Structure-Activity Relationships and Mechanism of Action of Eph-ephrin Antagonists: Interaction of Cholanic Acid with the EphA2 Receptor

Article abstract of DOI:10.1002/cmdc.201200102

The Eph-ephrin system, including the EphA2 receptor and the ephrinA1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist that is able to inhibit EphA2 receptor activation; it is therefore potentially useful as a novel EphA2 receptor-targeting agent. Herein we explore the structure-activity relationships of a focused set of lithocholic acid derivatives based on molecular modeling investigations and displacement binding assays. Our exploration shows that while the 3-α-hydroxy group of lithocholic acid has a negligible role in recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrinA1 to EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits the EphA2-ephrinA1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand binding domain of EphA2, with a steady-state dissociation constant (K_D) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 as well as cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrinA1 ligand. These findings suggest that cholanic acid can be used as a template structure for the design of effective EphA2 antagonists, and may have potential impact in the elucidation of the role played by this receptor in pathological conditions.

Full text of DOI:10.1002/cmdc.201200102

MED
DOI: 10.1002/cmdc.201200102
Structure–Activity Relationships and Mechanism of Action
of Eph–ephrin Antagonists: Interaction of Cholanic Acid
with the EphA2 Receptor
Massimiliano Tognolini,[b] Matteo Incerti,[a] Iftiin Hassan-Mohamed,[b] Carmine Giorgio,[b]
Simonetta Russo,[a] Renato Bruni,[c] Barbara Lelli,[d] Luisa Bracci,[d] Roberta Noberini,[e]
Elena B. Pasquale,[e] Elisabetta Barocelli,[b] Paola Vicini,[a] Marco Mor,[a] and Alessio Lodola*[a]
The Eph–ephrin system, including the EphA2 receptor and the
ephrinA1 ligand, plays a critical role in tumor and vascular
functions during carcinogenesis. We previously identified
(3a,5b)-3-hydroxycholan-24-oic acid (lithocholic acid) as an
Eph–ephrin antagonist that is able to inhibit EphA2 receptor
activation; it is therefore potentially useful as a novel EphA2
receptor-targeting agent. Herein we explore the structure–ac-
tivity relationships of a focused set of lithocholic acid deriva-
tives based on molecular modeling investigations and dis-
placement binding assays. Our exploration shows that while
the 3-a-hydroxy group of lithocholic acid has a negligible role
in recognition of the EphA2 receptor, its carboxylate group is
critical for disrupting the binding of ephrinA1 to EphA2. As
a result of our investigation, we identified (5b)-cholan-24-oic
acid (cholanic acid) as a novel compound that competitively
inhibits the EphA2–ephrinA1 interaction with higher potency
than lithocholic acid. Surface plasmon resonance analysis indi-
cates that cholanic acid binds specifically and reversibly to the
ligand binding domain of EphA2, with a steady-state dissocia-
tion constant (KD) in the low micromolar range. Furthermore,
cholanic acid blocks the phosphorylation of EphA2 as well as
cell retraction and rounding in PC3 prostate cancer cells, two
effects that depend on EphA2 activation by the ephrinA1
ligand. These findings suggest that cholanic acid can be used
as a template structure for the design of effective EphA2 an-
tagonists, and may have potential impact in the elucidation of
the role played by this receptor in pathological conditions.
Introduction
The 14 erythropoietin-producing hepatocellular carcinoma
(Eph) receptors represent the largest family of receptor tyro-
sine kinases. The Eph receptors and their eight ephrin ligands
are divided into two subclasses, A and B, depending on their
affinities for one another and sequence homology. Generally,
EphA receptors (EphA1–A8 and EphA10) bind to glycosylphos-
phatidylinositol-anchored ephrinA ligands (ephrinA1–A5), while
the EphB receptors (EphB1–B4 and EphB6) interact with trans-
membrane ephrinB ligands (ephrinB1–B3), which have a short
cytoplasmic domain.[1]
sues. In fact, these proteins modulate cell movements in mor-
phogenetic processes such as gastrulation, segmentation, an-
giogenesis, axonal pathfinding, and neural crest cell migra-
tion.[4,5] Moreover, in the adult, they are involved in the mainte-
nance of cellular architecture in various epithelia[6] and play
key roles in neural plasticity[7] and regeneration of the adult
nervous system.[8]
[a] Dr. M. Incerti, S. Russo, Prof. P. Vicini, Prof. M. Mor, Dr. A. Lodola
Dipartimento Farmaceutico, Universitꢀ degli Studi di Parma
Viale delle Scienze 27/A, 43124 Parma (Italy)
EphA and EphB receptors have a similar modular structure,
consisting of a globular N-terminal ephrin binding domain, fol-
lowed by a cysteine-rich region and two fibronectin type III re-
peats in the extracellular region. The intracellular region is
composed of a juxtamembrane segment, a conserved tyrosine
kinase domain (responsible for signal transduction), a sterile
a motif (SAM) domain, and a PDZ binding motif, which serves
as a docking site for interacting signaling proteins.[2,3]
E-mail: alessio.lodola@unipr.it
[b] Dr. M. Tognolini, I. Hassan-Mohamed, Dr. C. Giorgio, Prof. E. Barocelli
Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche applicate
Universitꢀ degli Studi di Parma
Viale delle Scienze 27/A, 43124 Parma (Italy)
[c] Dr. R. Bruni
Dipartimento di Biologia Evolutiva e Funzionale
Universitꢀ degli Studi di Parma
Viale delle Scienze 11/A, 43124 Parma (Italy)
The result of membrane localization of both ephrins and
Eph receptors is their ability to transduce “reverse” signals into
the cells in which the ephrins are expressed, as well as a “for-
ward” signal into Eph receptor-expressing cells. As a conse-
quence, the Eph–ephrin signaling system is responsible for
modulation of several biological activities involving cellular
contact, both during embryonic development and in adult tis-
[d] Dr. B. Lelli, Prof. L. Bracci
Dipartimento di Biotecnologie, Universitꢀ degli Studi di Siena
Via Fiorentina 1, 53100 Siena (Italy)
[e] Dr. R. Noberini, Prof. E. B. Pasquale
Sanford-Burnham Medical Research Institute
10901 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200102.
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Increasing evidence supports the notion that the Eph–
ephrin system, including the EphA2 and EphB4 receptors,
plays a critical role in tumor and vascular functions during car-
cinogenesis. EphA2 in particular is overexpressed in many
types of tumors, such as breast, prostate, urinary, bladder, skin,
lung, ovary, and brain cancers[2] The modulation of EphA2 ac-
tivity by recombinant proteins, such as monoclonal antibodies
or soluble EphA receptor–Fc fusion proteins, has been shown
to block tumor growth, metastasis, and angiogenic processes
in animal models.[9] Moreover, genome-wide or kinome screens
for somatic mutations in cancer have identified mutations in
essentially all of the Eph receptors, suggesting that mutations
affecting Eph receptor function play a role in cancer initiation
or progression.[10–14] Therefore, the Eph–ephrin system is
emerging as a novel target for the development of antitumori-
genic and antiangiogenic therapies.[15]
The development of small molecules capable of blocking
the biological activity of EphA2 represents an attractive alter-
native to antibodies, peptides, and recombinant proteins.[16–19]
Few examples of EphA2 inhibitors targeting the intracellular
kinase domain have been recently reported in the literature.[20]
As these compounds block EphA2 activity by occupying the
ATP binding pocket, they suffer from lack of selectivity, which
limits their use as pharmacological tools in vivo. Conversely,
compounds acting on the extracellular ligand binding domain
of the Eph receptors have some advantages with respect to
standard tyrosine kinase inhibitors, as they can block Eph re-
ceptor activity without having to penetrate the cell and have
the potential to be more selective than ATP-mimicking
agents.[21]
Figure 1. EphA2 ligand binding domain (white ribbons with gray side chain
carbon atoms) in complex with ephrinA1 (red ribbons with orange side
chain carbon atoms). The crucial salt bridge between Arg103 (EphA2) and
Glu119 (ephrinA1) is shown.
