Prostaglandins and Other Lipid Mediators

Prostaglandins & other Lipid Mediators 122 (2016) 18–27

Contents lists available at ScienceDirect

Original research article

In silico modelling of prostacyclin and other lipid mediators to nuclear

receptors reveal novel thyroid hormone receptor antagonist properties

∗

Noelia Perez Diaz, Mire Zloh, Pryank Patel, Louise S. Mackenzie

Life and Medical Sciences, University of Hertfordshire, Hatﬁeld AL10 9AB, UK

a r t i c l e i n f o a b s t r a c t

Article history: Prostacyclin (PGI2) is a key mediator involved in cardiovascular homeostasis, acting predominantly on

Received 10 August 2015

two receptor types; cell surface IP receptor and cytosolic peroxisome proliferator activated receptor

Received in revised form

(PPAR) ␤/␦. Having a very short half-life, direct methods to determine its long term effects on cells

19 November 2015

is difﬁcult, and little is known of its interactions with nuclear receptors. Here we used computational

Accepted 7 December 2015

chemistry methods to investigate the potential for PGI2, beraprost (IP receptor agonist), and GW0742

Available online 11 December 2015

␤ ␦

(PPAR / agonist), to bind to nuclear receptors, conﬁrmed with pharmacological methods.

In silico screening predicted that PGI , beraprost, and GW0742 have the potential to bind to different

Abbreviations: 2

nuclear receptors, in particular thyroid hormone ␤ receptor (TR␤) and thyroid hormone ␣ receptor (TR␣).

MLS, MLS000389544

Docking analysis predicts a binding proﬁle to residues thought to have allosteric control on the TR ligand

NSAID, non-steroidal anti-inﬂammatory

drug binding site. Luciferase reporter assays conﬁrmed that beraprost and GW0742 display TR␤ and TR␣

−5 −6

× ×

PPAR␤/␦, peroxisome proliferator activated antagonistic properties; beraprost IC50 6.3 10 mol/L and GW0742 IC50 4.9 10 mol/L. Changes to

receptor triiodothyronine (T3) induced vasodilation of rat mesenteric arteries measured on the wire myograph − −

PGI , prostacyclin 5 5

2 were measured in the presence of the TR antagonist MLS000389544 (10 mol/L), beraprost (10 mol/L)

PAH, pulmonary artery hypertension −5

and GW0742 (10 mol/L); all signiﬁcantly inhibited T3 induced vasodilation compared to controls.

T3, triiodothyronine

We have shown that both beraprost and GW0742 exhibit TR␤ and TR␣ antagonist behaviour, and sug-

TR␣, thyroid hormone ␣ receptor

gests that PGI2 has the ability to affect the long term function of cells through binding to and inactivating

TR␤, thyroid hormone ␤ receptor

thyroid hormone receptors.

Thyroid hormone

Mesenteric artery

Triiodothyronine

1. Introduction monary artery and bronchi. PGI2 has a very short half-life of 42 s,

which makes direct pharmacological analysis difﬁcult, although

Prostacyclin (PGI2) is a prostaglandin that was ﬁrst described by there are a large number of PGI2 mimetics available such as iloprost,

Sir John Vane in 1976 as a potent vasodilator and anti-thrombotic beraprost and treprostinil, which have half-lives of 30 min, 40 min

agent of the vasculature [1]. The long term effects of PGI2 is known and 4 h respectively. Prostanoid signalling is of key importance

to be important in different organ systems, for example PGI2 is in the lung, as evidenced by the use of PGI2 mimetics in pul-

known to suppress airway remodelling in experimental asthma monary artery hypertension and non-steroidal anti-inﬂammatory

models [2], and has important anti-inﬂammatory effects in pul- drug (NSAID) induced asthma [3], and involvement in the devel-

opment of asthma and COPD [4]. Lung diseases such as pulmonary

artery hypertension (PAH) are treated with PGI2 mimetics, which

greatly improve the symptoms but fail to reverse or cure the vas-

∗

Corresponding author at: Pharmacy, Pharmacology and Postgraduate Medicine, cular remodelling. The use of PGI2 mimetics in the clinic has so far

School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hat- only attempted to replace the acute IP receptor mediated changes

ﬁeld AL10 9 AB, UK.

related to PGI2, and have not yet attempted to mimic the genomic

E-mail addresses: [email protected], [email protected]

changes affected by PGI2 binding to nuclear receptors. Before this (L.S. Mackenzie).

http://dx.doi.org/10.1016/j.prostaglandins.2015.12.002

N. Perez Diaz et al. / Prostaglandins & other Lipid Mediators 122 (2016) 18–27 19

can be attempted we need to address our understanding of how the Luciferase Detection Reagent added. Light emission from each

PGI2 and mimetics signal via nuclear receptors. sample well was quantiﬁed using a plate reading luminometer.

