Targeting Protein-Protein Interactions of Tyrosine Phosphatases with Microarrayed Fragment Libraries Displayed on Phosphopeptide Substrate Scaffolds

Article abstract of DOI:10.1021/acscombsci.8b00122

Chemical library screening approaches that focus exclusively on catalytic events may overlook unique effects of protein-protein interactions that can be exploited for development of specific inhibitors. Phosphotyrosyl (pTyr) residues embedded in peptide m

Full text of DOI:10.1021/acscombsci.8b00122

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Article
Targeting protein-protein interactions of tyrosine
phosphatases with microarrayed fragment libraries
displayed on phosphopeptide substrate scaffolds
Megan Hogan, Medhanit Bahta, Kohei Tsuji, Trung Xuan Nguyen, Scott Cherry, George T. Lountos,
Joseph E. Tropea, Bryan Zhao, Xue Zhi Zhao, David S Waugh, Terrence R. Burke, and Robert G Ulrich
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00122 • Publication Date (Web): 10 Jan 2019
Downloaded from http://pubs.acs.org on January 16, 2019
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Targeting protein-protein interactions of tyrosine phosphatases with microarrayed
fragment libraries displayed on phosphopeptide substrate scaffolds
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Megan Hogan†, Medhanit Bahta‡, Kohei Tsuji‡, Trung X. Nguyen‡, Scott Cherry§, George T.
Lountos§,¥, Joseph E. Tropea§, Bryan M. Zhao†, Xue Zhi Zhao‡, David S. Waugh§ , Terrence
R. Burke, Jr. ‡, Robert G. Ulrich†*
†Molecular and Translational Sciences Division, United States Army Medical Research
Institute of Infectious Diseases, Frederick, MD 21702, USA; ‡Chemical Biology Laboratory,
Center for Cancer Research, National Institutes of Health, National Cancer Institute at
§
Frederick, Frederick, Maryland 21702, United States; Macromolecular Crystallography
Laboratory, National Cancer Institute, National Cancer Institute at Frederick, Frederick,
¥
Maryland 21702, USA; Basic Science Program, Frederick National Laboratory for Cancer
Research sponsored by the National Cancer Institute, Frederick, Maryland 21702, USA
KEYWORDS
Aminooxy phosphopeptide, catalytic assay, fragment-based drug design, kinetic assay,
microarray, oxime-containing phosphopeptide, protein tyrosine phosphatase, substrate affinity
screening, surface plasmon resonance
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ABSTRACT
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Chemical library screening approaches that focus exclusively on catalytic events may overlook
unique effects of protein-protein interactions that can be exploited for development of specific
inhibitors. Phosphotyrosyl (pTyr) residues embedded in peptide motifs comprise minimal
recognition elements that determine substrate specificity of protein tyrosine phosphatases
(PTPases). Using solid-phase synthesis, we incorporated aminooxy-containing amino acid
residues into a 7-residue epidermal growth factor receptor (EGFR)-derived phosphotyrosine-
containing peptide and subjected the peptides to on-resin oxime diversification by reacting with
aldehyde-bearing drug-like functionalities. The pTyr residue remained unmodified. The
resulting derivatized peptide library was printed in microarrays on nitrocellulose-coated glass
surfaces for assessment of PTPase catalytic activity, or on gold monolayers for analysis of
kinetic interactions by surface plasmon resonance (SPR). Focusing on amino-acid positions
and chemical features, we first analyzed dephosphorylation of the peptide pTyr residues within
the microarrayed library by the human dual-specificity phosphatases (DUSP) 14 and DUSP22,
as well as by PTPases from poxviruses (VH1) and Yersinia pestis (YopH). In order to identify
the highest affinity oxime motifs, the binding interactions of the most active derivatized
phosphopeptides were examined by SPR using noncatalytic PTPase mutants. Based on high-
affinity oxime fragments identified by the two-step catalytic and SPR-based microarray screens,
low molecular weight non-phosphate-containing peptides were designed to inhibit PTP
catalysis at low micromolar concentrations.
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INTRODUCTION
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Protein tyrosine phosphatases (PTPases) dephosphorylate tyrosine residues within proteins and
work in concert with protein tyrosine kinases to regulate signal transduction pathways. Because
of the critical involvement of signal transduction pathways in regulating pathological processes
in cancer and infectious diseases,1-2 PTPases have emerged as important targets for
development of therapeutic inhibitors. Despite growing successes in the development of small
molecules that inhibit kinases, modulators of PTPases have proven to be far more challenging,
and no inhibitor has yet achieved clinical approval.3 Effective PTPase targeting by small
molecule inhibitors is complicated by the relatively smooth protein surfaces and in the case of
dual-specificity PTPases (DUSPs), shallow catalytic pockets that accommodate
phosphotyrosine (pTyr), phosphoserine (pSer) and phosphothreonine (pThr) residues. In
addition, because recognition and binding of PTPase substrates involves a network of hydrogen
and ionic bonds within a highly conserved catalytic pocket [(H/V)C(X5)R(S/T)], preferred
ligands are typically negatively-charged, which results in limited specificity, cell permeability
and bioavailability. An alternative approach to increase the likelihood of finding druggable
features of PTPases is to exploit interactions with protein features that extend beyond the
catalytic pocket. Synthetic peptide substrates have previously been used as display platforms
for non-hydrolyzable pTyr-mimicking residues,4 which led to the identification of the
difluorophosphonomethyl-aryl moiety as a starting point for the design of small molecule
inhibitors by converting a good substrate into a high affinity inhibitor.4,5 In the approach
described herein, we used a high-affinity peptide substrate as a scaffold for presenting
microarrayed libraries of drug-like fragments to identify motifs for inhibitor design.6-9 The goal
of this work was to devise a method for developing inhibitors of protein-protein interactions
(PPIs) that are intrinsic to PTPase catalytic specificities. In order to investigate substrate
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binding interactions in proximity to the PTPase catalytic cleft, we sequentially examined
modified amino acid residues adjacent to the unaltered the pTyr residue of the peptide. A
diversified library was created by incorporating 300 different drug-like fragments at six
different positions on the EGFR-derived peptide, "VDADEpYL". Detailed interactions with
the substrate library were examined using PTPases from smallpox virus [Variola major H1
(VH1)], the plague bacillus [Yersinia outer protein H (YopH)], and the human enzymes
DUSP14 and DUSP22. In order to identify fragments that could be useful in inhibitor design,
primary results were obtained from catalytic assays with the microarrayed library, and kinetic
binding data were obtained from the microarrayed library by using a biosensor assay. As proof-
of-principal, a high-affinity oxime fragment identified by the two-step catalytic and SPR-based
microarray screens was employed to design low molecular weight, non-phosphate-containing
peptides, which were able to inhibit PTP catalysis at low micromolar concentrations.
