Utilization of nitrophenylphosphates and oxime-based ligation for the development of nanomolar affinity inhibitors of the Yersinia pestis outer protein H (YopH) phosphatase

Article abstract of DOI:10.1021/jm200022g

Our current study reports the first K_M optimization of a library of nitrophenylphosphate-containing substrates for generating an inhibitor lead against the Yersinia pestis outer protein phosphatase (YopH). A high activity substrate identified by this method (K_M = 80 μM) was converted from a substrate into an inhibitor by replacement of its phosphate group with difluoromethylphosphonic acid and by attachment of an aminooxy handle for further structural optimization by oxime ligation. A cocrystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a conserved water molecule proximal to the aminooxy group that was subsequently employed for the design of furanyl-based oxime derivatives. By this process, a potent (IC₅₀ = 190 nM) and nonpromiscuous inhibitor was developed with good YopH selectivity relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a noncytotoxic concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH. This article not subject to U.S. Copyright. Published 2011 by the American Chemical Society.

Full text of DOI:10.1021/jm200022g

ARTICLE
pubs.acs.org/jmc
Utilization of Nitrophenylphosphates and Oxime-Based Ligation for
the Development of Nanomolar Affinity Inhibitors of the Yersinia
pestis Outer Protein H (YopH) Phosphatase†,‡
Medhanit Bahta,§ George T. Lountos,|| Beverly Dyas,^ Sung-Eun Kim,§ Robert G. Ulrich,^ David S. Waugh,||
and Terrence R. Burke, Jr.*,§
§Chemical Biology Laboratory, Molecular Discovery Program, Center for Cancer Research, National Cancer Institute, National
Institutes of Health, NCI—Frederick, Frederick, Maryland 21702, United States
Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
NCI—Frederick, Frederick, Maryland 21702, United States
^Laboratory of Molecular Immunology, United States Army Medical Research Institute of Infectious Diseases, Frederick,
Maryland 21702, United States
S Supporting Information
b
ABSTRACT: Our current study reports the first KM optimiza-
tion of a library of nitrophenylphosphate-containing substrates
for generating an inhibitor lead against the Yersinia pestis outer
protein phosphatase (YopH). A high activity substrate identi-
fied by this method (KM = 80 μM) was converted from a
substrate into an inhibitor by replacement of its phosphate
group with difluoromethylphosphonic acid and by attachment
of an aminooxy handle for further structural optimization by
oxime ligation. A cocrystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a
conserved water molecule proximal to the aminooxy group that was subsequently employed for the design of furanyl-based oxime
derivatives. By this process, a potent (IC50 = 190 nM) and nonpromiscuous inhibitor was developed with good YopH selectivity
relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a noncytotoxic
concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH.
’ INTRODUCTION
In theory, avoiding promiscuous behavior could be achieved
through the use of substrates as templates for inhibitor design.
This is because substrates must interact with their enzyme hosts
in nonpromiscuous fashions in order for productive catalysis to
occur. Employing small nonpeptidic arylphosphates to identify
potential leads for PTP inhibitor design has been known for
some time.12ꢀ15 However, the explicit application of “substrate
activity screening” for the purpose of minimizing misleading
promiscuous inhibition has only more recently been proposed by
Ellman for protease16ꢀ20 and PTP targets.21 This approach
consists of first identifying substrates that exhibit high affinity,
structurally enhancing these substrates, and then converting the
optimized substrates to inhibitors by replacement of their labile
phosphoryl groups with suitable nonhydrolyzable phosphoryl
mimetics. Additional structural variations can then be performed
to further increase inhibitory potency.
Maintaining proper levels of tyrosyl phosphorylation through
the reversible actions of protein tyrosine kinases (PTKs) and
protein tryrosine phosphatases (PTPs) is vital for cellular
processes ranging from growth and metabolism to adhesion
and differentiation.1,2 Deregulation of PTPs can be linked to
diseases such as diabetes and cancer, and accordingly, this class of
enzymes represents a new source of potential drug targets.3ꢀ6 The
Gram-negative enterobacterium Yersinia pestis (Y. pestis) has played
an important role in human history as the causative agent of plague,7
and more recently it has gained attention because of its possible use as
a biological warfare agent. For pathogenicity, Y. pestis requires the
virulence factor Yersinia pestis outer protein H “YopH”, a highly active
PTP.8 Accordingly, potent and selective YopH inhibitors could
provide a basis for new antiplague therapeutics. One difficulty
encountered in the development of PTP inhibitors is a high
incidence of “false positives” that can arise through inhibition of
enzyme function by “promiscuous” mechanisms attributable to
nonspecific factors such as protein aggregation.9,10 It is generally
believed that promiscuous inhibitors do not represent valid leads,
and avoiding promiscuous mechanisms is an important compo-
nent of current drug development.11
In the identification of high affinity substrates for the devel-
opment of PTP inhibitors, advantage can be taken of the
hydrolytic action of a PTP on an arylphosphate, which produces
Received: January 10, 2011
Published: March 28, 2011
This article not subject to U.S. Copyright.
Published 2011 by the American Chemical Society
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’ RESULTS AND DISCUSSION
Nitrophenylphosphate Substrates. A total of 48 o- and p-
nitrophenylphosphate-containing substrates (2) were prepared
by phosphorylation (reaction with HPO3(Bn)2) of either com-
mercially available or synthetic nitrophenols, followed by TFA-
mediated cleavage of the resulting benzyl protecting groups. The
YopH affinities of these substrates were determined using an in
vitro assay that measured substrate turnover by monitoring the
yellow color arising from the reaction product nitrophenols.8
Color interference arising from sources other than the nitrophe-
nol products did not prove to be problematic. Assay results for a
subset of 11 selected substrates (2aꢀk, Table 1) show that the
3-aminooxymethyl-containing substrate 2e exhibited a 3.5-fold
decrease in its MichaelisꢀMenten constant (KM = 170 μM)
relative to pNPP (KM = 600 μM) while p-phenyl-o-nitrophenyl-
phosphate (2j) showed an approximate 4-fold decrease in its KM
(150 μM). The lowest KM was obtained with m-phenyl-p-
nitrophenylphosphate (2k, KM = 80 μM), which showed an
approximate 7.5-fold decrease relative to reference pNPP.
Lead Inhibitor Platform 5. When substrates are used as
structural models for inhibitor design, the interpretation of data
from the substrate enzyme assays can be an important factor. In
the current study, KM values were used to indicate substrate
affinity. Previous comparisons of KM and kcat/KM for small-
molecule non-peptidyl aryl substrates have shown that KM values
more closely reflect IC50 and Ki than do kcat/KM ratios.12ꢀ14,21
On the basis of this consideration, substrate 2k was selected for
conversion to an inhibitor because of its low KM.