PGI2 has been described as the putative endogenous ligand for In order to assess TR␣ and TR␤ antagonistic activities the proto-

peroxisome proliferator activated receptor ␤/␦ (PPAR␤/␦) nuclear col was adjusted; Reporter Cells were exposed to a sub-maximal

receptors [5]. Signalling via PPAR␤/␦ has a number of effects on concentration of a suitable agonist: dexamethasone for the GR

cell function and has been shown to be involved in fatty acid assay and T3 for TR␣ and TR␤ assays, followed by increasing con-

metabolism, lipid transport as well as proliferation and differentia- centrations of GW0742 and beraprost.

tion of cells. There are a large number of PPAR␤/␦ agonists, both

endogenously produced (PGI2, fatty acids including the ‘omega 2.3. Myography

3’ fatty acids) and synthetic compounds (GW0742, GW501516),

however the use of PPAR␤/␦ agonists in different cellular models Male Wistar rats (350–450 g) were housed in pairs, and killed

and at widely differing concentrations has led to a controversy in by CO2 asphyxiation. The care and use of the rats were carried

regards to the role of PPAR␤/␦ in the cell [6]. The PPAR␤/␦ agonist out in accordance with UK Home Ofﬁce regulations, UK Animals

GW501516 completed proof-of-concept clinical trials successfully (Scientiﬁc Procedures) Act of 1986 under PPL70/6788.

for dyslipidaemia [7] and hypercholesteraemia [8], although fur- First and second order mesenteric arteries were removed and

ther clinical trials were halted due to toxicology issues [9]. Since prepared as described previously [14]. Brieﬂy, artery segments

−3

this point, much interest has gathered as to the use of PPAR␤/␦ were dissected in Kreb’s buffer (pH 7.4, 1.18 10 mol/L NaCl,

−3 −3 −3

× × ×

agonists in various disease settings, as the nuclear receptor has 4.7 10 mol/L KCl, 1.2 10 mol/L MgSO4, 1.2 10 mol/L

−3 −2

× ×

multiple regulatory roles in cell function and differentiation from KH2PO4, 2.5 10 mol/L CaCl2, 2.5 10 mol/L NaHCO3 and

−2

controlling smooth muscle tone to remodelling of the pulmonary 1.1 10 mol/L glucose), and loaded onto isometric wire myo-

circulation [9–11]. graphs. The bath solution was continuously bubbled with 95% O2

There are fundamental questions as to the long term direct and 5% CO2. All vessels were allowed to equilibrate for 30 min prior

actions of PGI2 and related compounds on the cell and the wide to being set at a ‘normalised’ internal circumference 0.9.L100; this

variety of receptors which remain to be addressed. The aim of this is estimated to be 0.9 times the circumference they would main-

study was to identify novel binding targets of PGI2, and other lipid tain if relaxed and exposed to 100 mm Hg transmural pressure. This

mediators using computational chemistry methods, validated on was calculated for each individual vessel on the basis of passive

gene reporter assays and alterations to vascular function measured length-tension characteristics of the artery and the Laplace rela-

by myography. tionship [15]. The average diameter of the mesenteric arteries were

239.4 ± 10.9 ␮m.

−5 −5

Arteries were incubated with 10 mol/L MLS, 10 mol/L

2. Materials and methods −7

beraprost or 10 mol/L GW0742 for 30 min prior to addition of

−9 −6

increasing concentrations of U46619 (10 mol/L to 10 mol/L).

2.1. In silico methods

Arteries were washed four times with Krebs buffer, and once

tone had returned to basal levels, arteries were incubated with

OpenVirtualToxLab .5,21 [12] was used to predict toxic poten-

MLS, beraprost and GW0742 for a further 15 min. Arteries were

tial by predicting binding afﬁnities to 10 nuclear receptors. The −7

then pre-contracted with 3 × 10 mol/L U46619; once plataeu was

default values of the software for the predictions of toxic potentials

acheived, vasodilation in response to increasing concentrations of

for prostacyclin, beraprost and GW0742 were used as described −10 −7

l-triiodothyronine (T3; 10 to 3 × 10 mol/L) was measured.

previously [12].

