Discovery, design and synthesis of a selective S1P3 receptor allosteric agonist

Article abstract of DOI:10.1016/j.bmcl.2013.09.075

Potent and selective S1P₃ receptor (S1P₃-R) agonists may represent important proof-of-principle tools used to clarify the receptor biological function and assess the therapeutic potential of the S1P ₃-R in cardiovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide was identified by a high-throughput screening of MLSMR library as a promising S1P₃-R agonist. Rational chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide, a S1P₃-R agonist endowed with submicromolar activity and exquisite selectivity over the remaining S1P_1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide is an allosteric agonist and binds to the S1P₃-R in a manner that does not disrupt the S1P₃-R-S1P binding. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the molecular basis of the receptor function, and provides the bases for further rational design of more potent and drug-like S1P₃-R allosteric agonists.

Full text of DOI:10.1016/j.bmcl.2013.09.075

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6346–6349
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery, design and synthesis of a selective S1P3 receptor allosteric
agonist
Miguel Guerrero a, Ramulu Poddutoori a, Mariangela Urbano a, Xuemei Peng a, Timothy P. Spicer b,
Peter S. Chase b, Peter S. Hodder b,c, Marie-Therese Schaeffer d,e, Steven Brown d,e, Hugh Rosen d,e,f
,
Edward Roberts a,
⇑
a Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
b Scripps Research Institute Molecular Screening Center, Lead Identification Division, Translational Research Institute, 130 Scripps Way, Jupiter, FL 33458, United States
c Department of Molecular Therapeutics, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
d Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
e The Scripps Research Institute Molecular Screening Center, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
f Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2013
Revised 17 September 2013
Accepted 23 September 2013
Available online 1 October 2013
Potent and selective S1P3 receptor (S1P3-R) agonists may represent important proof-of-principle tools
used to clarify the receptor biological function and assess the therapeutic potential of the S1P3-R in car-
diovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide
was identified by a high-throughput screening of MLSMR library as a promising S1P3-R agonist. Rational
chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-
3-carboxamide, a S1P3-R agonist endowed with submicromolar activity and exquisite selectivity over the
remaining S1P1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis
and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxam-
ide is an allosteric agonist and binds to the S1P3-R in a manner that does not disrupt the S1P3-R–S1P bind-
ing. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the
molecular basis of the receptor function, and provides the bases for further rational design of more potent
and drug-like S1P3-R allosteric agonists.
Keywords:
S1P3 receptor
Allosteric agonist
Cardiovascular functions
Ó 2013 Elsevier Ltd. All rights reserved.
Sphingosine-1-phosphate (S1P) is a naturally occurring lyso-
phospholipid produced in most cell types, regulates fundamental
biological processes and functions including cell proliferation, cell
growth, angiogenesis, and inhibits apoptosis and lymphocyte traf-
ficking.1 The generation of S1P is mediated by two cytosolic sphin-
gosine kinase isoforms (SPK1 and SPK2) and occurs preferentially
in the plasma membrane.2 S1P has two distinct signaling roles,
as an intracellular second messenger and as an extracellular ligand
for a specific family of G-protein coupled receptors (GPCRs) named
S1P1–5-Rs.3 S1P can also affect cell function by either binding or
modifying putative intracellular targets or by affecting the relative
levels of other lipid products, particularly sphingomyelin and cer-
amide whose biological effects oppose those of S1P.4,2a
differential temporal and spatial pattern and intracellular signaling
pathways of each S1P-R enable S1P to exert its diverse biological
functions. S1P1–3-Rs are widely expressed in almost all organs in
mice and humans whereas S1P4-R and S1P5-R expression is re-
stricted to specific organs and cell types.6 In humans S1P3-R is
highly expressed in heart, lungs, spleen, kidney and pancreas.