In the conversion of a PTP substrate to an inhibitor, the choice
of phosphoryl mimetic can have a dramatic effect on the resulting
inhibitory potency.21 In our current work R,R-difluoromethyl-
phosphonic acid47 was used as a phosphoryl replacement, since it
is isosteric with the parent phosphate group and it has been
shown to be one of the highest affinity phosphoryl replacements
in PTP contexts (Scheme 1).48,49 A further consideration deals
with the fate of the nitro functionality, since its role as a
chromophore is no longer needed. Although an example has
been reported where protein hydrogen bonding exists for the
nitro group of a YopH-bound inhibitor,50 in the current work the
nitro functionality was removed at this stage. The transformation
of 2k to the inhibitor platform 5 was completed by introduction
of an aminooxy handle for use in preparing bidentate inhibitors
(6) using oxime-based click chemistry (Figure 2).34ꢀ42
Synthesis of Aminooxy-Containing Platform 5. The synth-
esis of 5 began with the CuBr and zinc promoted coupling
reaction of diethyl (bromodifluoromethyl)phosphonate with
3-iodobromobenzene51 to give the corresponding (difluoromethyl)
phosphonic acid diethyl ester product 7 (Scheme 1). Suzuki
coupling of 7 with 4-hydroxymethylboronic acid to give the
biphenyl product 8 was followed by Mitsunobu reaction with N-
hydroxyphthalimide and treatment of the resulting phthalimide
with hydrazine hydrate to yield the aminooxy-containing diethyl-
phosphonate 9 (79% yield). Finally, conversion of 9 to platform
5 was achieved through TMSBr-mediated phosphonate depro-
tection (Scheme 1).52
Crystal Structure of YopH in Complex with Platform 5. In
order to facilitate inhibitor optimization, the X-ray cocrystal
structure of 5 bound to YopH was determined. The orientation
of the phosphonodifluormethyl group of 5 within the catalytic
pocket was observed to be highly similar to that previously
reported for the phosphonodifluormethylphenylalanyl residue
Figure 1. Design progression leading from a library of nitrophenylpho-
sphates (1) to bidentate inhibitors (4).
both the corresponding phenol and inorganic phosphate. Tradi-
tionally, the released inorganic phosphate can be quantified using
colorimetric assays that employ phosphomolybdate22,23 or by
secondary enzyme assays, including the use of purine nucleotide
phosphorylase-mediated phosphate-dependent conversion of
2-amino-6-mercapto-7-methylpurine ribonucleoside to a deriva-
tive having an absorbance maximum at 360 nm.24 It is also
possible to spectrophotometrically measure the catalytically
produced phenol. A variety of easily detected fluorescence-based
substrates are known;25 however, these agents would be of little
value for the purpose of substrate activity screening and phenols
derived from the more structurally diverse arylphosphates
needed for substrate activity screening would typically exhibit
very low extinction coefficients.26 An exception to this is found
with nitrophenols, which exhibit intense yellow color because of
delocalization of the phenolate anionic charge. Because of this
property, p-nitrophenylphosphate (pNPP) has become a ubiqui-
tous substrate for monitoring the activity of phosphatases,
including YopH.8
In undertaking our current study, we desired to use direct
spectrophotometric monitoring of phenol reaction products. For
this purpose, we employed substrates (2) derived from either
o- or p-nitrophenols (1, Figure 1). These compounds allowed the
simple monitoring (absorbance at λ405 nm) of yellow color
resulting from the enzyme-catalyzed phosphoryl hydrolysis. Our
utilization of nitrophenylphosphates represents the first systema-
tic application of this structural class for PTP substrate
optimization.
Once an inhibitor platform has been identified through a
substrate activity approach, enhancement of affinity can be
undertaken by introducing additional functionality intended to
interact with sites proximal to the catalytic cavity.27ꢀ33
A
distinctive feature of the methodology in our current report is
its incorporation of aminooxy functionality into the lead inhibitor
platform (3) and the use of this handle for oxime-based
derivatization (4, Figure 1). Functional group ligation by means
of oxime bond formation can be considered to be a form of “click
chemistry”34 that we35ꢀ38 and others39ꢀ42 have shown can be
highly useful for the facile generation of compound libraries. In
the case of PTPs, azideꢀalkyne Huisgen cycloaddition click
reactions have been used previously for the rapid assembly of
bidentate libraries targeting protein tyrosine phosphatase 1B
(PTP1B) and Mycobacterium protein tyrosine phosphatase B
(mPTPB).43ꢀ45 However, a potential limitation of this type of click
chemistry is the requirement for high throughput syntheses of azide-
containing libraries of reactants.46 In contrast, oxime-based click
chemistry is advantageous because it can be conducted using
commercially available aldehydes and reaction products can be
directly evaluated biologically without purification. As reported in
our current paper, nitrophenylphosphate-based substrate activity
screening used in combination with oxime ligation proved to be
highly a successful approach that resulted in the development of a
nonpromiscuous YopH inhibitor exhibiting a nanomolar IC50.
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Table 1. In Vitro YopH KM for Selected Substrates
substrate
R1
R2
R3
R4
KM ( SE (mM)a
pNPP
2a
H
NO2
t-Bu
NO2
H
H
0.60 ( 0.10
0.47 ( 0.06
1.14 ( 0.19
1.13 ( 0.15
0.41 ( 0.05
0.17 ( 0.02
0.65 ( 0.11
no activity
NO2
H
H
H
2b
2c
t-Bu
H
H
NO2
H
ꢀCH2ONH2
H
2d
2e
2f
NO2
NO2
NO2
H
ꢀCH2ONH2
CH2ONdCHMe
H
H
H
H
H
H
H
H
H
Ph
Me
Me
H
2g
2h
2i
NO2
cyclohexyl
NO2
NO2
H
NO2
H
0.26 ( 0.02
no activity
cyclohexyl
2j
Ph
H
0.15 ( 0.03
0.08 ( 0.01
2k
NO2
ꢀH
a KM values were determined as indicated in the Materials and Methods.
Scheme 1. Synthesis of Inhibitor Platform 5a
Figure 2. Development of oxime-containing inhibitors (6) starting
from platform 2k.
a Reagents and conditions: (i) Zn, diethyl (bromodifluoromethyl)-
phosphonate, CuBr, DMF, 60 ꢀC to room temperature (96% yield);
(ii) 4-(hydroxymethyl)phenylboronic acid, Pd(PPh3)4, sat. K2CO3,
EtOH, PhMe, 70 ꢀC (45% yield); (iii) (a) N-hydroxyphthalimide,
Q446, and I443, similar to what is observed in the 1QZ0
structure (Figure 3 and Table 2).
PPh3, DIAD, THF, room temperature; (b) NH2NH2 H2O, EtOH,
3
room temperature (75% yield); (iv) TMSBr, CH2Cl2, room tempera-
Introduction of Oxime Functionality into 5. In the cocrystal
structure the aminooxyamine of 5 forms hydrogen bonds with
the side chain carboxyl of D231 and a water molecule (Wa43),
which also hydrogen-bonds to the D231 residue (Figure 4A).
The importance of this latter water is indicated by its presence
in the absence of inhibitor (designated as “Wa87” in PBD code
1LYV), suggesting that it could be used for inhibitor design.
