Effect of polygodial and its direct derivatives on the mammalian Na+/K+-ATPase activity

Article abstract of DOI:10.1016/j.ejphar.2018.04.031

The sesquiterpene polygodial is an agonist of the transient receptor potential vanilloid 1 (TRPV1). Our group recently reported the synthesis and anticancer effects of polygodial and its derivatives, and showed that these compounds retain activity against apoptosis- and multidrug-resistant cancer cells. Herein, we tested the inhibitory effect of these compounds on the activity of the enzyme Na⁺/K⁺-ATPase (NKA) from kidney (α₁ isoform) and brain (α₂ and α₃ isoforms) guinea pig extracts. Polygodial (1) displayed a dose-dependent inhibition of both kidney and brain purified NKA preparations, with higher sensitivity for the cerebral isoforms. Polygo-11,12-diol (2) and C11,C12-pyridazine derivative (3) proved to be poor inhibitors. Unsaturated ester (4) and 9-epipolygodial (5) inhibited NKA preparations from brain and kidney, with the same inhibitory potency. Nevertheless, they did not achieve maximum inhibition even at higher concentration. Comparing the inhibitory potency in crude homogenates and purified preparations of NKA, compounds 4 and 5 revealed a degree of selectivity toward the renal enzyme. Kinetic studies showed a non-competitive inhibition for Na⁺ and K⁺ by compounds 1, 4 and 5 and for ATP by 1 and 4. However, compound 5 presented a competitive inhibition type. Furthermore, K⁺-activated p-nitrophenylphosphatase activity of these purified preparations was not inhibited by 1, 4 and 5, suggesting that these compounds acted in the initial phase of the enzyme's catalytic cycle. These findings suggest that the antitumor action of polygodial and its analogues may be linked to their NKA inhibitory properties and reinforce that NKA may be an important target for cancer therapy.

Full text of DOI:10.1016/j.ejphar.2018.04.031

Author’s Accepted Manuscript
“Effect of polygodial and its direct derivatives on
+ +
the mammalian Na /K -ATPase activity”
Diogo Gomes Garcia, Cassiano Felippe Gonçalves-
de-Albuquerque, Camila Ignácio da Silva, Robert
Kiss, Ramesh Dasari, Sunena Chandra, Alexander
Kornienko, Patricia Burth
www.elsevier.com/locate/ejphar
PII:
S0014-2999(18)30254-1
https://doi.org/10.1016/j.ejphar.2018.04.031
EJP71780
DOI:
Reference:
To appear in:
European Journal of Pharmacology
Received date: 21 September 2017
Revised date: 6 April 2018
Accepted date: 26 April 2018
Cite this article as: Diogo Gomes Garcia, Cassiano Felippe Gonçalves-de-
Albuquerque, Camila Ignácio da Silva, Robert Kiss, Ramesh Dasari, Sunena
Chandra, Alexander Kornienko and Patricia Burth, “Effect of polygodial and its
+ +
direct derivatives on the mammalian Na /K -ATPase activity”, European
Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.04.031
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“Effect of polygodial and its direct derivatives on the mammalian Na+/K+-
ATPase activity”
Diogo Gomes Garciaa, Cassiano Felippe Gonçalves-de-Albuquerqueb,c,
Camila Ignácio da Silvad, Robert Kisse,f, Ramesh Dasarig, Sunena
Chandrag, Alexander Kornienkog1, Patricia Burthd1*
aDepartamento de Neurologia, Hospital Universitário Gaffrée e Guinle,
Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, RJ 20270-
330, Brazil
bLaboratório de Imunofarmacologia, Departamento de Bioquímica,
Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, RJ 20211-
010, Brazil
cLaboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Fundação Oswaldo
Cruz, Rio de Janeiro, RJ 21040-900, Brazil
dDepartamento de Biologia Celular e Molecular, Instituto de Biologia,
Universidade Federal Fluminense, Niterói, RJ 24020-141, Brazil
eLaboratorie de Cancérologie et de Toxicologie Expérimentale, Faculté de
Pharmacie, Université Libre de Bruxelles, Brussels, Belgium
fRetired – previously at the Fonds National de la Recherche Scientifique
(FNRS-FRS; Belgium).
gDepartment of Chemistry and Biochemistry, Texas State University, San
Marcos, TX 78666, USA
Diogo Gomes Garcia: diogoggarcia@ig.com.br
1 These authors contributed equally to this work.