S1P3-R couples with Gi/o, Gq, and G12/13. Interestingly, S1P3-R defi-
cient mice showed no obvious phenotype.7 However, clear associ-
ation of S1P3-R has been demonstrated in several cardiovascular
functions including regulation of heart rate and blood pressure in
rodents,8 vasorelaxation9 and cardiac fibrosis.10 Additionally, regu-
lation of myocardial perfusion and cardiomyocytes protection in
ischemia perfusion injury was dependent on the S1P3-R.11 Further-
more, studies with S1P3-R-null mice and PAR1-deficient dendritic
cells (DC) have shown that S1P3-R acts as a downstream compo-
nent in PAR1-mediated septic lethality.12 S1P3-R is also shown to
regulate the endothelial cells in splenic marginal sinus organiza-
tion13 and the stimulation of endothelial progenitor cells.14
S1P3-R has also been implicated in mediating and amplifying
inflammatory responses in various CNS disorders in autocrine
and paracrine fashion.15 Recently, it has been shown that S1P3-R
Increasing evidence supports that different subfamilies of ATP-
binding cassette (ABC) transporters contribute to, or are involved
in, secreting the S1P lipids across the plasma membranes.5 The
⇑
Corresponding author. Tel.: +1 858 784 7770; fax: +1 858 784 7745.
E-mail address: eroberts@scripps.edu (E. Roberts).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.075

M. Guerrero et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6346–6349
6347
is markedly up-regulated in a subset of adenocarcinoma cells, and
knock down of this receptor subtype inhibits proliferation and
growth of lung adenocarcinoma cells.16
A wealth of S1P-Rs agonists has been described in the literature.
However, the development of subtype selective S1P3-R agonists as
useful pharmacological tools has been limited as a consequence of
targeting the orthosteric binding site for receptor activation. The
orthosteric binding site displays a high level sequence homology
among the S1P-R family subtypes. S1P1-R and S1P3-R are the most
closely related by sequence, particularly at their agonist binding
pockets which consist of a lower hydrophobic and an upper polar
region where Leu-276 in S1P1-R and Phe-263 in S1P3-R are the
main difference. Receptor structure modeling and ligand docking
studies revealed that the S1P3-R binding pocket is contracted be-
tween the lower lipophilic area and the upper polar section by
1.5–1.8 Å compared to the S1P1-R due to the presence of
Phe-263. These differences in steric and space constrains in the
S1P3-R orthosteric binding site may explain the difficulty in
designing S1P3-R agonists devoid of S1P1-R agonist activity.17
Topologically distinct from the conserved orthosteric binding
site, an allosteric site provides a means to overcome important
selectivity issues associated with the orthosteric ligands, in partic-
ular within GPCRs in which the orthosteric site is highly conserved
between subtypes. In addition to offering a potential subtype-
selectivity, allosteric ligands may stabilize different conformations
and functional states, thus activating a distinct repertoire of recep-
tor signaling and regulatory properties that orthosteric ligands are
unable to initiate.18 The S1P binding site is highly conserved
among the S1P-R family thus selective allosteric ligands may be
useful pharmacological tools to decipher individual receptor bio-
logical functions.
Scheme 1. Synthesis of 1a–d. Reagents and conditions: (i) (a) 2 (1 equiv), SOCl2,
benzene, reflux, 3 h; (b) 3a–d (1.5 equiv), DIPEA (1.5 equiv), CH2Cl2, 0 °C–rt, 3 h, 70–
98% (over two steps).
Table 1
S1P3-R agonist activity of compounds 5a–i
a
Compd
Carboxylic acid
R
EC50 (nM)
S1P3-R
S1P1-R
5a
5b
5c
5d
5e
5f
5g
5h
5i
4a
4b
4c
4d
4e
4f
4g
4h
4i
Isobutyl
Isopropyl
Methyl
Cyclohexyl
Cyclopentyl
Cyclopropyl
Phenyl
4-Methoxyphenyl
3,4-Diethoxyphenyl
1375
959
6595
559
339
105
103
323
8070
6420
22,250
>50,000
>50,000
>50,000
33,300
662
859
1104
a
Data are reported as mean of n = 3 determinations.
