One-Bead-Two-Compound Thioether Bridged Macrocyclic γ-AApeptide Screening Library against EphA2

Article abstract of DOI:10.1021/acs.jmedchem.7b01280

Identification of molecular ligands that recognize peptides or proteins is significant but poses a fundamental challenge in chemical biology and biomedical sciences. Development of cyclic peptidomimetic library is scarce, and thus discovery of cyclic peptidomimetic ligands for protein targets is rare. Herein we report the unprecedented one-bead-two-compound (OBTC) combinatorial library based on a novel class of the macrocyclic peptidomimetics γ-AApeptides. In the library, we utilized the coding peptide tags synthesized with Dde-protected α-amino acids, which were orthogonal to solid phase synthesis of γ-AApeptides. Employing the thioether linkage, the desired macrocyclic γ-AApeptides were found to be effective for ligand identification. Screening the library against the receptor tyrosine kinase EphA2 led to the discovery of one lead compound that tightly bound to EphA2 (K_d = 81 nM) and potently antagonized EphA2-mediated signaling. This new approach of macrocyclic peptidomimetic library may lead to a novel platform for biomacromolecular surface recognition and function modulation.

Full text of DOI:10.1021/acs.jmedchem.7b01280

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Article
One-Bead-Two-Compound Thioether Bridged Macrocyclic
#-AApeptide Screening Library against EphA2
Yan Shi, Sridevi Challa, PENG SANG, Fengyu She, Chunpu Li, Geoffrey M. Gray, Alekhya
Nimmagadda, Peng Teng, Timothy Odom, Yan Wang, Arjan van der Vaart, Qi Li, and Jianfeng Cai
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01280 • Publication Date (Web): 07 Nov 2017
Downloaded from http://pubs.acs.org on November 9, 2017
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OneꢀBeadꢀTwoꢀCompound Thioether Bridged Macrocyclic
γꢀAApeptide Screening Library against EphA2
Yan Shi1,+, Sridevi Challa2,+, Peng Sang,1 Fengyu She,1 Chunpu Li,3 Geoffrey M. Gray,1
Alekhya Nimmagadda,1 Peng Teng,1 Timothy Odom,1 Yan Wang,3 Arjan van der Vaart,1 Qi
Li3,* and Jianfeng Cai1,*
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1Department of Chemistry, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620
2Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, 12902
Magnolia Drive, Tampa, FL 33612
3Department of Medical Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese
Medicine, Shanghai, 201203, China
+ These authors contributed equally to this work
jianfengcai@usf.edu and lzwf@hotmail.com
Abstract
Identification of molecular ligands that recognize peptides or proteins is significant, but poses a
fundamental challenge in chemical biology and biomedical sciences. Development of cyclic
peptidomimetic library is scarce and thus discovery of cyclic peptidomimetic ligands for protein
targets is rare. Herein we report the unprecedented OneꢀBeadꢀTwoꢀCompound (OBTC)
combinatorial library based on a novel class of the macrocyclic peptidomimetics γꢀAApeptides.
In the library, we utilized the coding peptide tags synthesized with Ddeꢀprotected αꢀamino acids,
which were orthogonal to solid phase synthesis of γꢀAApeptides. Employing the thioether
linkage, the desired macrocyclic γꢀAApeptides were found to be effective for ligand
identification. Screening the library against the receptor tyrosine kinase EphA2 led to the
discovery of one lead compound which tightly bound to EphA2 (Kd: 81 nM) and potently
antagonized EphA2ꢀmediated signaling. This new approach of macrocyclic peptidomimetic
library may lead to a novel platform for biomacromolecular surface recognition and function
modulation.
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Introduction
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Chemical biology and biomedical sciences are undergoing a new era of vigorous development
due to the rapid discovery of new protein targets and unveiling of their biological importance.
Consequently, it is an unmet need to identify ligands that recognize peptide or protein targets
with high specificity and affinity,1 since such an effort could help understanding function of
proteins and lead to potential therapeutic agents for diagnosis and treatment. Combinatorial
chemistry which serves as a powerful tool for ligand screening in basic medicinal chemistry, has
emerged to meet this challenge.2 The diverse library of compounds provides concurrent but
independent chances for target screening. Since it provides the possibility to yield important
therapeutic agents from various target proteins, the progress of drug discovery has been largely
accelerated.3 As peptides are versatile molecules, which have modular chemical diversity and
favorable binding activity, they are natural building blocks for combinatorial libraries for
screening against various targets. Among them, macrocyclic peptides which have enhanced
conformational constraint and binding affinity are widely recognized for exploring ligandꢀ
receptor interactions. Several general methods have been successfully developed to construct the
macrocyclic ring systems.4ꢀ5, 8ꢀ12
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Recent efforts have contributed to the creation of nonꢀnatural sequenceꢀspecific
peptidomimetics.13 These peptidomimetics are developed based on the mimicry of peptide
primary structure and possess a modified peptide backbone for the introduction of diverse
functional side chains. Compared with natural peptides, peptidomimetics possess improved
proteaseꢀresistance, chemodiversity, and bioavailability.14 The past decade has witnessed
noteworthy progress in the development of biomimetic oligomers, including βꢀpeptides,15ꢀ17, 19
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peptoids,18, 20ꢀ23 αꢀaminoxyꢀpeptides,24 α/βꢀpeptides,25,26 azapeptides,27 and others. However, to
date only a handful of peptidomimetic combinatorial libraries were systematically investigated
for protein ligand identification.28 The development of the macrocyclic peptidomimetic
combinatorial library is even rarer.29ꢀ31 Motivated by these findings, we have recently developed
the new class of peptidomimetics, γꢀAApeptides (oligomers of γꢀsubstitutedꢀNꢀacylatedꢀNꢀ
aminoethyl amino acids), which were inspired by the backbone structure of the chiral PNA.32,33
γꢀAApeptides are highly resistant to proteolytic degradation and possess cellular translocation
capability and have enhanced chemodiversity, making them ideal candidates as molecular probes
or therapeutic agents.34 Indeed, these features have been demonstrated by our previously
developed oneꢀbeadꢀoneꢀcompound (OBOC) linear γꢀAApeptidesꢀbased combinatorial libraries
capable of inhibiting Aβ aggregation and disrupting STAT3/DNA interaction.35,36 We thus
envisioned that the macrocyclic combinatorial library of γꢀAApeptides would be endowed with
enhanced conformational rigidity and constraints compared with the linear counterparts, could be
even more beneficial in the identification of bioactive ligands.