The three-dimensional structure of the EphA2–ephrinA1
complex has recently been resolved by X-ray crystallography.[22]
The interaction between these two proteins is primarily medi-
ated by the amino-terminal ligand binding domain of EphA2,
which forms a large hydrophobic cavity able to accommodate
a protruding loop from ephrinA1 (G–H loop, Figure 1).[23] The
binding interface is dominated by van der Waals contacts be-
tween two predominantly hydrophobic surfaces and is rein-
forced by a few salt bridges, including the salt bridge between
EphA2 Arg103 and ephrinA1 Glu119 (Figure 1). Despite the
large binding interfaces in the EphA2–ephrinA1 complex, it has
been shown that peptides of moderate size (12 amino acids),
as well as small molecules, exemplified by salicylic acid deriva-
tives such as compound 76D10 (Figure 2), can prevent Eph re-
ceptor–ephrin interactions, possibly by occupying the same
EphA2 receptor cavity as the G–H loop of the physiological
ephrin ligands.[24]
Figure 2. Recently identified antagonists of the EphA2 receptor.
rounding and retraction induced by EphA2 stimulation with
ephrinA1. These results indicate that the lithocholic acid scaf-
fold can be used to design effective EphA2 antagonists.
We report herein the characterization of the structure–activi-
ty relationship (SAR) of (5b)-cholan-24-oic acid derivatives,
leading to the identification of compounds with improved
binding affinity. We performed molecular modeling studies to
identify the putative binding mode of lithocholic acid (com-
pound 1) within the high affinity ephrin binding pocket of the
EphA2 receptor. Starting from this theoretical model, a focused
set of lithocholic acid derivatives, either commercially available
or obtained by chemical synthesis, were examined for their
ability to disrupt EphA2–ephrinA1 binding. This led to the dis-
covery of cholanic acid, which was more potent and selective
than lithocholic acid in both EphA2 binding and EphA2 phos-
phorylation inhibition assays.
In our search for novel EphA2 receptor modulators, we re-
cently screened an in-house chemical library of naturally occur-
ring compounds, identifying the secondary bile acid (3a,5b)-3-
hydroxycholan-24-oic acid (lithocholic acid, Figure 2) as a non-
peptidic ligand of the Eph receptors.[25] Investigation of the
mechanism of action of lithocholic acid revealed that this com-
pound acts as a competitive antagonist of the EphA2 receptor
(Ki =49ꢀ3 mm). Furthermore, functional experiments showed
that lithocholic acid inhibits EphA2 autophosphorylation in
a dose-dependent manner and blocks PC3 prostate cancer cell
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tion), establishing van der Waals contacts with the hydropho-
bic surface of the receptor through the b methyl groups
emerging from positions 18 and 19 of lithocholic acid (Fig-
ure 3b). These minor rearrangements of the lithocholic acid
binding mode did not significantly affect the salt bridge
formed by the carboxylic group of lithocholic acid with
Arg103 of EphA2, which persisted throughout the simulation
(figure S2, Supporting Information). On the other hand, the in-
teraction between the 3-hydroxy group and Arg159 was lost
in the initial phase of the MD simulation, suggesting that the
contribution of this interaction to binding affinity may be neg-
ligible.
Results and Discussion
Molecular modeling
The recent solution of the crystal structure of the ligand bind-
ing domain of the EphA2 receptor in complex with the ephri-
nA1 ligand[22] allowed us to investigate the binding mode of
lithocholic acid to EphA2 by docking and molecular dynamics
(MD) simulations. The application of these computational tech-
niques allows us to generate working hypotheses on the rec-
ognition process involving a ligand and its receptor, aiding in
the design of structural analogues.[26–30]
Figure 3a shows the best solution with regard to interaction
energy (see Experimental Section) obtained by docking litho-
cholic acid within the high affinity ephrin binding pocket of
The simulation also showed that the aromatic ring of
Phe156 came into close contact with the a hydrogen atoms at
positions 7 and 12 of lithocholic acid (Figure 3b), suggesting
that there is limited space to in-
troduce larger substituents at
these positions. Together, these
analyses suggest that the hydro-
phobic core of lithocholic acid
can mimic the ephrin G–H loop
in its interaction with the EphA2
binding site, with the carboxylic
group of lithocholic acid being
a
fundamental component of
EphA2 binding. Furthermore, the
computational results also sug-
gest that the a-hydroxy group at
position 3 may not be essential
for the binding activity.
Structure–activity relationships
of lithocholic acid derivatives
Figure 3. a) Docking of lithocholic acid (LCA, green carbon atoms) in the high-affinity ephrin binding pocket of
the EphA2 receptor (white ribbons with gray side chain carbon atoms). The G–H loop of ephrinA1 is also dis-
played (red ribbons). b) EphA2–LCA complex obtained at the conclusion of the MD simulation. LCA carbon atoms
are shown in green and hydrogen atoms in white, with the exception of those at positions 7a and 12a, highlight-
ed in pink. The a and b faces of LCA are also highlighted. EphA2 carbon atoms are shown in gray, with white hy-
drogen atoms.
Based on the computational re-
sults reported above, 15 deriva-
tives of lithocholic acid (com-
pound 1) were chosen and
tested for their ability to inter-
fere with the EphA2–ephrinA1
the EphA2 receptor. The compound occupies the same space
as the ephrinA1 G–H loop, inserting its cyclopenta[a]perhydro-
phenanthrene scaffold into a hydrophobic Eph receptor chan-
nel. The pentanoic acid fragment, emerging from position 17
of the lithocholic acid core, forms a salt bridge with Arg103,
mimicking the interaction with Glu119 from ephrinA1. Finally,
the 3-hydroxy group of lithocholic acid weakly interacts with
Arg159 of EphA2, which is usually engaged in a hydrogen
bond with Asp86 of ephrinA1.
interaction (Table 1). Compounds 2–6 were selected in order to
explore the interaction between the lipophilic scaffold of litho-
cholic acid and the EphA2 binding site. Compounds 7–12 and
13–16 were selected to examine the role played by the two
polar ends of lithocholic acid. The experimental procedures
employed to synthesize and characterize these compounds are
reported in the Experimental Section.
The potency values corresponding to inhibition of the
EphA2–ephrinA1 interaction, as indicated by the Ki values re-
ported in Table 1, revealed that lithocholic acid derivatives are
particularly sensitive to modulation of the cyclopenta[a]perhy-
drophenanthrene scaffold. Indeed, the introduction of an a-hy-
droxy group at positions 7 or 12 consistently results in inactivi-
ty, as exemplified by the naturally occurring cholic acid (2), de-
oxycholic acid (3), and chenodeoxycholic acid (4).[25] Similarly,
introduction of a 6- or 7- keto group is detrimental to the
binding affinity (compounds 5 and 6).
To evaluate the stability of the proposed binding mode,
a 30-ns MD simulation was carried out, beginning from the
structure shown in Figure 3a. After a few nanoseconds of sim-
ulation, the lithocholic acid moved from its initial position to
one deeper inside the EphA2 binding site, projecting its a face
toward the side chain of Phe156 (Figure 3b). The lithocholic
acid hydrophobic core stably oscillated around this position
until the end of the simulation (figure S1, Supporting Informa-
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a sterically hindered substituent, the compound became inac-
tive, as in the case of the sulfonic acid derivative 11. The re-
moval of the a-hydroxy group at position 3 yielded compound
12 (cholanic acid), the most potent compound of the series
(Table 1 and Figure 4) which disrupts the EphA2–ephrinA1 in-
teraction with a Ki value of 5.1ꢀ1.4 mm. Together, these data
indicate that position 3 of lithocholic acid projects toward a hy-
drophobic cavity of limited size, consistent with the binding
model shown in Figure 3b.
Table 1. Structure–activity relationship data for lithocholic acid deriva-
tives.
Compd
R1
R2
R3
R4
R5
Ki [mm][a]
49ꢀ3.0
Finally, esterification (compound 13), conjugation with
amine derivatives (compounds 14 and 15), or reduction to the
corresponding alcohol (compound 16) of the lithocholic acid
carboxylic group yielded inactive or weakly active compounds,
indicating that the presence of a negatively charged group at
this position is critical for binding the EphA2 receptor.
