing residues compared to cyclin a. examination of crystal 
 structures shows that cyclin d has a threonine residue in place of 
 an aspartate ( t62 vs d216 ) .
these results were further supported through 
 computational studies and reflected in the calculated interaction 
 energy values ( figure 2 ) for peptide analogs . 

incorporation of n , n--dimethyllysine 
 into the p21 pentapeptide resulted in greater loss of activity toward 
 cdk2a than does the replacement with citrulline .
this suggests that 
 despite the presence of an ion pairing group , its position and the 
 steric hindrance of the two methyl groups are unfavorable for binding . 

replacement again is tolerated to a greater degree than in the cyclin 
 d context , suggesting a larger volume of the binding pocket compared 
 to cyclin a. because of the important contributions of the side chain 
 of arg5 to binding , to determine the contribution of its amide nh 
 group , and to stabilize the peptide toward proteolysis , synthesis 
 of an n - methylated arginine containing p21 peptide was undertaken . 
 as this modification was shown to have little impact toward binding 
 in both cyclin contexts and even improved interaction with cdk4d by 
 2-fold
, highly useful information was obtained for improving druglikeness 
 of cyclin groove inhibitors requiring the guanidinium side chain .
nonconservative proteinogenic amino acid alternatives for the arg5 
 including gly , ala , and pro for the most part were ineffective replacements 
 due to loss of ion - pairing and h - bond interactions . the complete loss 
 of binding through proline incorporation almost certainly results 
 from the inability of this constrained residue to adopt the required 
 binding conformation with the loss of backbone h - bonding potentially 
 contributing also
again , the experimental binding observations were 
 validated structurally in modeling of these arginine replacements 
 and in terms of the predicted binding of the substituted peptides 
 ( figure 2 , supporting information ) . as a next step
, arg5 was replaced with a peptoid residue 
 to incorporate 
 a dimethylaminomethyl ( dmam ) group through n - alkylation ( table 2 ) .
inclusion of a peptoid moiety is a promising 
 strategy to retain peptide - like functionality while at the same time 
 improving overall stability .
since the side chain is moved from the 
  carbon onto the nitrogen , a tertiary amide resistant to proteolysis 
 will be formed upon coupling .
dmam was incorporated into the pentapeptide 
 as a substitution for arg5 in both a glycine and alanine context . 