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Results and Discussion
Design of the cyclic Library. The thioether moiety is widely found in nature. For instance,
methionine has a thioether side chain and is virtually present in all proteins. Other natural
products such as antibiotic lanthipeptides directly utilize thioether bridges to form
multimacrocylic ring structures. Heinis et al also adopted the thioether linkage to develop phageꢀ
display mediated bicyclic peptide libraries.4ꢀ7 This thioetherꢀbridge mediated cyclization has
proven to be highly efficient in cyclization, leading to cyclic peptides with not only rigidified
conformational freedom but also enhanced metabolic stability. These features make them
promising affinitive candidates for biological applications. By employing a thioetherꢀbridge
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approach, a novel cyclic γꢀAApeptide library could be synthesized and tested against target
proteins. To test our hypothesis, we embarked on the design of the cyclic γꢀAApeptide library by
introducing a Dmt (4,4′ꢀdimethoxytrityl) protected mercaptoethyl carbonyl group to the
secondary amine in the first γꢀAApeptide building block (Figure 1) on the solid phase. After
another three γꢀAApeptide building blocks were assembled subsequently, the 4ꢀ
(bromomethyl)benzoyl group was attached to the Nꢀterminal amino group of the sequence. Next,
the Dmt group was removed, the γꢀAApeptide could be cyclized under nearly neutral condition
with virtually quantitative yield due to the high efficiency of sulfurꢀmediated SN2 reaction.
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Figure 1. Synthesis of the thioether bridged oneꢀbeadꢀtwoꢀcompound macrocyclic γꢀAApeptide
library. Please see supporting information for details.
Molecular Dynamics (MD). In order to assess the potential conformational rigidity of this class
of cyclic γꢀAApeptides, we conducted molecular dynamics (MD) simulations of the cyclic
backbone and linear backbone in explicit water (Supporting information, Figure S5). Consistent
with our hypothesis, MD simulation (Supporting information, Figure S5) suggested the backbone
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of thioether bridged cyclic γꢀAApeptides is much more conformationally rigid that of the linear
one, leading to possibility of identification of novel ligands with high affinity.
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Library Diversity and decoding. To ensure the development of relatively unbiased cyclic
library, a diverse set of hydrophobic, cationic and negatively charged sidechains were chosen.
Briefly, due to the structural nature of γꢀAApeptides, 4 chiral side chains came from the side
chains of 5 different NꢀAlloc protected γꢀAApeptide building blocks (R, Figure 1). The other
three side chains were introduced by acylating the secondary amino group with 6 different
carboxylic acids or acyl chlorides after deprotection of the alloc group (R’, Figure 1). Thus, by
using the split and pool method, the theoretical diversity of the library was expected to be
135000 (Figure 1), and 405000 beads were used for the preparation of the library.
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Unlike linear γꢀAApeptide library,35,36 the direct sequencing of cyclic γꢀAApeptides was no
longer feasible using MS/MS. Instead, the strategy of a oneꢀbeadꢀtwoꢀcompound (OBTC) library
containing both ligands and analyzable coding sequences on the same bead was desired.37,38
Peptides consisted of αꢀamino acids could serve well as coding sequences since the MS/MS
pattern of amino acid fragments is unambiguous. To this end, we developed a highly effective
approach to synthesize coding peptides by using Dde ((1ꢀ(4,4ꢀdimethylꢀ2,6ꢀ
dioxacyclohexylidene) ethyl) protected αꢀamino acids. The deprotection of Dde was very mild
using NH2OH•HCl and imidazole39 and fully orthogonal to chemistry engaged in the synthesis of
thioetherꢀbridged cyclic γꢀAApeptides.