1
2
3
>200
>200
>200
4
Inhibition of EphA2–ephrinA1 by cholanic and isolithocholic
acids
5
114 ꢀ13
Of the 16 (5b)-cholan-24-oic acid analogues examined
(Table 1), we further analyzed isolithocholic acid (compound
10) and cholanic acid (compound 12), which were found to be
more potent inhibitors of EphA2–ephrinA1 interaction than
lithocholic acid (compound 1). These compounds inhibited the
binding of the biotinylated ephrinA1–Fc ectodomain to the im-
mobilized EphA2–Fc-ectodomain in a dose-dependent manner
(Figure 4a). The IC50 value for isolithocholic acid was 67 mm,
while cholanic acid appeared to be the most potent derivative
of the series (IC50 =9.6 mm).
In addition to determining the IC50 values, we also deter-
mined the saturation curves for EphA2–ephrinA1 binding in
the presence of increasing concentrations of isolithocholic or
cholanic acid (Figure 4b,d). We calculated the KD or apparent
KD for each curve and drew a Schild plot, in which log[DRꢁ1]
is a function of the ꢁlog10 [inhibitor][31] (DR=dose ratio; Fig-
ure 4b,e). Both isolithocholic and cholanic acids yielded well-
interpolated regression lines (r2 =0.98 and 0.99, respectively)
with slopes of 0.96 and 0.98, respectively, indicating competi-
tive binding. The intersection of the interpolated line with the
X-axis gives a pKi value of 4.60 (corresponding to a Ki value of
25 mm) for isolithocholic acid and 5.19 (corresponding to a Ki
value of 5.1 mm) for cholanic acid. We next performed EphA2–
ephrinA1 displacement experiments by incubating immobi-
lized EphA2 with 100 mm isolithocholic acid or cholanic acid for
1 h, then washing selected wells before adding 50 ngmLꢁ1 bio-
tinylated ephrinA1–Fc. Displacement of biotinylated ephrinA1–
Fc binding was observed only in the wells that were not
washed, indicating that the binding of isolithocholic and chol-
anic acids to EphA2 is fully reversible (data not shown)
6
138ꢀ20
157ꢀ47
114 ꢀ14
88ꢀ11
7
8
9
10
11
12
13
25ꢀ4.0
>200
5.1ꢀ1.4
>200
14
>200
>200
15
16
186ꢀ27
[a] Values are the mean ꢀSE from a minimum of three independent ex-
periments.
Modification of the two opposite ends of the hydrophobic
core of lithocholic acid produced significant results. Oxidation
of the a-hydroxy group at position 3 (compounds 7 and 8)
and acetylation of this group (compound 9) yielded com-
pounds with lower affinity than lithocholic acid (compound 1).
By contrast, inversion of the chiral center at position 3 yielded
compound 10 ((3b,5b)-3-hydroxycholan-24-oic acid or isolitho-
cholic acid, Ki =25ꢀ4 mm), which inhibits EphA2–ephrinA1 in-
teraction with a potency similar to compound 1. However,
when the b-hydroxy group of compound 10 was replaced by
Selectivity of cholanic and isolithocholic acids for different
Eph receptors
We next examined the ability of cholanic and isolithocholic
acids to inhibit ephrin binding to all EphA and EphB receptors
using biotinylated ephrinA1–Fc and biotinylated ephrinB1–Fc,
respectively, at their KD concentrations. In contrast to lithochol-
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region within the EphA receptor
ligand binding pocket that is es-
sential for the recruitment of
ephrin ligands.
Surface plasmon resonance
analysis of the binding of
cholanic acid to Eph receptors
To further characterize the mech-
anism of action of cholanic acid,
we investigated the properties
of its binding to the EphA2 re-
ceptor and other proteins using
a
surface plasmon resonance
(SPR) assay, implemented with
Biacore technology.[32] The dis-
solved compound was injected
over EphA2–Fc immobilized to
surfaces attached to an optical
biosensor surface, and binding
was determined based on the
change in mass at the sensor
surface.[33] The change in mass
depends linearly on the number
of molecules bound; thus, SPR is
a quantitative technique. Follow-
ing injection, running buffer was
allowed to flow over the surface,
and dissociation of cholanic acid
from the surface was observed.
This assay enabled assessment
of how cholanic acid associates
and dissociates from EphA2 in
real time, producing association/
dissociation rate constants (kon,
koff) as well as dissociation equi-
librium constants (KD).
^
Figure 4. Isolithocholic and cholanic acid competitively inhibit EphA2–ephrinA1 binding: a) Lithocholic ( ), isoli-
~
&
thocholic ( ) and cholanic acid ( ) displace ephrinA1–Fc from the immobilized EphA2–Fc ectodomain in a dose-
dependent manner. b),d) Binding of ephrinA1–Fc to immobilized EphA2–Fc in the presence of various concentra-
!
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*
*
tions of isolithocholic [(0 ( ), 12.5 ( ), 25 ( ), 50 ( ), 100 ( ), and 200 mm (ꢁ)] or cholanic acid [(0 ( ), 3 (+), 6 ( ),
As reported by SPR sensor-
grams (Figure 6), cholanic acid
was shown to bind the immobi-
lized EphA2 receptor in a concen-
tration-dependent manner. The
binding was saturable and well-
fitted by a 1:1 binding interac-
tion model, confirming that the
recognition process is specific.
!
^
12.5 ( ), and 25 mm ( )]. c),e) Dissociation constants (KD) from the displacement experiments shown in panels b
and d were used to calculate log[DRꢁ1] and to graph the Schild plots for isolithocholic (slope=0.96ꢀ007) or
cholanic acid (slope=0.98ꢀ002); pKi values were estimated by the intersection of the interpolated line with the
x axis. The slope of the interpolated line can be related to the nature of the binding. A slope between 0.8 and 1.2
indicates competitive binding, whereas a higher slope suggests nonspecific interactions (Ki =25ꢀ4 mm for isoli-
thocholic acid; Ki =5.1ꢀ1.4 mm for isolithocholic acid).
ic acid, which we recently demonstrated to be a promiscuous
ligand of all Eph receptors, cholanic and isolithocholic acids
demonstrated higher selectivity for the EphA receptor subfami-
ly. Particularly, cholanic acid displayed IC50 values for the EphA
receptors that were 3–30-fold lower than those calculated for
the EphB receptors (Figure 5). As cholanic acid was able to in-
hibit ephrin ligand binding to all members of the EphA recep-
tor subfamily in the low micromolar range (3.0–7.1 mm), this
suggests that cholanic acid interferes with the Eph receptor–
ephrin recognition process by occupying a highly conserved
The association between EphA2 and cholanic acid was reversi-
ble, as the protein-compound complex readily dissociated, re-
storing the baseline signal (Figure 6).
Kinetic analysis revealed good binding parameters for chol-
anic acid. From the sensorgrams, it was possible to determine
an association rate (kon) of 4.4ꢁ104 mꢁ1 sꢁ1 and a dissociation
rate (koff) of 3.69ꢁ10ꢁ1 sꢁ1, corresponding to an affinity con-
stant (KD) of 8.45ꢁ10ꢁ6 m. This was consistent with the KD
value of 1.16ꢁ10ꢁ6 m obtained from steady-state analysis (i.e.,
by plotting the binding at equilibrium versus the ligand con-
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tein (figure S4, Supporting Infor-
mation). Remarkably, cholanic
acid binds the EphA2 receptor at
this
same
concentration
(Figure 6).
Cholanic and isolithocholic
acids inhibit Eph receptor tyro-
sine phosphorylation at non-
cytotoxic concentrations
To evaluate the functional effects
of cholanic and isolithocholic
acids on Eph receptors, we per-
formed phosphorylation studies
using PC3 human prostate ade-
nocarcinoma cells, which endo-
genously express the EphA2 re-
ceptor, and T47D human mam-
mary carcinoma cells, which en-
dogenously express the EphB4
receptor. Similar to lithocholic
acid, the two compounds did
not stimulate Eph receptor tyro-
sine phosphorylation (activation)
on their own (data not shown).
However, they inhibited EphA2
and EphB4 phosphorylation in-
duced by ephrinA1–Fc or eph-
rinB2–Fc, respectively, in a dose-
dependent manner (Figure 7).
