A FRET-Based High Throughput Screening Assay to
Identify Inhibitors of Anthrax Protective Antigen Binding
to Capillary Morphogenesis Gene 2 Protein
Michael S. Rogers1, Lorna M. Cryan1, Kaiane A. Habeshian1, Lauren Bazinet1, Thomas P. Caldwell2, P.
Christine Ackroyd2, Kenneth A. Christensen2*
1Department of Surgery, Vascular Biology Program, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America, 2Department
of Chemistry, Clemson University, Clemson, South Carolina, United States of America
Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits
in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-
pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell
surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules
that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe
development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high
throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the
screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay
to screen for inhibitors of this protein–protein interaction is sensitive and robust, with observed Z’ values as high as 0.92.
Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors
of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In
contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-
angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput
screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.
Citation: Rogers MS, Cryan LM, Habeshian KA, Bazinet L, Caldwell TP, et al. (2012) A FRET-Based High Throughput Screening Assay to Identify Inhibitors of
Anthrax Protective Antigen Binding to Capillary Morphogenesis Gene 2 Protein. PLoS ONE 7(6): e39911. doi:10.1371/journal.pone.0039911
Editor: Rory Edward Morty, University of Giessen Lung Center, Germany
Received June 24, 2010; Accepted June 3, 2012; Published June 29, 2012
Copyright: ? 2012 Rogers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health (grants 1R03NS053690-01 to KAC and 1R01EY018829-01 to MSR), and the Department of
Defense (grant W81XWH-08-1-0710 to MSR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Angiogenesis is the process of blood vessel formation that occurs
when new capillaries sprout from pre-existing vessels . It is a
biological process that is normally only seen in the female
reproductive system, in fetal development, and in wound healing
[1–4]. Angiogenesis is required for any process that results in the
accumulation of more than a few microns of new tissue, as well as
many processes involving tissue remodeling. As such, it is a
characteristic of multiple common disease pathologies that involve
inappropriate tissue development , including cancer [6,7],
cardiovascular disease, arthritis, psoriasis, several rare genetic
diseases , and a variety of eye disorders, including macular
degeneration , diabetic retinopathy , herpetic keratitis,
trachoma, and retinopathy of prematurity . Therapies that
target angiogenesis can thus be used to halt or slow the
development of these disorders, and have been shown to be
effective in a variety of diseases [12–15].
We have previously demonstrated that protective antigen (PA),
a non-pathogenic component of the anthrax toxin which binds to
endothelial cell surface receptors, can inhibit angiogenesis .
Treatment with a PA mutant (PASSSR), with three altered amino
acids , increased inhibition of vessel growth in both VEGF-
and bFGF-induced corneal neovascularization assays, inhibited
migration of endothelial cells, and resulted in pronounced ($40%)
reductions in tumor growth . Anthrax toxin binds and co-opts
two endothelial cell surface receptors, anthrax toxin receptor 1
(ANTXR1; also called tumor endothelial marker 8, TEM8) ,
and anthrax toxin receptor 2 (ANTXR2; also called capillary
morphogenesis gene 2 protein, CMG2) . Significantly, PA
mutants that do not bind these receptors do not inhibit
angiogenesis, and the binding affinity of individual PA mutants
for the receptors correlates with their degree of inhibition .
These data strongly suggest that interaction with an anthrax
receptor is responsible for the anti-angiogenic effects of PASSSR.
The normal biological function(s) of TEM8 and CMG2 have
not been fully described, although the existing data indicates that
these receptors are involved in angiogenic processes, consistent
with the observed impact of PASSSRbinding on angiogenesis. Both
receptors contain a von Willebrand A or integrin-like inserted I
domain, with 60% identity in this region, and are the closest
related proteins to integrins, which are involved in cell binding to a
variety of extracellular matrix components. TEM8 was initially
PLoS ONE | www.plosone.org1June 2012 | Volume 7 | Issue 6 | e39911
identified as a protein expressed on colon tumor endothelium, but
not on normal endothelial cells , and was subsequently
detected in a variety of angiogenic or cancerous endothelial cell
types [21,22]. TEM8 knockout mice demonstrate alterations in
extracellular matrix deposition, and changes in the growth rate of
specific tumors . Importantly, TEM8 expression is upregu-
lated in tumor-associated endothelial cells, and receptor expression
is linked to disease progression in several cancer types [22,24,25].
Protein overexpression and gene knockdown experiments demon-
strate that TEM8 is involved in endothelial cell migration and tube
formation  via interactions with the extracellular cellular
matrix component collagen a3(VI) , and linkage to the actin
cytoskeleton . Finally, TEM8-specific antibodies strongly
inhibit the growth of a variety of solid tumors, but have no effect
on either the matrigel plug angiogenesis assay, or on wound
healing, suggesting some tumor specificity in TEM8 expression
. CMG2 is similarly involved in antiangiogenic processes. The
receptor was initially identified as the product of the capillary
morphogenesis gene 2, which is upregulated in endothelial cells
during capillary formation in collagen gels . CMG2 binds both
laminin and collagen type IV , suggesting that like TEM8, this
receptor’s physiological role involves interactions with the
extracellular matrix that are required for angiogenesis. Indeed,
the receptor is highly expressed in both normal and cancerous
vasculature, and its pattern of expression colocalizes with collagen
type IV . Genetic mutations in CMG2 result in the related
disorders juvenile hyaline fibromatosis and infantile systemic
hyalinosis  that are characterized by multiple recurring tumors
and inappropriate deposition of hyalin, an extracellular matrix
material. Like TEM8 knockout mice, female mice which lack the
CMG2 receptor do not give birth, an effect apparently mediated
by defects in uterine extracellular matrix remodeling [33–35].