A high-throughput screening (HTS) of the Molecular Libraries-
Small Molecule Repository (MLSMR) library identified the
N,N-dicyclohexyl-5-propylisoxazole-3-carboxamide 1a (Fig. 1) as
a S1P3-R agonist with acceptable in vitro potency/selectivity pro-
file.17,19 The structural integrity of the hit was corroborated by
the re-synthesis (Scheme 1) of the title compound that showed
confirmed EC50’s of 434 nM at S1P3-R, 7.87
agonist activity at S1P2,4-Rs at concentrations up to 50
l
M at S1P1-R and no
Scheme 2. Synthesis of 5a–i. Reagents and conditions: (i) (a) 4a–h (1 equiv), SOCl2,
benzene, reflux, 3 h; (b) 3a (1.5 equiv), DIPEA (1.5 equiv), CH2Cl2, 0 °C–rt, 3 h, 85–
98% (over two steps).
lM (Fig. 1).
Our SAR studies commenced varying the amide region C. The
synthesis and biological results of 1b–d are outlined in Scheme 1.
The carboxylic acid 2 was transformed into the corresponding acid
chloride and coupled with amines 3b–d to furnish the amide prod-
ucts 1b and 1d. Surprisingly, exchanging a cyclohexyl from 1a for a
cyclopentyl group (1b) led to loss in potency of 18-fold for the
S1P3-R but only of three-fold for the S1P1-R. Furthermore, changing
a cyclohexyl for a phenyl (1d) or hydrogen (1c) led to complete loss
of potency at both receptors. These results underscore the key role
played by the N,N-dicyclohexyl amide for binding to the S1P3-R.
Next we explored region A of the HTS-hit while keeping regions
B and C constant. Compounds 5a–i (Table 1) were synthesized
from a series of isoxazole carboxylic acids 4a–i commercially avail-
able (4a–c and 4f–i) or readily obtained (4d and 4e) according to
literature procedures (Scheme 2).20
Interestingly, the isobutyl 5a was approximately three-fold less
potent than the hit at the S1P3-R and four-fold less selective
against the S1P1-R. The isopropyl derivative 5b was approximately
two-fold less active than 1a for the S1P3-R, but the selectivity
against the S1P1-R remained similar. The methyl derivative 5c
was 15-fold less potent than 1a. Remarkably, the cyclohexyl (5d)
and cyclopentyl (5e) analogs were slightly less and more active
at the S1P3-R and inactive at the S1P1-R. Of note, the cyclopropyl
derivative 5f (CYM5541) was found four-fold more potent than
the hit and nearly 18-fold more selective against the S1P1-R. The
phenyl derivative 5g (CYM5544) was found equipotent to 5f for
the S1P3-R but significantly less selective against the S1P1-R. Inter-
estingly, the 4-methoxyphenyl 5h was slightly more potent than
the hit compound at the S1P3-R, but its selectivity against the
S1P1-R was less than three-fold. Intriguingly, 3,4-diethoxyphenyl
5i was 18-fold less potent than the hit at the S1P3-R but more ac-
tive (ꢀ7-fold) at the S1P1-R. All this information together indicates
that an aromatic ring at position five of the isoxazole hit (portion
A), although favorable for the S1P3-R activity, is also detrimental
for the selectivity against the S1P1-R.
Based on the obtained results, we focused our attention on the
SAR studies of 5f, particularly on the amide region C. The synthesis
of 7a–l is depicted in Scheme 3. The biological results are listed in
Table 2.
Figure 1. HTS S1P3-R agonist hit 1a.

6348
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6346–6349
Scheme 3. Synthesis of 7a–l. Reagents and conditions: (i) (a) 4d (1 equiv), SOCl2,
benzene, reflux, 3 h; (b) 3a, 3c, 6a–j (1.5 equiv), DIPEA (1.5 equiv), CH2Cl2, 0 °C–rt,
3 h, 65–98% (over two steps).
