Discovery of Mer specific tyrosine kinase inhibitors for the treatment and prevention of thrombosis

Article abstract of DOI:10.1021/jm4013888

The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC₅₀ value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.

Full text of DOI:10.1021/jm4013888

Article
pubs.acs.org/jmc
Discovery of Mer Specific Tyrosine Kinase Inhibitors for the
Treatment and Prevention of Thrombosis
Weihe Zhang,†,‡,# Andrew L. McIver,†,‡,# Michael A. Stashko,†,‡ Deborah DeRyckere,⊥
Brian R. Branchford,⊥ Debra Hunter,∥ Dmitri Kireev,†,‡ Michael J. Miley,§ Jacqueline Norris-Drouin,†,‡
Wendy M. Stewart,†,‡ Minjung Lee,⊥ Susan Sather,⊥ Yingqiu Zhou,‡ Jorge A. Di Paola,⊥ Mischa Machius,§
William P. Janzen,†,‡ H. Shelton Earp,∥,§ Douglas K. Graham,⊥ Stephen V. Frye,†,‡,∥
and Xiaodong Wang*,†,‡
‡
†Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry, Eshelman
School of Pharmacy §Department of Pharmacology ∥Lineberger Comprehensive Cancer Center, Department of Medicine, School of
Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
⊥Department of Pediatrics, School of Medicine, University of Colorado Denver , Anschutz Medical Campus, Aurora, Colorado 80045,
United States
S
* Supporting Information
ABSTRACT: The role of Mer kinase in regulating the second phase of platelet
activation generates an opportunity to use Mer inhibitors for preventing thrombosis
with diminished likelihood for bleeding as compared to current therapies. Toward
this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine
scaffold using a structure-based drug design and a pseudo ring replacement strategy.
The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct
activity have been determined. Subsequent SAR studies identified compound 23
(UNC2881) as a lead compound for in vivo evaluation. When applied to live cells,
23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM.
Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric
receptor containing the intracellular domain of Mer fused to the extracellular domain
of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation,
suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.
ones that do not increase the risk for bleeding and other side
effects associated with current antithrombotic agents.
INTRODUCTION
■
Platelets are small cells derived from precursor megakaryocytes.
The physiologic procoagulant activity of platelets helps prevent
excessive bleeding, while increased platelet activation and
overactive coagulation can lead to pathologic thrombus
formation which may result in stroke or heart attack.
Antiplatelet compounds are therefore an important family of
drugs for cardiovascular diseases and for certain surgical
procedures where a risk of stroke or thrombosis is prevalent.
However, current antiplatelet therapies are often complicated
by significant bleeding and other side effects. For example,
aspirin, an antiplatelet drug that functions through inhibiting
the production of thromboxane, has a baseline major bleeding
risk (gastrointestinal or intracranial) of 1−4%1 and is associated
with other toxicities including gastric side effects. The addition
of clopidogrel (Plavix), another widely used antiplatelet drug, as
a combination therapy further increases bleeding risk by 1%.2
Moreover, not all patients respond to aspirin and clopidogrel:
the nonresponse rates are 5.5−60% in patients treated with
aspirin3 and 4−30% in those treated with clopidogrel4 based on
meta-analyses. Consequently, there is still a significant need to
develop novel therapies for treatment of thrombosis, especially
Mer is a member of the TAM (Tyro3, Axl and Mer) receptor
tyrosine kinase (RTK) subfamily with growth-arrest-specific-6
(Gas6) as one of the endogenous ligands.5 Elevated Mer
activation has been strongly associated with the oncogenesis of
a number of human cancers.6 Recently, Mer has also been
shown to play important roles in regulating macrophage activity
and platelet aggregation.7 Mer knockout mice have decreased
platelet aggregation while maintaining normal bleeding times
and coagulation parameters. Consequently, these mice are
protected from thrombosis without increased spontaneous
bleeding.7a,8 These observations indicate that small molecule
Mer kinase inhibitors have potential as new antiplatelet drugs
with decreased bleeding complications, a profile that confers a
major advantage over currently available antiplatelet therapies.
