Cardiac glycoside activities link Na+/K+ ATPase ion-transport to breast cancer cell migration via correlative SAR

Article abstract of DOI:10.1021/cb500665r

The cardiac glycosides ouabain and digitoxin, established Na⁺/K⁺ ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na⁺/K⁺ ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure-activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na⁺/K⁺ ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein-small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na⁺/K⁺ transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1-M6 correlates with the Na⁺ pump activity and cell migration. Other Na⁺/K⁺ ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na⁺/K⁺ ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.

Full text of DOI:10.1021/cb500665r

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Article
Cardiac glycoside activities link Na+/K+ ATPase ion-
transport to breast cancer cell migration via correlative SAR
Anniefer N. Magpusao, George Omolloh, Joshua Johnson,
José Gascón, Mark W. Peczuh, and Gabriel Fenteany
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500665r • Publication Date (Web): 21 Oct 2014
Downloaded from http://pubs.acs.org on October 26, 2014
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Cardiac glycoside activities link Na+/K+ ATPase ion-transport to breast cancer cell
migration via correlative SAR
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Annie Magpusao, George Omolloh, Joshua Johnson, José Gascón, Mark W. Peczuh* and Gabriel Fenteany*
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Department of Chemistry, University of Connecticut, 55 N. Eagleville Road, U3060, Storrs, CT 06269, USA
Abstract:
The cardiac glycosides ouabain and digitoxin, established Na+/K+ ATPase inhibitors, were
found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical
genetics screen for cell motility. The Na+/K+ ATPase acts both as an ion-transporter and a
receptor for cardiac glycosides. To delineate which function is related to breast cancer cell
migration, structure-activity relationship (SAR) profiles of cardiac glycosides were established
at the cellular (cell migration inhibition), molecular (Na+/K+ ATPase inhibition), and atomic
(computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a
similar profile - a decrease in potency when the parent cardiac glycoside structure was
modified - for each activity investigated. Since assays were done at the cellular, molecular and
atomic levels, correlation of SAR profiles across these multiple assays established links
between cellular activity and specific protein-small molecule interactions. The observed
antimigratory effects in breast cancer cells are directly related to the inhibition of Na+/K+
transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation
path formed by transmembrane helices αM1-M6 correlates with the Na+ pump activity and cell
migration. Other Na+/K+ ATPase inhibitors that are structurally distinct from cardiac glycosides
also exhibit antimigratory activity, corroborating the conclusion that the antiport function of
Na+/K+ ATPase, and not the receptor function is important for supporting the motility of MDA-
MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific
biochemical functions and higher-level cellular processes, particularly for proteins with multiple
functions and small molecules with unknown or various modes of action.
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Cardiac glycoside activities link Na+/K+ ATPase ion-transport to breast cancer cell
migration via correlative SAR
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Anniefer N. Magpusao, George Omolloh, Joshua Johnson, José Gascón, Mark W. Peczuh* and Gabriel
Fenteany*
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Department of Chemistry, University of Connecticut, 55 N. Eagleville Road, U3060, Storrs, CT 06269, USA
Abstract:
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The cardiac glycosides ouabain and digitoxin, established Na+/K+ ATPase inhibitors, were
found to inhibit MDAꢀMBꢀ231 breast cancer cell migration through an unbiased chemical
genetics screen for cell motility. The Na+/K+ ATPase acts both as an ionꢀtransporter and a
receptor for cardiac glycosides. To delineate which function is related to breast cancer cell
migration, structureꢀactivity relationship (SAR) profiles of cardiac glycosides were established
at the cellular (cell migration inhibition), molecular (Na+/K+ ATPase inhibition), and atomic
(computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a
similar profile ꢀ a decrease in potency when the parent cardiac glycoside structure was
modified ꢀ for each activity investigated. Since assays were done at the cellular, molecular and
atomic levels, correlation of SAR profiles across these multiple assays established links
between cellular activity and specific proteinꢀsmall molecule interactions. The observed
antimigratory effects in breast cancer cells are directly related to the inhibition of Na+/K+
transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation
path formed by transmembrane helices αM1ꢀM6 correlates with the Na+ pump activity and cell
migration. Other Na+/K+ ATPase inhibitors that are structurally distinct from cardiac glycosides
also exhibit antimigratory activity, corroborating the conclusion that the antiport function of
Na+/K+ ATPase, and not the receptor function is important for supporting the motility of MDAꢀ
MBꢀ231 breast cancer cells. Correlative SAR can establish new relationships between specific
biochemical functions and higherꢀlevel cellular processes, particularly for proteins with multiple
functions and small molecules with unknown or various modes of action.
Introduction
Correlative SAR is a strategy to expand the use of existing biologically active
compounds to address some of the major challenges in the postgenomic era: annotating
protein functions and linking specific protein function to an observed phenotype. Traditional
SAR studies guide improvements of small molecule potency and selectivity for a target.