Cassiano Felippe Gonçalves-de-Albuquerque: cassiano.albuquerque@unirio.br
Camila Ignácio da Silva: camila_ignacio@id.uff.br
Robert Kiss: rkiss2012@gmail.com
Ramesh Dasari: r_d127@txstate.edu
Sunena Chandra: schandra@txstate.edu
Alexander Kornienko: a_k76@txstate.edu
Patrícia Burth: burth@vm.uff.br
*Corresponding author: Patricia Burth. Universidade Federal Fluminense,
Outeiro de São João Batista, S/n, Centro, Niterói, RJ, Brazil. ZC: 24020-141.
Phone/Fax: 55 21 2629-2288. E-mail: burth@vm.uff.br
ABSTRACT
The sesquiterpene polygodial is an agonist of the transient receptor
potential vanilloid 1 (TRPV1). Our group recently reported the synthesis and
anticancer effects of polygodial and its derivatives, and showed that these
compounds retain activity against apoptosis- and multidrug-resistant cancer
cells. Herein, we tested the inhibitory effect of these compounds on the activity
of the enzyme Na+/K+-ATPase (NKA) from kidney (α1 isoform) and brain (α2 and
α3 isoforms) guinea pig extracts. Polygodial (1) displayed a dose-dependent

inhibition of both kidney and brain purified NKA preparations, with higher
sensitivity for the cerebral isoforms. Polygo-11,12-diol (2) and C11,C12-
pyridazine derivative (3) proved to be poor inhibitors. Unsaturated ester (4) and
9-epipolygodial (5) inhibited NKA preparations from brain and kidney, with the
same inhibitory potency. Nevertheless, they did not achieve maximum inhibition
even at higher concentration. Comparing the inhibitory potency in crude
homogenates and purified preparations of NKA, compounds 4 and 5 revealed a
degree of selectivity toward the renal enzyme. Kinetic studies showed a non-
competitive inhibition for Na+ and K+ by compounds 1, 4 and 5 and for ATP by 1
and 4. However, compound 5 presented a competitive inhibition type.
Furthermore, K+-activated p-nitrophenylphosphatase activity of these purified
preparations was not inhibited by 1, 4 and 5, suggesting that these compounds
acted in the initial phase of the enzyme’s catalytic cycle. These findings suggest
that the antitumor action of polygodial and its analogues may be linked to their
NKA inhibitory properties and reinforce that NKA may be an important target for
cancer therapy.
Graphical abstract

Na+/ K+ -ATPase:Potential
target for cancer therapy
Polygo-11,12-diol(2)
C11,C12-pyridazine (3)
Non-inhibitory effect
Inhibitory effect
Polygodial (1)
(1) and (4) non-competitive inhibition
for Na+, K+ and ATP
(5) non-competitive inhibition for Na+
and K+ but competitive for ATP
Unsaturated ester (4)
9-epipolygodial (5)
KEYWORDS: Na+/K+-ATPase; Polygodial; Sesquiterpene; Anticancer agents
1. INTRODUCTION
The Na+/K+-ATPase (NKA) is a plasma membrane protein complex
transferring the stored energy of ATP to the active transport of Na+ and K+ ions
across the cell membrane. The ionic transport performed by this enzyme
creates an electrochemical gradient through the cell plasma membrane, which
is essential for the maintenance of muscle and nerve cell excitability (Skou,
1965, 1957). The NKA also participates in cell signaling events, which are
mediated via the NKA caveolar pool, affecting cell–cell interactions, as well as
cell proliferation, differentiation, and death (Gonçalves-de-Albuquerque et al.,
2017; Jaitovich and Bertorello, 2006; Liu et al., 2003; Skou, 2004).