The multikinase inhibitor dasati-
nib (1 mm), used as a control,
completely blocked EphA2 phos-
phorylation (data not shown).
Figure 5. Lithocholic acid derivatives partially discriminate between EphA and EphB receptor subclasses: a) Isoli-
thocholic acid and b) cholanic acid displace the binding of ephrinA1–Fc and the ephrinB1–Fc ectodomain from
immobilized EphA–Fc or EphB–Fc ectodomains, respectively, in a dose-dependent manner. Tested concentrations:
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3 mm ( ), 10 mm ( ), 30 mm ( ), and 100 mm ( ). IC50 values are the means from at least three independent experi-
ments; error bars represent standard errors.
According to binding data, cholanic acid inhibited Eph recep-
tor activation induced by ephrins more potently than lithochol-
ic acid, with IC50 values of 12 mm (EphA2) and 38 mm (EphB4)
compared with 46 and 74 mm for lithocholic acid.
Interestingly, despite the similarity between the lithocholic
and isolithocholic acid binding profiles, isolithocholic acid was
a more potent inhibitor of EphA2 and EphB4 phosphorylation
in cells, with IC50 values of 17 mm (EphA2) and 71 mm (EphB4).
This suggests an additional inhibitory effect of isolithocholic
acid on the intracellular kinase domain (see below). Compound
concentrations which inhibited receptor tyrosine phosphoryla-
tion were not cytotoxic after 2 h incubation with cells (fig-
ure S5, Supporting Information).
Figure 6. SPR sensorgrams for the interaction of cholanic acid with immobi-
lized EphA2–Fc on sensor chips. The colored lines denote different cholanic
acid concentrations: 3 mm (orange), 6 mm (green), 12.5 mm (pink), and 25 mm
(maroon).
Cholanic acid does not inhibit EphA2 kinase activity
centration and assuming that the KD equals the concentration
which yields 50% of the maximum response,[34] figure S3, Sup-
porting Information).
To rule out the theory that the observed inhibition of EphA2
phosphorylation by cholanic acid was due to a direct interac-
tion with the EphA2 kinase domain, the recombinant EphA2
kinase domain was incubated in the presence of a peptide
substrate, with or without 100 mm cholanic acid. The levels of
phosphorylated peptide were detected with a europium-la-
Finally, SPR analysis was applied to assess the specificity of
cholanic acid for the EphA2 receptor relative to other members
of the Eph–ephrin signaling system. This showed that cholanic
acid at 6 mm does not bind the EphB1 receptor or the Fc-pro-
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Figure 8. Cholanic acid does not inhibit EphA2 kinase activity. The enzymatic
activity of the recombinant human EphA2 kinase domain was evaluated
with the LANCE method using ATP and Ulight-TK peptide as substrate.
EphA2 was incubated for 30 min with the indicated compounds at concen-
trations of 100 mm, 1 mm staurosporine, or 1% DMSO as a control; **p<0.01
relative to control by one-way ANOVA, followed by Tukey’s multiple compar-
ison test.
~
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*
Figure 7. Lithocholic ( ), isolithocholic ( ), and cholanic acid ( ) inhibit Eph
receptor phosphorylation in a dose-dependent manner: Inhibition of
a) EphA2 phosphorylation and b) EphB4 phosphorylation. EphA2 phosphory-
lation was induced by 0.25 mgmLꢁ1 ephrinA1–Fc in PC3 cells, whereas
EphB4 phosphorylation was induced with 3 mgmLꢁ1 ephrinB2–Fc, pre-clus-
tered with 0.3 mgmLꢁ1 anti-Fc antibodies in T47D cells. Cells were pretreated
for 20 min with 1% DMSO or the indicated concentrations of compounds
and then stimulated for 20 min with ephrinA1/B2–Fc. Data are the means
ꢀSE of at least three independent experiments.
Figure 9. Isolithocholic and cholanic acids do not affect EGF receptor activi-
ty. PC3 cells were pretreated for 20 min with 100 mm lithocholic, isolithochol-
ic, or cholanic acid, 10 mm gefitinib, or 1% DMSO as a control, and were
stimulated for 20 min with 30 ngmLꢁ1 EGF. Phospho-EGF receptor levels are
relative to the EGF+DMSO condition. Data are the means ꢀSE of at least
three independent experiments; **p<0.01 relative to EGF+DMSO by one-
way ANOVA, followed by Tukey’s multiple comparison test.
beled antiphosphotyrosine antibody. Cholanic acid did not
affect EphA2 kinase activity, confirming that the effect in cells
is due to inhibition of Eph–ephrin protein–protein interaction.
In contrast, isolithocholic acid significantly decreased EphA2
kinase activity (Figure 8), explaining the unexpectedly high po-
tency of this compound in inhibiting EphA2 phosphorylation
induced by ephrin stimulation. On the other hand, the general
kinase inhibitor staurosporine fully inhibited the kinase activity
of EphA2 at 100 mm (Figure 8).
The specificity of lithocholic acid as an Eph receptor antago-
nist was previously determined by demonstrating the lack of
effects on other receptor tyrosine kinases, such as the EGF re-
ceptor, the VEGF receptor, the insulin receptor, and the insulin-
like growth factor receptor 1.[25] Similar to lithocholic acid, chol-
anic and isolithocholic acids also did not interfere with EGF re-
ceptor activation induced by EGF at concentrations up to
100 mm (Figure 9).
Cholanic and isolithocholic acids inhibit EphA2-mediated
cell retraction in PC3 cells
Cholanic and isolithocholic acids inhibited EphA2-mediated
cell retraction and rounding of PC3 cells stimulated with ephri-
nA1–Fc at concentrations as low as 25 mm (Figure 10), suggest-
ing that these compounds can be used to counteract the func-
tional effects mediated by EphA2.[35] As expected, isolithocholic
and cholanic acids inhibited cell retraction at concentrations
similar to those required to inhibit EphA2 phosphorylation.
None of the compounds affected cell morphology in the ab-
sence of ephrinA1 stimulation (Figure 10), confirming their lack
of toxicity.
Conclusions
The identification of small molecules able to disrupt protein–
protein interfaces is a challenging task. Complications include
the presence of wide protein–protein interacting surfaces,
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hydrogen bond acceptor group (represented by a carboxylate
functionality) are pivotal for effective disruption of EphA2–eph-
rinA1 binding, consistent with the predicted binding mode for
the EphA2–lithocholic acid complex. Notably, SPR experiments
indicated that cholanic acid interacts with the ligand binding
domain of the EphA2 receptor, in agreement with our working
hypothesis. SPR was also used to kinetically characterize the
binding of cholanic acid to EphA2, yielding a steady-state
binding constant in the low micromolar range (KD =1.16ꢁ
10ꢁ6 m).
Cholanic acid competitively displaces biotinylated ephrinA1
from the EphA2 receptor. Indeed, the shift in EphA2–ephrinA1
saturation curves obtained with increasing concentration of
cholanic acid produces a Schild plot consistent with competi-
tive antagonism. The inhibitory activity of cholanic acid toward
EphA2 was also confirmed by cell-based assays, where the ad-
dition of compound inhibited the ephrinA1-dependent tyro-
sine phosphorylation of EphA2 and retraction in PC3 cells in
a dose-dependent manner. Cholanic acid is less potent in
blocking the ephrinB1-dependent phosphorylation of EphB4,
paralleling the results obtained in the in vitro displacement
assay. Furthermore, cholanic acid has no effect on the EphA2
kinase domain, which is weakly inhibited by isolithocholic acid.
The reasonable binding affinity of cholanic acid, together
with its ability to block EphA2 activity in cell lines, supports
the notion that the (5b)-cholan-24-oic acid scaffold can be
used as a template structure to design an improved genera-
tion of EphA2 inhibitors. On the other hand, cholanic acid suf-
fers from high lipophilicity, which may hamper its bioavailabil-
ity in vivo. However, bile acid derivatives are known to be
a good reservoir of biologically active compounds, as in the
case of obeticholic acid (INT-747).[36,37] This farnesoid X receptor
(FXR) agonist has recently advanced to phase III clinical trials
for the treatment of chronic liver diseases (clinical trial
NCT00570765, study of INT-747 as monotherapy in patients
with primary biliary cirrhosis). Thus, a lead optimization pro-
gram aimed at the improvement of the physicochemical prop-
erties of cholanic acid may yield a small molecule able to effec-
tively inhibit the activity of EphA2 in vivo.