the loss of activity for both molecules ( 17 and 18 ) most likely stems from the steric and conformational properties 
 of the resulting molecule , despite the modeling results and design 
 suggesting the close interaction of the basic nitrogen of the dmam 
 group with asp283 ( figure 1 ) .
further refinement 
 of the side chain spacer may result in improvements in binding however . as discussed above ,
leu6 and phe8 of the c - terminal motif are critical 
 for binding as shown by replacement with alanine and through observed 
 contacts of these residues with the primary hydrophobic pocket ( table 3 ) . in the p21 sequence
, ile represents a spacing 
 residue that keeps both leu and phe in an orientation for optimal 
 complementarity with the major lipophilic binding site .
deletion of this residue results in 20-fold 
 lower activity for cdk2a ( rrlf , 20 ) , thereby further 
 demonstrating its importance .
not surprisingly , a similar affect was 
 observed with cdk4a due to the similarity of the two major hydrophobic 
 pockets .
extension of this sequence with glycine to generate the p107 
 pentapeptide sequence ( rrlfg , 19 ) resulted in a slight 
 potency gain , since the carboxylate group of this residue is in closer 
 proximity to arg250 .
it was hypothesized that replacement of the native 
 leu residue with its one carbon homolog , -homoleucine , would 
 provide additional flexibility for the phe side chain to adopt greater 
 complementarity with the major lipophilic site and therefore mimic 
 the critical side chain and the ile spacer residue in a single residue .
inclusion of a -amino acid should also 
 provide better pharmacokinetic properties through decreased proteolytic 
 degradation .
the potency increase observed in the octapeptide p107 
 series in the phe ( 4 , table 1 ) 
 and especially the 3ta ( 5 ) contexts confirmed this .
for 
 the 3ta replacement , the dramatic improvement in binding observed 
 suggests excellent complementarity of the thiophene side chain with 
 the hydrophobic pocket .
furthermore , significant activity enhancements 
 of greater than 10-fold occurred for the truncated peptide counterparts 21 and 22 ( cdk2a binding relative to 9 and 19 , table 3 ) , therefore 
 warranting further investigation in the context of optimized inhibitors .
a peptide analog containing -leu and also n - methylphenylanine ( 24 ) therefore resulted in greater 
 potency relative to its counterpart 20 in both the c - terminal 
 acid and amide context ( 25 ) .
n - methylation of amino acids 
 and their incorporation into peptides have been shown to promote cell 
 permeability by removing the amide h - bond donor , thus decreasing the 
 desolvation penalty occurring during transduction through a biological 
 membrane while imparting improved lipid solubility by addition of 
 the hydrophobic methyl group .
n - methylation also imparts greater resistance 
 to degradation , since tertiary amides are poor substrates for protease 
 enzymes . through the verification 
 that incorporation of the non - natural 
 amino acids designed to improve stability and druglikeness , into the 
 c - terminal motif , resulted in enhanced activity of binding to the 
 cyclin groove
, these substitutions were combined with the n - capping 
 groups previously identified.27 and 28 included the 35dcpt group and nmephe and 3ta , 
 respectively , as the phenylalanine replacement ( figure 3 and table 3 ) .
of these two compounds , 
 inclusion of the thiophene side chain ( 28 ) was found 
 to generate a 2-fold advantage in activity compared to the n - methylphe ( 27 ) .
this results from greater 
 complementarity of the sulfur containing ring with the primary hydrophobic 
 pocket relative to the phenyl side chain . since both of these compounds 
 have substantially reduced tpsa ( 220 , 28 , vs 303 , 9 ) increased clogp ( 2.57 , 28 , vs 2.75 , 9 ) and considerably fewer h - bond acceptors and donors ( table 3 ) ,
their druglike properties are dramatically enhanced 
 relative to the native p21 pentapeptide ( according to lipinski s 
 and other guidelines for oral drugs , tpsa should be less than 140 
  and clogp between 0.4 and 5.6 ) the effectiveness of 
 these compounds in cellular antiproliferative assays and therefore 
 their ability to cross the biological membrane and induce cell death 
 confirmed the validity of design and incorporation of druglike functionality 
 ( table 3 ) .
although the modified arginine residues 
 substituted into the 28 scaffold generated compounds 
 of similar or even better binding potency , improved cellular activity 
 was unexpectedly not observed .
the requirement for the free guanidine 
 group suggests that steric hindrance may preclude interaction with 
 the negatively charged headgroups of the bilayer and prevent the initial 
 interactions required for transduction across the membrane .
although 2932 series did not enhance cellular 
 activity relative to 28 , it is reassuring that the two 
 most active inhibitors showed the best antiproliferative activity 
 of the four tested .
the observation that the lead compound 28 resulted in a significant g1 blockage ( figure 4 ) , a cell cycle effect consistent with cdk2 and cdk4 inhibition through 
 the cyclin groove , demonstrates on - target effects of this novel approach 
 to cancer therapeutic development .
furthermore , these data confirm 
 that replace is an effective strategy for the iterative conversion 
 of potent peptide inhibitors into compounds that are more appropriate 
 for drug development .
all n - terminally capped 
 flips were synthesized and purified using standard fmoc chemistry 
 by genscript ( piscataway , nj ) .
hplc and ms were used to confirm the 
 purity and structure of each peptide ( supporting 
 information table 2 ) .
a sample procedure is given as follows : 
 an amount of 5 equiv of the c - terminal amino acid ( e.g. , fmoc - phe , 
 202.7 mg , 0.5 mmol ) was coupled to rink resin ( 200 mg , 0.1 mmol ) after 
 activation with diisopropylethylamine ( dipea ) ( 6 equiv , 77.54 mg , 
 0.6 mmol ) and hbtu ( 4.4 equiv , 166.86 mg , 0.58 mmol ) in 2 ml of dmf 
 for 1 h. the fmoc protecting group of the c - terminal amino acid was 
 removed using 20% piperidine in 3 ml of dmf for 10 min before addition 
 of 5 equiv of the next amino acid using diea ( 0.082 ml ) and hbtu ( 189.6 
 mg ) in 5 ml of dmf .
wash cycles ( 5  10 ml of dmf + 5  
 10 ml of dcm ) were applied to each step between coupling and deprotection 
 of fmoc . upon completion of assembly , side chain protecting groups 
 were removed , and peptides were cleaved from rink resin using ( 2 ml ) 
 90:5:5 mixtures of tfa / h2o / tips .
crude peptides were purified 
 using reverse - phase flash chromatography and semipreparative reverse - phase 
 hplc methods .
pure compounds were lyophilized and characterized using 
 mass spectrometry and analytical hplc ( see supporting 
 information table 1 ) . modeled complexes of peptidic cyclin groove inhibitors 
 bound to cyclin 