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RESULTS AND DISCUSSION
Methodology. A flowchart depicting the general approach used is presented in Figure 1. We
synthesized a library comprised of 1800 distinct oxime-modified phosphopeptides based on
the EGFR-derived heptapeptide sequence (biotin-linker-VDADEpYL-NH2, 5), which includes
the autophosphorylation site Tyr 992.10 The EGFR-derived pTyr-containing peptide is a
reliable substrate for many PTPases.11-12 Along with 5, the library was deposited by inkjet
printing of microarrays on either a nitrocellulose-coated or a gold-coated glass slide for use in
a fluorescent-based catalytic assay or surface plasmon resonance (SPR) based kinetic assay,
respectively. The percent dephosphorylation (compared to the parent peptide 5) and the binding
interaction of each library component with PTPases was assessed with VH1, DUSP14,
DUSP22) and YopH. Combining the data from both experiments allowed us to identify
fragments that may have the greatest utility as building blocks for design of potential inhibitors.
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Active, wild-type PTPases were used for all catalytic assays, while catalytically inactive mutant
enzymes were used in SPR binding-interaction studies to avoid dephosphorylation of the pTyr
residue. These PTPase substrate-trapping mutants, which do not possess measurable catalytic
activities but still bind tightly to pTyr-substrates,13 can be used to identify physiological
substrates in vivo and in vitro,14 as well as to elucidate binding modes by crystallography.15
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Figure 1. A flowchart depicting the methodology used in this study.
Synthetic Discussion
Synthesis of aminooxy-containing amino acid reagent 4. The preparation of aminooxy-
containing peptides 6(a – f) (Figure 2) by standard Fmoc-based solid-phase protocols entailed
use of the orthogonally-protected amino acid 4 (Scheme 1). The synthesis of 4 started from
compound 1, which was obtained from Cbz-(Asp)-OBn by literature procedures.16 Reaction of
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1 with N-hydroxyphthalimide under Mitsunobu coupling conditions gave the adduct 2 in good
yield. Similar Mitsunobu coupling on the carboxymethyl ester variant of 1 had been reported
to give low yields and cyclized byproducts.17 This necessitated a two-step protocol that
involved conversion of the side chain hydroxyl to a mesyl ester followed by nucleophilic
displacement with N-hydroxyphthalimide.17 Starting from 1 a similar two-step approach has
been reported to prepare 2.16 In the current work, treatment of 2 with methylhydrazine removed
the phthalimide group to provide the free aminooxy group, which was derivatized with Boc
anhydride to give the globally-protected analog 3 (Scheme 1). Hydrogenolytic removal of the
N-Cbz and O-Bn groups, followed by treatment with Fmoc succinimidyl carbonate (Fmoc-OSu)
and NaHCO3 in aqueous dioxane, gave the desired N-Fmoc-protected 4 in good yield. The
synthesis of 4 has been previously reported by a slightly different protocol.17-18 Reagent 4 was
employed in solid-phase peptide synthesis (SPPS), using standard Fmoc procedures to prepare
the biotinylated aminooxy-containing peptides 6a – 6f (Figure 2).
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Scheme 1. Synthesis of the orthogonally-protected amino acid reagent 4.
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Figure 2. Structures of the parent peptide 5 and the biotinylated aminooxy-containing
peptides 6(a-f).
Synthesis of a library of biotinylated oxime-containing peptides. Peptides and proteins
undergo clean oxime-forming reactions rapidly and in near quantitative without the need of
sidechain protection. The resulting products are highly stable.19-21 We have previously
prepared libraries of oxime-diversified peptides by incorporating aminooxy-containing
residues within parent peptides and then reacting the peptides in parallel with aldehydes in
DMSO in the presence of acetic acid. The oxime-forming reactions were nearly quantitative
and products of >90% purity were routinely obtained. These products are stable and can be
subjected directly to biological evaluation without a need for purification. 22-25 In our current
work we employed a collection of 300 aldehydes (structures are shown in Supporting
Information Tables S1 – S3) that were reacted with the biotinylated aminooxy-containing
peptides 6a – 6f. The optimized reaction conditions for oxime ligation used DMSO as
solvent and acetic acid as catalyst at room temperature overnight, with molar concentrations
of aminooxy: RCHO: acetic acid; 1:1:5 (Scheme 2).18, 19 This resulted in the formation of a
library of 1800 distinct oxime-containing phosphopeptides at final concentrations of 100 μM.
In light of well-established precedence, the libraries were used without purification and the
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solutions were used directly to prepare microarrays on nitrocellulose slides26 for screening as
described below.
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Scheme 2. Synthesis of oxime-containing peptide library. The aldehydes are listed in
Supporting Information Tables S1 – S3.