Importantly, venous endothelial cells that overexpress CMG2
show increased proliferation and formation of capillary-like
networks, while CMG2 knockdown cells demonstrate significantly
impaired endothelial cell proliferation . Together, the TEM8
and CMG2 data suggest that binding of endogenous ligands to
anthrax toxin receptors is involved in angiogenic processes in vivo,
and that inhibition of these interactions by competing ligands
should inhibit vascular growth. Hence, ANTXR-targeted small
molecule angiogenesis inhibitors represent a new strategy for anti-
Inhibition of the receptor-ligand interaction could also be used
as an anthrax therapy. Anthrax intoxication begins when PA binds
to CMG2 or TEM8 on the cell surface, prompting cleavage of PA
by cell-surface proteases , oligomerization to a heptameric
species, complex formation with the pathogenic toxin components,
Lethal Factor (LF) and Edema Factor (EF) , and their
subsequent delivery to the interior of the cell via endocytosis [38–
40]. The low pH of the mature endosome then prompts toxin
rearrangement and translocation of EF and LF to the cytosol .
Since receptor binding is the first step in this process, inhibition of
receptor binding is a plausible treatment for anthrax intoxication,
and small molecules that block the interaction(s) of PA with its
receptors would be effective anthrax toxin inhibitors. Previous
attempts to generate anthrax therapies [42–56] have targeted
several individual steps in the process of anthrax endocytosis, but
only a single study addresses receptor inhibition via receptor
binding . Importantly, unlike other proposed therapies,
receptor-targeted therapies can circumvent the traditional diffi-
culty of testing anthrax inhibitors with active toxin in vivo, because
compounds could be initially tested for their safety and cytotoxicity
in the context of ocular or cancer related angiogenesis.
We have developed a high throughput screening assay to
identify potential inhibitors of the interaction between PA and the
ATR2 (CMG2) receptor. The assay is based on fluorescence or
Fo ¨rster resonance energy transfer (FRET) observed upon interac-
tion of dye-labeled PA and a dye-labeled CMG2 truncation.
Steady-state FRET-based assays like this are ideally suited for high
throughput screening (HTS) methodology because they are
simple, sensitive, and easily automated. When conducted ratiome-
trically, these measurements yield a quantitative readout of the
macromolecular association state that is corrected for experimen-
tal fluctuations occurring between or across well-plates, including
differences in excitation power, pathlength, and photobleaching.
Here we demonstrate a ratiometric steady-state FRET-based
screening assay that is highly effective and capable of identifying
potential PA-CMG2 inhibitors. The anti-angiogenic effects of
compounds identified from preliminary screens of small molecule
libraries are characterized and described.
FRET screening assay design
FRET is the highly distant-dependent through-space transfer of
energy from a fluorescent donor to an acceptor molecule. FRET
between dye-labeled molecules occurs in cases when the donor
dye’s fluorescence emission spectrum overlaps with an acceptor
dye’s excitation spectrum. In this case, when the fluorescence of
the donor molecule is excited directly, a portion of that excitation
energy can be funneled to the acceptor molecule instead of being
followed solely by emission at the characteristic emission
wavelength of the donor dye. What is observed, instead, is
decreased emission of the donor, accompanied by increased (or
sensitized) emission for the second, acceptor, dye. Importantly,
such energy transfer can only occur when the two dyes are in close
spatial proximity (typically less than 100 A˚). Hence, FRET is a
sensitive probe of macromolecular association. FRET has been
used previously to measure the kinetics and stoichiometry of PA
binding to a soluble truncation of the extracellular domain of
CMG2 . Here we describe an adaptation of this assay for high
Using directly labeled protein reagents in a simple homoge-
neous assay format, we have developed a nearly ideal high
throughput FRET screening assay to identify small molecules and
natural product extracts that inhibit the PA-CMG2 protein-
protein interaction. As with other FRET assays, the screening
assay uses interactions between fluorescent labels on the two
proteins to report binding. We expressed, purified, and covalently
labeled a single cysteine PA mutant, PAE733C, with an Alexa Fluor
488 label (AF488; lex=488 nm; lem=525 nm). This labeled PA
was designed to be the FRET donor. A truncated soluble version
of CMG2 consisting of amino acid residues 40–217 containing the
mutations R40C and C178A (CMG2R40C
expressed, purified, and covalently labeled with Alexa Fluor 546
(AF546; lex=546 nm; lem=570 nm). This labeled CMG2 was
designed to be the FRET acceptor. To show energy transfer
between dye-labeled PA and CMG2, we made an equimolar
mixture (10 nM) of the two labeled proteins that was excited at
485 nm, which predominantly excites PAE733C*AF488, the donor
molecule. Specific spectral changes relative to free protein were
observed under these conditions. Figure 1A shows the emission
spectrum of 10 nM PAE733C*AF488alone (solid line), overlaid with
the emission spectrum of an equimolar mixture of the PAE733-
C*AF488and CMG2R40C*AF546(dashed line). A clear decrease in
PAE733C*AF488emission intensity was observed at , 525 nm
(donor quenching) for the mixture, together with enhancement of
) was also
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the emission intensity of the CMG2R40C*AF546acceptor dye at
,570 nm (sensitized emission). These significant spectral changes
strongly suggest energy transfer between the dyes, reflecting PA-
CMG2 binding. Rather than simply using either the decreased
donor emission or the increased acceptor emission to assess the
degree of binding, we have used a ratio of their values (i.e. Iacceptor
emission/Idonor emission =IAF546/IAF488) to gauge binding; the ratio
corrects for instrumental fluctuations and helps minimize possible
systematic errors in the fluorescence measurements.