The Pharmmapper, freely available web server (http://59.78.96.

2.4. Materials

61/pharmmapper), was used to predict potential target candidates

for all drugs. The mol2 ﬁles for all molecules were submitted to

All chemicals and reagents were obtained from Sigma Aldrich

the Pharmmapper server by using default settings and limiting the

unless otherwise stated. Drugs were dissolved in DMSO to a stock

target set to human targets [13]. −2

of 10 mol/L, and then further dilutions made in water for further

The ability of drugs to bind into protein active sites was investi-

dilutions.

gated using Glide (Small-molecule Drug Discovery Suite 2014-3:

Glide, Version 6.4 Schrödinger, LLC, New York, NY (2014)) with

2.5. Data analysis

Maestro as a graphical user interface. The protein preparation wiz-

ard was utilized to adjust charges and protonation states of proteins

Reporter assay data are expressed as a mean ± SEM percent

using relevant data bank entries, 1Q4X for TR␤, 1NAV and 4LNX

induction, with 100% induction deﬁned as the activity achieved

for TR␣, and 3GZ9 for, PPAR␤␦. Prepared protein structures were −7

with 10 mol/L of T3.

used to build energy grids with enclosing boxes of default sizes

Contractile data are expressed in changes in tension (mN) minus

centred on co-crystalized ligands. The molecules PGI2, beraprost

baseline tension. Vasodilation is expressed as percentage of the

and GW0742 were docked ﬂexibly using XP docking protocol;

U46619 mediated pre-contraction obtained prior to the addition

ligands were minimized onto OLSA-2005 non-bonded interaction

of T3. Data are presented as mean ± SEM; n refers to the number

grid, with all other parameters set to their default values.

of rats used. Statistical analysis was performed by two way anal-

ysis of variance using GraphPad Prism 5.0, and differences were

2.2. Reporter assays considered to be signiﬁcant when P was less than 0.05.

Human TR␣, TR␤ and glucocorticoid reporter (GR) assay sys- 3. Results

tems were purchased from INDIGO Bioscience (State College, PA).

Assays were performed according to the manufacturer’s instruc- 3.1. Virtualtox screening and docking programmes

tions for both agonist and antagonist activity. Brieﬂy, reporter cells

were dispensed into the wells of the assay plate and immediately Using the Virtualtox screening programme, structures for PGI2,

dosed with l-triiodothyronine (T3), beraprost and GW0742. Fol- beraprost and GW0742 were assessed for the potential binding to a

◦

lowing 24 h incubation at 37 C, treatment media was discarded and series of target protein known to be correlated with the side effects,

20 N. Perez Diaz et al. / Prostaglandins & other Lipid Mediators 122 (2016) 18–27

Table 1

predicted binding proﬁle of the three molecules with PPAR␤␦

In silico modelling of PGI2, beraprost and GW0742 to nuclear receptors.

(Table 3), that indicated a propensity that all three molecules will

␤␦

Predicted EC50 (mol/L) have afﬁnity for a binding site of crystal structure of PPAR (PDB

entry 1Y0S). However, a more recent structure of PPAR␤␦ (PDB

PGI2 Beraprost GW0742

entry 3GZ9) with resolution of 2 Å was selected for the docking

−7 −7 −8

AR 7.39 × 10 4.56 × 10 9.75 × 10

−5 −7 −6 analysis as a suitable model for in silico studies [16].

Er␣ 2.76 × 10 7.45 × 10 1.35 × 10

␤ × −5 −6 −6 All three ligands have a propensity to form polar interactions

× × Er 2.93 10 5.35 10 2.09 10

−9 −10 −9

␤␦

GR 1.36 × 10 9.25 × 10 4.18 × 10 with residues of PPAR binding site by forming hydrogen bonds

−8 −8 −9

× × ×

LXR 2.50 10 2.13 10 1.33 10 with H323, H449 and Y473 residues (Fig. 4), however, minor differ-

−7 −6 −7

MR 9.06 × 10 2.81 × 10 1.50 × 10

ences were observed in their spatial occupancy of the active site.