Synthesis of sidechain-modified peptides 11a,b and 12. Oxime-containing peptides that
displayed the aldehydes w33 and w202 (Supporting Table S2, Supporting Figure S2) were
selected for further examination from the high-affinity substrates observed in the microarray
assays. The original oxime-aminooxy oxygens of w33 and w202 were replaced by methylene
groups, while the remainder of the oxime functionality was replaced by structurally
homologous amides. This resulted in the design of peptides 11a (w33) and 11b (w202) as the
monosubstituted congeners, and peptide 12 as a disubstituted congener that combined features
of peptides w33 and w202 (Figure 3). An essential feature of 11a,b and 12 was the inclusion
of ornithine residues as homologous variants of the aminooxy-containing residue 4. By
employing 4-methyltrityl (Mtt) sidechain amine protection of the ornithine residues in
combination with Rink amide MBHA resin, it was possible to remove the Mtt protection by
treatment with a low concentration of TFA (1%) that preserved peptide attachment to the resin.
Amidation of the ornithine sidechain was then achieved using the appropriate carboxylic acid
prior to Fmoc removal and peptide chain extension. Cleavage of the completed peptide from
the resin was then accomplished using 95% TFA. Although the 3,5-dichloropyridine-4-
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carboxylic acid needed to prepare the ornithine amide Zzz in peptides 11b and 12 (Figure 3)
was commercially available (Aldrich), it was necessary to synthesize the 4-(4-
(trifluoromethyl)pyridin-2-yl)benzoic acid (10, Scheme 3), which was required to prepare the
ornithine amide Yyy in peptides 11a and 12 (Figure 3).
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Figure 3. Structures of sidechain-modified substrates based on peptide 5.
The synthesis of 10 was accomplished in two steps by initial Suzuki coupling of 2-bromo-4-
(trifluoromethyl)pyridine (7) and (4-(methoxycarbonyl)phenyl)boronic acid (8) to yield the
methyl ester 9, followed by hydrolysis of the ester under alkaline conditions (Scheme 3).
Scheme 3. Synthesis of reagent 10.
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Fluorometric array. The PTPase activity of each enzyme was measured against the peptide
libraries using a fluorescence-based microarray assay as a primary screening tool. The
unmodified EFGR-derived heptapeptide 5 served as a baseline to compare the effect of each
oxime-containing peptide within the library. The level of dephosphorylation was assessed by
using an antibody that detects phosphotyrosine within the substrate peptide and a fluorescently-
labeled secondary antibody (anti-mouse IgG). Figure 4 shows a representative slide of the
oxime-phoshopeptide library and controls that were spotted in triplicate and treated with
PTPase under fixed conditions. The magnified section compares the difference between a
buffer-treated slide (no PTPase) versus a PTPase-treated slide. Each spot represents an
individual phosphopeptide, and dephosphorylation is observed by a decrease in fluorescence
signal (Figure 4) as compared to the corresponding spot on the non-enzyme treated slide. The
results from each PTPase are depicted in the heat map diagram of Supplemental Figure S1,
which shows dephosphorylation values for individual components of the entire oxime-
containing phosphopeptide library, peptide 5, and peptides 6(a – f).
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Representative
slide
Buffer
treated
Enzyme
treated
Figure 4. A representative slide spotted with the oxime-containing phosphopeptide
library and treated with PTPase or buffer solution. After probing with an
Alexafluor647®-conjugated detection antibody, the fluorescent signal from each spot is
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measured. Strong signals are colored red and white and signify a greater amount of pTyr
remaining, while weaker signals are light red and black and signify less pTyr remaining. The
magnified images show the contrast between the buffer-treated and enzyme-treated slides.
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Figure 5 shows the change in dephosphorylation for the oxime-containing phosphopeptides
when treated with each PTPase (averaged for each spot), as compared to 5 (the baseline). The
peptides are grouped according to the position of each amino-acid residue relative to pTyr.
Protein concentrations and incubation times were determined from test experiments of the
PTPases with 5 and 6(a – f), in which approximately 40% dephosphorylation was obtained
(data not shown). This was done in order to ascertain whether fragments particular to individual
oxime-containing peptides enhanced or reduced the dephosphorylaiton of pTyr residues in
comparison to the parent peptide 5. When tested against the entire peptide library, the PTPase
activities of each enzyme with peptide 5 as substrate were as follows: VH1: 24%, DUSP14:
28%, DUSP22: 6%, YopH: 96%. This apparent change in dephosphorylation may be a result
of scaling up enzyme solutions for the larger peptide library, slight errors in measuring enzyme
concentration (by UV/Vis), and/or changes in the specific activity of the enzyme over time.
Because we were primarily interested in fragments associated with enhancing
dephosphorylation for use in the design of inhibitors, the dephosphorylation levels
corresponding to peptide 5 against VH1, DUSP14, and DUSP22 were acceptable. Although
the high activity of YopH across the entire library was not unexpected,27 this made it difficult
to control cleavage conditions for YopH in this assay. As seen in figure 5, large differences
from the baseline (peptide 5) for YopH are generally only apparent for decreases in percent
dephosphorylation. This inhibitory effect is most notable with the oxime-containing peptides
from 6d and 6e, in which the respective Asp and Glu residues have been modified. This is
consistent with findings from other studies showing that these two residues are crucial for
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substrate-enzyme binding interactions.28 The data set for YopH remains biased towards
decreases in dephosphorylation, due to the high amount of dephosphorylation observed with
the parent peptide 5. For this reason, YopH was used as a positive control in subsequent binding
experiments and analyses. In Figure 5, VH1 demonstrates varying degrees of activity towards
the entire peptide library, with the exception of those in group 6f, in which the Leu residue C-
proximal to the pTyr was modified.
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Figure 5. The effect of residue position of the peptide on enzymatic cleavage of pTyr.
The change in percent dephosphorylation as compared to parent peptide 5 for each oxime-
containing peptide, grouped according to the amino-acid residue that was modified 6(a – f).