Prior to screening, we were concerned about potential
photoluminescence or autofluorescence of compounds in the
screening library. Hence, we also tested the protein reagents
labeled with an alternate red-shifted dye-pair, in case we observed
competing luminescence from significant numbers of library
compounds using 485 nm excitation. Similar to the results for
the AF488/AF546 dye-pair labeled proteins, we observed
significant energy transfer in the observed fluorescence emission
spectra when 10 nM PAE733C*AF568and CMG2R40C*AF647were
mixed (Figure 1B). Again, we observed a clear decrease in intensity
of AF568 dye-labeled PA donor emission intensity at , 605 nm
(donor quenching) and enhancement of the intensity of AF647
dye-labeled CMG2 acceptor at ,675 nm (sensitized emission) was
observed. Slightly less energy transfer was observed for AF568/
AF647 dye-pair relative to the original AF488/AF546 dye-pair,
presumably due to differences in the relative Fo ¨rster distances for
the two dye pairs. Only a small percentage of the tested library
compounds had sufficient luminescence to interfere with assay
performance in the pilot screens reported here; therefore, we used
the slightly better performing AF488/AF546 dye pair in all
subsequent screening experiments. We expect that this alternate
dye pair could be used to achieve excellent assay performance in
conditions where compounds have significant short wavelength
The goal of our high throughput screen was to identify
compounds that inhibit the PA-CMG2 interaction. Because the
observed FRET is sensitive to the PA-CMG2 interaction, it is
dramatically reduced in the presence of compounds that inhibit
the interaction. For example, addition of EDTA, which shifts the
Kdby orders of magnitude , causes a two-fold reduction in the
fluorescence emission ratio (R=IAF488-PA/IAF546-CMG2;/R=I590
nm/I535 nm), reflecting substantially reduced PA-CMG2 binding
under the conditions of the validation assay (see below). This
reduction in FRET ratio in the presence of inhibitors became the
basis for the subsequent high throughput assay. Using the AF488/
AF546 dye pair described above, we measured the FRET ratio for
PAE733C*AF488and CMG2R40C*AF546in the presence of possible
inhibitors, and compared that value to that for labeled PA and
CMG2 alone. Significant reductions in observed FRET ratio were
then used to infer inhibition of the PA-CMG2 interaction by
individual library compounds.
More specifically, the high throughput screening assay measures
the FRET between CMG2 and PA in 384 well-plates in the
presence of potential inhibitors. Aliquots of CMG2 were added to
individual wells, followed by addition of small volumes of test
compound, incubation to allow possible interaction of the test
compound with CMG2, and addition of an aliquot of PA. After
further incubation, the fluorescence intensities at 535 nm (AF488-
labeled PA donor) and 595 nm (AF546-labeled CMG2 acceptor)
were read for each well. The ratio of these values (F595/F535) was
calculated for each well and compared to the corresponding ratio
for the negative control (no added compound) and positive control
(EDTA added with the CMG2 solution) on each well-plate. To
correct for possible day to day or plate to plate variation, a value
for the fraction inhibition could also be calculated relative to the
controls on each plate (Inorm=(Robs-Rneg)/(Rpos-Rneg); this value
reflects a normalized value for inhibition of the PA-CMG2
interaction. In this case, zero normalized inhibition reflects no
inhibition (equal to the negative control) and a normalized
inhibition value of 1 reflects complete inhibition (equal to the
EDTA positive control). To minimize use of library compounds
and the possibility of false positives, potential inhibitors were tested
in duplicate (i.e. were added to each of two wells). To determine
whether individual compounds qualified as ‘‘hits’’, the observed
FRET ratios (F595/F535) were averaged for all wells without
EDTA in a given plate, and the standard deviation of this mean
was calculated. Based on this standard deviation (sF595/F535), an
arbitrary cutoff FRET ratio was assigned that was three standard
deviations (i.e. 3*sF595/F535) lower than the negative control. When
both wells of an individual compound had FRET ratios below this
cutoff, the compound was assigned as a ‘‘hit’’. This cutoff
assignment could also be performed using a normalized fraction
inhibition rather than absolute FRET ratios.
High throughput screen optimization and validation
Within the context of the high throughput screen described
above, it was necessary to adjust experimental conditions to
optimize assay performance. The overall performance of a
screening assay is related to multiple parameters that include
stability, sensitivity, reproducibility, robustness, and well-to-well
variation across the screening plate. Most of these parameters
affect the observed Z’ value, which is calculated from the standard
deviation of the positive and negative control wells. As a result,
the Z’ value can be used to assess assay performance, based on
measurements of the positive and negative controls alone. As
described above, the negative control was assigned as the observed
FRET ratio in the absence of any inhibitor, while the positive
control was assigned as the observed FRET ratio in the presence of
EDTA, a potent inhibitor of CMG2 binding. Generally, a Z’.0.5
is the minimum acceptable value for an interpretable screening
assay while Z’.0.7 is considered a good result. Higher Z’-values
are even better. During the optimization and validation phases of
development, assay solution and acquisition parameters were
adjusted to optimize Z’.
First, the concentration of the labeled proteins was adjusted to
minimize reagent consumption and any inner-filter effect(s), while
Figure 1. Fluorescence emission spectra of labeled protein
reagents exhibit resonance energy transfer. A) Fluorescence
emission spectra of 10 nM PAE733C*AF488(solid line; donor alone) and
10 nM PAE733C*AF488+10 nM CMG2R40C*AF546(dashed line; donor +
acceptor) in HBST. Both spectra were acquired using 485 nm excitation.