a were generated using the following protocol which has previously 
 been validated : the crystal structure of rrlif 
 ( pdb code 1okv ) bound to cyclin a was used as the starting point following which 
 arg4 was mutated to the corresponding residue to generate cyclin groove 
 bound complexes for compounds 9 , 10 , 11 , 13 , and 16 ( table 2 ) .
all calculations were performed using discovery studio 
 3.0 and specifically using the macromolecules and simulations protocols . 

prior to minimization , the heavy atoms of the protein were set as 
 constraints , and the charmm force field was applied to the receptor  ligand 
 complex . for the energy minimization of the inhibitor cyclin complex , 
 the smart minimizer algorithm was used with 2000 minimization iterations 
 and a convergence criterion of 0.1 ( rms gradient ) .
the routine also 
 employed the gbsw ( generalized born with 
 a simple switching ) implicit solvent model using a dielectric constant 
 of 1 and an implicit solvent dielectric constant of 80 with all other 
 parameters being set as default .
after minimization of the complex 
 to convergence , the interaction energy between cyclin a and each peptide 
 was calculated using the receptor  ligand interaction module 
 again using the gbsw implicit solvent model . calculated interaction 
 energy values are shown in supporting information 
 table 1 .
u2os osteosarcoma cells 
 and du145 human 
 prostate cancer cell lines were obtained from atcc ( manassas , va ) . 

all cells were grown in dulbecco s modified eagle medium ( dmem , 
 fisher scientific ) supplemented with 10% fetal bovine serum ( fbs , 
 usa scientific ) and 100 g / ml streptomycin ( strep)/100 u / ml 
 penicillin ( pen ) .
cells were seeded in 96-well plates 
 at ( 2  10 cells / ml ) and allowed to adhere overnight 
 in dmem growth medium containing 10% nu serum and 1% pen / strep .
the 
 compounds 26 , 27 , and 28 were 
 dissolved in dmso and added in a dose dependent manner ( 1100 
 m ) .
the diluted compounds were added to the cells in triplicate 
 and incubated for 72 h at 37 c with 5% co2 .
absorbance readings were 
 obtained using a dtx880 multimode detector fitted with 595 nm filter . 

cell viability results were represented as the ratio of the absorbance 
 reading in treated vs untreated cells .
ic50 values were 
 obtained using a nonlinear regression line with graphpad prism 5.0 . 

cells were plated at 150  000 
 cells / ml overnight in six - well plates .
cdk2 inhibitors were added 
 in a dose dependent fashion for 48 h. the cells were then fixed with 
 70% ethanol overnight at 20 c .
the cells were washed twice with 1 
 pbs following staining with dapi ( 4,6 diamino-2-phenylindole ) 
 and analyzed using bd lsr ii ( becton dickenson ) with a blue filter . 