In silico docking studies performed using the cocrystal structure
of 5 with the inclusion of Wa4357,58 identified furanyl-based
oximes as providing favorable interactions with the D231 residue
through the intermediacy of the conserved water (Figure 4B).
Syntheses of a series of furanyl-based oxime inhibitors was
performed in DMSO by reacting 5 (24 mM) with commercially
available furanyl aldehydes and AcOH in the ratio (1:1:2). The
oxime products (6), which were typically of >90% purity as
shown by random HPLC analysis, were used directly for
biological evaluation. Inhibitory potencies (IC50) were obtained
spectrophotometrically in an in vitro YopH assay using pNPP as
substrate.38
ture (72% yield).
(F2Pmp) of the hexapeptide Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-
amide (PDB code 1QZ0) (Figure 3A).53 The protein backbones
of the two structures are nearly superimposable, and in both
structures the flexible “WPD loops” (residues 354ꢀ356) are held
in the “closed” conformation. For both structures the phosphor-
yl-mimicking difluoromethylphosphonic acid group is coordi-
nated within the conserved (H/V)CX5R(S/T) signature motif
“P loop” (residues 404ꢀ410) by six hydrogen bonds,54ꢀ56 while
the guanidine group of R409 forms two salt bridges with two
phosphonic acid oxygen atoms and indirect hydrogen bonds with
residues Q357, Q450, and Q446 are made through a conserved
water residue (designated as “Wa1” for ligand 5 and “cw” in PDB
1QZ0). The 1-phenyl ring of 5 is pivoted about the difluoro-
methylene carbon so that it is offset, yet within the same plane
relative to the F2Pmp aryl ring in the 1QZ0 structure. This allows
the 3-(40-methylphenyl) moiety of 5 to interact, similar to the
Leu side chain of the dipeptide unit F2Pmp-Leu in the 1QZ0
structure (Figure 3A). The aryl rings of 5 form extensive
hydrophobic contacts with residues F229, D231, I232, A405,
The 3-furanyloxime (6a) showed an IC50 of 3.69 μM, whereas the
2-furanyloxime (6b) was approximately 3-fold more potent (IC50 =
1.20 μM) (Table 3). In modeling studies, the furanyl oxygen in 6a
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Figure 3. Crystal structures of YopH-bound ligands. (A) Superposition of the complex YopHꢀ[Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-amide] (protein
backbone in red, ligand carbons in white; PDB code 1QZ0) with YopHꢀ5 (protein backbone in blue, ligand carbons in yellow) showing relative binding
orientations of the two ligands. (B) Residues providing key hydrophobic contacts with 5.
Table 2. Comparison of YopH Hydrophobic Contact Resi-
dues for Compound 5 and F2Pmp-Leu
It was also of interest to examine possible YopH selectivity of
6e, since significant structural homology exists among many
phosphases. For this purpose, the inhibitory activity of 6e was
measured against a panel of phosphatases that included the
classical tyrosine-specific phosphatases PTP1B and leukocyte
antigen related phosphatase (LAR),63 as well as the dual speci-
ficity phosphatases 14 and 2264 (DUSP14, DUSP22) and the
Variola phosphatase VH1. In these assays 6e showed an approx-
imate 17-fold selectivity for YopH relative to PTP1B and greater
than 2000-fold selectivity relative to the other phosphatases
examined (Table 4).
Evaluation of 6e in Biological Models. A cell-based assay was
used to assess toxicity of 6e. The mouse macrophage line J774
was cultured 40 h with 0.1ꢀ100 μM 6e, and toxicity was
measured by cellular ATP content. No toxicity for any com-
pound was observed for 6e below100 μM (data not shown).
Intracellular replication of Y. pestis was assessed by a previously
described human monocyte infection model.65 Primary human
monocytes were infected for 12 h with Y. pestis, using cell culture
media containing 0.1ꢀ100 μM inhibitors or control. Specific
inhibition of intracellular bacterial growth was observed with
10 μM 6e (Figure 5). An approximately 9-fold decrease in
intracellular bacteria resulted from treatment with 6e compared
to a negative control that showed no inhibition of YopH. The
positive control gentamycin (10 μM), which targets bacterial
ribosomes,66 produced nearly complete inhibition of intracellular
Y. pestis growth.
residue
5a
F2Pmp-Leua,b
F229
D231
I232
24.6
24.1
23.6
23.0
21.1
11.0
24.6
30.2
19.8
24.1
29.1
16.0
A405
Q446
I443
a Contact is given in Å2. b Data for F2Pmp-Leu was derived from PDB
code 1QZ0.
was seen to be at a greater distance from the conserved Wa43
than for 6b. Therefore, 6b was modified by sequential addition of
a 5-methyl group (6c, IC50 = 0.91 μM) and then by introduction
of a hydroxyl group onto this methyl (6d, IC50 = 0.73 μM) and
finally by oxidation of the 5-hydroxymethyl substitutent to a
carboxyl group (6e, IC50 = 0.19 μM). This sequence of mod-
ifications resulted in a 6-fold improvement relative to the parent
6b. The observed binding enhancement of 6e was consistent
with in silico docking studies that showed multiple interactions of
its carboxyfuranyl oxime with the conserved Wa43 and with the
backbone amide proton of R230 (Figure 4C).
Examination of Specificity of 6e. The development of YopH
inhibitors is less advanced than for several other phosphatases.
For example, while the literature contains numerous reports of
nanomolar-affinity PTP1B inhibitors,59 there are few examples of
YopH inhibitors exhibiting affinities in the submicromolar
range.30,60 Additionally, the development of PTP inhibitors is
plagued by a high incidence of false positives that are due to
nonspecific or promiscuous mechanisms arising from the forma-
tion of colloid protein aggregates,9,10,61,62 and for some YopH
inhibitors the possible roles of promiscuous mechanisms are
unclear. The nanomolar IC50 of 6e makes it one of the more potent
YopH inhibitors reported to date. In order to determine whether
promiscuous mechanisms are at work, assays were conducted in the
presence and absence of 0.01% TX-100, since it is known that
promiscuous inhibition can often be minimized by the addition of
such a detergent.62 These experiments showed that the inhibi-
tory potency of 6e is independent of detergent concentration,
providing strong evidence that YopH inhibition by 6e does not
arise through promiscuous mechanisms.
’ CONCLUSIONS
YopH has proven to be a difficult target for inhibitor devel-
opment. While there are numerous reports of nanomolar affinity
inhibitors against other phosphatases such as PTP1B, submicro-
molar affinity YopH inhibitors are very few. The current study
represents the first utilization of a library of nitrophenylpho-
sphate-containing substrates for the purposes of lead identifica-
tion. An attractive feature of this approach is that KM can be
calculated directly by colorimetric methods based on the enzy-
matic generation of nitrophenol chromophore-containing reac-
tion products. The current work is also characterized by its use of
oxime-based click ligation to optimize a substrate-derived lead.