B) 10 nM PAE733C*AF568(solid line; donor alone) and 10 nM PAE733C*AF568
+10 nM CMG2R40C*AF647(dashed line; donor + acceptor) in HBST. Both
spectra were acquired using 556 nm excitation.
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maximizing signal intensity. High signal-to-noise measurements
minimize instrument noise and decrease standard deviations to
improve Z’. The optimal concentrations were found to be 7.5 nM
PAE733C*AF488and 13 nM CMG2R40C*AF546. Given the high
affinity of the PA-CMG2 interactions (Kd$170 pM ), these
concentrations are consistent with quantitative binding of CMG2
to PA in the absence of inhibitor.
We monitored Z’ as a function of PA-CMG2 equilibration time
to ensure that the equilibration time was sufficient to achieve
optimal assay performance. These experiments revealed that
initial experimental conditions resulted in a time-dependent
decrease in Z’, as well as Z’ values below acceptable limits (data
not shown). We suspected that since we were using very dilute
protein solutions in the assay, non-specific binding of the labeled
protein reagents onto the well-plate was the cause of the observed
decrease in Z’. Consistent with this hypothesis, a stable Z’ was
observed when the experiment was repeated with the addition of
0.1 mg/ml BSA or 0.075% (v/v) Tween-20 to the CMG2 solution.
Addition of Tween-20 may have an additional beneficial effect,
since aggregating nonspecific compounds can create false ‘‘hits’’
during screening that can be ameliorated by addition of a
detergent . As a result, Tween-20 was included in all
subsequent experiments. Figure 2 shows Z’ as function of PA-
CMG2 equilibration time in the presence of Tween-20. These
data indicate that optimal assay performance is achieved with a 2–
4 hour incubation, presumably reflecting complete binding of PA
to CMG2 in this time frame. This observation is consistent with
the published association kinetics in this system (ka =1.1 x 105
M21s21), which predict complete binding of CMG2 by PA
within this time frame at these protein concentrations. PA was
incubated with CMG2 for 4 hours prior to fluorescence measure-
ment in all subsequent validation and pilot screening assays.
As part of our analysis of the effects of incubation time on assay
performance, we also tested the use of different well-plate materials
in the presence of Tween-20. We observed different assay
performance in polystyrene, polypropylene, and specialized
polypropylene plates designed to reduce nonspecific binding to
well surfaces (Figure 2). These data indicate that the material
composition of the well-plates affects assay performance, even in
the presence of Tween-20. Although the specialized low-binding
polypropylene plates produced the highest Z’ value, their higher
cost was prohibitive in our situation, and all subsequent screens
were carried out with polystyrene well-plates, the next best
As library compounds were to be added in DMSO solutions, we
also evaluated the effect of DMSO on assay performance. While
addition of 10% of the total assay volume of DMSO had a
significant but small effect on the assay (10% reduction in Z’), a
1% DMSO addition had no measureable effect on Z’, and was
used in the subsequent pilot and library screenings.
These data were used to develop an optimized screening assay
protocol (see Materials and Methods). Specifically, a CMG2
solution containing Tween-20 was added to individual wells of
polystyrene well-plates, followed by pin transfer of small volumes
of library compounds in volumes sufficiently small to keep total
DMSO concentrations low (,1% v/v) and an equilibration
designed to allow interaction of potential inhibitors with CMG2
prior to addition of PA. Addition of PA solution to each well was
followed by a 4 hour incubation of PA with CMG2. Delivered
concentrations of CMG2 and PA were chosen to ensure that their
final concentrations in solution reached optimal levels. Negative
controls (no added library compound) and positive controls
(EDTA added to the CMG2 solution to prevent PA binding)
were present on each plate. Comparison of measured FRET ratios
for inhibitor and control wells was then used to assess possible
inhibition of PA-CMG2 binding by library compounds.
These optimized conditions were used for validation of the PA-
CMG2 screening assay. In this validation assay, half of the wells in
each plate were assigned to positive control conditions, while the
other half were negative controls. These data are shown in
Figure 3. Two well-separated tight clusters of data are observed;
one for the positive control, and one for the negative control.
These same data are shown in a histogram format in Figure 3B,
expressed as a ratio of the fluorescence intensities. These data
demonstrate that binding and inhibition are extremely well
separated in our screen, a result which promotes sensitive
detection of binding inhibition. The validation assay results reflect
outstanding assay performance, with measured Z’ values consis-
tently at or above 0.9.
Figure 2. Time course of measured Z’. Z’ was measured over time
in polystyrene (N), polypropylene (.), and commercially available low-
binding (#) 384 well-plates. All wells contained 7.5 nM PAE733C*AF488
and 13 nM CMG2R40C*AF546in HBST plus either 5 mM NaCl (negative
control; 168 wells) or 5 mM EDTA (positive control; inhibitor; 168 wells).
Figure 3. PA-CMG2 high throughput screening assay perfor-
mance. A polystyrene 384 well-plate filled with 7.5 nM PAE733C*AF488
and 13 nM CMG2R40C*AF546in HBST plus either 5 mM NaCl (negative
control; 168 wells) or 5 mM EDTA (positive control; inhibitor; 168 wells).
A) Scatter plot of donor (lem=535 nm) and acceptor (lem=595 nm)
emission showing the shift induced by addition of EDTA (positive
inhibitor control). B) Histogram of the F595 nm/F535 nmratio taken from
the same data. For these data Z’=0.91.