As previously observed, removal of a cyclohexyl group (7a) led
to complete loss of activity. Similarly, exchanging a cyclohexyl for a
cyclopentyl (7b), 4-tetrahydropyranyl (7c) or cyclohexylmethyl
(7d) led to 16-, 33- and 32-fold loss of potency, respectively,
confirming the importance of the bicyclohexyl amide system.
Interestingly, the 2-methylcyclohexyl derivative 7e (CYM5558)
was three-fold less potent and selective than 5f, probably due to
conformational changes caused by the methyl group. Unexpect-
edly, when both cyclohexyls were substituted with a methyl group
(7f, CYM5556) the potency decreased by only two-fold compared
to 5f and the selectivity remained nearly similar. The N-(2-meth-
ylcyclohexyl)-N-cycloheptyl derivative 7g was almost two-fold
less potent than 7e while the 4-tetrahydropyranyl 7h was slightly
more potent than the cyclohexyl derivative 7c, although the
selectivity of both compounds decreased considerably. Surpris-
ingly, the cyclopentyl 7i was nearly equipotent to 7b. Furthermore,
the N-cyclopentyl-N-cycloheptyl derivative 7j was 26-fold less
potent than 5f. Not surprisingly, the installation of an aromatic
substituent (e.g., 7k and 7l) led to drastic loss in potency.
Next, we explored the middle region B holding constant the
bicyclohexyl amide region C as well as the cyclopropyl and phenyl
groups from region A. The synthesis and biological activity of rep-
resentative compounds 9a–c is depicted in Scheme 4. Interestingly
the oxazole 9a and the furan 9b were inactive at the S1P3-R; inter-
estingly, 9b showed micromolar activity for the S1P1-R. Fusing the
phenyl ring of 5g into the isoxazole moiety led to 9c, which was
inactive at the S1P3-R. This modification highlights the isoxazole
ring as an important binding motif of this chemotype.
Scheme 4. Synthesis of 9a–c. Reagents and conditions: (i) (a) 8a–c (1 equiv), SOCl2,
benzene, reflux, 3 h; (b) 2a (1.5 equiv), DIPEA (1.5 equiv), CH2Cl2, 0 °C–rt, 3 h, 70–
98% (over two steps).
Table 3
S1P2,4,5-Rs selectivity counter screen of selected compounds
a
Compd
EC50 (nM)
S1P2-R
S1P3-R
S1P1-R
S1P4-R
S1P5-R
3a
5b
5f
5g
5h
7f
434
959
105
103
323
202
337
535
7870
22,250
33,300
662
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
NT
NT
>25,000
NT
NT
NT
NT
NT
859
>50,000
33,100
4703
7e
7g
NT = not tested.
a
Data are reported as mean of n = 3 determinations.
relevant targets tested (stimulation/inhibition <50% at 30 lM). A
The functional activity of selected compounds was tested
against the S1P2,4-Rs subtypes (Table 3).17 Remarkably, all the com-
pounds were highly selective against the S1P2,4-Rs. Due to its selec-
tivity profile, 5f was also tested against the S1P5-R. Remarkably 5f
was found exquisitely selective against S1P1,2,4,5-R subtypes.
The selectivity profile of 5f was further investigated against the
Ricerca panel of off target proteins including GPCRs, enzymes and
borderline inhibition (P50%) was found for CYP450 2C19,
CYP450 3A4, norepinephrine transporter, cannabinoid CB1-R, his-
tamine H1-R, and sodium channel site two. Moderate activation
(50%) was found only for nicotinic acetylcholine receptor.