The combination of these two methodologies allowed the
identification of a nonpromiscuous nanomolar affinity YopH
inhibitor exhibiting good PTP selectivity that showed significant
inhibition of intracellular Y. pestis replication at a noncytotoxic
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Figure 4. Role of conserved water (Wa43) in the design of inhibitor 6e: (A) electrostatic potential surface rendering (blue = postive; red = negative) of
the YopHꢀ5 complex highlighting a key conserved water (Wa43); (B) predicted interaction of the furanyloxime oxygen of 6b with Wa43; (C) predicted
interaction of the 5-carboxyfuranyloxime group of 6e with Wa43 and the protein backbone.
concentration. The current work provides valuable insights into
the development of YopH inhibitors that may have broader
applicability in the discovery of inhibitors directed against other
phosphatases.
was prepared by mixing 25 mM Hepes buffer (pH 7.0ꢀ7.6), 50 mM
NaCl, 2.5 mM EDTA, and 5 mM dithiothreitol (DTT) with 1 mM fresh
DTT added right before an assay run. To each well was added 85 μL of
assay buffer, 0.25% BSA (5 μL) followed by 5 μL of nitrophenylpho-
sphate substrate in DMSO at dilutions of 1000, 500, 250, 125, 50, 25, 10,
and 5 μM. To the reaction mixtures was then added 5 μL of YopH in
buffer (25 μg/mL), and hydrolysis of substrate was monitored at 30 s
intervals over 15 min. MichaelisꢀMenten constants (KM) were deter-
mined using nonlinear regression with the equation y = Vmax[x/(KM þ x)].
Values for pNPP and synthetic substrates 2aꢀk are shown in Table 1.
Data curves are provided in the Supporting Information.
Determination of YopH IC50 Values. Total reactions volumes
of 100 μL/well of reaction volume were used in 96-well plates. Buffer
was prepared as above. To each well was added 79 μL of assay buffer,
0.25% BSA (5 μL) followed by 5 μL of inhibitors in DMSO at dilutions
of 400, 133, 44, 15, 5, 1.67, 0.56, 0.19, 0.063, 0.032, and 0 μM. To the
reaction mixtures was then added 5 μL of YopH in buffer (25 μg/mL)
followed by 6 μL of 10 mM pNPP buffer, and each plate was agitated
gently at 25 ꢀC for 15ꢀ20 min. Hydrolysis of the substrate was
immediately measured. IC50 values were determined by fitting the data
with a sigmoidal curve generated using the Boltzman equation. A parallel
independent assay was performed with 0.01% TritonX-100. Inhibition
constants for 6aꢀe are provided in Table 3. Data curves are provided in
the Supporting Information.
Toxicity Assay. Toxicity of 6e was assessed with the mouse
macrophage line J774 (American Type Culture Collection, Manassas,
VA), cultured in Eagle’s minimum essential medium supplemented with
4 mM L-glutamine, 4500 mg/L glucose, 1500 mg/L sodium bicarbonate,
and 7.5% fetal bovine serum (GIBCO/Invitrogen, Carlsbad, CA). Cells
were grown in 96-well (5 ꢁ 105 cells/100 mL) polystyrene plates
(opaque bottom; Corning, Lowell, MA), maintained in a 5% CO2,
humid air incubator (37 ꢀC). Inhibitor 6e was dissolved in DMSO to
produce a 10 mM stock solution and then added to cultures by diluting
in media to final concentrations of 0.1ꢀ100 μM. A negative control was
also used that shows no inhibition of YopH. Culture media for all cells
contained a final 1% DMSO, including control wells without chemical
compounds. Cell viability was assessed by ATP content 20 and 40 h after
treatment, using a commercial kit (ViaLight, Lonza, Basel, Switzerland)
and a luminometer (Wallac 1420 Victor; PerkinElmer, Shelton, CT) to
measure photon emission.
’ MATERIALS AND METHODS
General. The following reagents used for YopH enzyme assays were
obtained from Sigma-Aldrich: pNPP tablets, 30% BSA solution
(protease free), 1.0 M HEPES solution (pH 7.0ꢀ7.6), and dithiotreitol
(DTT). Aqueous ethylenediaminetetraacetic acid, sodium salt EDTA
(0.5 M, pH 8.0) was obtained from Invitrogen, and 96-well plates were
purchased from Costar. All reactions were carried out under argon
unless otherwise stated. All solvents were anhydrous and obtained from
Sigma-Aldrich. Final products were purified by a high pressure liquid
chromatography (HPLC) using a Waters Prep-LC 4000 system and
Phenomenex Gemini 10 μm, 110 Å C18 columns (250 mm ꢁ 21.20
mm, 10 μm) at a flow rate of 10 mL/min (preparative HPLC) with a
mobile phase of A = 0.1% aqueous TFA and B = 0.1% TFA in aqueous
acetonitrile. Typical gradients were from 10% B to 100% B over 40 min
with UV monitoring at 220, 254, and 280 nm. The purity of final
products was determined by analytical HPLC using a Waters Prep-LC
4000 system and Phenomenex Gemini 5 μm, 110 Å C18 columns (250
mm ꢁ 4.60 mm, 5 μm) at a flow rate of 1 mL/min with a mobile phase of
A = 0.1% aqueous TFA and B = 0.1% TFA in aqueous acetonitrile. All
final products were found to be g95% pure. NMR spectra were recorded
using a Varian 400 MHz spectrometer. Unit mass resolution LCꢀMS
results were obtained on synthetic intermediates, and high resolution
mass spectra were obtained for final products (University of California at
Riverside Mass Spectral Facility). Optical densities were measured with
Biotek Synergy 2 spectrophotometer at λabs = 405 nm using a kinetic
readout for determination of KM and absolute readout for determination
of IC50. The PTPse domain of YopH (residues 164ꢀ468) was expressed
in Escherichia coli according to a previously published procedure.53
Recombinant Proteins. The PTPase domain of YopH (residues
164ꢀ468) was expressed in Escherichia coli and purified as described
previously,8,53 as were the Variola major H1 (VH1)67 and human
DUSP-14 dual specificity phosphatases.68 Human DUSP-22, PTP1B,
and LAR catalytic domains were expressed and purified using generic
methodology.69
Intracellular Replication of Bacteria. Primary human mono-
cyte cultures65 were used to measure intracellular replication of the
plague bacterium. The Y. pestis strain CO92 pgmꢀ, plaꢀ, was previously
described.70 Colony-isolated bacteria were grown for 12 h in heart-infusion
Determination of YopH MichaelisꢀMenten Constants
(KM) for Nitrophenylphosphates. Total reactions volumes of
100 μL/well of reaction volume were used in 96-well plates. Buffer
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Table 3. In Vitro YopH IC50 for Selected Inhibitors
Figure 5. Effect of compounds on intracellular replication of Y. pestis.