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Pilot screening with the PA-CMG2 assay successfully
identified CMG2 inhibitors with anti-angiogenic
Using the optimized and validated assay, we screened a small
library of 2,640 known bioactive small molecules. The library was
selected for structural diversity and includes many classes of
compounds, including ion channel blockers, GPCR ligands,
second messenger modulators, nuclear receptor ligands, actin
and tubulin ligands, kinase inhibitors, protease inhibitors, gene
regulation agents, lipid biosynthesis inhibitors, as well as other
well-characterized compounds that perturb cell pathways. Ap-
proximately 50% of FDA-approved drugs are included in the
library, including a significant subset of substances known to
influence brain activity. Hence, the library was not biased towards
anti-angiogenic or anti-cancer activity. Figure 4 shows data from
the pilot screen of this library. Only two of the compound solutions
tested, cisplatin and tannic acid, had a F595 nm/F535 nm ratio
greater than 3 standard deviations from the plate mean for both of
the duplicate wells, indicating significant and repeatable inhibition
of the PA-CMG2 interaction. Both solutions were analyzed with
respect to inhibition of PA-CMG2 binding, disruption of
angiogenesis, and interaction with CMG2.
As shown in Figure 5A, cisplatin inhibits the PA-CMG2
interaction with an IC50of 34 mM, measured using the FRET
assay; the IC50curve had a Hill coefficient indistinguishable from
one, indicating that inhibition likely occurs by formation of a 1:1
complex. Cisplatin also had statistically significant anti-angiogenic
effects. Cisplatin inhibits cell migration in a dose-dependent
manner (Figure 6A), and has a modest but statistically significant
effect on corneal vessel growth (Figure 6C). However, since
cisplatin is a known DNA cross-linker with established cytotoxicity
, observed anti-angiogenic effects could result from mecha-
nisms unrelated to CMG2 binding. Indeed, cell proliferation
experiments designed to investigate cisplatin cytotoxicity showed
statistically significant reductions in cell proliferation following
long incubation with cisplatin (Figure 6B), suggesting the
possibility that cytotoxicity could contribute to cisplatin’s inhibi-
tion of cell migration and corneal vessel growth.
We carried out follow-up binding studies to provide support for
direct interaction of cisplatin with CMG2. We could not detect
cisplatin interaction with CMG2 by surface plasmon resonance
(SPR) (Figures 7A and B). However, since cisplatin is a relatively
small molecule (MW 300), and SPR detects intermolecular
association via mass increases, a negative SPR result under these
conditions does not completely rule out cisplatin binding. As a
result, we also conducted a kinetic assay to investigate interaction
of cisplatin with CMG2. In these experiments, labeled CMG2 was
first preincubated with cisplatin at concentrations likely to result in
significant interaction (i.e.10X the IC50measured by FRET), then
rapidly diluted with a solution of labeled PA to cisplatin
concentrations at least 10-fold below the measured IC50. Under
these conditions, any preformed cisplatin-CMG2 complex must
dissociate before the FRET positive PA-CMG2 complex can form.
As a result, reductions in the observed association rate of CMG2-
PA in the presence of cisplatin provides evidence for interaction of
cisplatin with CMG2. As shown in Figure 8, preincubation with
cisplatin had a profound effect on CMG2-PA binding, indicating
that the compound interacts with CMG2. However, similar kinetic
experiments also indicate interaction of cisplatin with PA;
preincubation of cisplatin with labeled PA before addition of
excess labeled CMG2 resulted in nearly identical disruption of
CMG2-PA binding. Hence, cisplatin is a nonspecific inhibitor of
PA-CMG2 binding that interacts with both CMG2 and PA.
Indeed, interaction of cisplatin with PA has been previously
reported . The inhibition appears irreversible for both
proteins, as evidenced by the failure of both samples to show
any association with an excess of the alternate protein after
incubation with cisplatin (Figure 8). As cisplatin is a known cross-
linker, it is possible that cisplatin inhibits the PA-CMG2
interaction via formation of covalent adducts. Thus, while cisplatin
is not a viable therapeutic lead compound, its isolation demon-
strates that the FRET high throughput screen was effective in
identifying an effective inhibitor of PA-CMG2 interaction.
We also characterized the binding and anti-angiogenic proper-
ties of the tannic acid solution. We used the FRET assay to assess
Figure 4. Results from screening a small library of known
bioactives. Scatter plot of screening data for a small library of known
bioactive small molecules using the PA-CMG2 screening assay. The pink
dashed line shows the arbitrary cutoff representing inhibition greater
than 3 standard deviations from the plate mean; a blue dashed line
showing 5 standard deviations from the plate mean has also been
drawn for comparison. Arrows point to compounds with inhibition
values larger than the cutoff for both wells (red lines connect duplicate
wells). Compounds that cluster in the top right hand corner have
significant compound autofluorescence and were not considered
Figure 5. IC50 of cisplatin and tannic acid for PA-CMG2
interaction. A) IC50was measured for cisplatin using the PA-CMG2
screening assay. Data were fit to the single site binding isotherm; B) IC50
was measured for tannic acid using the PA-CMG2 screening assay. Data
could not be fit to any binding model. Ratios presented here are
calculated differently than presented elsewhere in the paper (Robs
=F535nm/F595nm) in order to present a binding curve with standard
appearance, and are normalized against the positive and negative
control wells on each plate (Rnorm=(Robs-Rneg)/(Rpos-Rneg)).
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IC50of tannic acid on the PA-CMG2 interaction. As shown in
Figure 5B, the tannic acid IC50curve was complex, suggesting
multiple binding modes or the presence of impurities with a range
of binding affinities. Due to the complex binding isotherm, we
could not determine an IC50. However, these binding data
indicate that there are concentrations of tannic acid that clearly
inhibit PA-CMG2 interaction. Hence, isolation of tannic acid in
the high throughput screen was not a false positive. Binding of the
tannic acid solution to CMG2 was corroborated by SPR. As
shown in Figures 7C and D, addition of aliquots of the tannic acid
solution to CMG2 result in clear changes in SPR behavior relative
to CMG2, versus control sensor surface. These data were not
collected under conditions that allowed quantitative determination
of binding affinity; however, the data indicate significant
interaction of CMG2 with tannic acid or other solution
components at a total concentration of 1 mM, suggesting
dissociation constants similar to or lower than this value.
