Iterative conversion of cyclin binding groove peptides into druglike CDK inhibitors with antitumor activity

Article abstract of DOI:10.1021/jm5015023

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.

Full text of DOI:10.1021/jm5015023

Article
pubs.acs.org/jmc
Iterative Conversion of Cyclin Binding Groove Peptides into Druglike
CDK Inhibitors with Antitumor Activity
Padmavathy Nandha Premnath,† Sandra N. Craig,† Shu Liu,†,§ Erin L. Anderson,†
Asterios I. Grigoroudis,‡ George Kontopidis,‡ Tracy L. Perkins,† Michael D. Wyatt,† Douglas L. Pittman,†
and Campbell McInnes*,†
†Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina,
Columbia, South Carolina 29208, United States
‡Department of Biochemistry, Faculty of Veterinary Science, University of Thessaly, Trikalon 224, Karditsa 43100, Greece
S
* Supporting Information
ABSTRACT: 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 pharmaceuti-
cally 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.
suggest that its nonessential role is a result of the substitution of
one CDK for another.1,11−13 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 transcrip-
tional inhibition of CDK7 and CDK9.14,15 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. Targeting of the protein−protein
INTRODUCTION
■
Cyclin dependent kinases (CDKs) and their natural inhibitors
(CDKIs) are central to cell cycle regulation, and their functions
are commonly altered in tumor cells.1 Deregulation of CDK2
and CDK4 through inactivation of CDKIs such as p16INK4a
,
p21WAF1 (p21), p27KIP1, and p57KIP2 provides a means for
cancer cells to override the G1 checkpoint.2,3 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.4,5 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.6 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.7
Inhibition of CDK activity with cyclin groove inhibitors (CGI)
therefore results in tumor selective induction of apoptosis in
cells already possessing deregulated E2F.8−10
Special Issue: New Frontiers in Kinases
Received: September 29, 2014
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
© XXXX American Chemical Society
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Table 1. Structure−Activity Relationship of p21 and p107 Derived Peptide Analogs
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 inhib-
itors.16,17 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.8,10
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 develop-
ment.18−21 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.
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.18
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 μM) and CDK4D (0.88 vs 0.66 μM), 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 μM for CDK2A and 1.67 μM for
CDK4D, suggesting a 2-fold decrease in binding to both
contexts.
RESULTS
■
Structure−Activity Relationship of Octapeptide Ana-
logs. While structure−activity relationships for the cyclin
binding motif (CBM) have previously been described,
contributions of key residues have not been adequately
investigated.22−24 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
β-Homoleucine (β-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
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Table 2. Cyclin A/CDK2 and Cyclin D1/CDK4 Binding Activity for Arg Replacement Peptides
of LFG containing peptides. Preliminary proof of concept for
this has been previously obtained.25 Exploring the β-Leu
replacement in the p107 context (SAKRR[βLeu]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 β-
Leu substitution was examined in the 3TA containing peptide.
A dramatic enhancement in binding affinity for CDK2A was
also determined for SAKRR {βLeu}{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 μM for CDK2A and 0.18 μM 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 (174−432 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 μM and therefore was very similar to the result obtained
in the FP competitive binding assay validating both sets of
observed activities.
Structure−Activity Relationship of Pentapeptide
Analogs Based on p21. 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
μM and IC50 = 25.12 μM 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.
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 μM) but a lesser impact for the CDK4D context (3 fold
decrease, 40.46 μM).
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 Arg4.24,25 The
citrulline replacement at this position (11) confirmed this with
activity values of 5.19 μM for CDK2A and 15.76 μM for
CDK4D, suggesting a 5-fold decrease in affinity compared to
RRLIF and an increase in binding to CDK4D. Substitution with
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with the interaction energy obtained for the peptide complexes
with CDK2A, and a good correlation between these two
parameters was observed with an R2 value of 0.855 as shown
(Figure 2). These results demonstrate that the loss of ion
Figure 2. 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 (R2 = 0.857) suggests that the
computational method is predictive of actual binding.
pairing interactions occurring in the citrulline and other
nonconservative replacement derivatives was accurately re-
flected 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.