Alterations in the expression and activity of the NKA are implicated in the
pathophysiology of several diseases such as leptospirosis (Burth et al., 1997;
Gonçalves-de-Albuquerque et al., 2014), cardio-metabolic diseases (Liu and
Songu-Mize, 1997; Obradovic et al., 2017; Tsimaratos et al., 2001), Alzheimer’s
disease (Dickey et al., 2005; Ohnishi et al., 2015), and cancer (Chen et al.,
2006; Lefranc et al., 2008; Yu, 2003). Moreover, suppression or over-
expression of some NKA isoforms were detected at early stages of
tumorigenesis (Mijatovic et al., 2008; Sakai et al., 2004). The involvement of the
NKA in the pathogenesis of many other diseases and its signal transduction
properties make this enzyme an attractive target for drug development (Aperia,
2007; Gonçalves-de-Albuquerque et al., 2017).
Cardiac glycosides, which are classical NKA selective inhibitors, have
been widely used in patients with chronic cardiovascular diseases. However,
due to their high toxicity, the search for inotropic drugs having a better
therapeutic index is of great importance (Pôças et al., 2003). Incidentally, the
first indication of their anticancer properties emerged from the fact that a low
breast cancer mortality rate was detected in cardiac women treated with such
glycosides due to their cardiovascular problems (Stenkvist, 1999). This fact
generated new interest in cardiac glycosides as anticancer drugs (Kometiani et
al., 2005; Lefranc and Kiss, 2008; Mijatovic et al., 2007b; Winnicka et al., 2010).
These observations have been confirmed in several in vitro and in vivo studies,
and substances based on the structures of cardiac glycosides have already
been tested in clinical trials for cancer treatment (Gonçalves-de-Albuquerque et
al., 2017; Mijatovic et al., 2007b, 2007a; Prassas and Diamandis, 2008).

On the other hand, the importance of NKA in cancer therapy has also
been suggested using compounds unrelated to the cardiac glycoside structure
(Garcia et al., 2015; Lefranc et al., 2013). In this sense, the current investigation
describes the inhibitory effect on the NKA activity by polygodial and its synthetic
derivatives (Fig. 1). Polygodial is a terpenenoid dialdehyde isolated from
Persicaria hydropiper (L.) (Polygonaceae), a plant once used as a pepper
substitute in Europe and still a popular condiment for sashimi in Japan (Ohsuka,
1963). It is a known agonist of the transient receptor potential vanilloid 1
(TRPV1) showing cytotoxic effects on cancer cells. We found, however, that
several polygodial derivatives exert their antiproliferative action mainly through
cytostatic effects. The anticancer evaluation of these compounds revealed their
promising activity against human cancer cells displaying various forms of
resistance, including resistance to proapoptotic agents and multidrug resistance
(Dasari et al., 2015a, 2015b).
Present work investigated the inhibitory properties of polygodial and its
derivatives on the NKA from guinea pig kidney and brain tissues.
2. MATERIALS AND METHODS
2.1 Compound Synthesis
General: All reagents, solvents, and catalysts were purchased from
commercial sources (Acros Organics and Sigma–Aldrich) and used without
purification. All reactions were performed in oven-dried flasks open to the
atmosphere or under nitrogen or argon and monitored by thin-layer