In our efforts to develop novel therapeutic agents for cancer
treatment, we have discovered several potent small molecule
Mer inhibitors. However, the first generation of these molecules
possess either low selectivity for Mer relative to other TAM
Received: September 9, 2013
© XXXX American Chemical Society
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family members (1 (UNC569))9 or low solubility and poor
pharmacokinetic (PK) properties, such that they are not
suitable for in vivo study (UNC1062).10 While efforts to
optimize these molecules to yield a Mer selective, in vivo active
pyrazolopyrimdine are ongoing, here we report the discovery of
a new substituted-pyrimidine scaffold with significantly
improved selectivity to further validate Mer as a potential
target for thrombosis prevention.
Figure 1A and retain the potency observed with compound 1.
One such design is shown in Figure 1B, where the
intramolecular hydrogen bond in 2 will be formed between
the carbonyl oxygen of the amide group and the hydrogen on
the adjacent amino side chain. The other substituents are not
modified and will likely occupy the same regions as in 1.
However, because the pseudo ring is less rigid and larger in size
than the pyrazole ring, this new scaffold may have a distinct
kinase specificity profile or pharmacokinetic (PK) properties
due to subtle conformational and physical property changes.
Furthermore, the synthesis of 2 is straightforward making
efficient structure−activity relationship (SAR) studies feasible.
In the cocrystal structure of Mer in complex with compound
1 (Figure 1A),9 the inhibitor is fully confined to the relatively
CHEMISTRY
■
The syntheses of pyrimidine analogues are shown in Scheme 1.
An amide coupling reaction is used to introduce the R1 group
while an SNAr reaction is used to introduce the R2 and R3
groups. Path A is designed for SAR exploration of the R2 and R3
positions, while path B is designed for diversifying the R1
position.
RESULTS AND DISCUSSION
■
To test our pseudo ring replacement hypothesis, a small set of
compounds were synthesized using the route depicted in
Scheme 1 (compounds 3 and 5 started with 2,4-dichloropyr-
imidin-5-amine and 4-fluorobenzoyl chloride) (Table 1).
Inhibition of Mer kinase activity by these compounds was
tested using a microfluidic capillary electrophoresis (MCE)
assay.12 Indeed, compound 2 was very potent against Mer while
its close analogue 3 exhibited only weak activity. The only
structural difference between 2 and 3 was the regiochemistry of
the key hydrogen bond, enabling amide functionality. The
reverse amide bond in 3 is unable to form the pseudo ring
forming intramolecular hydrogen bond with the hydrogen on
the amino side chain at the R2 position resulting in greatly
diminished Mer activity. Comparison of the activity of 4 and 5
further confirmed the important role of the intramolecular
hydrogen bond and validated our design of the pseudo ring
replacement. To monitor selectivity within the TAM family, the
ability of these analogues to inhibit Axl and Tyro3 was also
tested, and they were significantly more active against Mer than
Axl and Tyro3 (Table 1). In addition, the trans-4-amino-
cyclohexylmethylamino group at the R2 position in 2 can be
replaced by a trans-4-hydroxycyclohexylamino group in 4 and
the activity against Mer is retained. In our previous studies, the
Figure 1. Structure-based design of a scaffold that features pseudo ring
formation through an intramolecular hydrogen bond. (A) X-ray
structure of 1 complexed with Mer protein (kinase domain) (PDB ID
code 3TCP). (B) Docking model (based on X-ray structure PDB ID
code 3TCP) of the designed molecule 2.
small adenine pocket, forming three hydrogen bonds: two with
the hinge region of Mer using one nitrogen of the pyrimidine
ring (with residue Met674) and the NH from the butyl amino
side chain (with residue Pro672) and another one with the
carbonyl of Arg727 via the methylcyclohexylamino group.