−7 −8 −8

PPAR␥ 4.81 × 10 5.93 × 10 1.02 × 10

␤␦

−8 −9 −9 The PGI2 binding to its putative nuclear receptor PPAR indicates

PR 4.31 × 10 2.51 × 10 9.87 × 10

− − −

6 8 8 a similarity in interaction proﬁle (Fig. 5B) to that of the known ago-

␣ × ×

TR 3.44 10 65.5 10 1.28 × 10

−8 −8 −9

␤

× ×

TR 16.6 10 5.51 10 7.73 × 10 nist GW0742, yet some differences were observed between binding

Charge −1 −1 −1

proﬁles of PGI2 and beraprost (Fig. 5A), which does not activate

ToxPot 0.67 0.70 0.67

PPAR␤␦ (data not shown). However, these differences in the inter-

actions may not be a main reason to lack of activation of PPAR␤␦ by

beraprost, it can be hypothesised that beraprost has higher afﬁnity

and a normalized toxicity potential was calculated (Table 1). The

towards other intracellular targets.

results suggest that PGI2, beraprost and GW0742 have the potential

to bind most nuclear receptors, albeit with various afﬁnities. The

3.3. Reporter assays: genomic effects

overall predicted toxicity potentials were 0.67 for PGI2, 0.70 for

beraprost and 0.67 for GW0742. These values indicate that have

In silico data indicated a potential for PGI2 to bind a wide range

potential to induce side effects to similar extent as chlomazone,

of nuclear receptors, most notably AR, GR, LXR, PR, TR␣ and TR␤

and bisphenol B.

(Table 1) although further analysis in vitro was not possible due

Data presented indicates the predicted EC50 of PGI2, beraprost

to the short half-life of PGI2 (42 s). Although our results indicated

and GW0742 to 10 nuclear receptors; Androgen Receptor (AR),

a potential for beraprost and GW0742 to bind to glucocorticoid

Estrogen receptor ␣ (ER␣), Estrogen receptor ␤ (ER␤), Gluco-

receptor, none of the compounds tested exhibited agonist (data not

corticoid receptor (GR), Liver X receptor (LXR), Mineralcotocoid

shown) or antagonist properties using the GR luciferase reporter

receptor (MR), PPAR␥, Progesterone receptor (PR), Thyroid hor-

assay (Figs. 6A and B ).

mone receptor ␣ (TR␣) and Thyroid hormone receptor ␤ (TR␤).

Similarly, a potential for beraprost and GW0742 to bind to

Binding potential is indicated by mol/L and ToxPot is a measure of

TR␣ and TR␤ were predicted, none of the compounds elicited

a toxic potential, a normalized binding afﬁnity in respect to a series

agonist properties using the TR␣ and TR␤ luciferase reporter

of protein models with known adverse effects.

assays (data not shown). However, further TR␣ and TR␤ reporter

The results indicated that the initial assumption that PGI2 and

assays were run using an antagonist protocol, incubating reporter

mimetics may interact with nuclear receptors was correct and war- −7

cells with 10 mol/L T3 (a TR␣ and TR␤ agonist) and then

ranted further investigation. As proof of principle, we investigated

increasing concentrations of beraprost and GW0742. Interestingly,

the potential for PGI2, beraprost and GW0742 to bind to TR␤. The

beraprost inhibited T3 induced TR␣ luciferase activity with an

predicted binding afﬁnities of drugs to TR␤ by PGI2, beraprost and −5 −5

IC50 3.15 × 10 mol/L and GW0742 IC50 2.6 × 10 mol/L (Figs.

GW0742 were in the nanomolar range (Table 1).

6C and D). Results from the TR␤ luciferase activity indicate that

The possibility that PGI2, beraprost and GW0742 could bind −5

beraprost exhibits an IC50 4.1 × 10 mol/L and GW0742 IC50

to these thyroid hormone receptors was further investigated by −6

1.02 × 10 mol/L (Figs. 6E and F); this indicates that the binding

using the reverse docking platform Pharmmapper. PGI2, beraprost

afﬁnities of beraprost and GW0742 to TR␣ and TR␤ may be greater

and GW0742 structures were submitted to the webserver and

to the binding afﬁnities of agonist T3.