chromatography (TLC) on TLC pre-coated (250 mm) silica gel 60 F254 glass-
backed plates (EMD Chemicals Inc.). Visualization was accomplished with UV
light, iodine, and p-anisaldehyde stains. 1H and 13C NMR spectra were recorded
on a Bruker 400 & 500 spectrometer. Chemical shifts (d) are reported in ppm
relative to the residual solvent signal internal standard. Abbreviations are as
follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), HRMS
spectra were recorded on a Waters Synapt G2 UPLC Mass Spectrometer.
Polygodial (1) was purchased from VWR.
Compound 2: To a solution of 1 (3 mg, 0.0128 mmol) in MeOH (2 ml) was
added sodium borohydride (1.0 mg, 0.027 mmol). The mixture was stirred at
room temperature for 2 h. After completion of the reaction, as monitored by
TLC, added water to the reaction mixture and evaporated MeOH. The reaction
mixture was diluted with ethyl acetate and organic phase was washed with 1N
HCl and water, dried over anhydrous sodium sulphate and concentrated under
reduced pressure. The crude product was purified by preparative TLC (10/90
EtOAc/Hexane) to obtain 2.4 mg of 2 (80% yield); 1H NMR (400 MHz, CDCl3) δ
5.83 – 5.78 (m, 1H), 4.38 – 4.32 (m, 1H), 3.99 (d, J = 12.1 Hz, 1H), 3.91 (dd, J =
10.9, 2.2 Hz, 1H), 3.69 (dd, J = 10.9, 8.2 Hz, 1H), 2.86 (brs, 1H), 2.14 (brs, 1H),
2.13 – 2.04 (m, 1H), 2.01 – 1.83 (m, 2H), 1.59 – 1.39 (m, 3H), 1.28 – 1.11 (m,
4H), 0.88 (s, 3H), 0.87 (s, 3H), 0.76 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
136.9, 127.6, 67.5, 61.5, 54.5, 49.4, 42.0, 39.3, 35.6, 33.2, 33.0, 23.6, 21.9,

18.8, 14.5; HRMS (ESI) calcd for C15H26NaO2 (M+Na) 261.1830, found
261.1829.
Compound 3: To a solution of 1 (3 mg, 0.0128 mmol) and hydrazine hydrate
(0.7 µl, 0.014 mmol) in MeOH (1 ml) were added 4A molecular sieves. The
mixture was stirred at room temperature for 20 h. After completion of the
reaction, as monitored by TLC, the reaction mixture was filtered and filtrate was
concentrated under reduced pressure. The crude product was purified by
preparative TLC (4/96 MeOH/CHCl3) to obtain 2.8 mg of 2 (95% yield); 1H NMR
(400 MHz, CDCl3) δ 9.02 (s, 1H), 8.84 (s, 1H), 2.99 – 2.90 (m, 1H), 2.82 (ddd, J
= 18.7, 11.1, 7.7 Hz, 1H), 2.37 – 2.31 (m, 1H), 2.06 – 1.98 (m, 1H), 1.86 – 1.65
(m, 3H), 1.58 – 1.51 (m, 1H), 1.42 (td, J = 12.7, 3.9 Hz, 1H), 1.31 – 1.23 (m,
2H), 1.23 (d, J = 0.7 Hz, 3H), 0.98 (s, 3H), 0.95 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 152.2, 148.3, 145.8, 135.8, 49.5, 41.2, 36.95, 36.4, 33.4, 33.0, 26.9,
24.2, 21.5, 18.6, 17.7; HRMS (ESI) calcd for C15H23N2 (M+H) 231.1861, found
231.1861.

Compound 4: To a solution of 1 (3 mg, 0.0128 mmol) in toluene (3 ml) was
added Wittig reagent (12.8 mg, 0.0384 mmol). The resultant mixture was stirred
at room temperature for 20 h. After completion of the reaction, as monitored by
TLC, the reaction mixture was filtered and the filtrate was concentrated under
reduced pressure. The crude product was purified by preparative TLC (9/91
EtOAc/Hexane) to obtain 4 (94% yield); 1H NMR (400 MHz, CDCl3) δ 9.47 (d, J
= 4.8 Hz, 1H), 7.33 (d, J = 16.3 Hz, 1H), 6.53 – 6.49 (m, 1H), 5.50 (d, J = 16.3
Hz, 1H), 3.71 (s, 3H), 2.83 (s, 1H), 2.37 – 2.16 (m, 2H), 1.87 – 1.80 (m, 1H),
1.53 – 1.44 (m, 3H), 1.38 – 1.30 (m, 1H), 1.23 – 1.16 (m, 2H), 1.00 (s, 3H), 0.94
(s, 3H), 0.90 (s, 3H); 13C NMR (100 MHz, C6D6): δ 203.8, 167.2, 146.9, 141.1,
130.7, 116.8, 62.9, 51.2, 48.3, 41.9, 40.2, 37.2, 33.2, 32.9, 24.7, 22.2, 18.3,
15.3; HRMS (ESI) calcd for C18H26NaO3 (M+Na) 313.1780, found 313.1779.
Compound 5: To a solution of 1 (3 mg, 0.0128 mmol) in dry toluene (1.5 ml)
were added 4A molecular sieves and PTSA (cat.). The mixture was stirred at
room temperature for 40 h. Solid NaHCO3 was added to the reaction mixture
and stirred for 10 minutes, filtered and the filtrate was concentrated under
reduced pressure. The crude product was purified by preparative TLC (8/92
EtOAc/Hexane) to obtain 1.2 mg of 5 (40% yield); 1H NMR (400 MHz, C6D6) δ
9.69 (d, J = 2.4 Hz, 1H), 9.19 (s, 1H), 6.20 (dd, J = 4.9, 2.4 Hz, 1H), 1.96 – 1.88
(m, 1H), 1.62 – 1.51 (m, 1H), 1.45 – 1.39 (m, 2H), 1.34 – 1.25 (m, 2H), 1.20 –
1.12 (m, 2H), 0.96 – 0.87 (m, 2H), 0.65 (s, 3H), 0.63 (s, 3H), 0.57 (d, J = 0.7 Hz,