Because the pyrazole ring does not appear to interact with the
Mer active site directly, its major role may be to rigidify the
molecule. Therefore, replacement of the pyrazole ring with a
pseudo ring11 constrained by an intramolecular hydrogen bond
while maintaining functionality to create the three hydrogen
bonds observed with 1 may mimic the binding conformation in
Scheme 1. Synthetic Routes for Pyrimidine Analogues
B
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Table 1. Key SAR Observations
Table 2. Preliminary SAR at the R1 Position
a
Values are the mean of two or more independent assays.
primary amino group at the R2 position could result in
undesired hERG activity.10a Therefore, the trans-4-hydroxycy-
clohexylamino group was used at the R2 position for further
SAR exploration at the R1 site.
Next, a focused library of compounds with different R1
groups was synthesized while keeping R3 fixed as a butylamino
group (Table 2). Interestingly, the amide nitrogen at the R1 site
could not be further substituted. Even simple methylation of
the amide bond almost abolished the Mer activity (6 vs 7 and 8
vs 9). This may be due to weaker binding of the ligand to the
hinge motif (residues 672−677). Not only does amide
methylation of the ligand preclude a hydrogen bond to the
Met674-backbone carbonyl, it also contributes to a less
favorable orientation of the aminopyrimidine group with
respect to Pro672 and Met674. Because 8 was more active
than the open chain analogue 6, our focus was initially on
further modification of 8. However, the addition of extra groups
i
such as Pr (10) and pyrimidine (11) to the nitrogen of the
piperidine ring did not boost the activity. Exchange of the
piperidine nitrogen with oxygen (12) or carbon (13) also did
not significantly affect the activity. Interestingly, when the
piperidine ring was replaced with an aromatic ring, the activity
of the analogues was modulated by the substituents on the
aromatic ring: an electron-withdrawing group such as CF3 on
the aromatic ring yielded a much weaker Mer inhibitor (14)
compared to 8, while an electron-donating group like MeO on
the aromatic ring produced an analogue with similar activity
(15). Inhibition of Mer was not sensitive to the substitution
pattern on the aromatic ring (15−17). In addition, larger
groups such as morpholine (18), morpholinomethyl (19), and
morpholinosulfonyl (which was the R1 group of UNC106210a
)
(20) could be tolerated on the aromatic ring. This is consistent
with the prediction that the R1 group interacting with the
solvent front based on the docking model. Furthermore, a
methylene group could be inserted between the amide bond
and the aromatic ring without a change in activity (4 vs 21,
Table 1). However, the activity of the analogues was increased
by introducing a larger group on the aromatic ring as in 22 and
23 (UNC2881). In general, these analogues are significantly
a
Values are the mean of two or more independent assays.
C
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more Mer-selective than 2, especially compound 23 (84-fold
over Axl and 58-fold over Tyro3).
group (32) decreased the activity slightly. However, a
morpholine at the R2 position (33) almost abolished the Mer
activity due to the absence of the pseudo ring forming
intramolecular hydrogen bond. In general, these analogues have
good selectivity for Mer over Axl and Tyro3.
In addition, we explored SAR at the R2 site while keeping R1
fixed as morpholinosulfonylphenyl carboxamide and R3 fixed as
butylamino group. A few examples are included in Table 3.
The SAR at the R3 position was further explored while fixing
R1 as a morpholinosulfonylphenyl carboxamide group and R2 as
a trans-4-hydroxycyclohexylamino group (Table 4). Absence of
Table 3. SAR Study of R2
Table 4. SAR Study of R3
a
Values are the mean of two or more independent assays.