Primary human monocytes were infected with Y. pestis and cultured for
12 h with 6e or a negative control (10 μM each). Gentamycin was
included as a positive control. Results are presented as viable intracellular
bacteria recovered per well of monocytes. Standard errors of the mean
were <10%. The assay was performed on three separate occasions using
three independent monocyte donors.
AB sera (Life Technologies, Carlsbad, CA). The monocytes were
incubated (1 h, 37 ꢀC) with a 1:1 ratio of Y. pestis in a 5% CO2, humid
air incubator(37ꢀC). The wells were pulsedwith gentamycin(10μg/mL,
20 min) and rinsed with warm media to remove remaining extra-
cellular bacteria. The cells were then cultured for 12 h in a 5% CO2,
humid air incubator (37 ꢀC) in media (100 μL of RPMI 1640
supplemented with 5% human AB sera) containing 0.1ꢀ100 μM
6e or a negative control that shows no inhibition of YopH. As a
positive control, gentamycin was added to wells containing no other
inhibitors. All cultures contained 1% DMSO by volume. The wells
were then gently washed with warm medium, followed by addition of
120 μL of sterile distilled water to lyse the cells. The cell lysates were
serially diluted (1:5) in HIB, and 200 mL of each dilution was placed
into duplicate wells of a culture plate (Honeycomb; Growth Curves
USA, Piscataway, NJ) and placed in a growth-monitoring incubator
(Bioscreen; Growth Curves USA) with constant agitation (37 ꢀC).
Bacterial growth was measured by optical density at 600 nm every
20 min for 16 h. The amount of Y. pestis was quantified by comparison
to a standard curve of bacteria.
X-ray Crystallography. The purified protein was pooled and
concentrated by diafiltration to 17.6 mg/mL in 100 mM sodium acetate,
pH 5.7, 100 mM NaCl, and 1 mM EDTA. Crystals of YopH were
obtained with condition D8 (0.1 M buffer system 2, pH 7.5, 0.12 M
alcohols, 12.5% v/v MPD, 12.5% w/v PEG 1000, and 12.5% w/v PEG
3350) from the Molecular Dimensions (Apopka, FL) Morpheus
screen.71 A 1:1 ratio of protein (17.6 mg/mL) to well solution was used
for crystallization at room temperature. Platelike crystals grew within 3
days. To obtain the proteinꢀinhibitor complex, compound 5 was
dissolved in DMSO and added to the crystallization solution to obtain
a final concentration of 10 mM (10% DMSO). The crystals were added
to the soaking solution and soaked for 48 h at room temperature.
Crystals were flash-frozen in liquid nitrogen without the need of an
additional cryoprotectant.
a IC50 values were determined as indicated in Materials and Methods.
Table 4. Inhibitory Potencies of 6e against a Panel of
Phosphatasesa
phosphatase
IC50 (μM)
fold difference
YopH
0.19
ref value
11.7
PTP1B
LAR
2.23
>400
>400
>400
>400
>2000
>2000
>2000
>2000
DUSP-14
DUSP-22
VH1
a Values were determined as described in Materials and Methods.
X-ray diffraction data for the YopHꢀ5 complex were collected at
beamline 22-ID of the SER-CAT facilities at the Argonne National
Laboratory utilizing remote data collection. By use of 1.0 Å X-ray
wavelength, 180 frames of data were collected using an exposure time
of 3 s and oscillation angle of 1ꢀ. The X-ray diffraction data were
processed with HKL3000.72 Data collection and refinement statistics are
outlined in Table 5. The structure was solved by molecular replacement
using the MOLREP program73 from the CCP4 suite74 and the co-
ordinates of the previously solved YopH structure (PDB code 1QZ0)
broth (HIB; Difco Laboratories, Detroit, MI) supplemented with 0.2%
xylose and 2.5 mM CaCl2. A dilution of the culture was grown (26 ꢀC) to
mid-log phase, and the bacteria were pelleted by centrifugation (600g)
before rinsing with RPMI 1640 medium. Human peripheral blood
monocytes (CD14þ) were isolated as previously described65 and added
(2 ꢁ 105/well) to tissue culture plates (96-well, flat bottom;
Corning) in 100 μL of RPMI 1640 supplemented with 5% human
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Table 5. Data Collection and Refinement Statistics
were removed from the enzyme with the exception of the catalytically
conserved water (Wa1) and a conserved water proximal to the ligand
aminooxy group (Wa43) (see Figure 4A). A 2-furanyl-based oxime
group was added to the aminooxyamine of 5, and the resulting oxime
structure (6a) was redocked using the “re-dock” option under the
“Ligand” menu (see Figure 4B). All docking experiments were per-
formed using the standard “re-dock” command, which utilizes a rigid
receptor protocol. The docked 6a was then modified by addition of a
carboxyl group to the furanyl 5-position, and the resulting structure (6e)
was redocked as described above (see Figure 4C).
parameter
X-ray source
22-ID, SER-CAT
1.0
wavelength (Å)
resolution (Å)
50.0ꢀ1.78 (1.80ꢀ1.78)a
P 212121
space group
unit cell dimensions
a (Å)
49.2
b (Å)
55.5
General Procedure for the Synthesis of Nitrophenylpho-
sphate Substrates (2aꢀk). To a solution of o- or p-nitrophenol
(1.0 mmol) in CH2Cl2 (5 mL) was added CCl4 (5.0 mmol) at ꢀ15 ꢀC,
and the reaction mixture was stirred at ꢀ15 ꢀC (5ꢀ10 min). To the
mixture was added N,N-diisopropylethylamine (DIEA) (2.0 mmol) and N,
N-dimethylaminopyridine (DMAP) (0.1 mmol). Then dibenzyl phosphite
was added dropwise at ꢀ15 ꢀC, and the mixture was stirred at ꢀ15 ꢀC
(1.5 h). The reaction was quenched by stirring with 0.5 M aqueous KH2PO4
(20 mL) at room temperature (5 min). The aqueous phase was extracted
with EtOAc, and the combined organic extract was dried (MgSO4) andtaken
to dryness under reduced pressure. The resulting residue was stirred with a
solution of TFA/CH2Cl2 (1:1, 5 mL) for 2ꢀ3 h. Volatiles were removed by
evaporation and crude products were subjected to HPLC purification to
provide final products in yields of 75ꢀ100%.
c (Å)
100.1
total reflections/unique reflections
completeness (%)
Rsym (%)b
148922/25241
92.8/74.8
10.6 (49.8)
21.9 (2.3)
5.9 (4.1)
I/σ(I)
redundancy
refinement statisticsc
resolution (Å)
50.0ꢀ1.78
23875/25241
16.5
no. of reflections working set/test set
Rwork (%)
Rfree (%)
no. of atoms, mean B-factor (Å2)
21.0
4-(tert-Butyl)-2-nitrophenyl Dihydrogen Phosphate (2a).