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
Figure 1. 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.
isosteric non-natural amino acids including lysine with
dimethylation of the ε-amino group (12; R(N,N-ε-
dimethyllys)LIF, IC50 = 20.62 μM) resulted in approximately
20-fold potency loss against CDK2A. Incorporation of N-Me
Arg (13, R(NMeArg)LIF, 1.70 μM) 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, nonconservative mutations were
incorporated (Table 2). Proline substitution (14, RPLIF) was
shown to be detrimental to activity with IC50 values greater
than 100 μM for both CDK/cyclin complexes. Glycine
substitution (15) led to a similar activity cliff. The alanine
surrogate (16, RALIF) retained some activity with a
competitive binding IC50 of just over 20 μM 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 primary hydrophobic pocket. RRLFG (19, IC50
=
18.88 μM) 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 μM). 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 β-Leu as a leucine
replacement resulted in recapitulation of activity for both
CDK2A (RRβLeuF, 21, IC50 = 2.81 μM) and CDK4D (21.17
μM). Readdition of the glycine residue resulted in a slight
further potency improvement (RRβLeuFG, 22;, IC50 = 1.73
μM CDK2/cyclin A, 20.24 μM CDK4D). Interchange of the
native residue with 4-fluorophenylalanine generated a 2-fold
binding increase to both CDK/cyclin complexes (RRLNpfF,
D
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Table 3. Conversion of p21 Peptides into More Druglike Cyclin Groove Inhibitors
23, IC50 = 0.70 μM CDK2/cyclin A, 9.26 μM 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 βLeu while also exploring the
role of the C-terminal functional group. RR{βLeu}NMeF-OH
(24, IC50 = 1.35 μM) and R{βLeu}NMeF-NH2 (25, IC50
=
5.82 μM) showed that N-methylation of the Phe backbone
resulted in greater inhibition of CDK2A.
Synthesis and Antitumor Effects of Druglike Cyclin
Groove Inhibitors. 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.18 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-tria-
zole-3-carboxamide (35DCPT) N-capped peptides, 35DCPT-
Cit-βLeu-NMePhe (26, IC50 >100 μM), 35DCPT-Arg-βLeu-
NMePhe (27, IC50 = 8.95 μM), and 35DCPT-Arg-βLeu-3TA
(28, IC50 = 5.70 μM). 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,
Figure 3. Binding mode of two druglike cyclin groove inhibitors. (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.
E
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Figure 4. Cell cycle analysis of asynchronous U2OS cells treated with flavopiridol and 28. 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. (A) Analysis of total DNA content. (B) Comparison of percentage of
cells in G1. (C) Comparison of percentage of cells in G2.
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 μM,
respectively) and 28 (18.3 and 21.8 μM, 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.
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 were undertaken (Table 3). It has been reported that
incorporation of modified arginine derivatives into peptides
results in enhancements both in ion-pairing ability and in cell
permeability.26 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
μM), dimethylated (G11, 30, IC50 = 3.69 μM), monoethylated
(G12, 31, IC50 = 16.39 μM), and cyclized (G19, 32, IC50 = 4.50
μM). 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 con-
sequences 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.8,9 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. The latter
result is consistent with previously published data for pan CDK
inhibitors.1
DISCUSSION
■
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
F
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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 proper-
ties, the structure−activity was determined for key binding
determinants of these sequences. 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.22 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-Σ-dimethyllysine into the p21 pentapep-
tide 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 replace-
ments 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 α 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.16 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, β-homoleucine, would provide additional
flexibility for the Phe side chain to adopt greater comple-
mentarity with the major lipophilic site and therefore mimic the
critical side chain and the Ile spacer residue in a single
residue.25 Inclusion of a β-amino acid should also provide
better pharmacokinetic properties through decreased proteo-
lytic 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 β-Leu and also N-methylphe-
nylanine (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
G
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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).
decreasing the desolvation penalty occurring during trans-
duction 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.27
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.18
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 Å 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 29−32 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.
Energy Minimization and Interaction Energy Calculation.
Modeled complexes of peptidic cyclin groove inhibitors bound to
cyclin A were generated using the following protocol which has
previously been validated:18,22 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 GBSW28 (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.