Figure 6. In vitro and in vivo effects of identified PA-CMG2 inhibitors. A) The effect of various concentrations of cisplatin or tannic acid on
endothelial cell migration toward serum-containing medium; effects of PA administration are shown for comparison. Error bars represent standard
deviation of the mean (SD; n=12, 4 10X fields from 3 membranes). Statistically significant differences between inhibitor-containing and inhibitor-free
conditions (as determined by the Holm–Bonferroni method) are shown by asterisks (*) preceding the concentration on the horizontal axis. Each of
these experiments was repeated at least twice with concordant results. B) The effect of various concentrations of cisplatin and tannic acid on cell
survival and proliferation. Proliferation assays were performed for 24 (dark bars) and 72 hours (light bars) in the indicated concentration of cisplatin
and tannic acid and cell numbers assessed using Cyquant and normalized to controls fixed in ethanol at time 0. At 24 hours, the highest
concentration of each molecule resulted in statistically significant decreases (as determined by t-test); however following Bonferroni correction, these
differences were not significant. At 72 hours all cisplatin doses resulted in statistically significant decreases in cell number (as determined by the
Holm–Bonferroni method). Error bars represent SD, n=3 wells. For DMSO, 7 concentrations from 0.001% to 1% were assessed and no significant
difference was observed among conditions. Therefore these data were pooled and thus for this sample n=21 wells. No significant difference
between DMSO-containing and media-only samples was observed (t-test). C) Effect of treatment with 4 mg/kg/day cisplatin on vessel area in the
corneal micropocket assay. Control mice were treated with vehicle alone. Error bars represent SEM (n=10 eyes). The observed difference is
statistically significant (p,0.03 by t-test). No animal weight loss with cisplatin administration was observed. D) Representative image of control eye
vessel area. E) Representative image of cisplatin treated mouse eye vessel area.
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PLoS ONE | www.plosone.org6 June 2012 | Volume 7 | Issue 6 | e39911
We assessed the anti-angiogenic effects of this tannic acid
solution using the endothelial cell migration assay (Figure 6A) and
observed statistically significant inhibition. This inhibition did not
appear to reflect cytotoxicity, as no statistically significant effect on
cell proliferation was observed over the range of concentrations
that inhibited cell migration (Figure 6B). Hence, tannic acid, or
impurities in this tannic acid solution, both bind CMG2 and
inhibit angiogenesis. We also measured in vivo anti-angiogenic
effects of tannic acid using the corneal pocket assay. While
inhibition of corneal vessel growth was observed (data not shown),
solution concentrations that inhibited corneal angiogenesis also
resulted in animal weight loss, indicating that the observed
reduction in angiogenesis is likely a result of compound toxicity,
rather than a specific effect. Thus, we do not cite this data as
supportive of tannic acid’s ability to inhibit angiogenesis.
We have developed a high throughput screening assay to
identify possible inhibitors of the interaction between PA and the
CMG2 (ATR2) receptor. CMG2 has at least ten-fold higher
affinity for PA than does TEM8 , is expressed alongside
TEM8 in tumor cells, and has four-fold higher expression in
endothelial cells  than is TEM8. Hence, while both TEM8
and CMG2 are of potential interest as targets for anti-angiogenic
therapies, our first screens focused on CMG2. Data described here
outlines the development and validation of a sensitive mix and
measure homogeneous high throughput screening assay for
inhibitors of PA-CMG2 interactions, based on FRET between
dye-labeled proteins. Assay performance was characterized based
on measured Z’ values. A consistently high Z’ value (Z’$0.9) was
observed for this high throughput screening assay, indicating
exceptional performance. This assay is therefore highly sensitive to
compounds that inhibit PA-CMG2 interactions and could be used
to effectively generate lead compounds for cancer, other angio-
genesis related diseases, and anthrax toxin therapies. In addition,
small molecule inhibitors identified by this assay could potentially
be used to investigate both the biological function of CMG2 and
the molecular recognition processes operating at the cell surface.
Initial screening of a relatively small library resulted in
identification of two compounds as inhibitors of the PA-CMG2
interaction. The first compound hit, cisplatin, has an established
history as an anti-cancer agent and a wide range of documented
physiological effects, largely mediated by DNA adduct formation.
However, secondary binding analysis of cisplatin indicates that this
compound inhibits PA-CMG2 binding by interaction with PA, as
well as CMG2, and any observed anti-angiogenic effects caused by
cisplatin administration may reflect mechanisms other than
CMG2 binding, including formation of possible DNA adducts.
The second hit, tannic acid, both binds CMG2 and inhibits
angiogenesis as measured by endothelial cell migration. Tannic
acid has multiple previously reported bioactivities including anti-
angiogenic properties such as inhibition of cell migration and
angiogenesis  and anti-cancer action in a variety of cancer cell
 and tumor types [65–68]. Our identification of this tannic
acid solution as a PA-CMG2 inhibitor suggests that one
mechanism of tannic acid bioactivity may involve interactions
with CMG2 that impact angiogenesis.