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Most of the peptides in 6f appear to have an enhancing effect on VH1 PTPase activity. From a
drug design viewpoint, it may be advantageous to target areas on the protein that are reachable
from this residue, keeping in mind the more polar oxime-handle that replaced the bulkier alkyl
chain of the Leu. In contrast, DUSP14 appears to be less sensitive to residue positioning, with
the exception of the C-terminal Leu, as seen in Figure 5. Nearly all modifications to residues
in the N-terminal direction of pTyr produce an increase in dephosphorylation. Modification of
either of the Asp residues (groups 6b or 6d) appears to enhance DUSP22 activity (Figure 5),
indicating that negative chemical functionalities are disfavored at these positions for this
PTPase.
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The fragments for each oxime-containing phosphopeptide were grouped into 11
categories according to chemical structure and functional group. For each of the 11 structural
categories, the median difference in percent dephosphorylation between each oxime-
phosphopeptide and 5 are depicted graphically in Figure 6. Differences in the abilities of the
functional groups to affect the PTPase dephosphorylation of the pTyr-containing peptides are
readily apparent. For clarity, the effects of each fragment have also been arranged according to
residue proximity to the pTyr residue and by PTPase. Bubble sizes represent the standard
deviations of the difference in percent dephosphorylation for the fragment-containing peptides
associated with that category (i.e., a smaller bubble signifies a tighter agreement between the
fragments in that category). The red dotted lines intersect the x-axis at zero to better visualize
effects that groups have on the ability of the PTPase to dephosphorylate the phosphopeptide
(bubbles above the line signify a median increase in percent dephosphorylation when compared
to 5, while bubbles below the line signify a median decrease within the group). Because the
fragment library was chosen based on drug-like properties, many of the members possess
qualities of multiple functional/structural groups. Therefore, a high degree of overlap is not
surprising. However, a number of notable features can be discerned from these graphs. For
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example, in the case of VH1 (Figure 6B), fragments containing nitrile groups seem to be
particularly disfavored at the Val (-5) and Asp (-2) positions. Based on these results, it would
be beneficial to avoid nitrile groups when designing VH1 inhibitors, at least in the chemical
space associated with the areas on the protein that interact with these Val and Asp residues. In
addition, although there is a high degree of overlap among many of the bubbles in the graph
for DUSP22 (Figure 6D), there is a clear differentiation between the phosphate-mimetics and
zwitterions versus the remaining groups at the Glu (-1) position. When designing inhibitors of
DUSP22, it may be beneficial to place functional groups that possess phosphate-like qualities
in close proximity. The large differences observed between each PTPase suggest that the
enzymes may be selectively targeted using specifically designed small-molecules and the
results could serve as a baseline for further structure-activity relationship studies.
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Figure 6. The effect of functional group/chemical structure on enzymatic cleavage of
pTyr. The change in percent dephosphorylation as compared to parent peptide 5 for each
oxime-containing peptide, grouped according to functional group/chemical structure as
named in the legend.
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PTPase kinetic interactions. The binding interactions between a select number of oxime-
containing phosphopeptides and PTPases were calculated using an SPR-based biosensor that
permitted continuous measurement in real-time. For these kinetic assays, a subset of peptides
was chosen according to their range of activities observed in the catalytic assay. A total of 45
oxime-containing phosphopeptides were selected in order to accommodate controls and allow
for replicate spots. The chosen peptides include six that showed little to no dephosphorylation;
five peptides that showed specific activity towards a particular enzyme and 17 peptides that
showed the highest levels of dephosphorylation (representing at least one peptide from each of
the 6(a-f) groups so that each residue position was included). The biotinylated-peptides and
controls were mixed with NeutrAvidin and each mixture was spotted onto a gold-coated SPR
slide that was derivatized with a self-assembled monolayer of carboxyl-terminated alkane
thiols
followed
by
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC)/N-
hydroxysuccinimide (NHS) coupling chemistry. Kinetic binding studies were then performed
for each PTPase, with anti-pTyr antibody and histidine-tagged maltose binding protein (His-
MBP) acting as positive and negative binding controls, respectively. Figure 7 shows
representative sensorgrams from the kinetic assays for the control proteins, VH1 C110S, and
DUSP14 C111S. For binding interactions between peptide 5 and the positive control proteins,
anti-pTyr mouse antibody and YopH, noticeable binding curves were observed, although the
slope of each curve was different. The on-rate (ka) and off-rate (kd) for the antibody were slower
than for YopH, indicating a slower but stronger complex formation. The binding curves
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between VH1 C110S and peptides 5 and 6f-w202 (nomenclature indicates the parent peptide
with the associated oxime group as shown in Supporting Information Tables S1 – S3) as well
as the negative control, PEG [a 12-chain poly-(ethylene glycol) (PEG) with a terminal
ethanolamide] are shown in Figure 7B. As depicted, negligible binding was observed between
the PTPase and PEG, indicating little non-specific binding. The higher binding affinity for
VH1 and peptide 6f-w202 (KD = 73 (± 27) nM) versus peptide 5 (KD = 96 (± 21) nM) was
consistent with the higher level of dephosphorylation in the catalytic assay (43% and 24% for
peptides 6f-w202 and 5, respectively). Binding curves for DUSP14 C111S and peptides 5, 6f-
w99, and 6f-w202 are shown in Figure 7C, with the associated KD values for each peptide being
as follows: 5, 1.4 ± 1.3 μM; 6f-w202, 642 ± 147 nM and 6f-w99, 94 ± 94 nM. Binding curves
for 5 and 6f-w99 are atypical and probably are not best fit to a Langmuir binding curve (also
demonstrated by the high standard deviation values for both). However, the trend in measured
binding affinities are consistent with data from the corresponding catalytic assays, in which
dephosphorylation values of 28%, 87%, and 99% were observed, respectively. The high
binding affinity, and likely also the potency, of peptide 6f-w99 can be attributed to the slow
dissociation rate, which indicates a stable complex with the PTPase. Under the conditions used
for the other PTPases, binding interactions were not observed between DUSP22 and the
phosphopeptides, including the unmodified parent peptide. Figure 8 presents the kinetic data,
ka (association rate or on-rate) and kd (dissociation or off-rate), as it relates to the catalytic data
(percent dephosphorylation), for VH1 and DUSP14 with a subset of the oxime-containing
phosphopeptide library. From a drug design perspective, peptides that reside at the top of the
graph in the upper and lower left quadrants include fragments with ideal inhibitor qualities of
high catalytic activity and a long residence time (slow off-rate).