Cell Culture. 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 μg/mL streptomycin (strep)/100 U/
mL penicillin (pen). Cells were incubated at 37 °C with 5% CO2.
Viability Assay. Cells were seeded in 96-well plates at (2 × 103
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 (1−100 μM). The diluted compounds were added to the cells
in triplicate and incubated for 72 h at 37 °C with 5% CO2. The cell
viability was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay.29 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.
Experiments were performed in triplicate.
Cell Cycle Analysis. 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 centrifuged, and ethanol
was removed. 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. Experiments were
performed in triplicate.
Fluorescence Polarization Binding Assay. CDK4D1 FP Assay.
This assay was performed using black 384-well plates using a
previously described procedure30 with the following modifications.
To each well were added the following: 5 μL of CDK4D1 (0.5 μg/well
purified recombinant human kinase complex from BPS Bioscience), 5
μL of compound solution, 5 μL 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
MATERIALS AND METHODS
■
Peptide and FLIP Synthesis. 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). Peptides
were assembled by using standard solid-phase synthesis methods. 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
H
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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.
Notes
The authors declare the following competing financial
interest(s): C.M. recently formed a company that aims to
commercialize future versions of the compounds described
herein.
relative mp
mp(compound) − mp(DMSO, protein, tracer)
mp(DMSO, protein) − mp(DMSO, protein, tracer)
ACKNOWLEDGMENTS
■
=
We thank Drs. Michael Walla and William Cotham in the
Department of Chemistry and Biochemistry at the University of
South Carolina for assistance with mass spectrometry and
Helga Cohen and Dr. Perry Pellechia for NMR spectroscopy.
Dr. Lim Chang assisted with running cell cycle analysis
experiments. We also acknowledge the contribution of Dr.
Joshua Bolger in providing expertise in synthetic chemistry.
This work was funded by the National Institutes of Health
through Research Project Grant R01CA131368.
CDK2A2 FP Assay. This assay was performed using black 384-well
plates. To each well were added the following: 5 μL of CDK2A2 (0.27
μg/well purified recombinant human kinase complex), 5 μL of
compound solution, 5 μL 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.
Determination of Dissociation Constant (Kd) from Fluo-
rescence Measurements of Cyclin A2 Binding Activity. For the
binding assay of 6xHis-cyclin A2 with compound 8, a previously
developed method was exploited31 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 (0.1−0.65 μM) was equilibrated in a
cuvette at 25 °C for 1 h. After equilibration, small increments (2−10
μL) 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. Measured
intensities were corrected for blank signals. The corresponding Kd
values of the binding reactions were determined using Prism
(GraphPadSoftware, San Diego, CA).
REFERENCES
■
(1) Shapiro, G. I. Cyclin-dependent kinase pathways as targets for
cancer treatment. J. Clin. Oncol. 2006, 24, 1770−1783.
(2) Sherr, C. J. Cancer cell cycles. Science 1996, 274, 1672−1677.
(3) Malumbres, M.; Barbacid, M. To cycle or not to cycle: a critical
decision in cancer. Nat. Rev. Cancer 2001, 1, 222−231.
(4) Ball, K. L.; Lain, S.; Fahraeus, R.; Smythe, C.; Lane, D. P. Cell-
̊
cycle arrest and inhibition of Cdk4 activity by small peptides based on
the carboxy-terminal domain of p21WAF1. Curr. Biol. 1997, 7, 71−80.
(5) Chen, J. S. P.; Kornbluth, S.; Dynlacht, B. D.; Dutta, A. Cyclin-
binding motifs are essential for the function of p21CIP1. Mol. Cell.
Biol. 1996, 16, 4673−4682.
(6) Kitagawa, M.; Higashi, H.; Suzuki-Takahashi, I.; Segawa, K.;
Hanks, S. K.; Taya, Y.; Nishimura, S.; Okuyama, A. Phosphorylation of
E2F-1 by cyclin A-cdk2. Oncogene 1995, 10, 229−236.
(7) Phillips, A. C.; Vousden, K. H. E2F-1 induced apoptosis.
Apoptosis 2001, 6, 173−182.