Figure 10. Inhibition of EphA2-dependent retraction and rounding of PC3
prostate cancer cells: a) Dose–response data for isolithocholic acid (isoLCA)
&
&
in the presence of ephrinA1–Fc ( ) using Fc ( ) as a control. b) Dose–re-
&
sponse data for cholanic acid in the presence of ephrinA1–Fc ( ) using Fc
&
( ) as a control. Histograms of panels a) and b) show the average percent-
age of retracting cells. Cells with a rounded shape and decreased spreading
were scored as retracting. The percentages of cell retraction under various
conditions were compared with those under Fc control conditions by one-
way ANOVA and Dunnett’s post test. c) Effects on cell morphology: PC3
cells, pretreated for 15 min with the indicated concentrations of isoLCA or
cholanic acid, were stimulated with 0.5 mgmLꢁ1 ephrinA1–Fc (+) using Fc as
a control (ꢁ) for 20 min in the continued presence of compounds. Cells
were stained with rhodamine–phalloidin to label actin filaments (red) and
4’,6-diamidino-2-phenylindole (DAPI) to label nuclei (blue); DMSO was used
as a control.
which lack deep cavities where small molecules can bind with
good affinity.[21] The ephrin binding pocket of the EphA2 recep-
tor, however, seems to present favorable features for high af-
finity binding of small molecules, as shown here and in other
recent papers.[24,35] In the present work, we report the discov-
ery of a small molecule, cholanic acid ((5b)-cholan-24-oic acid),
which binds the ligand binding domain of the EphA2 receptor
with an affinity in the low micromolar range. This compound
was identified through a focused medicinal chemistry effort
aimed at the optimization of lithocholic acid, a weak antago-
nist of the Eph–ephrin system that was recently discovered by
our group.[25]
Experimental Section
Molecular modeling
Molecular modeling simulations were performed, beginning from
the crystal structure of the EphA2–ephrinA1 complex (3HEI.pdb),[22]
using Maestro software[38] and OPLS2005 force field.[39] The EphA2–
ephrinA1 complex was submitted to a protein preparation proce-
dure which includes addition of missing side chains and hydrogen
atoms, assignment of the tautomeric state of histidine residues to
maximize the number of hydrogen bonds, and geometric optimi-
zation of the entire system to a root-mean-square displacement
(RMSD) value of 0.3 ꢂ.[40] At the end of this procedure, the ephri-
nA1 ligand was deleted from the EphA2 active site. A molecular
model of lithocholic acid (1) was also built using Maestro, and its
geometry was optimized by energy minimization using OPLS2005
to a gradient of 0.01 kcal(mol ꢂ)ꢁ1. Docking simulations were then
performed using Glide5.5,[41] beginning with placement of the
minimized structure of lithocholic acid in an arbitrary position
A computationally driven exploration of lithocholic acid de-
rivatives allowed us to build a clear structure–activity relation-
ship profile and identify the stereoelectronic requirements for
EphA2 binding. In particular, we found that the simultaneous
presence of a large hydrophobic region (represented by the
cyclopenta[a]perhydrophenanthrene scaffold) and an anionic
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Eph–ephrin Antagonists
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within a region centered on the surface of an EphA2 channel, de-
limited by Arg103, Phe156 and Arg159, using enclosing and
bounding boxes of 20 and 14 ꢂ on each side, respectively. van der
Waals radii of the protein atoms were not scaled, while van der
Waals radii of the ligand atoms with partial atomic charges lower
than j0.15j were scaled by 0.8. Standard precision mode was ap-
plied. The resulting binding poses were ranked according to the G-
score, and the best docking solution was selected for MD simula-
tions. The selected EphA2–lithocholic acid docking complex was
1) solvated by ~14000 SPC water molecules in a simulation box of
78ꢁ78ꢁ78 ꢂ, 2) neutralized by adding 5Na+ ions, and 3) equili-
brated by 30 ns of MD simulations. The simulation was performed
in the NPT ensemble under a constant pressure of 1 atm and tem-
perature of 300 K. All bond lengths to hydrogen atoms were con-
strained using M-SHAKE.[42] Short-range electrostatic interactions
were cut off at 9 ꢂ, whereas long-range electrostatic interactions
were computed using the Particle Mesh Ewald method.[43] A RESPA
integrator[44] was used with a time-step of 2 fs, and long-range
electrostatics were computed every 6 fs. Snapshots were saved
every 10 ps, for a total of 3000 structures. The MD simulation was
performed with the OPLS2005 force field, using Desmond package
v22623.[45]
(3a,5b)-3-Acetoxycholan-24-oic acid (9): A modification of a de-
scribed procedure[46] was used (scheme S1, Supporting Informa-
tion) in which lithocholic acid (1) (2.654 mmol) and 4-dimethylami-
nopyridine (DMAP) (0.409 mmol) were dissolved in anhydrous pyri-
dine (10 mL). Acetic anhydride (21.58 mmol) was added dropwise
to this solution, and the reaction mixture was stirred at room tem-
perature under nitrogen for 3 h. Ice and water were added, then
the mixture was acidified with concentrated HCl, and the white
precipitate was removed by filtration and washed with water. The
resulting solid was purified by flash chromatography [SiO2, CH2Cl2/
HCOOH/C2H5OH, 89:1:10 (300 mL)]. The crude product was recrys-
tallized from EtOH and water to give 9 as a white powder (0.730 g,
1
65%): H NMR (400 MHz [D6]DMSO): d=0.60 (s, 3H, CH3), 0.86 (d,
3H, J=6.4 CH3); 0.89 (s, 3H, CH3); 0.96–1.67 (m, 26H); 1.72–1.83
(m, 4H); 1.90–1.93 (m, 1H); 1.95 (s, 3H, CH3), 2.04–2.12 (m, 1H),
2.18–2.25 (m, 1H), 4.58–4.54 (m, 1H), 11.96 ppm (brs, 1H, OH);
13C NMR (100 MHz [D6]DMSO): d=12.30, 18.57, 20.88, 21.48, 23.45,
24.26, 26.40, 26.70, 27.04, 28.16, 31.14, 32.33, 34.60, 34.96, 35.26,
35.75, 41.63, 42.72, 56.02, 56.38, 73.89, 170.14, 175.25 ppm; MS
(ESI) calcd for C26H42O4: 418.61, found: 417 [Mꢁ1]ꢁ.
Methyl (3a,5b)-3-hydroxycholan-24-oate (13): Compound 13 was
synthesized following a literature protocol[47] (scheme S2, Support-
ing Information) in which concentrated sulfuric acid (0.5 mL) was
added to a stirred suspension of lithocholic acid (1) (3.04 mmol) in
MeOH (15 mL). The reaction mixture was stirred at room tempera-
ture for 3 h, then diluted with water. The resulting white precipi-
tate was removed by filtration under vacuum and was washed
with water. The crude product was recrystallized from EtOH and
water to give 13 as a colorless solid (1.118 g, 94%): 1H NMR
(400 MHz CDCl3): d=0.60 (s, 3H, CH3); 0.85–0.91 (m, 6H), 1.02–1.98
(m, 28H), 2.17–2.36 (m, 2H), 3.57–3.64 (m, 1H), 3.67 ppm (s, 3H,
CH3); 13C NMR (100 MHz CDCl3): d=12.04, 18.26, 20.82, 23.38,
24.20, 26.42, 27.20, 28.19, 30.54, 31.00, 31.06, 34.57, 35.36, 35.84,
36.45, 40.17, 40.43, 42.10, 42.73, 43.73, 51.48, 55.95, 56.49, 71.84,
174.79 ppm; MS (ESI) calcd for C25H42O3: 390.59, found: 413 [M+
Na+]+.