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A
Controls
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Anti-pTyr (10
nM)
His-MBP (1 μM)
YopH C403A/D356A (1μM)
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0 100 200 300 400
Time (sec)
0 100 200 300 400
0 100 200 300 400
B
VH1 C110S
6f-w202
5
PEG
0 100 200 300 400
0 100 200 300 400
0 100 200 300 400
Time (sec)
C
DUSP14 C111S
6f-w99
6f-w202
5
0 100 200 300 400
0 100 200 300 400
Time (sec)
0 100 200 300 400
Figure 7. Biomolecular interactions of phosphatases and phosphopeptide substrates.
Representative sensorgrams depicting the kinetic interaction between each PTPase or control
protein and the parent peptide 5, negative control PEG, or oxime-containing peptide 6f-w202
and 6f-w99.
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Figure 8. Depiction of catalytic and kinetic affinity. Mapping the on-rate (ka) and off-rate
(kd) of each ligand-enzyme interaction with respect to catalytic activity (%
dephosphorylation). Labels are included for select oxime-containing peptides.
Substrate enhancement and PTPase inhibitors. Peptide 12 was synthesized in order to test
whether multiple fragments that increased dephosphorylation and binding activity (as
compared to 5) could be combined to generate an additive effect (Figure 3). Using ornithine
amides to stabilize the fragment bonds, peptide 12 merges two fragments (w33 and w202) that
showed moderate-to-high affinity for VH1 and DUSP14 in both assays. The dually-
functionalized phosphopeptide 12 was spotted onto a nitrocellulose slide, along with the mono-
substituted peptides (11a and 11b), the original oxime-containing phosphopeptides 6d-w33
and 6f-w202, and the parent peptide 5. The catalytic assay was performed in the same manner
as with the oxime-containing library described previously. The percent dephosphorylation
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results are presented in Figure 9 as fold-change relative to the percent dephosphorylation of 5.
Peptides modified with 4-(4-(trifluoromethyl)pyridin-2-yl) benzoic acid (w33) presented the
highest activities, especially with VH1, while the ornithine amides reduced substrate activity
levels. However, there was no clear enhancement of catalytic activity towards the bisubstituted
peptide in comparison with the monosubstituted peptide. Though not further tested, it is
conceivable that other fragment combinations besides w33 and w202 may result in increased
PTPase activity. Since the fragment motifs were envisioned to afford enhanced interactions
with regions proximal to but outside the catalytic cleft, we wondered whether reasonable
affinity could be retained in the absence of the pTyr residue. In order to examine this hypothesis,
peptides 13 – 15 were synthesized (Scheme 4) by tethering two w33 fragments in
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Figure 9. Enhanced peptide substrates. Peptides substituted with fragments w33 and w202
were examined as oxime or ornithine amides in a PTPase assay with VH1 (dark bars) and
DUSP14 (open bars). Fragments w33 and w202 merged onto a single peptide backbone (12)
were compared to mono-substituted peptides (11a, b). Data ± SD are presented as substrate
activity (fold change) compared to the unmodified peptide 5.
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the absence of a pTyr residue. In order to enhance the hydrolytic stability of the oxime-ether
moiety, the original oxime aminooxy oxygen of 6d-w33 and 6f-w202 was replaced by a
methylene group, while the remainder of the oxime functionality was replaced by a structurally
homologous amide (compound 10, Scheme 3). The best inhibitory potency (Figure 10) was
achieved by 14 against VH1 (IC50 = 4 µM), while the same compound exhibited the poorest
inhibitory potency against DUSP14. Both VH1 and DUSP14 were inhibited equally by 13 (IC50
= 38 µM and 31 µM, respectively), while inhibition by 15 was greater for VH1 than DUSP14.
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Scheme 4. Synthesis of peptides 13 – 15. Reagents and conditions: (a) 20% piperidine/NMP
(rt, 15 min).; (b) HATU, HOAt, DIEA, NMP (rt, 3 h); (c) 1% TFA, DCM (rt, 15 min); (d)
TFA/TIPS/H2O (95 : 2.5 : 2.5) (rt, 1 h). Tpb = 4-(4-trifluoromethyl)pyridin-2-yl)benzoic acid
(10).
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Figure 10. Transformation of high affinity substrates to inhibitors by substitution of two
fragments onto a single peptide scaffold. Side chains of peptides 13, 14, and 15 were
replaced with a hydrolytically-stable mimic of the oxime group from 6d-w33 and tested in a
PTPase assay with peptide 5 as the substrate. Potencies for inhibition of peptide 5
dephosphorylation (IC50 ± SD) are shown.
CONCLUSION
Combinatorial synthetic efforts that targeted key substrate interactions with the catalytic site
were previously reported to develop potent PTPase inhibitors29,30. In the study described here,
diverse chemical fragments were introduced into all residues of a short peptide substrate as the
basis of a combinatorial screening approach. We synthesized a tethered fragment library of
1800 unique oxime phosphopeptide derivatives of a high affinity heptapeptide scaffold that is
widely employed as a general PTPase substrate. The entire oxime-containing peptide library
was spotted onto microarray slides and evaluated against a panel of PTPases. This allowed us
to measure a total of 7200 different catalytic interactions in a single assay. The results were
analyzed according to amino acid position on the peptide chain, as well as by chemical structure
or functional group of each oxime-derived fragment. The potencies of select oxime-containing
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peptides were confirmed by a microarray-based kinetic assay that simultaneously measured the
association and dissociation rates of up to 200 peptide-enzyme complexes in real-time. By
combining the results from the catalytic and the kinetic assays, we identified multiple
fragments that were preferred in different locations on the peptide. We removed the pTyr
residue to form tethered bivalent constructs 13 – 15 and found that these exhibited micromolar
PTPase inhibitory potencies. We rationalized that this reflected enhanced non-pTyr-dependent
interactions with regions proximal to, but outside, the catalytic cleft. Collectively, these results
suggest that chemical fragments identified by the described peptide-display method can be used
as building blocks for designing non-peptidyl inhibitors of PTPases.