the ability to bind to TR␤ was demonstrated as a list of possi-

In order to determine whether beraprost and GW0742 antag-

ble protein data bank ﬁles in whose active sites these two drugs

onism of TR non-genomic mediated activities, we performed

could favourably bind (Table 2). The docking analysis was carried

isometric wire myography of rat mesenteric arteries. The TR antag-

out using TR␤ crystal structure with three different ligands, PGI2,

onist MLS has no effect on U46619 mediated contraction (Fig. 7A).

beraprost and GW0742 (Fig. 1) which identiﬁed a shared afﬁnity −4

The presence of 10 mol/L l-NAME signiﬁcantly increased U46619

to residue R282 within the ligand binding TR␤ pocket. Due to the

mediated contraction of rat mesenteric arteries (Fig. 7B) whilst

similarities between TR␤ and TR␣, docking analysis was also car- −5 −7

10 mol/L beraprost (Fig. 7C) and 10 mol/L GW0742 signiﬁ-

ried out with the TR␣ crystal structure with PGI2, beraprost and

cantly reduced U46619 contraction (Fig. 7D).

GW0742. TR␣ has two known binding sites, so docking analysis

was conducted for both separate binding sites (Figs. 2 and 3).

3.4. Myography: non-genomic effects of TR

Having two published ligand binding sites, docking analysis

using TR␣ crystal structure was carried out with three different

In order to test the antagonist potential of MLS, GW0742 and

ligands, PGI2, beraprost and GW0742 on both sites. The ﬁrst site

beraprost on T3 induced vasodilation, arteries were pre-contracted

is located in a similar pocket within the protein, and is similar in

with U46619. Addition of the compounds effected the EC80 U46619

structure to TR␤ ligand binding site (Fig. 2). Analysis predicts the

induced tone, control 11.95 ± 2.5mN, l-NAME 14.96 ± 2.3 mN,

binding of residues R228 and R262 is common to PGI2, beraprost

beraprost 4.435 ± 1.9 mN and GW0742 7.8 ± 3.7 mN.

and GW0742. On the other hand, the second TR␣ predicted binding −9 −7

Cumulative addition of T3 (10 to 3 × 10 mol/L) to U46619

proﬁle shared the residues S326, Q342, W364, T372 and R375.

pre-contracted arteries induced a signiﬁcant reduction in tension

in mesenteric arteries (Fig. 8). This vasodilatory effect was signiﬁ-

3.2. PPARˇı docking cantly inhibited by incubation with the selective TR antagonist MLS

(Fig. 8A). Previous reports have indicated that T3 induced vasodi-

The Pharmamodels picked for further analysis were those that lation is mediated by a rapid non-genomic event involving eNOS

−4

shared 9 features in the predictive binding proﬁle, similar to the activation [17]. In order to test this hypothesis, 10 mol/L l-NAME

N. Perez Diaz et al. / Prostaglandins & other Lipid Mediators 122 (2016) 18–27 21

Fig. 1. In silico docking analysis of ligands to TR␤. Docking analysis showing predicted binding poses of (A) PGI2, (B) beraprost and (C) GW0742 ligands in human TR␤ (PDB

1Q4X). Ligands are represented by cylinders, side-chain residues of TR␤ are represented by sticks; dashed lines represents hydrogen bond interactions to amino acid residues

in TR␤; transparent surfaces are coloured by electrostatic charges; side-chain residues shown are within 4 Å of bound ligand.

Fig. 2. In silico docking analysis of ligands to TR␣. Docking analysis showing predicted binding poses of (A) PGI2, (B) beraprost and (C) GW0742 ligands in human TR␣ (PDB

1NAV). Ligands are represented by cylinders, side-chain residues of TR␣ are represented by sticks; dashed lines represents hydrogen bond interactions to amino acid residues

in TR␣; transparent surfaces are coloured by electrostatic charges; side-chain residues shown are within 4 Å of bound ligand.

22 N. Perez Diaz et al. / Prostaglandins & other Lipid Mediators 122 (2016) 18–27

Fig. 3. In silico docking analysis of ligands to the second binding site of TR␣. Docking analysis showing predicted binding poses of (A) PGI2, (B) beraprost and (C) GW0742

ligands in human TR␣ (PDB 4LNX). Ligands are represented by cylinders, side-chain residues of TR␣ are represented by sticks; dashed lines represents hydrogen bond

interactions to amino acid residues in TR␣; transparent surfaces are coloured by electrostatic charges; side-chain residues shown are within 4 Å of bound ligand.