When introducing the trans-4-aminocyclohexylamino group at
the R2 site, the corresponding analogue 24 had activity similar
to 20 (Table 2). However, removal of the polar group NH2 on
the cyclohexyl ring (25) reduced the activity dramatically (over
280-fold). This is consistent with the SAR observed with
analogues of 1: a hydrogen bond between a polar substituent at
the R2 position with the carbonyl of Arg727 of the Mer protein
is important.9 Compared to 25, secondary amines such as 4-
piperidinylmethylamino (26), (R)-3-piperidinylmethylamino
(27), and 4-piperidinylamino (28) at the R2 position increased
the activity compared to 25 but were less suitable than the
primary amine group. Interestingly, introduction of an (S)-3-
pyrrolodinylamino group in (30) led to a 9-fold higher activity
compared to the (R)-3-pyrrolodinylamino substitution (29).
The structural analysis suggests that this 9-fold activity gain is
most likely due to an optimal interaction of the pyrrolidine
nitrogen in 30 with the backbone carbonyl of Leu593, while the
same nitrogen in 29 would not be able to optimally approach
Leu593. A 4 substituted-pyridine ring at the R2 position
resulted in a much less active analogue 31 than the
corresponding 4 substituted-piperidine ring (26). Replacement
of the 4-piperidinylamino group (28) with a 4-pyranylamino
a
Values are the mean of two or more independent assays.
substitution on the amino group at the R3 position resulted in
only a weak Mer inhibitor (34), and even a methyl group at this
position significantly increased the activity (16-fold) (35 vs 34).
Analogues were progressively more active with addition of
longer alkyl side chains (36 and 37) until the chain reached 4-
carbons in length (20 (Table 2); the activity began to decrease
when the chain was 5-carbons (38) or longer. A cyclopropyl
group (39) was tolerated at the end of the propyl side chain,
while a polar group, such as trifluoromethyl (40) or a hydroxyl
group (41), was less tolerated. A branched alkyl side chain did
not significantly affect activity (42 vs 36). However, cyclohexyl
(43), 4-tetrahydropyranyl (44), or 4-fluorobenzyl (45) at this
D
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Figure 2. X-ray structures of compounds 22 (PDB ID code 4MHA) (a) and 7 (PDB code 4MH7) (b) in complex with the Mer kinase domain.
Table 5. PK Profile of 23
a
route
T1/2 (h)
Tmax (h)
Cmax (ng/mL)
AUClast (h·ng/mL)
Cl_obs (mL/min/kg)
94.5
Vss (L/kg)
1.65
%F
IV
0.80
2609
90.0
527
PO
0.30
71.7
14
a
A dose of 3 mg/kg for both routes.
position decreased Mer activity. Furthermore, when the amino
group at this position was fully substituted, as in 46 and 47,
Mer activity was completely eliminated. This result is consistent
with our design hypothesis where the nitrogen of the amino
group of R3 forms a hydrogen bond with residue Pro672 at Mer
protein hinge area. Again, these analogues are Mer-selective as
compared to Axl and Tyro3.
On the basis of its solubility, inhibitory activity against Mer,
and remarkable intrafamily selectivity, 23 was chosen as a lead
compound in this series. The in vivo PK properties of 23 were
assessed in mice via both intravenous (IV) and oral (PO)
administration (Table 5). 23 had high systemic clearance (94.5
mL/min/kg) and 14% oral bioavailability. The terminal half-life
was 0.80 h. The volume of distribution was 2-fold greater than
the normal volume of total body water (0.70 L/kg). Although
the PK properties of 23 are not yet ideal and need to be further
improved to enable chronic in vivo studies, this compound is
sufficient for in vitro or short-term in vivo studies.
The inhibitory activity mediated by 23 against a panel of 30
kinases was also determined at a concentration 100-fold above
its Mer IC50 (Figure 3) (details in Supporting Information).
This experiment provides a broad survey of kinase families
emphasizing tyrosine kinases along with a selection of serine/
threonine kinases. Six kinases were inhibited by greater than
50% in the presence of 430 nM 23, while none of the serine/
threonine kinases were appreciably inhibited.