Synthesis of the Library. To prepare these desired OBTC cyclic γꢀAApeptide combinatorial
library, TentaGel beads (200ꢀ250 ꢁm; 1.5 nmol/bead)) were soaked overnight in water and then
exposed to 1:1 (v/v) DCM/Et2O containing 0.5 equiv diꢀtertꢀbutyl decarbonate (Boc2O) (Figure
1).40,41 This was expected to possess Boc protection of amino groups on the outer surface of the
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beads since the interior of the beads still remains in water. After washing with DMF, the interior
of the beads was allowed to react with the Met, the amino acid facilitating coding peptide
cleavage upon cyanogen bromide (CNBr) treatment. After removal of the Fmoc protecting
group, the beads were split into 5 equal aliquots and reacted with 5 different Dde protected
amino acids respectively, so as to establish a coding tag representing the first γꢀAApeptide
building block on the outer layer. Subsequently, the Boc group of the outer layer was removed by
TFA, followed by the attachment of 5 Alloc γꢀAApeptide protected building blocks. Next, the
Alloc group was removed by Pd(PPh3)4 and Me2NH·BH3, and the Dmt protected 3ꢀ
mercaptopropanoic acid was added to react with the secondary amino group. The beads were
pooled and split into 5 aliquots again. After the Dde group was removed, the second set of the
Dde protected amino acids was added to introduce the coding tag for the second γꢀAApeptide
building block on the outer layer of the beads. These steps were repeated 3 more times.
Compared with the cycle of the first building block, the only difference in the subsequent
synthetic process on the outer layer was that after the Alloc group was removed, the N group of
the γꢀAApeptide building block were reacted with a different carboxylic acid or acyl chloride to
introduce diverse side chains. Since each γꢀAApeptide building block bears two side chains, two
Dde protected amino acids were used to code each building block (Figure 1). Finally, the 4ꢀ
(bromomethyl)benzoyl chloride was used to cap the Nꢀterminus of γꢀAApeptides on the outer
layer, followed by selective removal of the Dmt group on the thiol linker with 2% TFA in DCM.
The cyclization of γꢀAApeptides was achieved in the presence of the ammonium carbonate
((NH4)2CO3), which occurred on the surface of the beads only due to the lack of cyclization
linker in the interior of the beads. The deprotection of side chain protecting groups was finally
conducted in 94% TFA, 2% Triisopropylsilane, 2% water and 2% Thioanisole (v:v:v:v). Quality
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of the beads was excellent, as evidenced by the fact that 8 out 10 randomly selected beads
showed unambiguous MS/MS fragmentation patterns by MALDI and were able to provide
information of the coding peptide sequence almost instantly.
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Library Screening and Binding Affinity. With the success of obtaining the cyclic γꢀAApeptide
combinatorial library, we moved forward to examine its potential for identification of valuable
biological ligands. EphA2 (Ephrin typeꢀA receptor 2) was chosen due to its prominent role in the
pathogenesis of various tumors.42 EphA2 (Figure 2) belongs to the family of Eph receptor
tyrosine kinases which regulate tissue development, patterning of the visual system, and play a
critical role in mediating the cellꢀcell communication and angiogenesis.43,44 Recent findings
suggest that EphA2 overexpression is a key factor contributing to multiple cancers such as
melanoma, ovarian, lung, and breast.45ꢀ47 Therefore, EphA2 is a promising target for cancer
therapeutic development. Considerable efforts have started to be made to identify inhibitors that
block the capability of EphA2 for the phosphorylation of its downstream protein substrates,
thereby dampening EphA2 mediated cell signaling. However, to date only limited success was
achieved.48 Thus, it is compelling to identify ligands from our macrocyclic γꢀAApeptide library
that bind to EphA2 with high affinity.
Figure 2. EphA2 signalling and inhibition pathway.
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To conduct the library screening, we first incubated beads with GST tagged intracellular domain
of EphA2 protein (Figure 3A and supporting information). After the thorough wash and the
following incubation with the mouse antiꢀGST antibody, the beads were washed again and
treated with the Alexa Flour 594 labeled goatꢀanti mouse secondary antibody. Ten beads emitting
red fluorescence were identified under the fluorescence microscope and picked up as the putative
positive hits. Subsequently, these beads were incubated with guanidium chloride (GdmCl) to
denature any potential proteins stuck on the surfaces of the beads. The coding peptides in the
inner layers of the beads were then cleaved off the beads by treatment with CNBr, and
subsequently sequenced by tandem MS/MS of MALDI. Among the ten hits, seven different
peptide structures could be determined unambiguously (The rest three hits had poor
fragmentation which were very difficult for us to work out the structures. This may be caused by
the cleavage step.). Next, the corresponding sequences of the seven cyclic γꢀAApeptides
conjugated with fluorescein isothiocyanate (FITC) labels were resynthesized and measured for
their binding affinity toward EphA2 by fluorescence polarization (FP) assay. Fortunately, the
most potent hit, AApeptide 2 (FITC labeled AApeptide 1) (Figure 3B), exhibited excellent
binding affinity to EphA2 with a Kd value of 81 nM (Figure 3C). The potential binding to the
GST tag was excluded as 2 only showed negligible binding affinity to GST protein with a Kd of
14.2 ꢁM, which is ~170ꢀfold weaker than that of EphA2 binding (Figure 3D). Besides, the GST
protein, another two kinases AKT1ꢀPKBα and NFKBIA were chosen to test the selectivity of the
1, which showed Kd of 3.62 μM and 0.741 μM (Supporting information, Figure S8), respectively,
demonstrating the binding of 1 toward EphA2 is selective. Moreover, we treated C13 cells with 1
μM and 5 μM 2, and the diffuse fluorescence was clearly visible in 1 h (Supporting information,
Figure S4), suggesting the compound is cell permeable.
