Distinct molecular requirements for activation or stabilization of soluble guanylyl cyclase upon haem oxidation-induced degradation.

RESEARCH PAPER
Distinct molecular requirements for activation or
stabilization of soluble guanylyl cyclase upon haem
oxidation-induced degradation
LS Hoffmann1,2, PM Schmidt3, Y Keim1, S Schaefer1, HHHW Schmidt4 and JP Stasch1,2
1Pharma Research Centre, Bayer HealthCare, Aprather Weg 18a, Wuppertal, Germany, 2Martin-Luther-University, School of
Pharmacy, Halle, Germany, 3CSIRO Molecular & Health Technologies, 343 Royal Parade, Parkville, Vic., Australia, and
4Department of Pharmacology & Centre for Vascular Health, Monash University, Melbourne, Clayton, Vic., Australia
Background and purpose: In endothelial dysfunction, signalling by nitric oxide (NO) is impaired because of the oxidation and
subsequent loss of the soluble guanylyl cyclase (sGC) haem. The sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)
oxy]phenethyl}amino)methyl[benzoic]acid (BAY 58-2667) is a haem-mimetic able to bind with high affinity to sGC when the
native haem (the NO binding site) is removed and it also protects sGC from ubiquitin-triggered degradation. Here we investigate
whether this protection is a unique feature of BAY 58-2667 or a general characteristic of haem-site ligands such as the
haem-independent sGC activator 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-
benzamide sodium salt (HMR 1766), the haem-mimetic Zn-protoporphyrin IX (Zn-PPIX) or the haem-dependent sGC stimulator
5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272).
Experimental approach: The sGC inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was used to induce
oxidation-induced degradation of sGC. Activity and protein levels of sGC were measured in a Chinese hamster ovary cell line
as well as in primary porcine endothelial cells. Cells expressing mutant sGC were used to elucidate the molecular mechanism
underlying the effects observed.
Key results: Oxidation-induced sGC degradation was prevented by BAY 58-2667 and Zn-PPIX in both cell types. In contrast,
the structurally unrelated sGC activator, HMR 1766, and the sGC stimulator, BAY 41-2272, did not protect. Similarly, the
constitutively haem-free sGC mutant b1H105F was stabilized by BAY 58-2667 and Zn-PPIX.
Conclusions: The ability of BAY 58-2667 not only to activate but also to stabilize oxidized/haem-free sGC represents a unique
example of bimodal target interaction and distinguishes this structural class from non-stabilizing sGC activators and sGC
stimulators such as HMR 1766 and BAY 41-2272, respectively.
British Journal of Pharmacology (2009) doi:10.1111/j.1476-5381.2009.00263.x
Keywords: soluble guanylyl cyclase; BAY 58-2667; HMR 1766; BAY 41-2272; oxidative stress; nitric oxide; cGMP; haem
Abbreviations: BAY 41-2272, 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine; BAY
58-2667, 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid; CHO,
Chinese hamster ovary; EC, endothelial cell; HMR 1766, 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-
N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt; HSP90, heat shock protein 90; ODQ,
1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; ONOO-, peroxynitrite; PPIX, protoporphyrin IX; RLU, relative
light unit; ROS, reactive oxygen species; sGC, soluble guanylyl cyclase; WT, wildtype; Zn-PPIX,
zinc-protoporphyrin IX
Introduction
The second messenger cyclic guanosine monophosphate
(cGMP), synthesized from guanosine triphosphate (GTP) by
soluble guanylyl cyclase (sGC), is a key regulator of vascular
smooth-muscle cell relaxation and inhibition of platelet
aggregation (Lucas et al., 2000). sGC is a heterodimeric
enzyme consisting of an a and a haem-containing b subunit,
which represents the intracellular receptor for the gaseous
messenger, nitric oxide (NO). However, other mechanisms of
sGC regulation have been described such as membrane asso-
ciation or binding to the chaperone heat shock protein 90
(HSP90; Zabel et al., 2002; Agulló et al., 2005; Nedvetsky et al.,
2008). The prosthetic haem group is non-covalently bound to
Correspondence: Dr Johannes-Peter Stasch, Cardiovascular Research, Bayer
HealthCare AG, Aprather Weg 18a, Wuppertal D-42096, Germany. E-mail:
johannes-peter.stasch@bayerhealthcare.com
Received 1 December 2008; revised 30 January 2009; accepted 18 February
2009
British Journal of Pharmacology (2009), , ••–••
© 2009 The Authors
Journal compilation © 2009 The British Pharmacological Society All rights reserved 0007-1188/09
www.brjpharmacol.org

the b1 subunit via the proximal haem ligand H105 (Wedel
et al., 1994; Zhao et al., 1998) and the haem-binding motif
Y-x-S-x-R (Pellicena et al., 2004; Schmidt et al., 2004; 2005; Ma
et al., 2007). Binding of NO to the haem group activates sGC
and results in a considerable increase (up to 200-fold) in the
formation of cGMP (Ignarro et al., 1982). In turn, cGMP
affects various downstream targets such as protein kinases,
cyclic nucleotide-gated channels or phosphodiesterases
(Lucas et al., 2000; Feil et al., 2003), making the NO-sGC-
cGMP signalling one of the most important vasoprotective
signalling pathways.