When 23 was tested in cell-based assays, it mediated
inhibition of Mer phosphorylation in 697 B-ALL cells with
an IC50 value of 22 nM (Figure 4). In addition to this effect on
steady-state levels of phosphorylated Mer, 23 efficiently
inhibited ligand-dependent phosphorylation of a chimeric
protein consisting of the extracellular domain from the
epidermal growth factor (EGF) receptor and the intracellular
domain of Mer (Figure 5). Moreover, treatment with 23
inhibited platelet aggregation by greater than 25% in human
platelet-rich plasma in response to stimulation with fibrillar type
I equine collagen (Figure 6). ATP release, which is a marker of
platelet activation and degranulation, was decreased to a similar
extent. These data are the first to demonstrate functional
inhibition of platelet activity mediated by a Mer-selective small
molecule tyrosine kinase inhibitor and suggest the utility of Mer
inhibitors for this therapeutic application.
To characterize the binding interactions of the reported
pyrimidine inhibitors with Mer and provide a structural basis
for future directions in chemical optimization, cocrystal
structures of Mer in complex with compounds 7 and 22 were
obtained. The structures in Figure 2 demonstrate a binding
mode in which the pyrimidine ring inserts into the adenine
binding site and mimics interactions of the adenine with the
backbone atoms of the hinge (residues Pro672 and Met674).
Similar to the previously published Mer-1 complex,9 the butyl
amino side chain (R3 substituent) is folded into the relatively
small adenine site instead of protruding into the lipophilic back-
pocket. However, unlike the previously published structure, the
hydroxyl group of the R2 substituent forms a hydrogen bond
with the side chain of Asp741 instead of the nearby Arg727 (as
does the R2 amino group of 1). It is noteworthy that one of the
gate-forming residues, Ile650, is not conserved among other
members of the TAM subfamily, Axl and Tyro3, which feature
methionine and alanine in this position, respectively. This
variability is quite likely the reason for the significant intrafamily
selectivity observed. It is remarkable that despite sharing
identical core motifs, compounds 7 and 22 display very
different IC50 values of 5.9 and 0.0052 μM, respectively. The
most likely explanation for this significant difference is that the
methyl group substituted on the amide of compound 7
precludes a hydrogen bond to the Met674 backbone carbonyl
(present in the compound 22:Mer complex) and may also
prevent the aminopyrimidine group from binding to Pro672
and Met674 in the most favorable orientation. Consistent with
the SAR discussed above, the R1 substituent interacts mostly
with the solvent and does not significantly impact the activity.
Consequently, it can be utilized for tuning solubility and other
physical or PK properties.
CONCLUSIONS
■
We have successfully applied a pseudo ring replacement
strategy to discover a potent substituted-pyrimidine series as
new Mer kinase selective inhibitors. The cocrystal structures of
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Figure 5. 23 inhibits ligand-stimulated activation of a chimeric EGFR-
MerTK. 32D cells expressing a chimeric receptor consisting of the
extracellular ligand-binding domain of the EGF receptor and the
intracellular domain of Mer were treated with 23 or vehicle for 1 h
prior to stimulation with 100 ng/mL EGF ligand for 15 min. Mer was
immunoprecipitated from whole cell lysates and phospho-tyrosine
containing and total Mer proteins were detected by Western blot.
Results shown are representative of two independent experiments.
Figure 3. Kinase tree.
Figure 6. 23 inhibits collagen-stimulated platelet aggregation. Human
platelet-rich plasma was normalized to a platelet count of 250000/μL
with platelet-poor plasma obtained by differential centrifugation.
Samples were treated with 3 μM 23 or DMSO vehicle for 1 h at room
temperature prior to stimulation with 1 μg/mL type I equine fibrillar
collagen and assessment of aggregation and ATP release using an
aggregometer. (A) Representative aggregation from samples treated
with 3 μM 23 or DMSO. (B) Maximum percent aggregation and ATP
release are shown for samples treated with 23 or DMSO vehicle. Mean
values SEM derived using plasma collected from five (ATP release)
or seven (aggregation) different individuals are shown. Statistically
significant results were determined using the student’s paired t test (*
p = 0.05 and ** p = 0.01 relative to vehicle control).