Correlative SAR, on the other hand, takes advantage of a distinct pattern of structural
requirements for activity by a group of related molecules (SAR profile) across a series of
assays. The SAR profiles are then used to establish relationships between a protein or cellular
system and a bona fide biological processes, i.e. linking specific protein function to a cellular
phenotype. Direct correlation of SAR profiles for protein function and a cellular phenotype then
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implicate a role for the protein in the phenotype. This is especially useful for multifunctional
proteins and moonlighting proteins.1 Correlative SAR employs small molecule probes and thus
shares the complementarity and advantages of small molecules over genetics and RNA
interference techniques for annotating protein functions. In this study, we illustrate the
correlative SAR approach by exploring the involvement of Na+/K+ ATPAse ion transport
function in breast cancer cell migration.
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Cell migration is a fundamental biological process of physiological and pathological
phenomena such as wound healing, tissue remodeling, angiogenesis and cancer metastasis.
In response to soluble signaling factors and mechanical cues, the cytoskeleton generates
protrusive forces and adhesion receptorꢀlinked traction against the extracellular matrix.2
Because of the highly complex nature of the mechanisms of cell migration, no complete model
that incorporates all of its components currently exists. One of the major problems in
determining the precise roles of each of the proteins involved in cell migration is the lack of
potent and selective inhibitors for the functions of these proteins.3 Beside their therapeutic
potential, smallꢀmolecule inhibitors of cell migration serve as valuable tools to probe the
molecular basis of cell motility.4ꢀ6
In the course of screening libraries of ca. 12,000 total compounds for cell migration
inhibitors using a woundꢀclosure assay, cardiac glycosides were found to inhibit the migration
of the human tripleꢀnegative breast cancer cell line MDAꢀMBꢀ231 at subꢀmicromolar
concentrations. The MDAꢀMBꢀ231 line is a cellular model for tripleꢀnegative breast cancer,
which is refractory to current chemotherapeutic options.7 Cardiac glycosides are a group of
tripartite natural products consisting of a steroid core, an αꢀβꢀunsaturated lactone ring and a
sugar moiety. They are used in the treatment of congestive heart failure,8 acting through
inhibition of the Na+ and K+ antiport pumping activity of the Na+/K+ ATPase.9 Over the last 20
years, cardiac glycosides have also garnered increasing interest as potential antiꢀcancer
agents.10 The minimum pharmacophore (Scheme 1) for inhibition of the Na+/K+ ATPase
consists of the steroid core, which possesses a distinct cisꢀconfiguration of the junctions
between the AB and CD rings. The lactone is not essential for activity but increases the affinity
between the steroid core and the protein.11 The sugar moiety affects the potency as well as the
pharmacokinetic and pharmacodynamic properties of cardiac glycosides.12ꢀ14
Upon binding of cardiac glycosides at the wellꢀconserved binding site in the α1 subunit
of the Na+/K+ ATPase,15 various effects are observed depending on the concentration of
cardiac glycosides and the cell type used.16 At concentrations >100 nM, cardiac glycosides
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cause “classical” inhibition of the ion transport function of the Na+/K+ ATPase, leading to an
increase in intracellular Na+ ion concentration; this activates the Na+/Ca2+ exchanger resulting
in an increased intracellular Ca2+ concentration, explaining the positive inotropic effect of
cardiac glycosides.17 Recent studies have shown that the Na+/K+ ATPase also acts as a
receptor for endogenous cardiac glycosides as natural ligands.18 At concentrations too low to
affect the ion transport function of the pump (i.e., 10ꢀ100 nM), cardiac glycosides have a
distinctly different effect: they activate signaling cascades, one of which involves Src tyrosine
kinase in a complex with the Na+/K+ ATPase that affects diverse cellular functions such as cell
proliferation.19 A pool of nonꢀpumping Na+/K+ ATPase exists in the caveolae and forms a
complex with caveolinꢀ1 and Src. Binding of cardiac glycosides to this pool of the Na+/K+
ATPase activates Srcꢀdependent signaling pathways, leading to activation of the mitogenꢀ
activated protein kinase cascade.20
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Other cardiac glycosides inhibit the migration and proliferation of various cell types
without affecting the ionꢀtransport function of the Na+/K+ ATPase, thus supporting the nonꢀ
classical mode of action. UNBSꢀ1450, for example, a novel cardenolide (structure in
Supplementary scheme 1), inhibits the proliferation and migration of several types of cancer
cells,21ꢀ23 at concentrations that do not affect the ion transport activity of the Na+/K+ ATPase.