One of the crucial prerequisites of the NO-mediated sGC
activation is the presence of the reduced haem moiety. Its
oxidation or loss renders the enzyme insensitive to NO. Oxi-
dative stress, a hallmark of many cardiovascular diseases,
impairs the NO-cGMP signalling (Melichar et al., 2004). Pro-
posed mechanisms include direct chemical scavenging of NO
by reactive oxygen species (ROS) such as O2-, resulting in a
reduced bioavailability of NO and, in parallel, the formation
of the strong oxidant peroxynitrite (ONOO-). In turn, this
reactive intermediate is able to further inhibit NO signalling
by oxidizing the sGC prosthetic haem group to its
NO-insensitive Fe3+ state (Gladwin, 2006; Stasch et al., 2006;
Chirkov and Horowitz, 2007). In addition to this acute inac-
tivation of sGC, oxidation of the haem group facilitates the
degradation of the enzyme (Stasch et al., 2006; Meurer et al.,
2007). Oxidation-induced impairment of protective
NO-cGMP signalling is likely to contribute to endothelial
dysfunction in different vascular diseases such as arterial
hypertension, atherosclerosis, heart failure and erectile
dysfunction (Evgenov et al., 2006; Kemp-Harper and Feil,
2008).
For more than a century, provision of NO, as via
NO-releasing organic nitrates, has been a major therapeutical
approach for the treatment of cardiovascular diseases.
However, this class of drugs suffers from several drawbacks
including the development of tolerance and, unlike endog-
enous NO, the lack of any antithrombotic effect on platelets.
Moreover, blood vessels suffering from oxidative stress condi-
tions become increasingly unresponsive to NO, a situation
that is further aggravated by the fact that organic nitrates
increase oxidative stress and have been shown to directly
oxidize sGC (Artz et al., 2002; Munzel et al., 2005; 2007).
As an alternative therapeutic approach, two structurally
distinct classes of NO-independent, sGC-activating com-
pounds have been discovered, with the potential to overcome
some if not all of the above-mentioned shortcomings
(Evgenov et al., 2006). Haem-dependent sGC stimulators,
including 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo
[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272),
show a strong synergism with NO but lose their ability to
stimulate sGC once the prosthetic haem group is oxidized or
lost (Stasch et al., 2001). In contrast, haem-independent sGC
activators (e.g. BAY 58-2667) activate the NO-insensitive
oxidized/haem-free form of the enzyme (Stasch et al., 2002a)
via binding at the enzyme’s haem pocket (Schmidt et al.,
2004; Roy et al., 2008). Although this mechanism of binding
is generally accepted it is still unclear whether BAY 58-2667 is
able to actively compete with the weakly bound haem moiety
or if the compound binds solely to the haem-free sGC after
the enzyme has lost its oxidized prosthetic group as suggested
very recently by Roy et al.
By activating sGC and thereby increasing the amount of
released cGMP, BAY 58-2667 has different vascular effects. BAY
58-2667 lowers systemic blood pressure, has beneficial effects
in hypertension-induced cardiac hypertrophy and inhibits
platelet aggregation. In experimental pulmonary hyperten-
sion, treatment with BAY 58-2667 leads to a reduction of
ventricular systolic pressure and selective pulmonary vasodi-
latation. Furthermore, BAY 58-2667 decreases the load on the
heart and increases cardiac output as well as renal blood flow
in experimental congestive heart failure. Preload- and
afterload-reducing effects of BAY 58-2667 have been observed
in a phase I clinical study and in a phase II clinical trial with
patients suffering from acute decompensated heart failure (see
Evgenov et al., 2006; Schmidt et al., 2009).
In addition to its activating effect, BAY 58-2667 is able to
rescue the oxidation-impaired sGC from enhanced ubiquitin-
mediated degradation, thus accumulating its receptor in a
positive feedback loop (Stasch et al., 2006; Meurer et al.,
2007). Compounds mimicking the porphyrinic structure of
haem, for example zinc-protoporphyrin IX (Zn-PPIX) and
BAY 58-2667, protect sGC from oxidation-induced degrada-
tion (Stasch et al., 2006). With respect to the structurally
unrelated sGC activator, 5-chloro-2-(5-chloro-thiophene-2-
sulphonylamino -N - (4- (morpholine - 4 - sulphonyl) - phenyl)-
benzamide sodium salt (HMR 1766), competition with
different porphyrins suggests an interaction with the
enzyme’s haem pocket, as shown for BAY 58-2667 (Schindler
et al., 2006; Stasch et al., 2006). This raises the possibility that
protection against oxidation-induced degradation is a general
feature of haem-independent sGC activators. To test this
hypothesis and to further substantiate the mechanisms
leading to the observed stabilization of sGC protein levels, we
investigated the effects of both compounds and the high
affinity metallo-porphyrin, Zn-PPIX, under normal and
haem-oxidizing conditions. As the sGC stimulator BAY
41-2272 does not bind to the haem pocket, a stabilizing effect
on sGC protein levels was not anticipated, making this com-
pound suitable as negative control.