Analysis of the MD trajectory was performed by evaluating the
root mean square deviation (RMSD) of the EphA2 receptor and lith-
ocholic acid, using the first frame of the production phase as a ref-
erence structure. For EphA2, the RMSD was measured with regard
to the Ca of the amino acid residue, while for lithocholic acid, the
RMSD was measured while only accounting for its heavy atoms
after their optimal superposition. The interaction between the criti-
cal Arg103 of EphA2[22] and lithocholic acid was evaluated by plot-
ting the shortest of six possible distances between the three nitro-
gen atoms of the guanidinium group of Arg103 and the two
oxygen atoms of the carboxylic group of lithocholic acid for each
snapshot recorded during the simulation.
Chemistry
Unless otherwise noted, reagents and solvents were purchased
from commercial suppliers (Aldrich and Fluka) and were used with-
out purification. Melting points were determined on a Gallenkamp
melting point apparatus and were not corrected. Final compounds
1–16 were analyzed on a ThermoQuest (Italia) FlashEA 1112 Ele-
mental Analyzer for C, H, and N. The percentages found were
Methyl (3b,5b)-3-benzoyloxycholan-24-oate (10a): Compound
10a was synthesized using a modification of a described proce-
dure[48] (scheme S3, Supporting Information) in which triphenyl-
phosphine (0.648 mmol) was dissolved in anhydrous THF (5 mL)
and cooled to 08C. A solution of diisopropyl azodicarboxylate
(DIAD) (0.637 mmol) in anhydrous THF (1 mL) was added dropwise
to the stirred solution under nitrogen, maintaining the tempera-
ture at 08C. After the addition was complete, the reaction mixture
1
within ꢀ0.4% of the theoretical values. H NMR and 13C NMR spec-
tra were recorded on
a Bruker Avance 400 spectrometer
(400 MHz); chemical shifts (d scale) are reported in parts per mil-
lion (ppm). 1H NMR spectra are reported in the following order:
multiplicity, number of protons and approximate coupling con-
stants (J value) in Hertz (Hz); signals are characterized as s (singlet),
d (doublet), t (triplet), m (multiplet), brs (broad signal). Mass spec-
tra were recorded on an Applied Biosystem API-150 EX system
spectrometer with an ESI interface. Progress of reactions was moni-
tored by thin-layer chromatography with F254 silica gel pre-coated
sheets (Merck KGaA, Darmstadt, Germany). UV light and potassium
permanganate solution (10% w/v) were used for detection. Flash
chromatography was performed using Merck silica gel 60 (Si 60,
40–63 mm, 230–400 mesh ASTM). Tetrahydrofuran (THF) was dried
by distillation over Na/benzophenone. All reactions were carried
out using flame-dried glassware under a nitrogen atmosphere.
Compounds 1–8 and 12 were purchased from Sigma and charac-
terized by elemental analysis (see table S1, Supporting Informa-
tion). Compounds 9–11 and 13–16 were synthesized according to
the procedures described below.
was warmed to room temperature and
a solution of 13
(0.510 mmol) and benzoic acid (0.510 mmol) in anhydrous THF
(5 mL) was added dropwise. After stirring overnight at room tem-
perature, the solvent was removed under reduced pressure, and
the residue was purified by flash chromatography [SiO2, CH2Cl2/n-
hexane from 90:10 (200 mL) to 100% CH2Cl2 (100 mL)]. The crude
product was recrystallized from EtOH and water to give 10a as
1
a white powder (0.245 g, 97%): H NMR (400 MHz CDCl3): d=0.66
(s, 3H, CH3), 0.92 (d, 3H, J=6.4 CH3), 1.02–1.21 (m, 9H), 1.27–1.45
(m, 9H), 1.73–2.10 (m, 11H), 2.18–2.26 (m, 1H), 2.32–2.40 (m, 1H),
3.67 (s, 3H, CH3), 5.34 (s, 1H, OH), 7.44 (t, 2H, J=7.6 Ar), 7.55 (t,
1H, J=7.4 Ar), 8.05 (d, 2H, J=8.4 Ar); 13C NMR (100 MHz CDCl3):
d=12.08, 18.29, 21.14, 24.04, 24.20, 25.22, 26.19, 26.57, 28.19,
30.80, 31.02, 31.07, 31.09, 35.00, 35.38, 35.70, 37.74, 39.96, 40.19,
42.78, 51.48, 55.99, 56.57, 71.40, 121.31 129.51, 131.19, 132.68,
165.92, 174.77 ppm; MS (ESI) calcd for C32H46O4: 494.70, found: 517
[M+Na+]+.
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(3b,5b)-3-Hydroxycholan-24-oic acid (10): Compound 10 was syn-
thesized following a modification of a described procedure[49]
(scheme S4, Supporting Information) in which a solution of NaOH
(15% w/v, 50 mL) was added to a solution of 10a (1.96 mmol) in
EtOH (75 mL), and the mixture was stirred at reflux overnight.
EtOH was removed under vacuum, and the solution was acidified
with concentrated HCl until a precipitate formed. The resulting sus-
pension was extracted with CH2Cl2 (3ꢁ100 mL), and the organic
extracts were washed with water and brine and dried over anhy-
drous Na2SO4. Evaporation of solvent under reduced pressure yield-
ed a white solid that was purified by flash chromatography [SiO2,
CH2Cl2/HCOOH/C2H5OH from 99.37:0.03:0.6 (100 mL) to 97.50:0.5:2
(150 mL)]. The crude product was recrystallized from EtOH and
(3b,5b)-3-Sulfocholan-24-oic acid (11): A peracetic acid solution
(40% w/w, 4.16 mmol) was added dropwise to a stirred solution of
11 b (1.27 mmol) under nitrogen at 08C (Scheme S7). The mixture
was allowed to warm to room temperature while stirring for 4 h.
Evaporation of the solvent under reduced pressure afforded
a white solid that was purified by flash chromatography [SiO2,
CH2Cl2/HCOOH/C2H5OH from 83:7:10 (250 mL) to 73:7:20 (200 mL)].
The crude product was recrystallized from EtOH and water to give
11 as a white amorphous solid (0.354 g, 63%): mp: 285–2888C;
1H NMR (400 MHz [D6]DMSO): d=0.60 (S, 3H), 0.84–0.86 (m, 6H),
1.01–1.90 (m, 30H), 2.15–2.20 (m, 1H), 2.25–2.29 (m, 1H), 2.54–
2.58 ppm (m, 1H); 13C NMR (100 MHz [D6]DMSO): d=12.32, 18.57,
21.03, 21.37, 23.97, 24.28, 26.20, 26.74, 26.85 28.15, 31.06, 31.09,
31.56, 34.54, 35.23, 35.65, 36.36, 40.20, 42.74, 54.35, 55.94, 56.02,
56.56, 60.10, 173.70 ppm; MS (ESI) calcd for C24H40O5S: 440.64,
found: 439 [Mꢁ1]ꢁ; Anal. calcd for C24H40O5S·1.4H2O: C 61.61, H
9.65; found: C 61.85, H 9.62.
1
water to give 10 as a white solid (1.02 g, 83%): H NMR (400 MHz
[D6]DMSO): d=0.60 (s, 3H, CH3); 0.85–0.91 (m, 6H), 1.02–1.98 (m,
28H), 2.17–2.36 (m, 2H), 3.57–3.64 (m, 1H), 3.67 ppm (s, 3H, CH3);
13C NMR (100 MHz [D6]DMSO): d=12.04, 18.26, 20.82, 23.38, 24.20,
26.42, 27.20, 28.19, 30.54, 31.00, 31.06, 34.57, 35.36, 35.84, 36.45,
40.17, 40.43, 42.10, 42.73, 43.73, 51.48, 55.95, 56.49, 71.84
174.79 ppm; MS (ESI) calcd for C24H40O3: 376.57, found: 375
[Mꢁ1]ꢁ.