(8) Chen, Y. N.; Sharma, S. K.; Ramsey, T. M.; Jiang, L.; Martin, M.
S.; Baker, K.; Adams, P. D.; Bair, K. W.; Kaelin, W. G., Jr. Selective
killing of transformed cells by cyclin/cyclin-dependent kinase 2
antagonists. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4325−4329.
(9) Mendoza, N.; Fong, S.; Marsters, J.; Koeppen, H.; Schwall, R.;
Wickramasinghe, D. Selective cyclin-dependent kinase 2/cyclin A
antagonists that differ from ATP site inhibitors block tumor growth.
Cancer Res. 2003, 63, 1020−1024.
(10) Mendoza, N.; Severin, C.; Marsters, J.; Wickramasinghe, D.
Cyclin A antagonists kill tumor cells in vitro and in vivo. Proc. Am.
Assoc. Cancer Res. 2004, 45, 834.
(11) Tetsu, O.; McCormick, F. Proliferation of cancer cells despite
CDK2 inhibition. Cancer Cell 2003, 3, 233−245.
(12) Malumbres, M.; Barbacid, M. Is Cyclin D1-CDK4 kinase a bona
fide cancer target? Cancer Cell 2006, 9, 2−4.
(13) Malumbres, M.; Sotillo, R.; Santamaria, D.; Galan, J.; Cerezo, A.;
Ortega, S.; Dubus, P.; Barbacid, M. Mammalian cells cycle without the
D-type cyclin-dependent kinases Cdk4 and Cdk6. Cell 2004, 118,
493−504.
(14) Lam, L. T.; Pickeral, O. K.; Peng, A. C.; Rosenwald, A.; Hurt, E.
M.; Giltnane, J. M.; Averett, L. M.; Zhao, H.; Davis, R. E.;
Sathyamoorthy, M.; Wahl, L. M.; Harris, E. D.; Mikovits, J. A.;
Monks, A. P.; Hollingshead, M. G.; Sausville, E. A.; Staudt, L. M.
Genomic-scale measurement of mRNA turnover and the mechanisms
of action of the anti-cancer drug flavopiridol. Genome Biol. 2001, 2,
research0041.1−0041.11.
(15) Lu, X.; Burgan, W. E.; Cerra, M. A.; Chuang, E. Y.; Tsai, M. H.;
Tofilon, P. J.; Camphausen, K. Transcriptional signature of
flavopiridol-induced tumor cell death. Mol. Cancer Ther 2004, 3,
861−872.
(16) Zheleva, D. I.; McInnes, C.; Gavine, A.-L.; Zhelev, N. Z.;
Fischer, P. M.; Lane, D. P. Highly potent p21WAF1 derived peptide
inhibitors of CDK-mediated pRb phosphorylation: delineation and
ASSOCIATED CONTENT
■
S
* Supporting Information
One table providing analytical characterization data for FLIPs
shown in Tables 1−3; one table showing the calculated data for
the interaction energies plotted in Figure 2; one figure showing
the plot and calculated data for the measurement of direct
binding affinity of HAKRRLIF to cyclin A using intrinsic
tryptophan fluorescence. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mcinnes@sccp.sc.edu. Phone: (803) 576-5684.
Present Address
§S.L.: Department of Surgery, University of California at San
Francisco, San Francisco, CA. E-mail, shu.liu@ucsfmedctr.org.
I
dx.doi.org/10.1021/jm5015023 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
structural insight into their interactions with cyclin A. J. Pept. Res.
2002, 60, 257−270.
(17) McInnes, C.; Andrews, M. J.; Zheleva, D. I.; Lane, D. P.; Fischer,
P. M. Peptidomimetic design of CDK inhibitors targeting the
recruitment site of the cyclin subunit. Curr. Med. Chem.: Anti-Cancer
Agents 2003, 3, 57−69.
(18) Liu, S.; Premnath, P. N.; Bolger, J. K.; Perkins, T. L.; Kirkland,
L. O.; Kontopidis, G.; McInnes, C. Optimization of non-ATP
competitive CDK/cyclin groove inhibitors through REPLACE-
mediated fragment assembly. J. Med. Chem. 2013, 56, 1573−1582.