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EXPERIMENTAL SECTION
Synthetic
General. The following reagents are commercially available from Sigma–Aldrich: 2-bromo-
4-(trifluoromethyl)pyridine (7) (cat. no. 661139); (4-(methoxycarbonyl)phenyl)boronic acid (8)
(cat. no. 594539). The following reagents are available from Chem–Impex, International:
Fmoc-Orn(Mtt)-OH (cat. no. 03729); Fmoc-L-Tyr(HPO3Bzl)-OH (cat. no. 03746); Fmoc-
Glu(OtBu)-OH (cat. no. 02413); Fmoc-Asp(OtBu)-OH (cat. no. 00494); Fmoc-Ala-OH (cat.
no. 02369); Fmoc-Val-OH (cat. no. 02470) and (+) biotinyl-6-aminohexanoic acid (cat. no.
14003). Preparative high-pressure liquid chromatography (HPLC) was conducted using a
Waters Prep LC4000 system having photodiode array detection and Phenomenex C18 columns
(250 mm × 21.2 mm, 10 µm particle size, 110 Å pore size) at a flow rate of 10 mL/min. Binary
solvent systems were employed consisting of A = 0.1% aqueous TFA and B = 0.1% TFA in
CH3CN. Purified samples were lyophilized to provide final products as white powders. The
mass spectra of final peptides were measured by electrospray ionization-mass spectra (ESI-MS)
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using an Agilent 1200 HPLC system equipped with a diode-array detector set to measure UV
absorption at 220 nm. Chromatography was carried out on a narrow-bore (100 mm × 2.1 mm),
small-particle (3.5-µm), Zorbax Rapid-Resolution reversed-phase C18 column coupled with a
C18 guard column of the same bonded phase (12.5 mm × 2.1 mm) eluted with a linear gradient
of MeOH : H2O at a flow rate of 300 µL/min. Solvent A consisted of 5% CH3OH/H2O
containing 0.1% CH3COOH and solvent B consisted of 90% CH3OH/H2O containing 0.1%
CH3COOH was employed in the following gradient: isocratic A for 3 min; linear gradient of B
to 100% B in 9 min; isocratic B for 5 min; linear reset to 100 % A in 5 min; isocratic A for 3
min to equilibrate.
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Synthesis of (S)-benzyl-2-(((benzyloxy)carbonyl)amino)-4-(((tert-
butoxycarbonyl)amino)oxy)butanoate (3).
To a solution of 2 (2.96 g, 6.1 mmol) in CH2Cl2 (10 mL) at 0 °C was added methyl hydrazine
(0.80 mL, 0.70 mmol) and the reaction mixture was stirred at 0 °C (2 h). The mixture was
filtered and the filtrate was concentrated and dried under high vacuum. The resulting crude
material was dissolved in THF (10 mL) and to this was added triethylamine (1.22 mL, 0.89
mmol) and Boc2O (1.9 g, 1.9 mmol) and the solution was stirred at room temperature
(overnight). The reaction mixture was diluted with EtOAc (20 mL) and washed with H2O (2 ×
20 mL) and brine (20 mL) and dried (MgSO4). The crude material was purified using silica gel
CombiFash (Hex : EtOAc, 2 : 1) to give product 3 as a colorless oil (1.86 g, 4.1 mmol, 72 %).
1H NMR (400 MHz, CDCl3) δ 7.23-7.31 (m, 10H), 5.12 (m, 2H), 5.08 (m, 2H), 4.52 (m, 1H),
13
3.81-3.92 (m, 2H), 2.09 (m, 2H), 1.42 (s, 9H). C NMR (400 MHz, CDCl3) δ 171.87 (1C),
156.89 (1C), 156.14 (1C), 136.36 (1C), 135.27 (1C), 128.52 (2C), 128.40 (2C), 128.35 (1C),
128.24 (2C), 127.97 (1C), 127.85 (2C), 85.10 (1C), 72.76 (1C), 67.17 (1C), 66.78 (1C), 51.72
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(1C), 30.25 (1C), 28.11 (3C). ESI -MS (m/z): Calcd. for C24H30N2O7 + Na, 481.2; Found, 481.2
5
6
[M + Na]+.
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(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(((tert-
butoxycarbonyl)amino)oxy)butanoic acid (4).
A mixture of 3 (1.86 g, 4.1 mmol) and 10% Pd•C (0.19 g) was dissolved in MeOH (5 mL) and
stirred under H2 (overnight). The mixture was passed through Celite and the solvent was
removed under reduced pressure. The crude material was dissolved in dioxane (10 mL) and
H2O (10 mL) and FmocOSu (1.64 g, 8.2 mmol) and NaHCO3 (0.68 g, 4.9 mmol) were added
and the reaction mixture was stirred at room temperature (overnight). The reaction mixture was
washed with Et2O (10 mL) and the remaining aqueous layer was acidified with 1N HCl to pH
3 – 4 and extracted with EtOAc (3 x 20 mL). The organic phase was dried (MgSO4), filtered
and the was solvent evaporated. The crude product was purified using silica gel CombiFlash
(MeOH/CH2Cl2 0-85%) and lyophilized from CH3CN : H2O (1 : 1 with 0.1%TFA) to provide
product 4 as a white solid (1.65 g, 3.6 mmol, 88 %). 1H NMR (400 MHz, CDCl3) δ 7.72 (d, J
= 7.2 Hz, 2H), 7.62 (d, J = 7.2 Hz, 2H), 7.41 (s, 1H), 7.36 (t, J = 7.2 Hz, 2H), 7.27 (td, J = 1.2,
7.2 Hz, 2H), 4.57 (m, 1H), 4.36 (m, 2H), 4.21 (m, 1H), 3.96 (m, 1H), 2.14 (m, 1H), 1.46 (s,
13
9H). C NMR (400 MHz, CDCl3) δ 174.86 (1C), 157.78 (1C), 156.69 (1C), 143.87 (1C),
143.73 (1C), 141.23 (2C), 127.64 (2C), 127.04 (1C), 127.02 (1C), 125.22 (2C), 119.88 (2C),
82.71 (1C), 73.24 (1C), 67.26 (1C), 51.79 (1C), 47.08 (1C), 29.85 (1C), 28.11 (3C). ESI -MS
(m/z): Calcd. for C24H28N2O7 + Na, 479.2; Found, 479.2 [M + Na]+.