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Figure 3. A). Screening of the γꢀAApeptide library. B). Structure of 2 (FITC labeled lead compound 1). C). The Kd
of the compounds to the EphA2. D). The Kd of the compounds to the GST.
Inhibition Kinase activity of EPhA2. Given the strong binding affinity of 1 (Figure 4A) toward
EphA2, we next performed assays to evaluate its biological activity. The initial in vitro kinase
assay (Figure 4B) show that 2 µM 1 could completely inhibit EphA2 kinase activity by
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preventing phosphorylation of its substrate poly(GluꢀTyr). Another compound 9 (Figure S1,
supporting information), with the same molecular scaffold, failed to inhibit activity of EphA2,
suggesting that recognition of 1 toward EphA2 was specific. At the same concentration, 1 also
greatly suppressed the autoꢀcatalytic activity of EphA2 (Figure 4C). Both abovementioned in
vitro kinase assays suggested 1 is a potent inhibitor of EphA2 kinase activity. Inspired by the
results, we further investigated the ability of 1 for the regulation of catalytic activity in an
ovarian cancer cell line C13 cells which displays high expression of EphA2.49 As shown in
Figure 4D, the phosphorylation level of EphA2 was inhibited on a doseꢀdependent fashion with
the increased concentrations of 1. The IC50 of 1 for the inhibition of EphA2 inhibition was
approximately 5 µM.
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Figure 4. A). Structure of lead compound 1. B). in vitro kinase assay was performed using 50 ng EphA2 and 50 µg
Poly(GluꢀTyr) as a substrate containing 2µM 1. Quantification of substrate phosphorylation was shown. C). Auto
phosphorylation of EphA2 was detected by incubating 50 ng EphA2 with 2µM 1 in the kinase assay buffer.
Immunoblot analysis with pTyr antibody was performed. D). C13 cells were treated with the indicated
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concentrations of 1 for 24 hours. Immunoblot analysis of EphA2ꢀSer897 was performed. Total EphA2 was used as a
control. The extent of phosphorylation was quantified using ImageJ software. Densitometric analysis was performed
by SPSS
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Cell Migration and Invasion. Since EphA2 was shown to play a critical role in cell migration,50
we next tested the effect of 1 on cell migration and invasion. 1 was applied to C13 cells and
analyzed using a scratchꢀwound motility assay. The data in Figure 5A showed a time and doseꢀ
dependent downregulation of cell migration in response 1 treatment, as shown by as much as
70% delay in wound closure at 24 hours postꢀtreatment with the highest dose of 1 (Figure 5A).
Similar results were seen in Matrigelꢀcoated transwell assays, treatment of 10 µM 1 led to
around 70% and 80% decrease in cell migration (Figure 5B) and invasion (Figure 5C),
respectively, suggesting EphA2 mediated cell signaling was significantly suppressed. The
inactive compound 9 was also tested for comparison (Supporting information, Figure S2), which
as expected did not show capability to prevent migration. A linear compound AApeptide 10 was
also tested (Figure S3), and the result was consistent with the MD simulation because 10 was
much less potent. Taken together, the ability of 1 for the strong inhibition of EphA2 activity in
cells suggests that cyclic γꢀAApeptides possess excellent cell permeability, augmenting their
future development in biomedical sciences.
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Figure 5. A). Woundꢀhealing assay. Images were taken 0, 12 and 24 hours after wound formation. Data are
presented as mean ± SD of triplicate experiments and using image J software. C13 cells treated with 10 µM 1 or
vehicle were subjected to migration B) and invasion C) assays. Transwell data was determined by the number of the
migrated cells and the value from parental cells was arbitrarily set at 100%.
Conclusion
In summary, a new class of macrocyclic peptidomimetic combinatorial library has been
developed. With the unique γꢀAApeptide backbone, which ensures the chemodiversity in the
library, this new method has great potential to be a rich source in protein/peptide ligands
identification. In addition, compared with previous linear peptide library developed by our
group, the thioether bridged macrocyclic γꢀAApeptide library has the dual advantages of
significantly enhanced conformal rigidity of the backbones and cell permeability. This led to
higher promise in the identification of more potent and useful ligands/molecular probes.
Furthermore, the new encoding approach of Dde peptide tags also greatly increases the
possibility and ease of the structural elucidation of putative hits. The promise of this macrocyclic
library was manifested by the identification of potent ligands that specifically target the EphA2
receptor tyrosine kinase. With a Kd value of 81 nM, the compound 1 was found to be a potent
inhibitor of the EphA2 signaling in both in vitro and cellular assays. Currently, the library is
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being used as a new platform for screening against various targets in our group, and the in vivo
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study of compound 1 is underway.