3H); 13C NMR (100 MHz, CDCl3) δ 202.2, 192.8, 153.4, 137.4, 58.5, 44.2, 42.0,
37.7, 37.1, 32.9, 32.7, 25.5, 21.9, 21.5, 18.4; HRMS (ESI) calcd for C15H22NaO2
(M+Na) 257.1517, found 257.1519.
2.2 NKA preparation
Guinea pig brain and kidney were used in the preparation of
homogenates and NKA-enriched fractions according to Jørgensen’s procedure
(Jørgensen, 1977). Animals were kept in compliance with the Guide for the
Care and Use of Laboratory Animals (US Department of Health and Human
Services, NIH) and according to the in-house regulations of our institution.
The final enzyme suspensions (adjusted to 0.35 mg of protein/ml) were
prepared in a solution containing 30 mM Tris-HCl pH 7.5, 1 mM EDTA and 1%
sucrose. Aliquots of these suspensions were poured into dark flasks, lyophilized
and stored at −20 °C. The lyophilized preparation could be stored for more than
6 months without losing its activity. Freeze-dried aliquots suspended in water
and kept at 0 °C could be used within 24 h after the suspension. Enzyme
specific activities were 112 and 120 μmol of Pi formed/h/mg of protein for kidney
and brain, respectively. Ouabain insensitive ATPase activity was not detected in
these purified preparations. NKA specific activities in whole kidney and brain
homogenates were 1.4 and 1.8 μmol of Pi formed/h/mg of protein, respectively.
2.3 NKA assays

Enzyme activity was determined in microtiter plates. The final incubation
volume contained 110 mM NaCl, 20 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl pH
7.6, compounds (absent in controls) dissolved in 100% dimethyl sulfoxide
(DMSO), enzyme preparation and 5 mM ATP. Assays were performed as
detailed previously (Burth et al., 1997). In all experiments (including controls
and blanks), the final DMSO concentration in the incubation mixture was kept
below 1% (v/v), a condition that did not affect the enzyme activity. The amount
of the enzyme preparation used in all experiments (including tissue
homogenates) corresponded to an activity of 0.12–0.13 μmol Pi formed/h in
control assays (100% enzyme activity). Preparations were preincubated for 10
min before starting the enzyme reaction with ATP and this reaction was stopped
40 min later. In the blanks, the enzyme was added only after stopping the
reaction. NKA activity in whole tissue homogenates was calculated from the
difference between the total ATPase activity (in the absence of ouabain) and
Mg++-ATPase activity (in the presence of 3.6 mM ouabain). For kinetic studies
varying Na+, K+ or ATP concentrations, the concentration of the compounds
were fixed about 50% the enzyme activity.
2.4 K+ activated-p-nitrophenylphosphatase (K+-pnppase) Assay
K+-pnppase assay was performed according to Rodriguez de Lores
Arnaiz et al. (2003). For basal measurements, KCl was omitted and substituted
by 1 mM ouabain. K+-pnppase activity was the difference between total and
basal activities.