Inhibition of the α1 subunit of the Na+/K+ ATPase by UNBSꢀ1450 leads to a sharp decrease in
intracellular ATP concentration, causing disorganization of the actin cytoskeleton and induction
of proautophagic cytotoxic effects.22
After discovering that cardiac glycosides inhibit migration of MDAꢀMB 231 cells, we
endeavored to define the specific role of the Na+/K+ ATPase (classical versus nonꢀclassical) in
cell migration using correlative SAR. Analogs of ouabain and digitoxin were synthesized and
SAR was established with respect to inhibition of cell migration (cellular), inhibition of Na+/K+
ATPase activity in vitro (molecular), and orientation in the cardiac glycoside binding site of
Na+/K+ ATPase (atomic). We evaluated whether inhibition of cell migration was separable from
inhibition of the Na+/K+ antiport pump by correlating the SAR profiles of ouabain in each of
these assays. In addition, to determine if the inhibition of cell migration resulted from general
inhibition of the Na+/K+ ATPase or is confined to an effect of cardiac glycosides, other
structurallyꢀunique Na+/K+ ATPase inhibitors were tested for their antimigratory activity. A
smallꢀmolecule inhibitor that does not activate the Src signaling pathway but inhibits the ion
pump24 was also tested in the cell migration assay. To our knowledge, no SAR study on the
antimigratory activity of cardiac glycosides in MDAꢀMB 231 breast cancer cells has been
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reported prior to this work. Furthermore, the current study shows the utility of correlative SAR
to establish links between specific protein functions and cellular phenotypes.
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Results and Discussion
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Scheme 1. Semisynthesis of ouabain analogs.
Ouabain and analogs – correlative SAR between inhibition of MDA-MB-231 breast
cancer cell migration and the Na+/K+ ATPase. Three analogs of ouabain 1 were synthesized
(2ꢀ4, Scheme 1) by methods that had been reported previously in the literature.25, 26 The
derivatives were prepared, starting from ouabain itself, by removal of the sugar moiety (2),
reduction of the lactone ring (3) and modification of the steroid core (4). Hydrolysis of the
glycosidic linkage between the Lꢀrhamnose and steroid core provided ouabagenin 2. Reduction
of the butenolide olefin by hydrogenation provided analog 3. Compound 4 contains an
acetonide group that links the C1 and C19 hydroxyl groups. The analogs were chosen
because they were synthetically accessible and because they consisted of variations of the
key structural segments of the parent compound in a way that would allow for a reasonably
comprehensive SAR profile. Ouabain 1 and analogs 2ꢀ4 were assayed for cell migration
inhibition using a quantitative scratchꢀwound assay27.
The MDAꢀMBꢀ231 breast cancer cell line was chosen for the scratchꢀwound cell
migration assay. MDAꢀMB 231, a cellular model for triple negative breast cancer, has an
invasive phenotype and is metastatic in animal models. The assay involved mechanical
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wounding of a confluent monolayer of cells with a pipet tip after treatment with different
concentrations of compounds or DMSO (vehicle) alone. The progress of wound closure was
monitored by analysis of digital microscope images of the wounds at different times postꢀ
wounding. The area of the wound at each time point was calculated with the publicꢀdomain
ImageJ program, and statistical significance was tested with a twoꢀtailed Student’s tꢀtest.
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1
100
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ꢀ8.5
ꢀ7.5
ꢀ6.5
ꢀ5.5
ꢀ4.5
ꢀ3.5
ꢀ2.5
Log [Ouabain Analog]
Figure 1. IC50 curve for cell migration inhibition by 1ꢀ4
Based on the IC50 values for inhibition of wound closure (Figure 1 and Table 1), none of
the synthesized analogs were more potent than the parent compound, ouabain 1 (with an IC50
of 173 nM). However, the potency of the analogs varied with respect to the type of modification
made to the structure of 1. Ouabagenin (2), which lacks the sugar moiety, had an IC50 value of
790 nM. The olefin of the butenolide was reduced to prepare compound 3; its IC50 of 4.3 ꢁM
demonstrates that a simple modification to the lactone segment results in a drop in potency by
a factor of 25. It further suggests that the olefin is relatively important to the ability of cardiac
glycosides to inhibit cell migration. A major decrease in potency (178ꢀfold) was observed for
ouabain analog 4, which contains a C1, C19 acetonide group. Modification to the steroid core
and specifically the introduction of a bulky group on the concave face of the cupꢀshaped
steroid greatly reduced the antimigratory activity of ouabain. The SAR profile (pattern of
structural requirement for activity modulation) for antimigratory activity is: modification on
steroid core > modification on lactone ring > modification on sugar in decreasing impact on
potency (Figure 2a).
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Table 1. IC50 values (ꢁM) for the inhibitory activity of ouabain and analogs in the Na+/K+ ATPase assay and cell
migration assay.
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Compound
Na+/K+ ATPase
Cell migration
0.173
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4
0.105
0.669
2.6
0.790
4.3
30.9
12.4
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* Using trypan blue assay, compounds 1ꢀ4 were not toxic to MDAꢀMBꢀ231 breast cancer
cells during the time frame of the woundꢀclosure assay. However, for compound 1, some
cells lifted off of the monolayer at concentrations above 60ꢁM.