Fig. 4. In silico docking analysis of ligands to PPAR␤␦ receptor. Docking analysis showing predicted binding poses of (A) PGI2, (B) beraprost and (C) GW0742 ligands in human

PPAR␤␦ (PDB 3GZ9). Ligands are represented by cylinders, side-chain residues of PPAR␤␦ are represented by sticks; dashed lines represents hydrogen bond interactions to

amino acid residues in PPAR␤␦; transparent surfaces are coloured by electrostatic charges; side-chain residues shown are within 4 Å of bound ligand.

N. Perez Diaz et al. / Prostaglandins & other Lipid Mediators 122 (2016) 18–27 23

Table 2

The ranked list of hit target pharmacophore models for PGI2, beraprost and GW06742 obtained using Pharmmapper webserver. The list is sorted by number of feature

interactions and ﬁltered to show PGI2, beraprost and GW0742 hits for TR␤, where Pharma Model indicates the relevant PDB entry.

Molecule Pharma model No. feature Fit No. hydro-phobic No. HB acceptor No. HB donor No. +ve No. −ve No. aromatic

PGI2 1nuo v 7 4.09 4 1 1 0 1 0

Beraprost 1n46 v 7 4.65 4 1 1 0 0 1

Beraprost 1nax v 7 4.40 4 1 1 0 1 0

Beraprost 1nq1 v 7 4.36 4 1 1 0 1 0

GW0742 1nuo v 7 4.23 4 1 1 0 1 0

GW0742 1nq1 v7 4.06 4 1 1 0 1 0

GW0742 1nq2 v 7 3.76 4 1 1 0 1 0

Beraprost 1r6g v 8 4.51 5 1 1 0 1 0

GW0742 1r6g v 8 3.75 5 1 1 0 1 0

PGI2 1q4x v 9 4.58 6 2 0 0 1 0

Beraprost 1q4x v 9 5.10 6 2 0 0 1 0

GW0742 1q4x v9 4.03 6 2 0 0 1 0

Table 3

The ranked list of hit target pharmacophore models for PGI2, beraprost and GW06742 obtained using Pharmmapper webserver. The list is sorted by number of feature

interactions and ﬁltered to show PGI2, beraprost and GW0742 hits for PPAR␤␦, where Pharma Model indicates the relevant PDB entry.

Molecule Pharma model No. feature Fit No. hydro-phobic No. HB acceptor No. HB donor No. +ve No. −ve No. aromatic

PGI2 1y0s v 9 4.76 8 0 0 0 1 0

Beraprost 1y0s v9 4.80 8 0 0 0 1 0

GW0742 1y0s v 9 3.65 8 0 0 0 1 0

Fig. 5. Overlays of ligands in favourable docking poses in PPAR␤␦. Superposition of the most favourable docking pose obtained for ligands into active site of PPAR␤␦ (PDB

3GZ9) with the docking poses of (A) PGI2 (stick representation with grey carbon atoms) and Beraprost (gold carbon atoms); (B) PGI2 (with grey carbon atoms) and GW0742

(green carbon atoms) and (C) beraprost (blue carbon bonds) and GW0742 (orange carbon bonds) ligands into same active site.

was added to the bath prior to mesenteric artery contraction to 4.1. Interactions of ligands with TRˇ and TR˛

U46619. l-NAME had no signiﬁcant effect on T3 induced vasodila-

tion (Fig. 8B). In contrast, incubation in the presence of beraprost The in silico binding proﬁles of beraprost, GW0742 and PGI2

(Fig. 8C) and GW0742 (Fig. 8D) signiﬁcantly inhibited T3 induced to TR␤ show remarkable similarities. Conﬁrmation with luciferase

vasodilation of mesenteric arteries. reporter assay indicates that beraprost inhibited T3 induced TR␤

−5

gene induction with an IC50 4.1 × 10 mol/L and GW0742 IC50

−6

1.02 × 10 mol/L, which is comparable to the anti-thyroid hor-

4. Discussion −5

mone activity of bisphenol A IC50 1.64 × 10 mol/L [19]. The

docking proﬁles indicate a common interaction of all three

A link between PGI2 and thyroid function has previously been

molecules with R282 found in the TR␤ Ligand–Binding Domain

established, whereby PGI2 increases thyroid hormone production

(LBD) which is involved in the genomic and non-genomic activity

[18]. The data presented here is the ﬁrst to show that PGI2 has

of the nuclear receptor [20,21].