Methyl 4-(4-(trifluoromethyl)pyridin-2-yl)benzoate (9). To a solution of 2-bromo-4-
(trifluoromethyl)pyridine (7) (0.547 mL, 4.42 mmol) in a solution of toluene : EtOH : H2O (5 :
1 : 5, 26.4 mL) was added Na2CO3 (4.22 g, 39.8 mmol), Pd(PPh3)4 (0.511 g, 0.442 mmol) and
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(4-(methoxycarbonyl)phenyl)boronic acid (8) (1.194 g, 6.64 mmol) and the mixture was stirred
at 110 °C (overnight). The mixture was cooled to room temperature, diluted with EtOAc (200
mL) and washed with H2O (50 mL) and brine (2 × 50 mL) then dried (Na2SO4), filtered and
concentrated. The resulting residue was purified by silica gel CombiFlash to provide 9 as a
white solid (805 mg, 65 % yield). 1H NMR (400 MHz, CDCl3) δ 8.83 (d, J = 5.0 Hz, 1H), 8.11
(d, J = 8.4 Hz, 2H), 8.05 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 0.6 Hz, 1H), 7.46-7.41 (m, 1H), 3.90
(s, 3H). DUIS-MS m/z: 282 (MH+).
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4-(4-(Trifluoromethyl)pyridin-2-yl)benzoic acid (10). To a solution of 9 (805 mg, 2.86 mmol)
in THF : H2O (2 : 1, 9 mL) was added LiOH•H2O (240 mg, 5.7 mmol) and the mixture was
stirred at room temperature (overnight). The mixture was acidified with ethereal HCl (3 mL,
2.0 M in Et2O) and concentrated and the resulting residue was suspended in MeOH (3 mL),
collected by filtration, washed (MeOH) and dried to provide 10 as a white solid (757 mg, 99
% yield). 1H NMR (400 MHz, DMSO-d6) δ 13.04 (brs, 1H), 8.93 (d, J = 5.0 Hz, 1H), 8.34 (s,
1H), 8.31-8.22 (m, 2H), 8.03 (dd, J = 8.2, 1.3 Hz, 2H), 7.74 (d, J = 5.0 Hz, 1H). ESI-MS m/z:
268.1 (MH+).
Synthesis of peptides 5 and 6(a – f). Peptides were synthesized on Rink amide MBHA resin
(Novabiochem, cat. no. 01-64-0037) by standard Fmoc solid-phase protocols using active ester
coupling methodology in N,N-dimethylformamide (DMF). In summary, coupling was achieved
by reacting each residue, amino acid or acid (5.0 equivalents based on resin loading), 1-
hydroxybenzotriazole (HOBt) (5.0 eq.) and N,N’-diisopropylcarbodiimide (DIC) (5.0 eq.)
(single couple, 2 h). Peptides 6(a – f) were prepared using reagent 4 where appropriate. The
final resin was washed with DMF, MeOH, CH2Cl2, and Et2O and then dried under vacuum
(overnight). Peptides were cleaved from the resin by treatment with TFA : triisopropylsilane
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(TIPS)/H2O (95: 2.5: 2.5, 5 mL, 4 h). The resin was removed by filtration, the peptide was
precipitated in cold Et2O and the precipitate was centrifuged and washed with Et2O. The
resulting white solid was dissolved in 50% aqueous CH3CN (4 mL) and purified by reverse
phase preparative HPLC. (Analytical HPLCs of purified peptides are shown in Supporting
Information Figure S1).
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Peptide 5. ESI -MS (m/z): Calcd. for C52H80N11O20PS - H, 1240.4; Found, 1240.3 [M - H]-.
Peptide 6a. ESI-MS (m/z): Calcd. for C51H79N12O21PS + Na, 1281.5; Found, 1281.3 [M + Na]+.
Peptide 6b. ESI-MS (m/z): Calcd. for C52H83N12O19PS + H, 1243.5; Found, 1243.3 [M + H]+.
Peptide 6c. ESI-MS (m/z): Calcd. for C53H83N12O21PS + H, 1287.5; Found, 1287.4 [M + H]+.
Peptide 6d. ESI-MS (m/z): Calcd. for C52H83N12O19PS+ Na, 1265.5; Found, 1265.4 [M + Na]+.
Peptide 6e. ESI-MS (m/z): Calcd. for C59H87N12O19PS + Na, 1251.5; Found, 1251.4 [M + Na]+.
Peptide 6f. ESI-MS (m/z): Calcd. for C50H77N12O21PS + Na, 1268.5; Found, 1268.2 [M + Na]+.