200
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100
50
A
)
B)
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r = 0.9979
10
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n = 4, p = 0.0021, 95% CI = 0.90-1.00
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Na+/K+ ATPase
assay
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antimigratory
assay
IC50 cell migration inhibition
ouabain and analogs
D)
C)
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1.5
1.0
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0.0
r = 0.9894
r = 0.8706
0.8
0.6
0.4
0.2
0.0
n = 32, p < 0.0001, 95% CI = 0.75-0.94
0.5 1.0 1.5
cell migration inhibition
n = 9, p < 0.0001, 95% CI = 0.95-1.00
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0.6
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1.1
cell migration inhibition
Figure 2. (A) Decrease in potency of ouabain analogs in the cell migration assay and the Na+/K+ ATPase assay
(SAR profile). (B) Pearson correlation plot for IC50 values of ouabain and analogs in the antimigratory assay and
the Na+/K+ ATPase assay (Correlative SAR). (C) Pearson correlation plot for ouabain’s inhibition of Na+/K+
ATPase and cell migration at different concentrations. (D) Pearson correlation plot for ouabain and analogs’
inhibition of Na+/K+ ATPase and cell migration at different concentrations.
Ouabain is a potent and specific inhibitor of the Na+/K+ ATPase28. The Na+/K+ ATPase
is a transmembrane antiport ion transporter that pumps three Na+ ions out of the cell for every
two K+ ions pumped into the cell against their concentration gradients for each molecule of
ATP hydrolyzed. Besides its ionꢀtransport function, this protein complex also functions as a
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receptor, for ouabain as ligand, that has been implicated in processes and key cellular
elements that affect cell motility such as cell tight junctions,29 cell contacts30 and actin
dynamics.31 We investigated whether the antimigratory effect of ouabain was linked to the
classical inhibition of Na+/K+ ATPase17 and/or stimulation of its receptor function20 using
correlative SAR. Specifically, we looked at the SAR profile of ouabain in an Na+/K+ ATPase
activity assay and correlated it to the SAR profile for antimigratory activity.
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The SAR profile in Na+/K+ ATPase inhibition is similar to inhibition of cell migration
(Table 1 and Figure 2A). Compound 2, with the cleaved sugar moiety, had a potency that is
relatively similar to the parent compound ouabain 1. This implies that for inhibition of Na+/K+
ATPase activity, as for inhibition of cell migration, the sugar moiety is less important than other
parts of the molecule. Compound 3, with the saturated lactone ring, displays the next lowest
decrease in potency, while 4, with the bulky acetonide group on the steroid core, shows the
greatest decrease in potency. The IC50 values of ouabain and its analogs for inhibition of
Na+/K+ ATPase activity and the values for inhibition of cell migration fall within the same range.
The distinct pattern of modulation in Na+/K+ ATPase activity by ouabain and the analogs, as
measured by both IC50 (Figure 2B) and doseꢀresponse for ouabain alone (Figure 2C) and for
combined ouabain and analogs (Figure 2D) correlates well with the pattern observed for their
effect on cell migration. The correlation suggests that the inhibition of cell migration is directly
related to the inhibition of Na+/K+ ATPase activity. This result was corroborated by the same
strong correlation (r = 0.9998, p = 0.0002, 95% CI = 0.99-1.00) observed for correlative SAR of
digitoxin, a hydrophobic digitalis cardiac glycoside (see Supporting Information). The direct
relationship between inhibition of Na+/K+ ATPase ion transport function with inhibition of cell
migration seemed to be applicable to other members of the cardiac glycoside family of natural
products.
Ouabain and analogs – ouabain-Na+/K+ ATPase interaction at atomic level
resolution via molecular modeling. The ouabain binding site on the extracellular side of the
αꢀsubunit of the Na+/K+ ATPase is located in a cleft between the transmembrane segments
αM1 and αM6.32ꢀ35 Two orientations of ouabain to this site were originally proposed, one with
the lactone ring facing toward the cytoplasmic side, as revealed by Xꢀray crystal structure
studies32ꢀ34 (Figure 3AꢀB) and the other one with the lactone ring facing toward the extracellular
side, as suggested by a lanthanideꢀbased resonance energy transfer (LRET) experiment.35
Regardless of the two orientations of ouabain, all structural and functional studies agreed that
the location of the cardiotonic steroid binding site is in the deep vestibule formed by the
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tansmembrane helices αM1ꢀM6. Most of these transmembrane helices constitute the cation
permeation path (mapped in Figure 3A as residues in cyan) that traverses the Na+/K+ pump
from one side of the membrane to the other and passes through the ionꢀbinding site II36 (Figure
3A – red circle), suggesting that ouabain acts by blocking the ion path and directly preventing
cations from accessing their binding sites. Another possibility is that ouabain binding may lock
the cation transport region into a configuration that prevents the pump from undergoing the
conformational change required for ion transport.37
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To our knowledge, no experimental evidence linking ouabain’s orientation to the cation
permeation path and inhibition of Na+/K+ ATPase currently exists. We investigated the
interactions of ouabain and its analogs to Na+/K+ ATPase at the atomic level (Figure 3BꢀE) by
molecular docking experiments in an effort to rationalize the SAR results with respect to
inhibition of Na+/K+ ATPAse ionꢀtransport function and cell migration. We explored how each
modification on the ouabain structure affects binding energy (evaluated in the form of a
docking score) and orientation relative to ouabain’s structure in the coꢀcrystal (evaluated in
terms of a rootꢀmeanꢀsquareꢀdeviation, RMSD) (Table 2). We then correlated the SAR profile
for calculated RMSD (Figure 4) and binding energy (Supplementary Figure S5) to the SAR
profile for inhibition of Na+/K+ ATPase activity and inhibition of cell migration.