this assay was performed 
 using black 
 384-well plates using a previously described procedure with the following modifications . to each well 

were added the following : 5 l of cdk4d1 ( 0.5 g / well 
 purified recombinant human kinase complex from bps bioscience ) , 5 
 l of compound solution , 5 l of 12 nm fluoresceinyl - ahx - pro - val - lys - arg - arg - leu-(3clphe)-gly 
 tracer peptide .
compounds and kinase complexes were diluted using 
 assay buffer ( 25 nm hepes , ph 7 , 10 nm nacl , 0.01% nonidet p-40 , 1 
 mg / ml bsa , 1 mm dithiothretiol ( dtt ) ) .
plate was centrifuged for 1 
 min at 500 rpm and then incubated with shaking for 45 min at room 
 temperature .
fluorescence polarization was read on dtx880 multimode 
 detector ( beckman coulter , brea , ca ) fitted with 485 nm/535 nm excitation / emission 
 filters and a dichroic mirror suitable for fluorescein .
relative mp 
 was calculated for each concentration tested using the equation showing 
 below .
ic50 values were determined by logarithmic regression 
 by correlating relative melting points and testing concentrations .
were added the following : 5 l 
 of cdk2a2 ( 0.27 g / well purified recombinant human kinase complex ) , 
 5 l of compound solution , 5 l of 12 nm fluoresceinyl - ahx - pro - val - lys - arg - arg - leu-(3clphe)-gly 
 tracer peptide .
compounds and kinase complexes were diluted using 
 assay buffer ( 25 nm hepes , ph 7 , 10 nm nacl , 0.01% nonidet p-40 , 1 
 mg / ml bsa , 1 mm dithiothretiol ( dtt ) ) .
plate was centrifuged for 1 
 min at 500 rpm and then incubated with shaking for 45 min at room 
 temperature .
fluorescence polarization was read on dtx880 multimode 
 detector ( beckman coulter , brea , ca ) fitted with 485 nm/535 nm excitation / emission 
 filters and a dichroic mirror suitable for fluorescein .
relative mp 
 was calculated for each concentration tested using the equation showing 
 above .
ic50 values were determined by logarithmic regression 
 by correlating relative melting points and testing concentrations . for the binding assay of 6xhis - cyclin a2 with compound 8 ,
a previously developed method was exploited in order to determine the dissociation constant ( kd ) of recombinant cyclin a2 with the ligand . 

differences in fluorescence intensity at 345 nm between the complex 
 ( cyclin a2/compound 8) and free protein ( excitation at 
 295 nm ) were analyzed by measuring the respective fluorescence intensities 
 with a hitachi f-2500 fluorescence spectrophotometer in 0.4 cm  
 1 cm quartz cuvettes at 25 c . the excitation and emission wavelengths 
 were 295 and 345 nm , respectively .
the slits were set at 5 and 20 
 nm for the excitation and emission , respectively .
briefly , 1.5 ml 
 of protein solution in fluorescence buffer ( 0.10.65 m ) 
 was equilibrated in a cuvette at 25 c for 1 h. after equilibration , 
 small increments ( 210 l ) of the ligand solution were 
 injected and binding was allowed to take place for 2 min , with shutter 
 closed to avoid protein deterioration , before fluorescence intensity 
 measurement . in order to determine the mandatory dilution effect due 
 to the addition of ligand and any fluorescence effect due to the unbound 
 ligand , a blank sample containing tryptophan with fluorescence signal 
 of a similar level was titrated with the addition of the exact ligand 
 injections .
the corresponding kd values of the binding 
 reactions were determined using prism ( graphpadsoftware , san diego , 
 ca ) .
 the cyclin groove is an important 
 recognition site for substrates 
 of the cell cycle cyclin dependent kinases and provides an opportunity 
 for highly selective inhibition of kinase activity through a non - atp 
 competitive mechanism . 
 the key peptide residues of the cyclin binding 
 motif have been studied in order to precisely define the structure  
 activity 
 relationship for cdk kinase inhibition . through this information , 
 
 new insights into the interactions of peptide cdk inhibitors with 
 key subsites of the cyclin binding groove provide for the replacement 
 of binding determinants with more druglike functionality through replace , 
 a strategy for the iterative conversion of peptidic blockers of protein  protein 
 interactions into pharmaceutically relevant compounds . as a result , 
 replace is further exemplified in combining optimized peptidic sequences 
 with effective n - terminal capping groups to generate more stable compounds 
 possessing antitumor activity consistent with on - target inhibition 
 of cell cycle cdks . 
 the compounds described here represent prototypes 
 for a next generation of kinase therapeutics with high efficacy and 
 kinome selectivity , thus avoiding problems observed with first generation 
 cdk inhibitors . 
Introduction
Results
Discussion
Materials and Methods