The properties of 5f were further examined through a compet-
itive binding experiment using [33P]S1P as the orthosteric binding
ligand and S1P as the competitive agonist in the control experi-
ment. We found that S1P competes with [33P]S1P for the S1P3-R
binding pocket. Remarkably, 5f does not compete with [33P]S1P
ion channels at a concentration of 30 lM. The compound was
found to be highly selective against the pool of therapeutically
Table 2
S1P3-R agonist activity of compounds 7a–l
a
Compd
Amine
R1
R2
EC50 (nM)
S1P3-R
S1P1-R
7a
7b
7c
7d
7e
7f
7g
7h
7i
3c
3b
6a
6b
6c
6d
6e
6f
H
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
>50,000
1683
3536
3430
337
>50,000
>50,000
>50,000
>50,000
33,100
>50,000
4700
Cyclopentyl
4-Tetrahydropyranyl
Cyclohexylmethyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
Cyclopentyl
Cyclohexyl
2-Methylcyclohexyl
Cycloheptyl
4-Tetrahydropyranyl
Cyclopentyl
Cycloheptyl
Benzyl
202
535
2820
1740
2753
7000
6563
3260
6g
6h
6i
>50,000
34,150
44,900
>50,000
7j
7k
7l
Phenyl
2-Pyridinyl
6j
Benzyl
a
Data are reported as mean of n = 3 determinations.

M. Guerrero et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6346–6349
6349
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for the binding pocket of the S1P3-R and binds in a manner that
does not disturb the [33P]S1P–S1P3-R binding.21 Site-directed
mutagenesis of S1P1-R/S1P3-R and docking studies performed on
an S1P3-homology model showed that 5f binds in an allosteric
binding pocket.
Even though 5f is a highly selective S1P3-R agonist with appro-
priate potency for further biological studies, its physicochemical
properties remain suboptimal (tPSA = 41.9, ClogP = 3.9, solubility
in PBS = 1.24 lM).
Based on the characterized ligand/binding pocket interactions,
our research group will embark on a medicinal chemistry program
aiming to further improve the potency and physicochemical prop-
erties while maintaining selectivity of 5f.
In summary, rational chemical modifications of the MLSMR hit
1a led to the development of S1P3-R agonists 5f, 7e and 7f
(CYM5541, CYM5558, CYM5556) exquisitely selective against the
remaining S1P1,2,4,5-R subtypes. Noteworthy, we have reported
the discovery, design and synthesis of the first small molecule
S1P3-R allosteric agonist based on a N,N-dicyclohexyl-5-alkylisox-
azole-3-carboxamide chemotype, 5f, that is highly selective
against a panel of therapeutically relevant targets. The studies
herein described provide novel pharmacological tools to decipher
the biological function and assess the therapeutic utility of the
S1P3-R. Further studies of our research program aiming to improve
physicochemical properties and potency while maintaining selec-
tivity will be communicated in due course.
16. Hsu, A.; Zhang, W.; Lee, J.; An, J.; Ekambaram, P.; Liu, J.; Honn, K. V.; Klinge, C.
M.; Lee, M. Int. J. Oncol. 2012, 40, 1619.
17. Schürer, S. C.; Brown, S. J.; Gonzales-Cabrera, P. J.; Schaeffer, M.; Chapman, J.;
Jo, E.; Chase, P.; Spicer, T.; Hodder, P.; Rosen, H. ACS Chem. Biol. 2008, 3, 486.
18. De Amici, M.; Dallanoce, C.; Holzgrabe, U.; Tränkle, C.; Mohr, K. Med. Res. Rev.
2010, 30, 463.