2.5 Statistics
Prism 5.0 software (GraphPad Inc., CA, USA) was used for graphical
presentation and statistical analysis. The statistical analyses included Student’s
t-test and Two-Way ANOVA followed by Bonferroni test. The data are
expressed as the means ± standard deviation (S.D.) of at least three
independent experiments. Significance was determined at P < 0.05.
3. RESULTS
3.1 NKA inhibition
3.1.1 Purified preparations
We evaluated the synthesized compounds’ inhibitory effect on the activity
of purified NKA from guinea pig kidney and brain. Fig. 2-A shows a polygodial
(1) dose-dependent inhibition of both purified NKA preparations. The IC50
values were 9.9 μM (± 0.1) for the brain enzyme and 53 μM (± 5.1) for the
kidney enzyme, indicating a higher sensitivity for the NKA cerebral isoforms (P
< 0.001, Two-Way ANOVA Bonferroni post-test). Compounds 2 and 3
presented a weak effect (Fig. 2-B and 2-C), reaching 50% inhibition of both
enzymes only at high concentrations: the IC50 for 2 was above 100 μM, while 3
did not achieve 100% enzymatic inhibition even at 1000 μM. Compounds 4 and
5 inhibited the activity of purified kidney and brain NKA preparations (Fig. 2-D

and 2-E) with similar potencies (IC50 ≈ 50 μM). Neither of these two compounds
achieved a NKA total inhibition at the concentrations tested.
3.1.2 Crude preparations
Inhibitory potencies in crude homogenates compared to purified
preparations of NKA are shown in Fig. 3. This approach can serve as an
indication of selectivity. In the crude homogenates, a nonspecific binding to
different targets may occur and it would decrease the inhibitory effects toward
the NKA. Our results suggest good selectivity level for compounds 4 and 5,
because the inhibition in the kidney homogenate was similar to the purified
enzyme (Fig. 3-B and 3-C). Compound 1 did not display the same pattern (Fig.
3-A). The results were different however when the brain tissue was used. The
compounds did not show selectivity for cerebral NKA isoforms (Fig. 3-D to 3-F).
3.2 Kinetic studies
Kinetic studies evaluating the influence of the compounds at fixed
concentrations on the activation of purified kidney NKA preparations by Na+, K+
and ATP can be observed from double reciprocal plots shown in Figs. 4, 5 and
6. A non-competitive inhibition for Na+ (Fig. 4-A to 4-C) and K+ (Fig. 5-A to 5-C)
was found for compounds 1, 4 and 5. Altering the ATP concentration,
compounds 1 and 4 also displayed a non-competitive inhibition (Fig. 6-A and 6-
B) but 5 exhibited a competitive inhibition (Fig. 6-C).

3.3 Influence on K+-activated p-nitrophenylphosphatase activity
Considering the two phases (I and II) of the NKA catalytic cycle, the K+-
activated p-nitrophenylphosphatase (K+-pnppase) measures phase II activity
(K+-activated enzyme dephosphorylation). As shown in Fig. 7, compound 1, 4
and 5 did not affect this phosphatase in a purified kidney NKA preparation.
4. DISCUSSION
Polygodial is an agonist of the transient receptor potential vanilloid 1
(TRPV1) (Andre et al., 2004; André et al., 2006; D’Acunto et al., 2010; Della
Monica et al., 2007; Iwasaki et al., 2009; Mendes et al., 1998; Sterner and
Szallasi, 1999), a temperature-sensitive ion channel with the preference for
Ca2+ ions (Caterina et al., 1997; Caterina and Julius, 2001). In addition to its
expression in sensory neurons, TRPV1 is upregulated in several human cancer
cells (Gkika and Prevarskaya, 2009; Hartel et al., 2006; Premkumar and
Bishnoi, 2011). As an example, in human glioma cells the activation of TRPV1
leads to cell death by endoplasmic reticulum stress (Stock et al., 2012). In our
previous investigations, polygodial and a series its analogues were investigated
for TRPV1-agonistic and anticancer activities, showing cytotoxic and cytostatic
effects depending on a specific derivative (Dasari et al., 2015a, 2015b).
Furthermore, our previous results suggested that in contrast to polygodial (1),
the antiproliferative activity of compounds 4 and 5 were non-TRPV1-mediated.
Inhibition of various types of ion channels is a strategy used to combat
several types of cancers, including those associated with dismal prognoses. We