1H NMR (400 MHz, CD3OD): δ 7.86 (d, J = 2.0 Hz, 1H), 7.67 (dd, J =
2.4 Hz, J = 8.8 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 1.31 (s, 9H). 13C NMR
(400 MHz, CD3OD): δ 149.99 (1C), 143.19 (1C), 143.04 (1C), 132.27
(1C), 123.66 (1C), 123.06 (1C), 35.69 (1C), 31.49 (3C). HRMS-ESI
(m/z): [M ꢀ H]ꢀ calcd for C10H14NO6P, 274.0486; found, 274.0490.
3-(tert-Butyl)-4-nitrophenyl Dihydrogen Phosphate (2b).
1H NMR (400 MHz, CD3OD): δ 7.42ꢀ7.46 (m, 2H), 7.18 (m, 1H),
1.39 (s, 9H). 13C NMR (400 MHz, CD3OD): δ 154.17 (1C), 149.26
(1C), 144.82 (1C), 126.89 (1C), 121.66 (1C), 119.79 (1C), 35.28 (1C),
29.36 (3C). HRMS-ESI (m/z): [M ꢀ H]ꢀ calcd for C10H14NO6P,
274.0486; found, 274.0490.
protein
2237/15.5
22/15.7
inhibitor
water
274/29.3
rms deviation from ideal geometry
bond length (Å)/bond angle (deg)
Ramachandran plot
most favored (%)
additionally allowed (%)
generously allowed (%)
disallowed (%)
0.014/1.5
93.2
6.4
0.4
0
MolProbity protein geometry score
PDB accession code
1.53 (92nd percentile)
4-((Aminooxy)methyl)-2-nitrophenyl Dihydrogen Phos-
phate (2c). H NMR (400 MHz, D2O): δ 8.09 (d, J = 2.2 Hz, 1H),
1
2Y2F
a Values in parentheses are for reflections in the highest resolution shell.
b Rsym = ∑hkl∑i|Ii(hkl) ꢀ ÆI(hkl)æ|/∑hkl∑iIi(hkl), where ÆI(hkl)æ is the
mean intensity of multiply recorded reflections. c R = ∑|Fobs(hkl) ꢀ
7.78 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 5.09 (s, 2H).
HRMS-ESI (m/z): [M ꢀ H]ꢀ calcd for C7H8N2O7P, 263.0075; found,
263.0077.
F
calc(hkl)|/∑ |Fobs(hkl)|. Rfree is the R calculated for 5% of the data set
3-((Aminooxy)methyl)-4-nitrophenyl Dihydrogen Phos-
phate (2d). 1H NMR (400 MHz, D2O): δ 8.27 (d, J = 9.2 Hz, 1H),
7.53 (s, 1H), 7.40 (d, J = 9.2 Hz, 1H), 5.41 (s, 2H). HRMS-ESI (m/z):
[M ꢀ H]ꢀ calcd for C7H8N2O7P, 263.0075; found, 263.0067.
(3-(((Ethylideneamino)oxy)methyl)-4-nitrophenyl Dihydro-
not included in the refinement.
after removing all solvent and ligand atoms. Cross-rotation and transla-
tional searches were performed using data up to 3.0 Å followed by rigid-
body refinement with REFMAC5.75 Iterative rounds of model rebuild-
ing and refinement were performed with COOT76 and REFMAC5, and
the location of the inhibitor was unambiguously identified using σA-
weighted 2mFo ꢀ DFc and mFo ꢀ DFc electron density maps.77 The
coordinates and refinement restraint files were prepared using the
Dundee PRODRG server.78 Water molecules were located using COOT
and refined with REFMAC5. The refinement was monitored by setting
aside 5% of the reflections for calculation of the Rfree value.79 Model
validation was performed using MolProbity.80 The electron density map
for YopH-bound inhibitor 5 is included in the Supporting Information.
The coordinates and structure factor files were deposited in the Protein
Data Bank with accession code 2Y2F.
In Silico Studies. Docking of inhibitors 6a and 6e onto YopH was
done with ICM Chemist Pro software57 running on a MacIntosh computer
(OSX, version 10.5.8) using default parameters and procedures.58 In
summary, modeling started with the X-ray crystal structure of YopH
in complex with 5. The “convert PDB” command was used to convert to
native ICM format, with optimization of hydrogens. All H2O molecules
1
gen Phosphate (2e). H NMR (400 MHz, CD3OD): δ 8.16 (d, J =
9.2 Hz, 1H), 7.36 (m, 1H), 7.27 (m, 1H), 6.98 (q, J = 5.6 Hz, 1H), 5.40 (s,
2H), 1.88 (d, J = 5.6 Hz, 3H). HRMS-ESI (m/z): [M þ H]þ calcd for
C9H12N2O7P, 291.0377; found, 291.0374.
2-Methyl-4-nitrophenyl Dihydrogen Phosphate (2f). 1H NMR
(400MHz, CD3OD): δ8.06(m, J= 4.0 Hz, 1H), 8.00 (dd, J=4.0Hz,J=8.0
Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 2.34 (s, 3H). 13C NMR (400 MHz,
CD3OD): δ 156.26 (1C), 145.43 (1C), 132.75 (1C), 127.32 (1C), 123.68
(1C), 121.33 (1C), 16.65 (1C). HRMS-ESI (m/z): [M ꢀ H]ꢀ calcd for
C7H8NO6P, 232.0016; found, 232.0011.
2-Methyl-6-nitrophenyl Dihydrogen Phosphate (2g). 1HNMR
(400 MHz, CD3OD): δ7.67 (dd, J = 1.2 Hz, J = 8.0 Hz, 1H), 7.50 (d, J = 9.5
Hz, 1H), 7.20 (td, J = 1.2 Hz, J = 8.0 Hz, 1H), 2.41 (s, 3H). 13C NMR (400
MHz, CD3OD): δ 145.15 (1C), 143.43 (1C), 136.75 (1C), 135.56 (1C),
126.02 (1C), 124.10 (1C), 17.04 (1C). HRMS-ESI (m/z):[Mþ H]þ calcd
for C7H8NO6P, 234.0162; found, 234.0160.
2-Cyclohexyl-4-nitrophenyl Dihydrogen Phosphate (2h).
1H NMR (400 MHz, CD3OD): δ 7.68 (dd, J = 1.6 Hz, J = 8.0 Hz, 1H),
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7.61 (dd, J = 1.2 Hz, J = 8.0 Hz, 1H), 7.30 (td, J = 1.2 Hz, J = 8.0 Hz, 1H),
3.09 (m, 1H), 1.75ꢀ1.88 (m, 5H), 1.30ꢀ1.53 (m, 5H). 13C NMR (400
MHz, CD3OD): δ 155.29 (1C), 145.93 (1C), 141.78 (1C), 123.91
(1C), 123.41 (1C), 121.50 (1C), 38.59 (1C), 34.19 (2C), 27.90 (2C),
27.20 (1C). HRMS-ESI (m/z): [M þ H]þ calcd for C12H16NO6P,
300.0642; found, 300.0642.