19. Chinese Hamster Ovary (CHO) cell line stably transfected with human S1P3-R,
nuclear factor of activated T-cell-beta lactamase (NFAT-BLA) reporter construct
and the Ga16 pathway coupling protein was used. Cells were cultured in T-
175 sq cm flasks at 37 °C and 95% relative humidity (RH). The growth medium
consisted of Dulbecco’s Modified Eagle’s Media containing 10% v/v heat
inactivated bovine growth serum, 0.1 mM NEAA, 1 mM sodium pyruvate,
Acknowledgments
25 mM HEPES, 2 mg/mL 5 mM
L
-glutamine, 0.2 mg/mL hygromycin B and 1Â
This work was supported by the National Institute of Health
Molecular Library Probe Production Center Grant U54 MH084512
(E.R., P.H. and H.R.) and AI074564 (M.O. and H.R.). We thank Mark
Southern for data management with PubChem; and Pierre Baillar-
geon for compound management efforts of the MLSMR.
penicillin-streptomycin. Prior to the start of the assay, cells were suspended to
a
concentration of 1.25 Â 106/mL in phenol red free Dulbecco’s Modified
Eagle’s Media containing 0.5% charcoal/dextran treated fetal bovine serum,
0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, and 5 mM L-glutamine.
The assay began by dispensing 10 mL of cell suspension to each test well of a
384 well plate. The cells were then allowed to incubate in the plates overnight
at 37 °C in 5% CO2 and 95% RH. The next day, 50 nL of test compound (50 lM
final concentration) in DMSO was added to sample wells, and DMSO alone (0.5
final concentration) was added to high control wells. Next, S1P prepared in 2%
BSA (0.7 micromolar final nominal concentration, corresponding to the EC80 of
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S1P) was added to the appropriate wells. After 4 h of incubation, 2.2 lL/well of
the GeneBLAzer fluorescent substrate mixture, prepared according to the
manufacturer’s protocol and containing 10 mM probenicid, was added to all
wells. The plates were then incubated for 2 h at room temperature. Plates were
read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at
an excitation wavelength of 405 nm and emission wavelengths of 535 and
460 nm.
20. Griffioen, G. WO2010/142801, 2010.
21. S1P3 jump-in CHO cells were cultured in T-175 sq cm flasks at 37 °C and 95%
RH. The growth media consisted of Dulbecco’s Modified Eagle’s Media (DMEM)
(with GlutaMAX) containing 10% v/v heat inactivated fetal bovine serum
(dialyzed), 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, and 1Â
penicillin–streptomycin–neomycin. On the day before the assay, cells were
suspended at a concentration of 0.2 Â 106/mL in the growth media and plated
at 0.1 Â 106/well in a 24-well plate. On the day of the assay, the growth
medium was replaced with serum-starvation medium consisted of DMEM
(with GlutaMAX) containing 0.1 mM NEAA, 1 mM sodium pyruvate, and no
antibiotics. The cells were incubated at 37 °C for 4 h. After 4 h of serum
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starvation, the medium was removed and the cells were rinsed with 200 lL of
ice-cold binding buffer. The binding buffer consisted of 20 mM Tris–HCl (pH
7.5), 100 mM NaCl, 15 mM NaF, 0.5 mM EDTA, 1 mM Na3VO4, 0.5% fatty acid-
free BSA, and 1Â protease inhibitor cocktail. Experiment 1: for the S1P3 agonist
competition assay, 30 lL of increasing concentrations of 5f (final 5f
concentrations of 0.001 nM to 10
containing [33P]S1P (final concentration of 0.1 nM) were added to the cells in
each well. Experiment 2: for the control assay, 30 of increasing
concentrations of cold S1P (final S1P concentrations of 0.001 nM to 10 M)
and 270
L ice-cold binding buffer containing [33P]S1P (final concentration of
lM) and 270 lL ice-cold binding buffer
l
L
l
l
0.1 nM) were added to the cells in each well. For both experiments, the cells
were incubated at 4 °C for 30 min. The cells were then washed three times with
500 lL ice-cold binding buffer. The cells were lysed with 300 lL 0.5% SDS and
transferred to scintillation vials. 5 mL of scintillation cocktail was dispensed
into each vial and the vial vortexed. The 33P radioactivity (cpm) was counted
for 5 min/vial in a Beckman LS 6000SC counter.