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Experimental Section
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General Information. Fmocꢀprotected amino acids were purchased from Chemꢀimpex (Wood
Dale, IL). TentaGel resin (0.23 mmol/g) was purchased from RAPP Polymere (Tuebingen,
Bermany). Rink AmideꢀMBHA resin (0.55 mmol/g) was purchased from GL Biochem
(Shanghai, China). 1ꢀHydroxybenzotriazole wetted with no less than 20% wt. water (HOBt), 1ꢀ
Ethylꢀ3ꢀ(3ꢀdimethylaminopropyl) carbodiimide (EDC), 5,5ꢀDimethylꢀ1,3ꢀcyclohexanedione and
4,4'ꢀDimethoxytrityl Chloride were purchased from Oakwood Chemical (Estill, SC). 4ꢀ
(Bromomethyl)benzoic acid was purchased from AKꢀScientific (Union City, CA). 3ꢀ
Mercaptopropionic Acid was purchased from TCI (Tokyo, Japan). Fluorescein isothiocyanate
(FITC) was purchased from Chemodex (Gallen, Switzerland). Solid phase synthesis was
conducted in peptide synthesis vessels on a Burrell WristꢀAction shaker. γꢀAApeptides were
analyzed and purified on a Waters Breeze 2 HPLC system, and then lyophilized on a Labcono
lyophilizer. The purity of the compounds was determined to be >95% by analytical HPLC.
Masses of γꢀAApeptides and the MS/MS analysis were obtained on an Applied Biosystems 4700
1
Proteomics Analyzer. H NMR spectra were recorded at 500 MHz using TMS as the internal
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standard. C NMR spectra were recorded at 125MHz using TMS as the internal standard. The
multiplicities are reported as follows: singlet (s), doublet (d), doublet of doublets (dd), triplet (t),
quartet (q), multiplet (m). Coupling constants are reported in Hertz (Hz). High resolution mass
spectra were obtained on an Agilent 6220 using electrospray ionization timeꢀofꢀflight (ESIꢀTOF).
Cell culture media was purchased from Gibco (Rockford, IL), fetal bovine serum (FBS) was
purchased from Peak Serum (Fort Collins, CO), penicillinꢀstreptomycin was purchased from
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Invitrogen (Carlsbad, CA). GSTꢀEphA2 recombinant protein, Poly(GluꢀTyr) and Alexa Fluor
594 goat antiꢀmouse antibody were purchased from Life Technologies (Carlsbad, CA) and
SigmaꢀAldrich (St. Louis, MO) respectively. PhosphoꢀTyrosine HRP conjugated antibody (pTyr)
was purchased from R&D Biosystems (Minneapolis, MN). pEphA2ꢀSer897 and EphA2
antibodies were purchased from Cell signaling technologies (Danvers, MA). AntiꢀGST antibody
was purchased from Santa Cruz Biotechnology (Dallas, TX). All solvents and other chemical
reagents were obtained from SigmaꢀAldrich (St. Louis, MO) and were used without further
purification.
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Synthesis of the Dmt protected mercaptopropionic acid (11). 4,4'ꢀDimethoxytrityl Chloride
(6.38g, 18.82 mmol) was dissolved in 40 mL CH2Cl2 containing 3ꢀMercaptopropionic Acid
(1.64ml, 18.82 mmol) at room temperature. The Triethylamine (3.93, 22.58 mmol) was slowly
added to the above solution. The solution was stirred at room temperature for 4 h to 6 h. After
that the mixture was evaporated under reduced pressure, and the residue was washed with
saturated citric acid and extracted with ethyl acetate (30 mL ×3). The organic layer was dried
over anhydrous Na2SO4 and the solvent was evaporated. The residue was purified by flash
column chromatography (Hexane/Ethyl acetate 1:1) to afford the desired product as a light
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yellow solid (80% yield). H NMR (500 MHz, CDCl3): δ 7.42ꢀ7.44 (d, J = 8.00 Hz, 2H), 7.34ꢀ
7.35 (d, J = 9.00 Hz, 2H), 7.29(t, J = 7.50 Hz, 2H), 7.21 (t, J = 7.50 Hz, 1H), 6.84ꢀ6.82 (d, J =
13
9.00 Hz, 2H), 3.79 (s, 6H), 2.49 (t, J = 7.50 Hz, 2H), 2.30 (t, J = 7.50 Hz, 2H); C NMR (125
MHz, CDCl3): δ 178.2, 158.1, 145.2, 137.0, 130.7, 129.4, 127.9, 126.6, 113.2, 66.0, 55.2, 33.5,
26.6.
Synthesis of the 4ꢀ(bromomethyl)benzoyl chloride (12). The 4ꢀ(bromomethyl) benzoic acid
(5g, 23.25 mmol) was dissolved in 10 mL thionyl chloride and reflux for 5 h. The excess thionyl
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chloride was removed under reduced pressure to afford the desired product as a white solid and
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directly use without purification (85% yield).51 1H NMR (500 MHz, CDCl3): δ 8.06 (d, J = 5.00
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Hz, 2H), 7.52 (d, J = 10.00 Hz, 2H), 4.50 (s, 2H); C NMR (125 MHz, CDCl3): δ 167.7, 145.3,
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132.9, 131.8, 129.6, 31.4.