Diethyl((40-((Aminooxy)methyl)-[1,10-biphenyl]-3-yl)difluoro-
methyl)phosphonate (9). To a mixture of 8 (184.0 mg, 0.50 mmol),
N-hydroxyphthalimide (98.1 mg, 0.60 mmol), and PPh3 (170.2 mg, 0.65
mmol) in anhydrous THF (5 mL) was added diisopropyl azodicarbox-
ylate (DIAD) (0.13 mL, 0.65 mmol). The mixture was stirred at room
temperature overnight. The reaction mixture was partitioned (H2O/
EtOAc), and the organic layer was dried over MgSO4 and solvent was
removed under reduced pressure. To a solution of the resultant product
(168 mg, 0.33 mmol) in CH2Cl2 (5 mL) and ethanol was added 50%
aqueous hydrazine hydrate (80 μL, 1.30 mmol). The mixture was stirred
at room temperature for 4 h. The resulting precipitate was removed by
filtration, and solvent was removed from the filtrate. The crude product
was purified by silica column chromatography (50ꢀ100% EtOAc in
hexanes) to yield 9 as an amorphous white solid (100 mg, 79% yield).
1H NMR (400 MHz, CD3OD): δ 7.81 (m, 2H), 7.59 (m, 4H), 7.46 (m,
2H), 4.73 (s, 2H), 4.20 (m, 4H), 1.30 (m, 6H). 13C NMR (400 MHz,
CD3OD): δ 141.17 (1C), 139.31 (1C), 137.26 (1C), 132.90 (1C), 132.76
(1C), 129.29 (1C), 129.27 (1C), 128.63 (2C), 128.23 (1C), 126.64 (2C),
124.63 (1C), 124.24 (1C), 76.98 (1C), 65.14 (1C), 65.07 (1C), 15.27
(1C), 15.21 (1C). APCI-MS (m/z): calcd for C18H22F2NO4P, 385.13;
found, 386.10 [M þ H]þ.
2-Cyclohexyl-6-nitrophenyl Dihydrogen Phosphate (2i).
1H NMR (400 MHz, CD3OD): δ 8.15 (m, 1H), 8.07 (dd, J = 2.8 Hz, J =
8.8 Hz, 1H), 7.52 (dd, J = 1.2 Hz, J = 8.8 Hz, 1H), 3.30 (m, 1H), 1.77ꢀ
1.90 (m, 5H), 1.32ꢀ1.51 (m, 5H). 13C NMR (400 MHz, CD3OD): δ
145.11 (1C), 144.68 (1C), 142.13 (1C), 133.31 (1C), 126.30 (1C), 123.85
(1C), 38.10 (1C), 34.77 (2C), 27.89 (2C), 27.20 (1C). HRMS-ESI (m/z):
[M þ H]þ calcd for C12H16NO6P, 300.0642; found, 300.0642.
3-Nitro-[1,10-biphenyl]-4-yl Dihydrogen Phosphate (2j).
1H NMR (400 MHz, CD3OD): δ 8.07 (dd, J = 0.8 Hz, J = 2.4 Hz,
1H), 7.85 (dd, J = 2.0 Hz, J = 8.4 Hz, 1H), 7.58ꢀ7.60 (m, 3H),
7.41ꢀ7.45 (m, 2H), 7.35 (m, 1H). 13C NMR (400 MHz, CD3OD): δ
155.48 (1C), 144.51 (1C), 139.62 (1C), 139.38 (1C), 130.29 (2C), 129.47
(1C), 128.01 (2C), 124.44 (1C), 124.41 (1C), 124.36 (1C). HRMS-ESI
(m/z): [M þ H]þ calcd for C12H11NO6P, 296.0319; found, 296.0315.
6-Nitro-[1,10-biphenyl]-3-yl Dihydrogen Phosphate (2k).
1H NMR (400 MHz, CD3OD): δ 7.95 (d, J = 8.8 Hz, 1H), 7.40ꢀ7.43
(m, 3H), 7.34ꢀ7.38 (m, 2H), 7.30ꢀ7.32 (m, 3H). 13C NMR (400
MHz, CD3OD): δ 155.48 (1C), 146.76 (1C), 139.79 (1C), 138.63
(1C), 129.76 (2C), 129.49 (1C), 128.90 (2C), 127.35 (1C), 129.32
(1C), 120.89 (1C). HRMS-ESI (m/z): [M þ H]þ calcd for
C12H11NO6P, 296.0319; found, 296.0316.
((40-((Aminooxy)methyl)-[1,10-biphenyl]-3-yl)difluoromethyl)
phosphonic Acid (5). To a solution of 9 (100 mg, 0.26 mmol) in
anhydrous CH2Cl2 (5 mL) under argon was added trimethylsilyl
bromide (0.13 mL, 0.93 mmol), and the mixture was stirred at room
temperature for 3 h. Solvent was removed, and HPLC purification was
performed as described in the general synthetic methods section
(retention time of 15.6 min) to provide 5 as an amorphous white solid
Diethyl ((3-Bromophenyl)difluoromethyl)phosphonate
(7). A suspended solution of Zn dust (1.27 g, 19.4 mmol) in 3 mL of
DMF was purged with argon. To this solution diethyl (bromodifluo-
romethyl)phosphonate (3.4 mL, 19.4 mmol) in 2 mL of DMF was
added dropwise by maintaining reaction temperature at 50ꢀ60 ꢀC
(reaction is exothermic). The reaction mixture was stirred at room
temperature for over 3 h. CuBr (2.79 g, 19.4 mmol) was added, and the
mixture was stirred for 30 min at room temperature. A solution of
bromo-3-iodobenzene (2.00 g, 7.1 mmol) in 1 mL of DMF was added
dropwise and was stirred for over 24 h at room temperature. Water
(10 mL) and ether (10 mL) were added, and mixture was passed
through Celite. The layers were separated, and the aqueous layer was
extracted by ether. The organic extract was dried over MgSO4, filtered,
and solvent was removed. The crude material was purified via silica gel
column chromatography (9:1 to 2:1 hexanes/EtOAc) to give pale yellow
oil product (2.3 g, 96%). 1H NMR (400 MHz, CDCl3): δ 7.71 (s, 1H),
7.58 (m, 1H), 7.52 (m, 1H), 7.29 (m, 1H), 4.19 (m, 4H), 1.29 (m, 6H).
13C NMR (400 MHz, CDCl3): δ 133.86 (1C), 129.99 (1C), 129.97
(1C), 129.85 (1C), 129.25 (1C), 129.97 (1C), 122.40 (1C), 64.92 (1C),
64.85 (1C), 16.29 (1C), 16.24 (1C). APCI-MS (m/z): calcd for
C11H14BrF2O3P, 342.0 and 344.0; found, 343.0 and 345.0 [M þ H]þ.