have been studying the NKA, especially its α1 subunit, which is overexpressed
in gliomas, melanomas, and non-small cell lung cancer (Garcia et al., 2015;
Lefranc et al., 2008, 2013; Mathieu et al., 2009; Mijatovic et al., 2007a). When
the activity of the NKA α1 subunit is impaired, cancer cells, including those with
multidrug resistance phenotype, undergo cell death through apoptosis or
autophagy (Gonçalves-de-Albuquerque et al., 2017; Lefranc et al., 2007;
Lefranc and Kiss, 2008; Mijatovic et al., 2009, 2012). These effects on cancer
cells have been observed using either natural or synthetic NKA inhibitors with
steroidal (e.g., cardiac glycosides, classical NKA inhibitors) or non-steroidal
structure (Garcia et al., 2015; Lefranc et al., 2013, 2008).
Herein, the brain enzyme, rich in NKA α2 and α3 isoforms, showed a
higher sensitivity to compound 1. The inhibition of this enzyme was ca. 500%
more potent than that of the kidney enzyme, expressing α1 isoform. On the
other hand, compounds 2 and 3 were found to be poor inhibitors of the NKA
enzyme activity. Furthermore, compounds 4 and 5 acted with the same potency
on the kidney and brain NKA, indicating that all isoforms are equally inhibited.
The inhibitory capacity of ouabain, a well-known NKA specific inhibitor,
was ca. 3.5 times higher for brain than for kidney preparations from guinea-pig
(Garcia et al., 2009), while this ratio is around 1000 for rat enzymes (Pôças et
al., 2003). In contrast to the rat, the affinity of all human NKA α-subunit isoforms
for ouabain is similar (Crambert et al., 2000; Wang et al., 2001). These results
indicate large differences of isoform sensitivity to cardiac glycosides among
animal species. Therefore, we cannot predict, at this time, the inhibition
behavior of human isoforms to these compounds.

Aiming to determine the magnitude of selectivity of these compounds
towards NKA, we performed another experiment using organ crude
homogenates instead of purified preparations. Using kidney homogenate,
polygodial (1) did not show selectivity. Compounds 4 and 5, however, seem to
selective for the NKA α1 isoform. Using brain homogenates, no level of
selectivity was found. The IC50 ratio between the kidney purified and crude
enzyme preparations for compounds 4 and 5 was 1.5 and 1.0, respectively.
Drawing a comparison with our previous work (Garcia et al., 2009), the
selectivity was similar to ouabain (1.2) and significantly lower for a nonselective
inhibitor, oleic acid (3.3) (Table 1). These data point to a high degree of
selectivity of compound 4 and, mainly, compound 5 in binding NKA. These
finding are consistent with the fact that polygodial (1) has other targets in the
cell, such as TRPV1, while additional interactions for compounds 4 and 5 have
not been revealed. Also consistent is the fact that compound 5 has
antiproliferative potency significantly exceeding that of 1 and was found to
maintain potency against apoptosis-resistant cancer cells as well as those
displaying the multidrug-resistant (MDR) phenotype (Dasari et al., 2015b).
Given the small number of compounds tested herein and the lack of information
on the structural requirements for binding to NKA, it is premature to speculate
on the structure-activity relationship in this series of compounds. This will be the
subject of future studies.
The possibility thus remains that the antiproliferative effect of polygodial,
which is not a cardiotonic steroid, and its derivatives could be mediated via the
NKA caveolar pool at lower concentrations, because it is independent of the
changes in intracellular Na+ and K+ concentrations. Importantly, cardiotonic