(19) McInnes, C. Progress in the development of non-ATP
competitive protein kinase inhibitors for oncology. In Annual Reports
in Medicinal Chemistry; Bernstein, P, Desai, M., Ed.; Elsevier:
Amsterdam, 2012; Vol. 47, pp 459−474.
(20) Andrews, M. J.; Kontopidis, G.; McInnes, C.; Plater, A.; Innes,
L.; Cowan, A.; Jewsbury, P.; Fischer, P. M. REPLACE: a strategy for
iterative design of cyclin-binding groove inhibitors. ChemBioChem
2006, 7, 1909−1915.
(21) Premnath, P. N.; Liu, S.; Perkins, T.; Abbott, J.; Anderson, E.;
McInnes, C. Fragment based discovery of arginine isosteres through
REPLACE: towards non-ATP competitive CDK inhibitors. Bioorg.
Med. Chem. 2014, 22, 616−622.
(22) Liu, S.; Bolger, J. K.; Kirkland, L. O.; Premnath, P. N.; McInnes,
C. Structural and functional analysis of cyclin D1 reveals p27 and
substrate inhibitor binding requirements. ACS Chem. Biol. 2010, 5,
1169−1182.
(23) Kontopidis, G.; Andrews, M. J. I.; McInnes, C.; Cowan, A.;
Powers, H.; Innes, L.; Plater, A.; Griffiths, G.; Paterson, D.; Zheleva, D.
I.; Lane, D. P.; Green, S.; Walkinshaw, M. D.; Fischer, P. M. Insights
into cyclin groove recognition: complex crystal structures and inhibitor
design through ligand exchange. Structure 2003, 11, 1537−1546.
(24) Castanedo, G.; Clark, K.; Wang, S.; Tsui, V.; Wong, M.;
Nicholas, J.; Wickramasinghe, D.; Marsters, J. C., Jr.; Sutherlin, D.
CDK2/cyclinA inhibitors: targeting the cyclinA recruitment site with
small molecules derived from peptide leads. Bioorg. Med. Chem. Lett.
2006, 16, 1716−1720.
(25) Kontopidis, G.; Andrews, M. J.; McInnes, C.; Plater, A.; Innes,
L.; Renachowski, S.; Cowan, A.; Fischer, P. M. Truncation and
optimisation of peptide inhibitors of cyclin-dependent kinase 2-cyclin a
through structure-guided design. ChemMedChem 2009, 4, 1120−1128.
(26) Kennedy, K. J.; Lundquist, J. T. I.; Simandan, T. L.; Kokko, K.
P.; Beeson, C. C.; Dix, T. A. Design rationale, synthesis, and
characterization of non-natural analogs of the cationic amino acids
arginine and lysine. J. Pept. Res. 2000, 55, 348−358.
(27) Chatterjee, J.; R, F.; Kessler, H. N-methylation of peptides and
proteins: an important element for modulating biological functions.
Angew. Chem., Int. Ed. 2013, 52, 254−269.
(28) Khandogin, J.; Brooks, C. L., 3rd. Constant pH molecular
dynamics with proton tautomerism. Biophys. J. 2005, 89, 141−157.
(29) Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth
and survival. Modifications to the tetrazolium dye procedure giving
improved sensitivity and reliability. J. Immunol. Methods 1986, 89,
271−277.
(30) Andrews, M. J.; McInnes, C.; Kontopidis, G.; Innes, L.; Cowan,
A.; Plater, A.; Fischer, P. M. Design, synthesis, biological activity and
structural analysis of cyclic peptide inhibitors targeting the substrate
recruitment site of cyclin-dependent kinase complexes. Org. Biomol.
Chem. 2004, 2, 2735−2741.
(31) Papaneophytou, C. P.; Grigoroudis, A. I.; McInnes, C.;
Kontopidis, G. Quantification of the effects of ionic strength, viscosity,
and hydrophobicity on protein-ligand binding affinity. ACS Med.
Chem. Lett. 2014, 5, 931−936.
J
dx.doi.org/10.1021/jm5015023 | J. Med. Chem. XXXX, XXX, XXX−XXX