the potential to bind to TR␤ and TR␣, and a range of other nuclear

While the LBD of TR␤ and TR␣ differ by only one residue,

receptors which require further in depth study. While direct in vitro

sequence homology of the second binding domain indicates that

analysis is not possible due to the very short half-life of PGI2, we

the two TR proteins have an identical second binding site; how-

investigated the ability of the PGI2 mimetic, beraprost and GW0742

ever, the crystal structure of the second domain has only been

to bind to nuclear receptors. Our results indicated a potential for

described from TR␣ [22]. Our ﬁndings predict that PGI2, beraprost

PGI2, beraprost and GW0742 to bind to numerous nuclear recep-

and GW0742 have the common binding proﬁle of R228 and R262

tors, notably AR, GR, LXR, PR, TR␣ and TR␤.

in the ﬁrst binding site of TR␣, and share binding of residues S326,

For proof of concept we chose to investigate GR, TR␤ and TR␣

Q342, W364, T372 and R375 in the second binding site of TR␣. There

further, due to the potential for beraprost and GW0742 to bind to

is an interesting commonality of the key residues involved in what

these nuclear receptors in the nanomolar range. The reporter assays

is thought to be a novel allosteric mechanism in which a high con-

indicated that beraprost and GW0742 lacked GR agonist and antag-

centration of T3 in the nucleus binds to R342 and R375 in the second

onist behaviour, indicating that the computer generated binding

site and interacts with T3 binding of R228 in the ﬁrst site of TR␣ (or

proﬁles can only provide guidance and not replace in vitro testing.

24 N. Perez Diaz et al. / Prostaglandins & other Lipid Mediators 122 (2016) 18–27

−7

Fig. 6. Luciferase reporter gene assay of nuclear receptor antagonism. Reporter cells were activated with EC50 l-triiodothyronine (T3; 10 mol/L), and (A) and (B) Glucocor-

ticoid receptor (GR); (C) and (D) TR␣; (E) and (F) TR␤ antagonist activity measured following incubation with increasing concentrations of beraprost and GW0742 for 24 h.

−7

Data are presented as mean ± SEM percent induction, with 100% induction deﬁned as the activity achieved with 10 mol/L T3; n = 4–8.

R282 in TR␤) [22]. This novel allosteric interaction has been sug- timing of experiments, since initiation of either event is quite dif-

gested to be a mechanism by which TR activation is suppressed by ferent. Examples include the enhancement of the antiviral activity

high concentrations of the thyroid hormones in the nucleus, and of interferon ␥ by TR␤ may be achieved by both genomic and non-

thereby reduce TR induction of transcription [22]. These similari- genomic routes; T4-dependent activation of the mitogen activated

ties in the predicted binding sites of PGI2, beraprost and GW0742 to protein kinase /extracellular signal-regulated kinase 1/2 (MAPK/

TR␣ and TR␤ proteins give weight to our ﬁndings that beraprost and ERK1/2) which can phosphorylate serine (Ser142) located on DNA

GW0742 display antagonistic properties to these nuclear receptors, binding domain of TR␤ [21]. This phosphorylation leads to dissocia-

evident especially at high concentrations. tion of TR␤ from corepressor proteins, which trans- activates target

Our data indicates that PGI2, beraprost and GW0742 all exhibit genes. Yet TR␤ antagonists which inhibit the non-genomic activity

both genomic and non-genomic TR␣ and TR␤ activities, thus mak- blocks the T4 potentiation of the antiviral and immune-modulatory

ing a clear distinction between genomic and non-genomic effects actions of the interferon ␥, even though these antagonists lack any

difﬁcult to make, and one which requires further exploration. The direct effect on the interferon-␥ action [24].