Synthesis of peptide 11a, 11b and 12. For peptides 11a, 11b and 12 active ester coupling was
employed using N-Fmoc-protected amino acid (4 eq.), HATU (3.8 eq.), and DIEA (8.0 eq.)) in
N-methyl-2-pyrrolidinone (NMP). Coupling of Fmoc-L-Orn(Mtt)-OH was achieved using
Fmoc-L-Orn(Mtt)-OH (2.0 eq.), HATU (1.9 eq.), and DIEA (4.0 eq.) in NMP by double
coupling. After the completion of coupling of Fmoc-L-Orn(Mtt)-OH, the resin was then
subjected to a global Mtt-deprotection by DCM/TFA/TIPS = 95:1:4 (10 min × 2). The resulting
resins were washed with 10% DIEA in NMP and were subsequently coupled with 3,5-
dichloroprridine-4-carboxylic acid or 4-(4-(trifluoromethyl)pridin-2-yl)benzoic acid (10).
Peptide chains were then elongated using standard SPPS. The completed resins were treated
with TFA/TIPS/H2O cocktail (95:2.5:2.5) for 3 h and then filtered. Cold Et2O was added to the
filtrates and the resulting precipitates were washed with cold Et2O (50 mL x 3). The crude
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product was dissolved in DMSO and purified by reverse-phase preparative HPLC. (Analytical
HPLCs of purified peptides are shown in Supporting Information Figure S1).
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Peptide 11a. Preparative HPLC conditions: linear gradient elution (79.9/20/0.1
H2O/acetonitrile/TFA to 29.9/70/0.1 H2O/acetonitrile/TFA over 30 minutes). ESI-MS (m/z)
Calcd. for C66H91F3N13O19PS – H: 1488.6; Found [M – H]– = 1488.4.
Peptide 11b. Preparative HPLC conditions: linear gradient elution (79.9/20/0.1
H2O/acetonitrile/TFA to 54.9/45/0.1 H2O/acetonitrile/TFA over 30 minutes). ESI-MS (m/z)
Calcd. for C57H80Cl2N13O21PS – H: 1414.4; Found [M – H]– = 1414.3.
Peptide 12. Preparative HPLC conditions: linear gradient elution (69.9/30/0.1
H2O/acetonitrile/TFA to 39.9/60/0.1 H2O/acetonitrile/TFA over 30 minutes). ESI-MS (m/z)
Calcd. for C71H91Cl2F3N15O20PS – H: 1662.5; Found [M – H]– = 1662.3.
Synthesis of peptides 13 – 15. Peptides 13 – 15 were synthesized on Rink amide MBHA
resin by standard Fmoc solid-phase protocols using active ester coupling methodology as
described above, except that in N-methyl-2-pyrrolidone (NMP) was used in place of DMF.
Initial coupling was achieved on Fmoc-deprotected resin with a solution of Fmoc-L-
Orn(Mtt)-OH (5.0 eq.), HATU (5.0 eq.), HOAt (5.0 eq.), and DIEA (10 eq.) in NMP (5 mL).
The mixture was shaken for 3 h and the solvent was then drained. The resin was washed with
NMP (5 mL × 3, in 5 min/wash, drained after each wash) and iPrOH (5 mL × 3, in 5
min/wash, drained after each wash). The above sequence was repeated for the coupling of
Fmoc-L-Gly-OH (5.0 eq.) (for peptides 14 and 15), Fmoc-L-Orn(Mtt)-OH (5.0 eq.), and
acetic anhydride in pyridine (1:9 v/v, 5 eq.). After the completion of peptide backbone, the
resin was then subjected to a global Mtt-deprotection in a wash sequence of 1% TFA in
CH2Cl2 (5 mL × 3, in 5 min/wash, drained after each wash), DIEA (5 mL × 3, in 5 min/wash,
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drained after each wash), and NMP (5 mL × 3, in 5 min/wash, drained after each wash). The
resin was then reacted with a solution of 4-(4-(trifluoromethyl)pyridin-2-yl)benzoic acid (10,
10 eq.), HATU (5.0 eq.), HOAt (5.0 eq.), and DIEA (10 eq.) in NMP (5 mL). After shaking
(3 h), the solvent was drained. The resin was washed with NMP (5 mL × 3, in 5 min/wash,
drained after each wash) and iPrOH (5 mL × 3, in 5 min/wash, drained after each wash). The
final product was cleaved from the resin by shaking in a TFA/TIPS/H2O cocktail (95:2.5:2.5)
(7.5 mL, 30 min). Another 7.5 mL portion of the cocktail was diluted with TFA (7.5 mL) to
make a 15 mL solution. This solution was used to wash the resin twice (7.5 mL/wash, in 15
min/wash). The filtrate was collected, combined and concentrated. Addition of cold Et2O
caused precipitation of a white solid. The mixture was centrifuged and decanted. The white
precipitate was collected and washed with cold Et2O (5 mL x 2). The crude product was
dissolved in CH3CN and purified by reverse-phase preparative HPLC.
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Peptide 13 (Ac-OtOt-NH2) 33 mg, 81% yield. ESI-MS (m/z) Calcd. for C38H37F6N7O5,
785.3; Found [M + H]+ = 786.3, [M + Na]+ = 808.2, [M + 2H]+ = 393.7.
Peptide 14 (Ac-OtGOt-NH2): 26 mg, 59% yield. ESI-MS (m/z) Calcd. for C40H40F6N8O6,
842.3; Found [M + H]+ = 843.3, [M + Na]+ = 865.3, [M + 2H]+ = 422.2.
Peptide 15 (Ac-OtGGOt-NH2): 16 mg, 34% yield. ESI-MS (m/z) Calcd. for C42H43F6N9O7,
899.3; Found [M + H]+ = 900.2, [M + Na]+ = 922.2, [M + 2H]+ = 450.7.
Biological
Materials. Single pad and 16-pad nitrocellulose-coated FAST slides were purchased from
KeraFAST, Inc. (Boston, MA, USA). Gold SPRi slides and index matching oil were purchased
from Horiba Scientific (Edison, NJ, USA). NeutrAvidin, biotin-LC-NHS, biotin-PEG12-NHS,
EDC, and NHS were purchased from Thermo Scientific (Rockford, IL, USA). Flexchip-
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