Synthesis of 2ꢀacetylꢀ5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione (13). To a 100 mL round bottom
flask was added 5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione (10 g, 71.34 mmol), N, Nꢀ
Diisopropylethylamine (14.91mL, 85.6 mmol), 4ꢀDimethylaminopyridine (435.76mg, 3.57
mmol) and 50 mL DCM. The mixture was stirred in an ice bath to which acetyl chloride (6.08
mL, 85.6 mmol) was added. The reaction was warmed up to room temperature and allowed to
stir for 8 h. The solvent was evaporated and the residue was washed with 1M HCl then extracted
with ethyl acetate (30 mL × 3). The organic layer was dried over anhydrous Na2SO4 and then
removed in vacuo. The residue was purified by flash column chromatography (Hexane/Ethyl
acetate 1:1) to afford the 2ꢀacetylꢀ5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione as a yellowish solid (11 g,
yield 85%).52 1H NMR (500 MHz, CDCl3): δ 2.54 (s, 3H), 2.48 (s, 3H), 2.3 (s, 2H), 1.01 (s, 6H);
13C NMR (125 MHz, CDCl3): δ 202.3, 197.8, 195.1, 112.3, 52.4, 46.8, 30.6.
Synthesis of Dde protected amino acids. The LꢀAmino acid (1 equiv) was suspended in a
solution of the 2ꢀacetylꢀ5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione (1.3 equiv) in absolute ethanol (ca.
50 mL). Triethylamine (1.5 equiv) was added and the reaction mixture was refluxed for 18 h.
The resulted yellow solution was cooled and concentrated under reduced pressure. The residue
was dissolved in CH2Cl2 (50 mL), and washed with 1 M HCl (50 mL × 2). The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo. Addition of Et2O (ca. 40 mL) to the
residue resulted in immediate white precipitate, which was filtered and washed with cold Et2O to
afford the title compound as an offꢀwhite crystalline solid (~ 70 %).53
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DdeꢀAlaꢀOH (14). White solid. H NMR (500 MHz, DMSOꢀd6): δ 13.51 (d, J = 5.00 Hz, 1H),
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4.61 (t, J = 5.00 Hz, 1H), 2.48 (s, 3H), 2.27 (s, 4H), 1.41 (d, J = 5.00 Hz, 3H), 0.92 (s, 6H); C
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NMR (125 MHz, CDCl3): δ 197.4, 172.9, 172.5, 107.6, 52.8, 51.6, 30.3, 28.3, 19.1, 18.1. HRMS
(ESI) ([M+H]+) Calcd. for C13H20NO4: 254.1392, found: 254.1396.
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DdeꢀValꢀOH (15). White solid. 1H NMR (500 MHz, CDCl3): δ 13.6 (d, J = 5.00 Hz, 1H), 10.97
(s, 1H), 4.61 (t, J = 5.00 Hz, 1H), 2.5 (s, 3H), 2.39 (s, 4H), 2.36 (m, 1H), 1.08 (d, J = 5.00 Hz,
3H), 1.04 (d, J = 5.00 Hz, 3H), 1.0 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 174.3, 171.6, 107.9,
62.3, 51.9, 31.1, 30.1, 28.1, 19.1, 18.7, 17.0. HRMS (ESI) ([M+H]+) Calcd. for C15H24NO4:
282.1705, found: 282.1717.
DdeꢀPheꢀOH (16). White solid. 1H NMR (500 MHz, CDCl3): δ 13.71 (d, J = 5.00 Hz, 1H), 7.18ꢀ
13
7.27 (m, 5H), 4.57ꢀ4.61 (m, 1H), 3.05ꢀ3.09 (m, 2H), 2.36 (s, 4H), 2.20 (s, 3H), 1.00 (s, 6H); C
NMR (125 MHz, CDCl3): δ 198.1, 173.6, 171.0, 135.5, 129.4, 128.6, 127.4, 107.9, 58.3, 52.4,
45.5, 39.3, 30.1, 28.0, 18.1, 8.5. HRMS (ESI) ([M+H]+) Calcd. for C19H24NO4: 330.1705, found:
330.1714.
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DdeꢀLeuꢀOH (17). White solid. H NMR (500 MHz, CDCl3): δ 13.60 (d, J = 10.00 Hz, 1H),
10.01 (s, 1H), 4.57ꢀ4.61 (m, 1H), 2.50 (s, 3H), 2.37 (s, 4H), 1.82 (m, 2H), 1.77 (m, 1H), 0.99 (s,
6H), 0.95 (d, J = 5.00 Hz, 3H), 0.89 (d, J = 5.00 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 198.9,
173.9, 107.9, 54.9, 52.3, 45.6, 41.3, 30.1, 28.2, 24.8, 22.7, 21.7, 18.7, 8.4. HRMS (ESI)
([M+H]+) Calcd. for C16H26NO4: 296.1862, found: 296.1873.
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DdeꢀGlu(OBn)ꢀOH (18). Pale yellow solid. H NMR (500 MHz, DMSOꢀd6): δ 13.77 (d, J =
5.00 Hz, 1H), 10.50 (s, 1H), 7.33 (s, 5H), 5.10 (s, 2H), 4.55 (m, 1H), 2.53ꢀ2.59 (m, 2H), 2.51 (s,
3H), 2.39 (s, 4H), 2.21ꢀ2.25 (m, 2H), 1.01 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 174.3, 171.9,
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171.2, 135.4, 128.6, 128.4, 128.3, 128.2, 66.7, 55.4, 52.3, 30.2, 29.6, 28.2, 27.7, 18.7. HRMS
(ESI) ([M+H]+) Calcd. for C22H28NO6: 402.1917, found: 402.1925.