We cannot unequivocally conclude that the inhibitor isolated in
this screen disrupts angiogenesis exclusively by interacting with
CMG2. For example, we cannot rule out an anti-angiogenic effect
exerted through a non-CMG2-mediated pathway, although its
observed angiogenic activity is consistent with inhibition of
CMG2. In addition, it is possible that the observed effects on
angiogenesis result from cross-reactivity with TEM8. Both
receptors are present in the corneal micropocket and endothelial
cell migration assays, and given the relatively high sequence
homology of TEM8 with CMG2, we suspect that some overlap in
Figure 7. SPR analysis of cisplatin and tannic acid binding
immobilized CMG2. A) SPR sensorgram using a streptavidin-
modified carboxydextran gold sensorchip where biotinylated CMG2
was immobilized in channel 2 (blue trace) and biotin-PEG was
immobilized in channel 1 (black trace) as a control. A solution of
500 mM cisplatin in HBST was flowed over the sensor surface. Three
independent injections are recorded. B) A difference sensorgram is
shown based on the data in Figure 7A. C) SPR sensorgram where
biotinylated CMG2 was immobilized in channel 2 (green trace) and
biotin-PEG was immobilized in channel 1 (black trace) as a control. A
solution of 1 mM tannic acid in HBST was flowed over the sensor
surface. Three independent injections are shown. D) A difference
sensorgram is shown based on the data in Figure 7C.
Figure 8. FRET kinetic assay for cisplatin binding to CMG2 and
PA. Fluorescently labeled CMG2R40.C C178A*AF546(1 mM) was preincu-
bated with vehicle alone (DMSO) for 30 minutes before being rapidly
diluted into PAE733C*AF488to a final concentration of 10 nM of both
proteins and the acceptor/donor fluorescence ratio was recorded over
time (black line). Similarly, fluorescently labeled CMG2 was also
preincubated with 500 mM cisplatin for 30 minutes before being rapidly
diluted into PA as in the vehicle control. Fluorescence was again
recorded over time (solid blue line). Fluorescent PA (1 mM) was also
preincubated with 500 mM cisplatin for 30 minutes before being rapidly
diluted into a solution of labeled CMG2. Fluorescence was recorded
over time (dashed blue line). Excitation was 485 nm.
HTS Assay for CMG2 Antagonists
PLoS ONE | www.plosone.org7June 2012 | Volume 7 | Issue 6 | e39911
recognition is likely to occur. We are currently involved in
additional high throughput screens to identify inhibitors of PA-
TEM8 binding, and are interested to correlate results from these
Given its large range of bioactivities, tannic acid may not be a
viable therapeutic lead. However, its identification in this screen is
proof of principle that a simple, sensitive, and robust high
throughput screening assay can identify compounds that inhibit
the PA-CMG2 interaction and have potential anti-angiogenic
properties. We anticipate that these observations will lay the
groundwork for additional high throughput screens for PA-CMG2
inhibitors based on substantially larger libraries.
Materials and Methods
Protein expression, purification, and labeling
cloned, expressed in BL21 DE3 Star E. coli (Invitrogen), and
purified using combinations of ion exchange (HP Q-Sepharose;
GE Healthcare), affinity (GST Bind Agarose; Novagen), and size
exclusion chromatography (Sephacryl 200HR; GE Healthcare)
similar to those methods previously reported . Protein purity
was determined to be $85% by SDS-PAGE with Coomassie
staining. These single cysteine mutants were labeled with either
Alexa fluor 488 C5maleimide, Alexa fluor 546 C5maleimide,
Alexa fluor 568 C5maleimide, Alexa fluor 647 C2maleimide
(Invitrogen), or maleimide-PEG2-biotin (Pierce) using manufac-
turer recommended methods. The dye: protein ratios of all protein
conjugates were determined by UV-VIS spectrophotometry or the
HABA assay for biotin to be between 0.85 and 1.15. Protein
activity for the FRET assay reagents was assessed using
fluorescence spectroscopy to measure resonance energy transfer
upon PA binding CMG2 in vitro.
C178Asite-directed mutants were
Fluorescence spectra and inhibitor IC50
All fluorescence spectra were acquired using a spectrofluorom-
eter (QM-4; Photon Technology International) with a 75W Xe arc
lamp excitation and photon counting photomultiplier detection.
Slits for both the excitation and emission monochromators were
set to achieve a 4 nm band pass. Briefly, fluorescence spectra
10 nM solutions of either PAE733C*AF488or mixtures of PAE733-
C*AF488and CMG2R40C C178A*AF546in HEPES buffered saline +
Tween-20 (HBST; 50 mM HEPES pH 7.4, 150 mM NaCl,
0.1 mM CaCl2, 0.1% Tween-20). Fluorescence emission spectra
of the red-shifted versions of PA and CMG2 were measured in a
IC50 for both tannic acid and cisplatin was determined by
addition of serial dilutions of the inhibitors in DMSO to a solution
of CMG2R40C C178A*AF546in HBST followed by addition of
PAE733C*AF488to a final concentration similar to that used in the
screening assay (13 nM CMG2R40C C178A*AF546, 7.5 nM PAE733-
C*AF488) in 96-well-plates. Plates were incubated for 4 hours and
read on a Genios (Tecan) plate reader with 485/10 nm excitation
filter and 535/13 nm and 585/11 nm emission filters. The
F585 nm/F535 nm fluorescence emission ratio was measured
and plotted as a function of final inhibitor concentration. The
cisplatin binding isotherm was fit to a single-site binding model
using SigmaPlot (Systat).