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Figure 3. (A) Crystal structure (4HYT)32 of Na+/K+ ATPase with bound ouabain (orange) and residues involved in
the cation permeation path36 (cyan) and the K+ binding site II. Below is the cupꢀshaped ouabain structure showing
the hydrophobic αꢀsurface and the polar βꢀsurface. Orientation of ouabain (B) and docked analog 2 (C), docked
analog 3 (D) and docked analog 4 (E) to cardiac glycoside binding site in the Na+/K+ ATPase.
The observations of the structural interactions of ouabain and its analogs with the
Na+/K+ ATPase are consistent with the observed experimental SAR profile for their inhibition of
Na+/K+ ATPase ionꢀtransport function and breast cancer cell migration. The docked analogs 2
and 3 (Figure 3C and 3D), which are the two most potent analogs (5x and 25x less potent than
ouabain respectively) adopted a similar orientation to the crystal structure of ouabain bound to
Na+/K+ ATPase (Figure 3B). The concave nonpolar αꢀsurface of ouabain32 and analogs 2 and 3
is stabilized by hydrophobic residues such as I315, F316, F783 and F786 (Figure 3 BꢀD). The
hydroxyl groups of the polar βꢀsurface, on the other hand, are stabilized by Hꢀbonding
interactions (between C19ꢀOH and the highly conserved C14ꢀOH of cardiac glycosides and
N111 and T797 of αM1 and αM6 respectively). The C14ꢀhydroxylꢀT797 interaction for analog 3,
however, was slightly shifted, and thus the Hꢀbonding angle was less than ideal
(Supplementary Figure S3), possibly due to reduction of lactone ring olefin. This could explain
the 25x decrease in potency observed for both the pump and cell migration inhibition. On the
other hand, the least potent analog 4 (Figure 3E) adopted a different pose in the ion
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permeation path altogether (see Supplementary Figure 2 for overlayed crystal structure of
ouabain and docked ouabain and analogs). Analog 4 took up a shallow position and was
oriented in the opposite direction with its polar βꢀsurface facing the helices M4 and M5, while
the apolar αꢀsurface faced the helices M1, M2 and M6. This orientation does not allow for Hꢀ
bonding interactions with Q111 and T797, residues identified by mutagenesis studies38, 39 to be
critical for ouabain’s inhibition of Na+/K+ ATPAse. However, the orientation of the docked
analog 4 is possibly stabilized by hydrophobic interactions of I315, F316, F783 with the steroid
rings C and D, and Hꢀbonding interaction of C19ꢀhydroxyl with E117 instead of Q111
(Supplementary Figure S4).
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The binding energy and RMSD were calculated for the docked compounds to quantify
the effect of each structural modification on the ouabainꢀNa+/K+ ATPase interaction (Table 2).
These parameters were then correlated to the experimental results from in vitro Na+/K+
ATPase inhibition and breast cancer cell migration assays. Considering that the docking score
is only an approximate measure of true free energies of binding, both the IC50 values for
inhibition of Na+/K+ ATPase and inhibition of cell migration by ouabain and analogs correlate
reasonably well with the calculated binding energy (Supplementary Figure S5) for analogs 2
and 3. That is, analogs 2 and 3 have less negative energy of binding compared to 1, although
with similar orders of magnitude, which is consistent with lower potency. A proper orientation
relative to the cardiac glycoside binding site seemed to be highly critical to Na+ꢀpump inhibition.
The more negative binding energy calculated for the least potent analog 4 probably results
from a stable analog 4ꢀNa+/K+ ATPase complex (possibly resulting from some hydrophobic
and Hꢀbonding interactions discussed earlier) but the orientation of analog 4 to the ion
permeation path might not be as effective in blocking ion permeation as that of ouabain. In
other words, despite analog 4 being more tightly bound, it is noticeably positioned in a region
of the binding site much closer to the surface, and therefore less able to stop the permeation of
ions deep inside the pocket (Figure 4E). On the other hand, both the IC50 values for inhibition
of Na+/K+ ATPase and inhibition of cell migration by ouabain analogs correlate very well with
the RMSD (Figure 4), implying that deviation from ouabain’s orientation in the cation
permeation of Na+/K+ ATPase is directly proportional to the decrease in its inhibitory activity.
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Table 2. Calculated binding energies and RMSD of ouabain and analogs from molecular docking experiments.