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DdeꢀAspꢀOH (19). White solid. 1H NMR (500 MHz, DMSOꢀd6): δ 13.56 (d, J = 10.00 Hz, 1H),
4.84 (m, 1H), 2.90 (dd, J = 15.00, 5.00 Hz, 1H), 2.78 (dd, J = 15.00, 5.00 Hz, 1H), 2.46 (s, 3H),
2.27 (s, 4H), 1.36 (s, 9H), 0.92 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 172.3, 170.7, 168.7,
107.7, 81.5, 52.5, 38.2, 30.1, 38.3, 28.0, 17.9. HRMS (ESI) ([M+H]+) Calcd. for C18H28NO6:
354.1917, found: 354.1929.
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DdeꢀGluꢀOH (20). White solid. 1H NMR (500 MHz, DMSOꢀd6): δ 13.54 (d, J = 10.00 Hz, 1H),
4.26 (q, J = 5.00 Hz, 1H), 2.43 (s, 3H), 2.28 (s, 4H), 2.24ꢀ2.26 (m, 2H), 1.90ꢀ2.10 (m, 2H), 1.36
(s, 9H), 0.93 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 172.7, 171.8, 107.4, 80.3, 54.2, 52.1, 45.2,
30.8, 30.1, 28.3, 28.1, 27.8. HRMS (ESI) ([M+H]+) Calcd. for C19H30NO6: 368.2073, found:
368.2075.
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DdeꢀLysꢀOH (21). White solid. H NMR (500 MHz, CDCl3): δ 13.69 (m, 1H), 10.24 (s, 1H),
4.80 (m, 1H), 4.40 (m, 2H), 3.09 (m, 2H), 2.50 (s, 3H), 2.34 (s, 4H), 1.95 (m, 2H), 1.50 (m, 2H),
1.40 (s, 9H), 1.01 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 173.3, 172.9, 172.0, 171.6, 155.9,
107.6, 80.9, 78.9, 60.1, 56.1, 40.9, 39.8, 32.1, 30.3, 29.1, 27.9, 21.6, 18.4, 13.9. HRMS (ESI)
([M+H]+) Calcd. for C21H35N2O6: 411.2495, found: 411.2497.
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FmocꢀLys(Dde)ꢀOH (22).54 Pale yellow solid. H NMR (500 MHz, CDCl3): δ 13.29 (s, 1H),
7.73ꢀ7.74 (d, J = 5.00 Hz, 2H), 7.58 (t, J = 5.00 Hz, 2H), 7.37 (t, J = 5.00 Hz, 2H), 7.27 (t, J =
5.00 Hz, 2H), 5.79 (d, J = 10.00 Hz, 1H), 4.42ꢀ4.46 (m, 1H), 4.35ꢀ4.37 (d, J = 10.00 Hz, 2H),
4.18 (t, J = 5.00 Hz, 1H), 3.38ꢀ3.39 (m, 2H), 2.53 (s, 3H), 2.35 (s, 4H), 1.78ꢀ1.80 (m, 2H), 1.70ꢀ
1.72 (m, 2H), 1.47ꢀ1.57 (m, 2H), 1.00 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 198.5, 174.7,
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174.3, 156.2, 143.8, 143.7, 141.3, 127.7, 127.0, 125.1, 119.9, 107.8, 67.1, 53.4, 52.2, 47.1, 43.4,
31.9, 30.2, 28.3, 28.2, 22.4, 21.1, 18.3, 14.2. HRMS (ESI) ([M+H]+) Calcd. for C31H37N2O6:
533.2652, found: 533.2661.
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Synthesis of Cyclic γꢀAAPeptides Hits. After structures of putative hits were determined by
MALDI MS/MS, the hits and its FITC labeled analogues were reꢀsynthesized on the Rink Amide
resin and confirmed by Applied Biosystems 4700 Proteomics Analyzer (Scheme S2). For the
synthesis of the fluorescent cyclic peptide, the FmocꢀLys(Dde)ꢀOH was first attached to the Rink
amide resin. The Fmoc protection group was then removed, followed by the desired building
blocks needed for the sequence synthesis. After the γꢀAApeptides were cyclized, the Dde group
was removed. Then FITC (2 equiv.) and DIPEA (6 equiv.) in DMF were added to the resin and
shaken for 12 h at room temperature. The FITC labeled cyclic γꢀpeptide was cleaved by 1:1 (v/v)
DCM/TFA containing 2% triisopropylsilane. The crude was purified by the Waters HPLC
system with flow rate of 0.8 mL/min with a linear gradient from 5% to 100% (CH3CN in water)
in 40 min (Figure S5).
Fluorescence Polarization (FP). The binding affinity (Kd) of the hits was obtained by
fluorescence polarization (FP). The FP experiment was performed by incubating 50 nM FITC
labeled AApeptide with EphA2 (0.0625 to 2 ꢁM) in 1×PBS. The binding affinity of the lead
compound to the GST protein (Kd) was obtained by incubating 50 nM FITC labeled AApeptide
in GST ranging from 0.3125 to 55 ꢁM. Dissociation constants (Kd) were determined by plotting
the fluorescence anisotropy values as a function of protein concentration, and the plots were
fitted to the following equation (Figure S7ꢀ8).37 The Lst is the concentration of the AApeptide
and the x stands for the concentration of the protein. The experiments were conducted in
triplicates and repeated for three times.