(3a,5b)-3-Hydroxycholan-24-hydroxamic acid (14): Compound 14
was synthesized following a modification of a described proce-
dure[51] (scheme S8, Supporting Information) in which KOH
(12.97 mmol) dissolved in MeOH (4 mL) was added dropwise to
a solution of hydroxylamine hydrochloride (6.48 mmol) in MeOH
(4 mL) under nitrogen at 08C. The mixture was stirred for a further
20 min, then a solution of 13 (0.64 mmol) in MeOH (7 mL) was
added dropwise while maintaining the reaction temperature at
08C. The reaction mixture was then warmed to room temperature
and stirred for 4 h. Finally, the mixture was diluted with water,
cooled, and acidified with 6n HCl to afford a white precipitate. The
solid was removed by filtration under vacuum and purified by
flash chromatography [SiO2, CH2Cl2/HCOOH/C2H5OH from
94,50:0,5:5 (200 mL) to 82:8:10 (150 mL)]. The crude product was
recrystallized from EtOH and water to give 14 as a reddish amor-
phous solid (0.140 g, 56%): mp: 169–1738C; 1H NMR (400 MHz
[D6]DMSO): d=0.59 (s, 3H, CH3), 0.86–0.91 (m, 6H), 1.01–1.92 (m
27H), 3.52–3.59 (m, 1H), 4.42 (s, 1H, OH), 4.42, 8.62 (s, 1H, NH),
10.30 ppm (s, 1H, OH); 13C NMR (100 MHz [D6]DMSO): d=12.36,
18.69, 20.88, 23.74, 24.31, 26.63, 27.36, 28.18, 29.67, 30.85, 31.90,
34.67, 35.33, 35.62, 35.85, 36.76, 41.99, 42.73, 56.00, 56.54, 70.32,
169.94 ppm; MS (ESI) calcd for C24H41NO3: 391.58, found: 390
[Mꢁ1]ꢁ.
Methyl (3b,5b)-S-acetyl-3-mercaptocholan-24-oate (11a): Triphe-
nylphosphine (0.648 mmol) was dissolved in anhydrous THF (5 mL)
and cooled to 08C (scheme S5, Supporting Information). A solution
of DIAD (0.637 mmol) in anhydrous THF (1 mL) was added drop-
wise to the stirred solution under nitrogen, maintaining the tem-
perature at 08C. After addition was complete, the reaction mixture
was warmed to room temperature, and
a solution of 13
(0.510 mmol) and thioacetic acid (1.02 mmol) in anhydrous THF
(5 mL) was added dropwise. After stirring overnight at room tem-
perature, the solvent was removed under reduced pressure, and
the residue was purified by flash chromatography [SiO2, CH2Cl2/n-
hexane, from 50:50 (300 mL) to 90:10 (100 mL)]. The crude product
was recrystallized from EtOH and water to give 11 a as a white
1
powder (0.148 g, 65%): mp: 128–1318C; H NMR (400 MHz CDCl3):
d=0.63 (s, 3H, CH3), 0.89–0.93 (m, 6H); 1.03–1.67 (m, 21H); 1.77–
1.96 (m, 5H, CH3); 2.19–2.26 (m, 1H); 2.29 (s, 3H, CH3); 3.65(s, 3H,
CH3), 4.09 ppm (s, 1H); 13C NMR (100 MHz CDCl3): d=12.05, 18.27,
20.96, 23.90, 24.17, 26.33, 26.38, 26.85, 28.17, 30.97, 31.00, 31.04,
32.01, 32.99, 35.21, 35.36, 35.72, 39.52, 40.13, 40.24, 42.73, 42.79,
51.48, 55.94, 56.49, 174.76, 195.77 ppm; MS (ESI) calcd for
C27H44O3S: 448.70, found: 471 [M+Na+]+; Anal. calcd for
C27H44O3S: C 72.27, H 9.88; found: C 72.49, H 9.53.
(3a,5b)-3-Hydroxycholan-24-hydrazide (15): Compound 15 was
synthesized following a modification of a described procedure[52]
(scheme S9, Supporting Information) in which hydrazine monohy-
drate (103 mmol) was added dropwise to
a solution of 13
(0.768 mmol) in MeOH (10 mL), and the reaction mixture was
stirred for 6 h at room temperature. The reaction was diluted with
water, and the resulting white precipitate was removed by filtra-
tion under vacuum and washed with water. The resulting white
solid was recrystallized from EtOH and water to give 15 (0.288 g,
96%): mp: 201–2068C; 1H NMR (400 MHz [D6]DMSO): d=0.59 (s,
3H, CH3), 0.85–0.91 (m, 6H), 1.01–2.04 (m 27H), 3.52–3.59 (m, 1H),
4.11 (s, 2H, NH2), 4.42 (d, 1H, J=4.4 OH), 8.88 ppm (s,1H, NH);
13C NMR (100 MHz [D6]DMSO): d=12.37, 18.76, 20.90, 23.71, 24.30,
26.63, 27.40, 28.12, 30.91, 31.99, 34.71, 35.38, 35.68, 35.92, 36.85,
42.09, 42.79, 56.14, 56.61, 70.38, 172.46 ppm; MS (ESI) calcd for
C24H42N2O2: 390.60, found: 389 [Mꢁ1]ꢁ.
(3b,5b)-3-Mercaptocholan-24-oic acid (11b): Compound 11 was
synthesized following a modification of a described procedure[50]
(scheme S6, Supporting Information) in which a solution of NaOH
(15% w/v, 35 mL) was added to a solution of compound 11 a
(1.55 mmol) in EtOH (60 mL) and the mixture was held at reflux for
2 h under nitrogen. EtOH was removed under vacuum, and the so-
lution was acidified with concentrated HCl until a precipitate
formed. The resulting suspension was extracted with CH2Cl2 (3ꢁ
100 mL). The organic extracts were washed with water and brine
and dried over anhydrous Na2SO4. Evaporation of solvent under re-
duced pressure yielded 11 b as a white solid that was sufficiently
pure for the next reaction step (0.583 g, 96%): 1H NMR (400 MHz
CDCl3 =0.67 (s, 3H, CH3), 0.95–1.05 (m, 6H), 1.08–1.61 (m, 22H),
1.80–2.00 (m, 6H), 2.22–2.43 (m, 3H), 3.59–3.65 (m,1H);13C NMR
(100 MHz CDCl3): d=12.08, 18.25, 20.93, 23.92, 24.17, 26.55, 26.72,
28.17, 28.70, 29.71, 30.24, 30.75, 31.04, 34.31, 35.31, 35.50, 35.71,
36.70, 37.46, 40.20, 40.24, 42.76, 55.96, 56.60, 180.66 ppm; MS (ESI)
calcd for C24H40O2S: 392.64, found: 391 [Mꢁ1]ꢁ.
(3a,5b)-Cholan-3,24-diol (16): Compound 16 was synthesized fol-
lowing a modification of a described procedure[53] (scheme S10,
Supporting Information) in which a solution of lithocholic acid (1)
(2.66 mmol) in anhydrous THF (30 mL) was added dropwise to
a suspension of LiAlH4 (10.64 mmol) in anhydrous THF (30 mL),
stirred at 08C under nitrogen. The reaction mixture was then al-
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Eph–ephrin Antagonists
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lowed to warm to room temperature and was stirred overnight.
The reaction was then chilled to 08C and carefully quenched by
dropwise addition of a 2n H2SO4 solution (30 mL), then stirred at
room temperature until the reaction mixture became clear. THF
was removed under reduced pressure, and the mixture was ex-
tracted with diethyl ether (3ꢁ40 mL). The organic phase was
washed with water and brine and dried over anhydrous Na2SO4.
Evaporation of the solvent under reduced pressure afforded
a white solid, which was recrystallized from EtOH and water to
give 16 (0.791 g, 81%): 1H NMR (400 MHz [D6]DMSO): d=0.62 (s,
3H, CH3), 0.88–0.92 (m, 7H), 0.95–1.93 (m, 31H); 4.32 (t, 1H, J=6.8
CH2-OH), 4.44 ppm (d,1H, J=6.4 OH); 13C NMR (100 MHz
[D6]DMSO): d=12.31, 18.99, 23.71, 24.30, 26.62, 27.37, 28.31, 29.59,
30.82, 32.24, 34.65, 35.57, 35.62, 35.85, 36.73, 42.00, 42.69, 56.27,
56.54, 61.75, 70.31 ppm; MS (ESI) calcd for C24H42O2: 362.59, found:
385 [M+Na+]+.
the negative log10 of the inhibitor concentration. The Hill coeffi-
cient was calculated using linear fitting to evaluate whether inhibi-
tion was competitive or uncompetitive.