steroids display in vitro growth inhibitory effects usually in nM ranges and, yet,
they have not been developed clinically to combat various types of cancer. The
fact remains that various types of cancer display significantly higher amounts of
“NKA receptors” (mainly the NKA alpha-1 subunit) when compared to their
normal tissues (Lefranc et al., 2008; Mathieu et al., 2009; Mijatovic et al., 2008,
2007a). Thus, on the one hand, the concentration expected to inhibit the NKA
activity in the type of assay used may be higher than the one required for
inhibition of cell proliferation. On the other hand, it is known that the high
potency of cardiotonic steroids in inhibition of the NKA activity is related to their
ability to link / bridge / block the fifth loop of the alpha-1 subunit in the plasma
membrane, as demonstrated with the alpha-1 NKA subunit in rodents, in which
it is doubly mutated and is therefore 1000 times less sensitive to the toxic
effects of cardiotonic steroids (Mijatovic et al., 2007b).
Kinetic studies show a non-competitive inhibition for Na+ and K+ by
compounds 1, 4 and 5. Moreover, the same type of inhibition was found for 1
and 4 when ATP concentration was changed. Remarkably, a competitive
inhibition was presented by compound 5. In competitive inhibition, the substrate
(ATP) and inhibitor compete for access to the active site of the enzyme. Thus,
the maximum velocity of the reaction was unchanged, while the apparent affinity
of the substrate to the binding site decreased. Although a direct interaction of 5
with a specific enzyme site may be involved, we cannot rule out the possibility
that this compound binds to the lipid microenvironment involving the enzyme.
Furthermore, compounds 1, 4 and 5 have no effect on the second phase of the
NKA catalytic cycle, the K+-activated p-nitrophenylphosphatase (K+-pnppase).
Consistent with that, unlike cardiac glycosides, polygodial and its derivatives

acted on the first phase of the NKA cycle, which starts with ATP binding to the
enzyme and is followed by Na+-activated enzyme phosphorylation by ATP
(Rodríguez de Lores Arnaiz et al., 2003).
In general, diverse mechanisms of cell death are triggered via NKA but
the induction of apoptosis has been reported more frequently (Garcia et al.,
2015; Kulikov et al., 2007; Yang et al., 2014; Yu et al., 2008). Nonetheless, the
cytostatic effects in different cancer cell models can be exerted by impairing the
NKA as well (Bloise et al., 2009; Schneider et al., 2017). Our manuscript
describes a new way of blocking the alpha-1-related NKA activity, which could
be successful in combating cancers with dismal prognoses, because all our
data suggest that the polygodial derivatives under study do not display their
antiproliferative activity through the sole inhibition of the alpha-1 NKA subunit,
and definitively not through the fifth loop of the alpha-1 NKA subunit.
5. CONCLUSION
The present communication provides for the first time the evidence that
polygodial and its derivatives are NKA inhibitors. Interestingly, the minor
structural differences found among these compounds, especially between 1 and
5, which are epimers, strikingly altered their inhibitory and selective properties
on NKA. Further experiments are necessary to define the effects of these
compounds on the activation of NKA-dependent signaling cascades leading to
cell death. It is important to note that we are not in search of NKA inhibitors
more potent than cardiotonic steroids, but of NKA inhibitors that could be
clinically manageable. Therefore, the effects of these compounds on NKA may

encourage the development of polygodial analogues showing superior inhibitory
properties in a search of new anticancer agents.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge to Programa de Fomento à Pesquisa
da Universidade Federal Fluminense (FOPESQ-UFF) for their financial support.
Diogo Gomes Garcia had a postdoctoral fellowship and Camila Ignácio da Silva
had a postgraduate Scholarship from Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES).
CONFLICTS OF INTEREST: None.
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FIGURE LEGENDS
Fig. 1. Synthesis of compounds 2-5.
Fig. 2: Inhibition of purified preparations of NKA from kidney (light gray) or brain
(dark gray) by compounds 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E). Each point is the
mean ± S.D. of three different experiments conducted in triplicate.