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9
Binding Energy
(Amber Score)
kcal/mol
RMSD
(Nonꢀhydrogen
atoms)
Na+/K+
ATPase
Cell
migration
Docked Ligand
1
2
3
4
–5420.51
–5399.66
–5412.08
–5584.89
0.0000
0.5807
0.9367
6.5933
0.105
0.669
2.6
0.173
0.790
4.3
12.4
30.9
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A)
B)
15
40
r = 0.9959
r = 0.9979
30
20
10
0
10
5
n = 4, p = 0.0021, 95% CI = 0.90-1.00
n = 4, p = 0.0041, 95% CI = 0.81-1.00
2
4
6
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0
-10
RMSD
0
2
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RMSD
Figure 4. (A) Pearson correlation plot for IC50 values of ouabain and analogs in the Na+/K+ ATPase assay and the
RMSD. (B) Pearson correlation plot for IC50 values of ouabain and analogs in the cell migration assay and the
RMSD
Inhibition of MDA-MB-231 breast cancer cell migration and Na+/K+ ATPase activity
by other Na+/K+ ATPase inhibitors and potential involvement of Na+/K+ ATPase in MDA-
MB-231 breast cancer cell migration - To explore whether the inhibition of cell migration
results from general inhibition of the Na+/K+ ATPase or is specific for cardiac glycosides, the
antimigratory effect of other Na+/K+ ATPase inhibitors that are structurally distinct from cardiac
glycosides was determined. All of the nonꢀcardiacꢀglycoside Na+/K+ ATPase inhibitors tested
also inhibited cell migration with varying potencies (Table 3). Compound 9, a nonspecific
inhibitor of Na+/K+ ATPase is the most potent; however, it was also toxic and its IC50 is very
close to its minimum lethal concentration which is likely due to its effect on other cellular
targets. The next most potent is compound 11. This Na+/K+ ATPase inhibitor is particularly
interesting because it was reported that, unlike ouabain, it does not activate the Na+/K+
ATPase/Src complex nor does it stimulate the protein kinase cascades.24 Compound 11 is a
useful small molecule probe since it can inhibit only one function (Na+/K+ ATPase enzymatic
activity) of the pump without activating its receptor function. This allows exploration and
differentiation of the functional consequences of the enzymatic versus receptor function of the
Na+/K+ ATPase. Since 11 also inhibits cell migration, the antimigratory activity of cardiac
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glycosides does not primarily entail activation of the receptor function of Na+/K+ ATPase.
Furthermore, any of the compound tested that inhibit Na+/K+ ATPase also inhibit MDAꢀMB 231
breast cancer cell migration. Therefore, the cardiac glycosides do not have a unique
antimigratory effect among inhibitors of the Na+/K+ ATPase.
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Table 3. Inhibitory effect of Na+/K+ ATPase inhibitors that are structurally different from cardiac glycoside in the
Na+/K+ ATPase assay and the cell migration assay
Literature IC50 for
IC50 for inhibition
of cell migration
Minimum lethal
concentration
Compounds
inhibition of the
Na+/K+ ATPase
40 nM40
5.6 ꢁM
15.6 ꢁM
(9)
Na3VO4
19 µM41
(10)
34.4 ꢁM
>250 ꢁM
(11)
1.5 ꢁM24
7.86 ꢁM
>250 ꢁM
The increase in [Ca2+]i, through the activation of the reverse Na+/Ca2+ exchanger
resulting from the “classical” inhibition of the ion transport function of the Na+/K+ ATPase, might
explain the observed inhibitory activity of Na+/K+ ATPase inhibitors in MDAꢀMBꢀ231 breast
cancer cell migration. Ca2+ signaling is a crucial coordinator of cell migration through various
mechanisms. Some of these mechanisms include activation of myosin light chain kinase
(MLCK), modulation of nascent focal adhesions, and retractionꢀadhesion of lamellipodia
through local calcium pulses near the leading edge of migrating cells42, 43. Consequently,
inhibition of key regulators of calcium homeostasis affects cell migration. Using woundꢀclosure
assay, thapsigargin, a specifc and irreversible inhibitor of sarco/endoplasmic reticulum Ca2+
ATPase (SERCA) inhibited the migration of bovine aortic endothelial cells (BAEC). In this
migrating BAEC, thapsigargin treatment elicit a rapid increase in [Ca2+]i due to depletion of
Ca2+ store sites44. Interestingly, through chemical genetic screening using the same
woundꢀclosure assay, we found that thapsigargin and analog inhibited MDAꢀMBꢀ231
breast cancer cell migration below their cytotoxic concentration (Figure S6). Besides
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small molecule inhibition, it was reported that siRNA knockdown of SERCA and other key Ca2+
regulators such as plasma membrane calcium ATPase (PMCA) and components of storeꢀ
operated Ca2+ (SOC) influx pathway also altered the basal [Ca2+]i. An inverse correlation
between the effect of small molecule and siRNA knockdown to [Ca2+]i and cell migration was
observed, where an increase in [Ca2+]i reduced the speed of human umbilical vein endothelial
cells (HUVECs) and vice versa45. The aim of the present study is to illustrate the utility of
correlative SAR as a strategy to link a specific protein function of a multifunctional protein to an
observed cellular phenotype. Although the present study did not explore the effect of Na+/K+
ATPase inhibitors in Ca2+ signaling, we showed that the antimigratory effect of Na+/K+ ATPase
inhibitors in MDAꢀMBꢀ231 breast cancer cell migration is directly related to inhibition of the ionꢀ
transport function of the Na+ pump. It is established that this classical inhibition of Na+/K+
ATPase leads to an increase in intracellular [Ca2+]I,17 and thus, it is possible that the observed
antimigratory effect of Na+/K+ ATPase inhibitors in MDAꢀMDꢀ231 breast cancer cells might be
due to the increase in [Ca2+]i, probably, in a similar manner that thapsigargin inhibited the
migration of this cell line.