Figure 4. 23 inhibits endogenous Mer tyrosine kinase activation in
acute lymphoblastic leukemia cells. 697 B-ALL cells were treated with
the indicated concentrations of 23 for 1 h. Pervanadate was added to
cultures for 3 min to stabilize the phosphorylated form of Mer. Mer
was immunoprecipitated from cell lysates, and total Mer protein and
Mer phosphoprotein were detected by immunoblot. (A) Representa-
tive Western blots. (B) Relative levels of phospho-Mer and Mer
proteins were determined. Mean values standard error derived from
three independent experiments are shown. IC50 = 21.9 nM with a 95%
confidence interval of 16.9−28.5 nM.
Microfluidic Capillary Electrophoresis (MCE) Assay.9 Activity
assays were performed in a 384 well, polypropylene microplate in a
final volume of 50 μL of 50 mM Hepes, pH 7.4 containing 10 mM
MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% bovine serum
albumin (BSA), containing 1.0 μM fluorescent substrate (Table 6) and
ATP at the Km for each enzyme (Table 6). All reactions were
terminated by addition of 20 μL of 70 mM EDTA. After a 180 min
incubation, phosphorylated and unphosphorylated substrate peptides
(Table 6) were separated in buffer supplemented with 1× CR-8 on a
7 and 22 with Mer protein have been determined and their
binding modes provide a rationale for the 1000-fold difference
in their IC50 values. The lead compound 23 inhibited Mer
kinase activity in cell-based assays and mediated functional
inhibition of human platelet activation, suggesting the utility of
Mer-selective small molecule inhibitors for treatment and/or
prevention of pathologic thrombosis.
Table 6. Assay Conditions for MCE Assays
kinase
peptide substrate
kinase (nM) ATP (μM)
2.0 5.0
120 65
10 21
EXPERIMENTAL SECTION
Details on the synthesis of all compounds are given in the Supporting
Information. The purity of all tested compounds was determined by
LC/MS to be >95%.
■
Mer
Axl
5-FAM-EFPIYDFLPAKKK-CONH2
5-FAM-KKKKEEIYFFF-CONH2
5-FAM-EFPIYDFLPAKKK-CONH2
Tyro
F
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LabChip EZ Reader equipped with a 12-sipper chip. Data were
analyzed using EZ Reader software.
ASSOCIATED CONTENT
* Supporting Information
Experimental details, characterization of all compounds, and
selectivity profiling. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
Cell-Based Assays for Mer Kinase Inhibition. 697 B-ALL Cell
Assay. 697 B-ALL cells were cultured in the presence of 23 or vehicle
only for 1.0 h. Pervanadate solution was prepared fresh by combining
20 mM sodium orthovanadate in 0.9× PBS in a 1:1 ratio with 0.3%
(w/w) hydrogen peroxide in PBS for 15−20 min at room temperature.
Cultures were treated with 120 μM pervanadate prior to collection for
preparation of whole cell lysates, immunoprecipitation of Mer, and
analysis by Western blot.
Accession Codes
The atomic coordinates for the X-ray crystal structures of 7 and
22 have been deposited with the RCSB Protein Data Bank
under the accession code 4MH7 and 4MHA.
Then 697 B-ALL cells were treated with pervanadate for 3.0, 5.0,
and 1.0 min, respectively. Cell lysates were prepared in 50 mM HEPES
pH 7.5, 150 mM NaCl, 10 mM EDTA, 10% glycerol, and 1% Triton
X-100, supplemented with protease inhibitors (Roche Molecular
Biochemicals, no. 11836153001). Mer protein was immunoprecipi-
tated with a monoclonal anti-Mer antibody (R&D Systems, no.