High throughput screening
For high throughput screening, 30 ml of a solution of 17 nM
CMG2R40C C178A*AF546in HBST was added to the wells of a
barcode labeled Corning 3710 384-well-plate using a WellMate
liquid handling robot (Matrix Technologies) with integrated
stacker. Next, 0.3 ml of test compound (5 – 10 mg/mL) diluted
in DMSO was added by pin transfer using a custom Epson robot
to duplicate plates. Following a 1–3 hour incubation, 10 ml of a
30 nM PAE733C*AF488solution in 50 mM HEPES pH 7.4,
150 mM NaCl, 0.1 mM CaCl2was then added to all wells using
the Wellmate and plates were incubated for 3–4 hours. Final
CMG2 concentration (13 nM) and PA concentration (7.5 nM)
were sufficient to promote quantitative binding of CMG2 in the
absence of effective inhibitors, based on the previously reported Kd
($170 pM) . Incubation lengths varied between individual
wells, as a function of the time required for delivery of library
compounds to individual positions in the well-plate. Following
incubation, plates were read on an Envision (PerkinElmer) plate
reader using a 485/14 excitation filter, with 535/25 and 595/60
emission filters incorporating a barcode reader to correlate
fluorescence measurements with plates. For each plate, 32 positive
control wells were generated by adding 10mM EDTA to the
CMG2 solution; 32 negative control wells were generated by
addition of 10 mM NaCl to the CMG2 solution. Control wells did
not receive addition of library compound(s).
Endothelial cell migration assay
Human microvascular endothelial cells (Lonza) were main-
tained in EGM-2 media (Lonza) according to the vendor’s
instructions and used before passage 7. Polycarbonate transwell
inserts, 6.5 mm diameter with 8.0 mm pores (Corning), were
coated with 20mg/ml fibronectin (Sigma) overnight at 4uC. Cells
were harvested and resuspended in EBM media (Lonza) contain-
ing 0.1% bovine serum albumin (Sigma). Cells (10,000 per well)
were plated onto wells and placed within wells containing full
serum EGM-2 medium alone or EGM-2 medium containing the
molecule to be tested. Cells were allowed to migrate for 4 h with
5% CO2at 37uC. Membranes were then rinsed once in PBS, and
fixed and processed using Diff-Quick (Dade Diagnostics). Cells on
the top of the membrane were removed using cotton-tipped
applicators. Membranes were removed from the insert using a
scalpel and mounted on slides, and the number of cells in 4 106
microscopic fields were counted.
Endothelial cell proliferation assay
Human microvascular endothelial cells (HMVECs; Cambrex)
were maintained in EGM-2 (Cambrex) according to the manu-
facturer’s instructions, and used before passage 7. Proliferating
cultures of cells were seeded at ,10% confluence into 96 well
plates. After attachment, medium was exchanged into that
containing the designated concentration of inhibitor (Cambrex).
Cells were allowed to grow for 24 and 72 h and then quantified
using CyQUANT (Invitrogen) according to the manufacturer’s
protocols. The degree of proliferation in culture was measured by
comparison of experimental wells with those fixed in absolute
ethanol at t=0.
Mouse corneal micropocket assay
The corneal micropocket assay was performed as previously
described , using pellets containing 80 ng of basic fibroblast
growth factor (bFGF) in C57BL/6J mice. The treated groups
received i.p. injections of compound in PBS. Treatment was
started on the day after pellet implantation; control mice received
vehicle alone i.p. The area of vascular response was assessed on
the 5th postoperative day using a slit lamp. Typically, 10 eyes per
group were measured.
HTS Assay for CMG2 Antagonists
PLoS ONE | www.plosone.org8June 2012 | Volume 7 | Issue 6 | e39911
Surface plasmon resonance binding assay
Surface plasmon resonance (SPR) was used to determine
binding of cisplatin and tannic acid to CMG2. CMG2R40C
R178Awas labeled with biotin-PEG-maleimide (Pierce) for immo-
bilization to a streptavidin-modified carboxydextran SPR sensor
surface (SA; GE Healthcare). All experiments were performed
using a Biacore X (GE Healthcare). Biotinylated CMG2 (100 nM
in HBST) was flowed across channel 2 of the on the SA chip for
5 minutes at of 5 ml/min followed by a HBST wash. As a control,
PEG-biotin was immobilized in channel 1 of the sensor chip under
identical conditions. Solutions of tannic acid (1 mM) or cisplatin
(500 mM) in HBST were flowed across the functionalized sensor
chip at 10 ml/min and the sensorgrams recorded. Sensorgrams
were recorded in triplicate.
FRET kinetic assay
A FRET-based kinetic assay was used to monitor the rate of PA-
CMG2 binding in the presence of cisplatin. First, fluorescently
labeled PAE733C*AF488(1 mM) or CMG2R40C C178A*AF546(1 mM)
were preincubated with 500 mM cisplatin in HBST for 30 min-
utes. This mixture was then rapidly diluted to a final concentration
of 10 nM of each fluorescently labeled binding partner (CMG2 or
PA respectively) in HBST and the donor and sensitized emission
ratio was monitored over time. Control experiments consisted of
preincubation with the DMSO vehicle alone.
Ethical treatment of animals
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. All
animal studies were conducted according to protocols approved by
the Institutional Animal Care and Use Committee of Children’s
Hospital Boston (Protocol A06-10-086R). All surgery was
performed under avertin anesthesia.
The author’s thank the ICCB-Longwood screening facility for access to
their libraries, equipment, screening supplies, and technical advice. The
author’s also thank Junhong He for her help with IC50measurements of
cisplatin and tannic acid and Dr. Yang Lin, Dr. Guzeliya Kornev and Dr.
Robert Latour for help with SPR experiments and use of their Biacore X.
Conceived and designed the experiments: MSR LMC PCA KAC.
Performed the experiments: MSR LMC KAH LB TPC. Analyzed the
data: MSR LMC LB TPC. Wrote the paper: MSR LMC PCA KAC.
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HTS Assay for CMG2 Antagonists
PLoS ONE | www.plosone.org10 June 2012 | Volume 7 | Issue 6 | e39911