Diethyl (Difluoro(40-(hydroxymethyl)-[1,10-biphenyl]-3-
yl)methyl)phosphonate (8). A mixture of 7 (500.0 mg, 1.46 mmol),
(4-(hydroxymethyl)phenyl)boronic acid (332.0 mg, 2.19 mmol), and
Pd(PPh3) (84.0 mg, 0.07 mmol) in 5 mL of a saturated solution of K2CO3,
2 mL of EtOH, and 5 mL of toluene was purged with argon and stirred at
70 ꢀC overnight. Water (20 mL) was added upon cooling. The aqueous
layer was extracted by EtOAc, and organic extract was dried over MgSO4,
filtered. The solvent was removed under reduced pressure. Crude material
was purified via silica gel chromatography (1.5:1 to 1:3 hexanes/EtOAc) to
give 8 as a colorless oil (308 mg, 57% yield). 1H NMR (400 MHz, CDCl3):
δ 7.83 (m, 1H), 7.79 (m, 1H), 7.58 (m, 3H), 7.52 (m, 1H), 7.43 (m, 2H),
4.71 (s, 2H), 4.21 (m, 4H), 2.42 (s, 1H), 1.31 (m, 6H). 13C NMR (400
MHz, CDCl3): δ 141.15 (1C), 140.78 (1C), 139.12 (1C), 133.11 (1C),
132.98 (1C), 129.36 (1C), 128.90 (1C), 127.40 (2C), 127.16 (2C), 124.95
(1C), 124.73 (1C), 64.90 (1C), 64.83 (1C), 16.31 (1C), 16.26 (1C). ESI -
MS (m/z): calcd for C18H21F2O4P, 370.11; found, 393.20 [M þ Na]þ.
1
(44.4 mg, 52% yield). Analytical HPLC gave 99% purity. H NMR
(400 MHz, DMSO-d6): δ 7.69ꢀ7.73 (m, 2H), 7.56 (m, 2H), 7.49 (m,
2H), 7.36 (m, 2H), 4.70 (s, 2H). HRMS-ESI (m/z): [M þ H]þ calcd for
C14H15NO4F2P, 330.0701; found, 330.0694.
(E)-5-((((30-(Difluoro(phosphono)methyl)-[1,10-biphenyl]-
4-yl)methoxy)imino)methyl)furan-2-carboxylic acid (6e). To
a solution of 5 (8.2 mg, 0.025 mmol) and 5-formylfuran-2-carboxylic
acid (4.2 mg, 0.030 mmol) in 2 mL of DMSO was added AcOH (2.9 μL,
0.050 mmol). The reaction mixture was agitated at room temperature
overnight. Product was purified via HPLC with a retention time of 18.4
min to give a white solid product (7.8 mg, 69%). 1H NMR (400 MHz,
CD3OD): δ 8.07 (s, 1H), 7.77 (s, 1H), 7.67 (d, J = 7.2 Hz, 1H),
7.56ꢀ7.59 (m, 2H), 7.41ꢀ7.52 (m, 5H), 7.22, 7.16 (d, J = 3.6 Hz, 1H),
7.18, 6.76 (d, J = 3.6 Hz, 1H), 5.26, 5.17 (s, 2H). 13C NMR (400 MHz,
CDCl3): δ 159.78 (1C), 150.55 (1C), 147.54 (1C), 145.59 (1C),
140.75 (1C), 139.89 (1C), 139.02 (1C), 136.89 (1C), 135.45 (1C),
128.55 (2C), 128.46 (1C), 128.44 (1C), 126.75 (1C), 126.66 (2C),
124.52 (1C), 118.78 (1C), 112.77 (1C), 76.01 (1C). HRMS-ESI (m/z):
[M ꢀ H]ꢀ calcd for C20H16F2NO7P, 450.0560; found, 450.0562.
’ ASSOCIATED CONTENT
S
Supporting Information. Curves for determination of
b
substrate YopH MichaelisꢀMenten constants (KM); YopH inhibi-
tion curves for oxime-containing inhibitors 6aꢀe; electron density
map for YopH-bound inhibitor 5 and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
†Coordinates and structure factor files have been deposited in the
Protein Data Bank with accession code 2Y2F.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (301) 846-5906. Fax: (301) 846-6033. E-mail: tburke@
helix.nih.gov.
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Journal of Medicinal Chemistry
ARTICLE
’ ACKNOWLEDGMENT
(8) Zhang, Z. Y.; Clemens, J. C.; Schubert, H. L.; Stuckey, J. A.; Fischer,
M. W.; Hume, D. M.; Saper, M. A.; Dixon, J. E. Expression, purification, and
physicochemical characterization of a recombinant Yersinia protein tyrosine
phosphatase. J. Biol. Chem. 1992, 267, 23759–23766.
Appreciation is expressed to Afroz Sultana (LMI) for technical
support and to Joseph Tropea and Scott Cherry (MCL) for
assistance with the production of phosphatases. Electrospray
mass spectrometry experiments were conducted on the LC/
ESMS instrument maintained by the Biophysics Resource in the
Structural Biophysics Laboratory, Center for Cancer Research,
National Cancer Institute at Frederick. X-ray diffraction data
were collected at the Southeast Regional Collaborative Access
Team (SER-CAT) beamline 22-ID at the Advanced Photon
Source, Argonne National Laboratory. Supporting institutions
may be found at http://www.ser-cat.org/members.html. Use of
the Advanced Photon Source was supported by the U.S. Depart-
ment of Energy, Office of Science, Office of Basic Energy
Sciences, under Contract No. W-31-109-Eng-38. This work
was supported in part by the Intramural Research Program of
the NIH, Center for Cancer Research, NCI—Frederick and the
National Cancer Institute, National Institutes of Health, and the
Joint Science and Technology Office of the Department of Defense.
The content of this publication does not necessarily reflect the views
or policies of the Department of Health and Human Services, nor
does mention of trade names, commercial products, or organiza-
tions imply endorsement by the U.S. Government.
(9) McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. A
common mechanism underlying promiscuous inhibitors from virtual
and high-throughput screening. J. Med. Chem. 2002, 45, 1712–1722.
(10) McGovern, S. L.; Helfand, B. T.; Feng, B.; Shoichet, B. K. A specific
mechanism of nonspecific inhibition. J. Med. Chem. 2003, 46, 4265–4272.
(11) Seidler, J.; McGovern, S. L.; Doman, T. N.; Shoichet, B. K.
Identification and prediction of promiscuous aggregating inhibitors
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’ ABBREVIATIONS USED
Y. pestis, Yersinia pestis; YopH, Yersinia pestis outer protein H;PTP,
protein tyrosine phosphatase; PTK, protein tyrosine kinase
pNPP, p-nitrophenylphosphate; PTP1B, protein tyrosine
phosphatase 1B; mPTPB, Mycobacterium protein tyrosine
phosphatase B; TFA, trifluoroacetic acid; F2Pmp, difluoro-
phosphonomethylphenyl; LAR, leukocyte antigen related;
DUSP 14, dual specificity phosphatase 14; DUSP 22, Dual
specificity phosphatase 22; BSA, bovine serum albumin; HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA,
ethylenediaminetetraacetic acid, sodium salt
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’ ADDITIONAL NOTE
‡ A preliminary account of this work has been reported: Bahta
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