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ꢑ
ꢂ
ꢈ
ꢊꢋ + Lꢌꢍ + x − ꢎꢂꢊꢋ + ꢏꢌꢍ + ꢐꢈ − 4ꢏꢌꢍꢐ
ꢁ
ꢂ
ꢈꢉ
ꢀ = FPmin + ꢃꢄꢅꢆꢇ − ꢃꢄꢅꢆꢇ
2ꢏꢌꢍ
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in vitro Kinase assays and immunoblotting. in vitro kinase assay to evaluate EphA2 kinase
activity was performed as described before.46 Briefly, 50µl kinase reaction containing 50 ng
recombinant EphA2, 50 µg Poly(GluꢀTyr) and 200 µM ATP and 2 µM of 1 and 9 (negative
control) were incubated at 30 oC for 40 min. The reaction was stopped by addition of gel loading
buffer. The samples were boiled for 5 min and SDS PAGE was performed. Immunoblotting with
pTyrꢀHRP antibody (1:2000 dilution) was performed to detect substrate phosphorylation. The
extent of phosphorylation was quantified using ImageJ software. For studying the activity of the
compounds in cells, C13 cells were treated at indicated doses for 24 h. The cells were lysed in
lysis buffer and immunoblot analysis was performed. Densitometric analysis was performed by
SPSS. The experiments were conducted in triplicates and repeated for three times.
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Scratchꢀwound motility assay. C13 cells were trypsinized and 105 cells were reseeded on a
twelveꢀwell tissue culture plate. After 12 h, the attached cells were scratched with a 200ml
pipette tip and cell migration was observed for up to 24h. 0 h images were captured using a
Nikon ECLIPSE microscope. The plates were placed back at 37 ºC and 5% CO2 for 24h, and
another set of images were captured of the same wounds. The wound widths were measured by
ImageJ (version 1.50). The experiments were conducted in triplicates and repeated for three
times.
Matrigelꢀcoated transwell assays. The assays were performed by transfected the C13 cell line
with control EphA2 for 48 h. After that the cells were plated into the top chambers and treated
with either vehicle or 10µM 1. 24 hours later cells in top chamber were scraped and migrated
cells were fixed with crystal violet staining. Transwell data was determined by the number of the
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migrated cells and the value from parental cells was arbitrarily set at 100%. The experiments
were conducted in triplicates and repeated for three times.
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ASSOCIATED CONTENT
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Supporting Information
Procedures for library preparation, library screening, the bioactivity assay, cell permeability test,
molecular dynamics simulations; the list of hits, the determination of decoding sequences, the Kd
data of other hits and kinases, 1H and 13C NMR spectra and HPLC traces.
Molecular formula strings (CSV).
AUTHOR INFORMATION
Corresponding Author
*J.C.: eꢀmail, Jianfengcai@usf.edu
*Q.L.: eꢀmail: lzwf@hotmail.com
Author Contributions
Y.S. and S.C. contributed equally to the work.
Note
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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This work was generously supported by NSF CAREER 1351265 (JC), NIH 1R01GM112652ꢀ
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01A1 (JC), National Natural Science Foundation of China (No. 81520108031，81473478),
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Shanghai Outstanding Academic Leaders Plan(No.16XD1403600); The Science Foundation for
Shanghai Committee of Science Project (14430722900) Shanghai Outstanding Medical
Academic Leader; Shanghai ThreeꢀYear Action Plan of Traditional Chinese Medicine (No.ZY3ꢀ
CCCXꢀ3ꢀ3012, No.ZY3ꢀCCCXꢀ2ꢀ1003). We thank Ning Ma for technical assistance with the
parameterization. Computer time was provided by USF Research Computing, sponsored in part
by NSF MRI CHEꢀ1531590.
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ABBREVIATIONS USED
EphA2, ephrin typeꢀA receptor 2; DMF, dimethylformamide; DCM, dichloromethane; HOBt,
hydroxybenzotriazole; DIPEA, N,Nꢀdiisopropylethylamine; DIC, N,N′ꢀdiisopropylcarbodiimide;
TFA, trifluoroacetic acid; FITC, fluorescein isothiocyanate; Kd, dissociation constant; Tris,
tris(hydroxymethyl)aminomethane.
REFERENCES
(1) Kodadek, T. The rise, fall and reinvention of combinatorial chemistry. Chem. Commun. 2011,
47, 9757ꢀ9763.
(2) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. A
new type of synthetic peptide library for identifying ligandꢀbinding activity. Nature 1991, 354,
82ꢀ84.
(3) Aquino, C.; Sarkar, M.; Chalmers, M. J.; Mendes, K.; Kodadek, T.; Micalizio, G. C., A
biomimetic polyketideꢀinspired approach to smallꢀmolecule ligand discovery. Nat. Chem. 2012,
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(4) Chen, S.; Rentero Rebollo, I.; Buth, S. A.; MoralesꢀSanfrutos, J.; Touati, J.; Leiman, P. G.;
Heinis, C. Bicyclic peptide ligands pulled out of cysteineꢀrich peptide libraries. J. Am. Chem.
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