ﬁnding that both beraprost and GW0742 exhibit TR␤ antagonist Thyroid hormones have been shown to act directly on coro-

properties highlights a strong possibility for PGI2 to also act as a nary artery smooth muscle cells [25] and rat mesenteric arteries

TR␤ and TR␣ antagonist at high concentrations, and that the activity [26] to induce vasodilation in a non-genomic manner, occurring

may also have non-genomic effects on function. within a few minutes of stimulation [17]. The mechanism and

Surprisingly for a nuclear receptor, phosphorylated TR␤ is receptors responsible for T3 mediated dilation is emerging in the

located at the plasma membrane or in the cytoplasm, which once literature, with one recent study in TR␣ knockout mouse mesen-

initiated may lead to complex, nucleus-mediated cellular events teric arteries lacking the T3 vasodilatory effect. Other studies have

leading to the transcription of genes including HIF1A MCL1 and shown that T3 activates TR␤ which cross-couples to the phospho-

GLUT1 [23]. A distinction between the genomic and non-genomic inositol 3-kinase/protein kinase pathway [17], which in arterial

activities of TR␤ may not be clear cut, since ligands such as T3 acti- endothelial cells, leads to eNOS activation and NO production of NO

vate both pathways. However a clear division can be made using the and thus induces dilation. Other members of the steroid hormone

N. Perez Diaz et al. / Prostaglandins & other Lipid Mediators 122 (2016) 18–27 25

−5 −4 −5 −7

Fig. 7. The effects of ligands on mesenteric artery contraction The effects of (A) 10 mol/L MLS, B. 10 mol/L l-NAME, (C) 10 mol/L Beraprost and (D)10 mol/L GW0742

on U46619 induced contraction. Data are presented as mean ± SEM, and signiﬁcance is represented as * = p < 0.05, ** = p < 0.01, *** = p < 0.001 by two way ANOVA and f = p < 0.05

and fff = p< 0.001 by Bonferroni post hoc test; ns = non-signiﬁcant.

−7

Fig. 8. The effects of ligands on mesenteric artery vasodilation. T3 induced vasodilation in mesenteric arteries pre-contracted with EC80 U46619 (3 × 10 mol/L), in the

−4 −5 −7

presence of (A) MLS, (B) 10 mol/L l-NAME, (C) 10 mol/L Beraprost and (D) 10 mol/L GW0742. Data are presented as mean ± SEM, and signiﬁcance is represented as

***p = <0.001 by two way ANOVA and f = p < 0.05, ff = p< 0.01 and fff =p < 0.001 by Bonferroni post hoc test, and non-signiﬁcance by ‘ns’.

superfamily have been shown to cross couple with the PI3- vasodilation of rat mesenteric arteries is not directly dependent

␤ ␦ kinase/Akt pathway, including PPAR / [27] and glucocorticoid on NO production [25,26]. Here we show that full dilation is

−7

receptors [28] indicating that non-genomic control of steroidal achieved at 3 × 10 mol/L T3, which was signiﬁcantly reduced by

receptors play an important role in vascular homeostasis. Our data incubation with the TR antagonist MLS. While MLS cannot differ-

is in agreement with a similar study showing that T3 mediated entiate between TR␣ and TR␤ [29], without further study we can

26 N. Perez Diaz et al. / Prostaglandins & other Lipid Mediators 122 (2016) 18–27

only conclude that T3 induces vasodilation in rat mesenteric arter- Source of funding

ies that is TR␣ and TR␤ mediated and not dependent on nitric oxide

synthase. The conduct of the research and preparation of the article was

As expected, MLS had no signiﬁcant effect on U46619 medi- funded by the University of Hertfordshire, UK.

ated contraction, indicating that TRs are not involved in contractile

responses. Beraprost is an IP agonist, so the signiﬁcant reduction Disclosure

in U46619 induced tone is likely to be due to the dilatory effect on

the smooth muscle cells. Contraction was achieved however, albeit The authors declare that there is no conﬂict of interest regarding

at a reduced level, and T3 mediated vasodilation was signiﬁcantly the publication of this article.

reduced. Taking the results together this suggests that beraprost

is binding to and inactivating TR non-genomic activity in smooth Acknowledgements

muscle cells.

In a similar manner, GW0742 reduced arterial contraction. This Authors thank to Biograf for the license to use OpenVirtual-

is a phenomenon previously reported [9,10], although not fully ToxLab software. We also thank Open Eye Scientiﬁc Software, Inc.,

explained. The addition of GW0742 signiﬁcantly reduced T3 medi- for the free academic license of the Open Eye Toolkits. We also

ated vasodilation, which may be due to the direct binding of thank Schrodinger Ltd., for their support in the ﬁnal stages of the

␤

GW0742 to TR in smooth muscle cells as predicted by the in silico manuscript revision by providing the license for their software.

modelling and reporter assays.

The normal physiological range of total T3 in the human plasma References

−9

is between 1 and 2.9 × 10 mol/L, concentrations which did not

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