MAB8912) and Protein G agarose beads (InVitrogen). Phospho-Mer
was detected by Western blot using a polyclonal antiphospho-Mer
antibody raised against a peptide derived from the triphosphorylated
activation loop of Mer. Nitrocellulose membranes were stripped and
total Mer protein was detected using a second anti-Mer antibody
(Epitomics Inc., no. 1633-1). For 697 B-ALL cells, relative
phosphorylated and total Mer protein levels were determined by
densitometry using Image J software and IC50 values were determined
by nonlinear regression.
32D-EMC Cell Assay. 32D-EMC suspension cultures were treated
with the indicated concentration of 23 or vehicle before stimulation
with 100 ng/mL EGF (BD Biosciences no. 354010) for 15 min. Cells
were centrifuged at 1000g for 5 min and washed with 1× PBS. Cell
lysates were prepared in 20 mM HEPES (pH 7.5), 50 mM NaF, 500
mM NaCl, 5.0 mM EDTA, 10% glycerol, and 1% Triton X-100,
supplemented with protease inhibitors (10 μg/mL leupeptin, 10 μg/
mL phenylmethylsulfonyl fluoride, and 20 μg/mL aprotinin) and
phosphatase inhibitors (50 mM NaF and 1.0 mM sodium
orthovanadate), and Mer protein was immunoprecipitated using a
custom polyclonal rabbit anti-Mer antisera raised against a GST
protein derived from the C-terminus of human Mer and Protein A
agarose beads (Santa Cruz Biotechnology). Phosphotyrosine-contain-
ing proteins were detected by Western blot with a monoclonal HRP-
conjugated antiphosphotyrosine antibody (Santa Cruz Biotechnology,
no. sc-508). Antibodies were stripped from membranes and total Mer
levels were determined using the custom polyclonal rabbit anti-Mer
antibody raised against a peptide derived from the catalytic domain of
Mer.
Platelet Aggregation Assay. Human whole blood (WB) was
collected from healthy volunteers as permitted by an Institutional
Review Board-approved protocol (COMIRB no. 09-0816). WB was
drawn by venipuncture into 3.8% sodium citrate and centrifuged at
200g for 20 min at room temperature. Platelet-rich plasma (PRP) was
separated, and the remaining solution was spun at 2400g for 10 min to
create platelet-poor plasma (PPP). PRP and PPP were mixed in
appropriate proportions to obtain a final concentration of 2.5 × 105
platelets/μL and then used within 3 h of the initial blood draw.
For platelet aggregation assays, plasma was incubated for 60 min at
RT with either 3 μM 23 or vehicle (20% DMSO in saline). Samples
were analyzed using a light-transmission aggregometer (CHRONO-
LOG Corporation, Havertown, PA) at 37 °C in the presence of
Chrono-lume reagent (CHRONO-LOG) with magnetic stirring at
1200 rpm and addition of 1 μg/mL equine type I fibrillar collagen
(CHRONO-LOG) as an aggregation agonist. Optical density was
determined as an indicator of aggregation. Emission of light by
Chrono-lume, a luciferin-luciferase compound, occurs as a result of
ATP binding and is determined relative to a 2 nM standard as a
measure of the amount of ATP released from activated platelets. ATP
release (nM) and the maximum percent aggregation were recorded 7
min after collagen addition.
AUTHOR INFORMATION
Corresponding Author
*Phone: 919-843-8456. E-mail: xiaodonw@unc.edu.
■
Author Contributions
#W.Z. and A.M. contributed equally.
Notes
The authors declare the following competing financial
interest(s): DD, DK, WPJ, HSE, DG, SF, and XW hold equity
of Meryx, Inc.
ACKNOWLEDGMENTS
■
This work was supported by the University Cancer Research
Fund and Federal Funds from the National Cancer Institute,
National Institute of Health, under contract no.
HHSN261200800001E. 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 organizations imply endorse-
ment by the U.S. Government.
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