Surface plasmon resonance (SPR): EphA2 (3000 RU), EphB1
(3000 RU), and Fc fragment (1000 RU) were immobilized via an
amine group on the dextran matrix of a CM4 sensor chip, on flow
cell 2, flow cell 3, and flow cell 4, respectively Blank immobilization
was performed on flow cell 1 in order to be used as reference sur-
face. Cholanic acid was dissolved into a solution of DMSO (final
concentration 1 mm), then diluted to 50 mm in PBS (0.05%, pH 7.4).
Subsequent dilutions from 25 mm to 3 mm were performed in 5%
DMSO–PBS (0.05%, pH 7.4), which was also used as running buffer.
Cholanic acid was injected over immobilized EphA2, EphB1, and Fc
fragment for 90 sec at a flow rate of 30 mLminꢁ1, followed by
a 300 sec dissociation. Kinetics were analyzed using Biacore T100
evaluation software and were calculated as a 1:1 binding model
and as steady state affinity.
Pharmacology
Cell lysates: PC3 or T47D cells were seeded in 12-well plates at
a concentration of 105 cells per mL, 1 mL per well, in complete
medium until they reached ~70% confluence and were serum-
starved overnight. The following day, cells were treated with the
compounds under study, vehicle, or standard drug, stimulated with
the proper agonist (ephrinA1–Fc or ephrinB2–Fc), rinsed with ster-
ile PBS, and solubilized in lysis buffer. The lysates were resuspend-
ed and rocked at 48C for 30 min, then centrifuged at 14000ꢁg for
5 min. The protein content of the supernatant was measured using
a BCA protein assay kit (Thermo scientific) and was standardized to
Reagents: all culture media and supplements were purchased
from Lonza. Recombinant proteins and antibodies were from R&D
systems. Cells were purchased from ECACC. Leupeptin, aprotinin,
NP40, MTT, tween20, BSA, and salts for solutions were from Appli-
chem; EDTA and sodium orthovanadate were from Sigma. Human
IgG Fc fragment was from Millipore (AG714).
Cell culture: PC3 human prostate adenocarcinoma cells were
grown in Ham F12 supplemented with 7% fetal bovine serum
(FBS) and 1% antibiotic solution. T47D human breast tumor cells
were grown in RPMI 1640 with 10% FBS and 1% antibiotic solu-
tion. All cell lines were grown in a humidified atmosphere of 95%
air and 5% CO2 at 378C.
200 mgmLꢁ1
.
Phosphorylation of EphA2, EphB4, and EGFR in cells: Phosphory-
lation of EphA2, EphB4, and EGFR was measured in cell lysates
using a DuoSet IC Sandwich ELISA (R&D Systems: #DYC4056,
#DYC4057, and #DYC1095, respectively) following the manufactur-
er’s protocol. Briefly, 96-well ELISA high-binding plates (Costar
#2592) were incubated overnight at room temperature with 100 mL
per well of the specific capture antibody diluted in sterile PBS to
the proper working concentrations. After blocking, the wells were
incubated for 2 h at room temperature with 100 mL lysates per
well, followed by a 2 h incubation at room temperature with the
detection antibody. Receptor phosphorylation was revealed using
a standard HRP format with a colorimetric reaction read at 450 nm.
ELISA assays and Ki/IC50 determinations: ELISA assays were per-
formed as previously described.[54] Briefly, compounds (Table 1)
were stocked as 20 mm solutions in dimethyl sulfoxide (DMSO)
and tested both in displacement and saturation studies, starting
from a concentration of 200 mm. ELISA 96-well high-binding plates
(Costar #2592) were incubated overnight at 48C with 100 mL per
well of 1 mgmLꢁ1 EphA2–Fc (R&D 639-A2), diluted in sterile phos-
phate buffered saline (PBS: 0.2 gLꢁ1 KCl, 8.0 gLꢁ1 NaCl, 0.2 gLꢁ1
KH2PO4, 1.15 gLꢁ1 Na2HPO4, pH 7.4). The following day, wells were
washed with washing buffer (PBS+0.05% tween20, pH 7.5) and
blocked with blocking solution (PBS+0.5% BSA) for 1 h at 378C.
Compounds were added to the wells at selected concentrations in
1% DMSO and were incubated at 378C for 1 h. Biotinylated ephri-
nA1–Fc (R&D Systems BT602) was added at 378C for 4 h at a con-
centration corresponding to its KD in displacement assays, or in
a range from 1 to 2000 ngmLꢁ1 in saturation studies. The wells
were washed and incubated with 100 mL per well streptavidin-HRP
(Sigma S5512) in blocking solution (0.05 mgmLꢁ1 in PBS supple-
mented with 0.5% BSA, pH 7.4) for 20 min at room temperature,
then washed again and incubated at room temperature with
0.1 mgmLꢁ1 tetramethylbenzidine (Sigma T2885), reconstituted in
stable peroxide buffer (11.3 gLꢁ1 citric acid, 9.7 gLꢁ1 sodium phos-
phate, pH 5.0) and 0.1% H2O2 (30% w/w in water), added immedi-
ately before use. The reaction was quenched with 3n HCl (100 mL
per well), and the absorbance was measured using an ELISA plate
reader (Sunrise, TECAN, Switzerland) at 450 nm.
EphA2 kinase assay: the ability of isolithocholic acid and cholanic
acid to interact directly with the intracellular kinase domain of
human EphA2 was assessed by measuring the phosphorylation of
the substrate Ulight-TK peptide (50 nm) in the absence and in pres-
ence of 100 mm of the tested compound. The LANCE detection
method was applied,[55] and the general kinase inhibitor stauro-
sporine was used as a reference compound.
LDH assay: the cytotoxicity of all compounds was evaluated using
the CytoTox 96 Non-Radioactive Cytotoxicity Assay following the
manufacturer’s protocol (Promega, #1780). Briefly, cells were
seeded in 96-well plates at a density of 105 cells per mL and the
following day, treated with compounds or lysis buffer for 2 h. After
incubation, released LDH in culture supernatants was measured
using a 30 min coupled enzymatic assay, which results in the con-
version of a tetrazolium salt (INT) into a red formazan product. The
resulting amount of color is proportional to the number of lysed
cells and is quantified using an ELISA plate reader (Sunrise, TECAN,
Switzerland) at 492 nm. The results were expressed as the ratio be-
tween absorbance of the cells treated with the compounds and
cells treated with lysis buffer.
IC50 values were determined using one-site competition nonlinear
regression, and KD values of the curves with or without antagonists
were calculated using one-binding site nonlinear regression analy-
sis with Prism software (GraphPad Software Inc.). Ki values were ob-
tained using the Schild plot,[31] in which log[DRꢁ1] is a function of
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The work was supported by the Ministero della Universitꢀ e della
Ricerca “Futuro in Ricerca” program (project code: RBFR10FXCP),
Associazione Italiana per la Ricerca sul Cancro (AIRC) “My first
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Received: February 20, 2012
Revised: March 23, 2012
Published online on && &&, 0000
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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FULL PAPERS
M. Tognolini, M. Incerti,
Well worth the Ephort! A combined ap-
plication of computational and experi-
mental techniques led to the identifica-
tion of (5a)-cholan-24-oic acid deriva-
tives that disrupt the ephrinA1–EphA2
complex by specific interaction with the
ligand binding domain of the EphA2 re-
ceptor. SAR studies provide a detailed
analysis of the requirements for small
molecules able to disrupt the Eph–
ephrin interaction. As this system plays
a critical role in tumor and vascular
functions during carcinogenesis, these
compounds could provide leads for
therapeutic agents.
I. Hassan-Mohamed, C. Giorgio, S. Russo,
R. Bruni, B. Lelli, L. Bracci, R. Noberini,
E. B. Pasquale, E. Barocelli, P. Vicini,
M. Mor, A. Lodola*
&& – &&
Structure–Activity Relationships and
Mechanism of Action of Eph–ephrin
Antagonists: Interaction of Cholanic
Acid with the EphA2 Receptor
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www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 14
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These are not the final page numbers!