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Conclusion:
In this study, correlative SAR of cardiac glycosides and analogs were used to link the
ion transport function of Na+/K+ ATPase to MDAꢀMB 231 breast cancer cell migration. Analogs
of representative cardiac glycosides ouabian and digitoxin that contain modifications in each
part of the tripartite cardiac glycoside were synthesized. The effect of each structural
modification on cardiac glycosides to their activities in both inhibition of Na+/K+ ATPAse
(molecular) and cell migration (cellular) provided a distinct pattern (SAR profile). Our results
showed that modification on steroid core > modification on lactone ring > modification on sugar
in decreasing impact on potency. These SAR profiles correlated in both experimental assays,
showing a direct relationship between inhibition of MDAꢀMB 231 breast cancer cell migration
and inhibition of Na+/K+ ATPase ion transport function. The interactions of ouabain and
analogs to Na+/K+ ATPAse were also investigated at the atomic level using molecular docking
experiments. Their activities in both inhibition of Na+/K+ ATPase and cell migration correlated
quite well with the degree of similarity in orientation with respect to the known ouabian
orientation, and reasonably well with binding energy calculations, provided that the analog
completely blocks the permeation path. A cardiac glycoside analog may have a higher binding
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energy to Na+/K+ ATPase but if its orientation does not allow for effective blocking of the ion
permeation path, binding of that analog may not result to efficient inhibition.
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Based on these results, we conclude that it is the ionꢀtransport function of Na+/K+
ATPase that is involved in MDAꢀMB 231 breast cancer cell migration and not the receptor
function. This conclusion is further corroborated by the observed inhibition of breast cancer cell
migration by other Na+/K+ ATPase inhibitors that are structurally distinct from cardiac
glycosides. In particular, a Na+/K+ ATPase inhibitor that does not activate Na+/K+ ATPase/Src
complexes nor the protein kinase cascades, still inhibited cell migration, implying that the
antimigratory effect of Na+/K+ ATPase inhibitors does not involve activation of its receptor
function. We speculate that a mechanism in which perturbation of intracellular Na+ and K+ and
therefore Ca+ ion concentrations leads to inhibition of breast cancer cell migration. Further
studies on how exactly perturbations of ion homeostasis affect the migration of MDAꢀMB 231
cells could help contribute to understanding the overall mechanism of triple negative breast
cancer cell migration. This report also provides an example of how correlative SAR can help
establish new relationships between specific biochemical functions and higherꢀlevel cellular
processes by providing a quantitative way to link the effect of small molecules at the cellular,
molecular and atomic level.
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Methodology
Quantitaive cell migration assay - Quantitative wound closure assay27 was performed
on compounds that were active at sub micromolar concentrations during the primary chemical
genetic screening assay to evaluate their concentrationꢀresponse profiles. Confluent
monolayers of MDAꢀMB 231 breast cancer cells were wounded 30 min after treatment with
different concentrations of these compounds or DMSO alone. The progress of the wound
closure was followed quantitatively by taking digital images of the wounds at 3 h, 6 h, 12 h and
24 h postꢀwounding. Using the NIH ImageJ software ( http://rsbweb.nih.gov/ij/), the remaining
open area of the wound at each time point was calculated by tracing the wound margin in each
images. Microsoft Excel and GraphPad Prism software were used to tabulate and analyze the
corresponding data. GraphPad Prism software was used to determine the IC50 for inhibition of
wound closure from doseꢀresponse data and twoꢀtailed Student’s tꢀtest (p > 0.05) was used to
statistically determine significant differences. Among the antimigratory compound hits from
screening of MDAꢀMB 231 breast cancer cell migration inhibitors, cardiac glycosides were
chosen for further characterization of their mechanism of action in relation to inhibition of MDAꢀ
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MB 231 breast cancer cell migration. Analogs of reperesentative cardiac glycosides, ouabain
and digitoxin were synthesized and analyzed for their effect on MDAꢀMB 231 breast cancer
cell migration using the same quantitative cell migration assay. Doseꢀresponse (using 9
concentrations starting from 1mM to 15 nM) for cardiac glycosides and analogs’ inhibition of
cell migration was obtained for each test compounds for 3 trials with 3 replicates each trial.
In vitro Na+/K+ ATPase assay – The enzymatic activity of Na+/K+ ATPase (purchased
from Sigma as lyophilized powder from porcine cerebral cortex) was measured by colorimetric
quantification of Pi released during ATP hydrolysis. A previously published procedure46 was
adapted with some modifications. The reaction was started by incubating 10ꢁL of Na+/K+
ATPase (600 units/mL) with 2.5 ꢁL of KCl/NaCl solution (45mM KCl and 2M NaCl) at 37oC for
30 minutes with either 5 ꢁL DMSO (control) or 5 ꢁL test compounds in 67.5 ꢁL buffer (24mM
Tris HCl buffer with 0.68 mM Ethylenediaminetetraacetic acid and 6.0 mM Magnesium chloride,
pH 7.8). 5ꢁL of 80mM ATP was then added and the reaction mixture was incubated again at
37oC for 15 minutes. 30 ꢁL of 100% w/v Tricholoroacetic acid was then added to the reaction
mixture followed by centrifugation for 3 minutes. 50ꢁL of the supernatant was transferred to a
96ꢀwell plate containing 100ꢁL of TausskyꢀShorr reagent. The absorbance at 660nm was read
after incubation at room temperature for 5 minutes. The concentration of phosphate released
in the enzymatic reaction was measured from the absorbance at 660nm using a standard
curve. The effect of test compounds on enzymatic activity (percent inhibition) was calculated
by taking the ratio of the concentration of liberated phosphate after compound treatment
against that of DMSO control. Doseꢀresponse (using 9 concentrations starting from 1mM to 15
nM) for Na+/K+ ATPase inhibition was obtained for each test compounds for 3 trials with 3
replicates each trial. GraphPad Prism software was used to determine the IC50 for Na+/K+
ATPase inhibition from doseꢀresponse data and twoꢀtailed Student’s tꢀtest (p > 0.05) was used
to statistically determine significant differences. GraphPad Prism software was also used to
calculate and generate correlation plots for the inhibitory effect of test compounds on the
Na+/K+ ATPase enzymatic activity and MDAꢀMB 231 breast cancer cell migration using
Pearson correlation.
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Molecular docking - A model of the complex between oubain bound sodiumꢀpotassium
transporting ATPase was obtained from the available crystallographic data at the RSCB
website (4HYT) and refined using Chimera GUI. Duplicate chains (C, D, and E) and chain G
were deleted, as they were not needed in the ligand docking. All residues were protonated
based on neutral pH after the assignment of bond orders using the Dock prep tool in Chimera.
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Histidine tautomers were determined based on the ability to form Hydrogen bonds with the
neighboring residues. Ions, solvent molecules, extraneous molecules and substitution codes
for the side chains were also deleted.
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The protein was assigned Amber force field parameters while antechamber was used to
assign the ligand and nonꢀstandard residues’ parameters. The charge set used for the ligand
was AM1ꢀBCC charges; the structure was then minimized and duplicated – one part had the
ligand deleted (the receptor) while the other had the protein part deleted (the ligand). The
ligand was duplicated again one part was modified and minimized, while the other served as a
control.
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Docking followed the standard docking procedure in the Dock6 manual. Molecular
surface was constructed using the dms program inbuilt in UCSF’s Chimera using a probe
radius of 1.4Å. The spheres were then generated using sphgen_cc implemented in Dock6
suite and the Xꢀray structure (reference) ligand was used to select the hotspot upto 10Å from
the position of the heavy atoms of the reference ligand. A site box and the generated spheres
were then prepared using the showbox and showsphere modules. The grid box enclosing
the selected spheres had an extra 5Å added in every dimension. Grids were computed using a
grid spacing of 0.3A, distance dielectric of 4 with a combination of contact and energy scores
using the Van der Waals parameters of Amber99 force field.
Docking was performed through grid scoring and automated ligand orientation and
matching first with a rigid ligand then choosing 10 best poses and redocking while keeping the
ligand flexible. To account for receptor flexibility an ensemble docking was attempted with
Amber force field parameters and running a molecular dynamics simulation; this instead did
not give an improvement in the docking poses but produced fluctuations in the structure. The
final rescoring was done on the top most poses from the grid score, using amber score
(binding energy)
Supporting Information
Figures S1ꢀS6, Table S1 and Scheme S1 that provide correlative SAR data for digitoxin and
additional details on the cardiac glycoside interactions with the Na+/K+ ATPase. The material is
available free of charge via the Internet at http://pubs.acs.org .
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Acknowledgement
This work was supported by National Institutes of Health Grant GM077622 (to G. Fenteany). J.
Johnson was a UConn Honors Scholar and Summer Undergraduate Research Fellowship
(SURF) awardee. Narenjan Perera is acknowledged for assisting in the generation of graphics
associated with docking experiments.
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