Experiments were conducted by using two cell models,
primary porcine endothelial cells (ECs) and Chinese hamster
ovary (CHO) cells expressing wild-type (WT) sGC or the con-
stitutive haem-free sGC mutants b1H105F and b1Y135A/
R139A. Our findings suggest that BAY 58-2667-like
compounds have a unique structural ability to reassemble the
spatial structure of the haem moiety within sGC and that this
feature allows to prevent sGC degradation in a hitherto not
reported drug-induced positive feedback loop on the expres-
sion level of its therapeutic target protein.
Methods
Cell culture
Primary ECs were obtained from fresh porcine aortae by col-
lagenase detachment as previously described (Stasch et al.,
2002b). Briefly, aortae were freed from surrounding tissue, cut
open and mounted on a framework with the intima facing
upwards. An amount of 20 mL sterile 0.14% collagenase
Molecular protection of sGC
2 LS Hoffmann et al
British Journal of Pharmacology (2009)  ••–••

solution (Biochrom AG, Berlin, Germany) was poured onto
the aorta’s luminal surface, and the aorta was incubated for
15 min. ECs were scraped from the tissue and cultured until
confluent.
cGMP reporter cells were generated and cultured as previ-
ously described (Schmidt et al., 2004; Wunder et al., 2005).
Briefly, the cGMP reporter cells consist of CHO cells stably
transfected with the cGMP-gated Ca2+-channel CNG2 and
aequorin, which translates increasing levels of intracellular
Ca2+ into bioluminescence. In addition, these cells have been
stably transfected with WT sGC (a1 and b1 subunits of the rat
lung enzyme), a1/b1H105F sGC or a1/b1Y135A/R139A sGC
(Becker et al., 1999; Schmidt et al., 2004).
Western blotting
Cells were seeded in six-well plates, grown until confluent and
subsequently incubated with 10 mmol·L-1 1H-(1,2,4)-
oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) alone or combined
with 0.01–10 mmol·L-1 BAY 58-2667, 10 mmol·L-1 BAY
41-2272, 5 mmol·L-1 Zn-PPIX or 10 mmol·L-1 HMR 1766 respec-
tively. After 24 h, cells were harvested and lysed, and protein
was extracted as described earlier (Rothkegel et al., 2007). An
amount of 15–30 mg of total protein was separated by SDS-
PAGE and transferred to nitrocellulose membrane. The indi-
vidual sGC subunits were detected by using polyclonal
antibodies directed against specific epitopes of the a1 subunit
(Sigma, Steinheim, Germany) and the b1 subunit (Cayman
Chemical Company, Ann Arbor, MI, USA). Actin was used as
loading control by using commercially available antibodies
(Sigma). Detection was performed by the ECL method
(Amersham/GE Healthcare, Buckinghamshire, UK). Protein
levels were determined by densitometric analysis of the spe-
cific protein bands (GS-800 Calibrated Densitometer, Quan-
tity One Analysis Software, BioRad, Munich, Germany).
Values were normalized to the respective control of sGC,
which was set to 100% as well as to the respective actin ratio.
Data shown in Figures 3 and 8 were obtained in independent
sets of experiments using different batches of cells.
sGC activity assays
cGMP concentrations of ECs were determined by a commer-
cially available radio-immuno assay kit (IBL, Hamburg,
Germany; Stasch et al., 2002a; Schmidt, 2009). In the cGMP
reporter cell line, sGC activity was determined 48 h after
seeding (Schmidt et al., 2004; Wunder et al., 2005). Briefly,
cells were incubated with increasing concentrations of the
respective test substances for 10 min. Subsequently,
10 mmol·L-1 CaCl2 was added, and the resulting biolumines-
cence directly correlated with intracellular cGMP concentra-
tions (Wunder et al., 2005). Values were expressed as relative
light units (RLUs).
The activity of haem-free recombinant rat sGC was assayed
via the formation of [32P]-cGMP from [a-32P]-GTP in the pres-
ence of Mg2+(Hoenicka et al., 1999; Schmidt, 2009). Removal
of the haem group was achieved by adding 2% Tween-20 to
the reaction buffer, as previously described (Foerster et al.,
1996; Schmidt et al., 2003). sGC was incubated with
100 nmol·L-1 BAY 58-2667 or 100 mmol·L-1 HMR 1766, which
resulted in similar fold stimulation. These fixed concentra-
tions of sGC activators were combined with increasing con-
centrations of Zn-PPIX.
Receptor binding assay
Homologous and heterologous competition binding studies
were performed by using a receptor binding assay, as
described previously (Schmidt et al., 2003). An amount of
1 mg sGC was incubated with 100 nmol·L-1 3H-BAY 58-2667
and increasing concentrations of unlabelled BAY 58-2667,
HMR 1766 or Zn-PPIX respectively. Free and bound radioli-
gands were separated via 96-well filter plates coated with
polyvinylpyrrolidone. Bound radioactivity was determined by
scintillation counting. Non-specific binding was measured by
the addition of a 1000-fold excess of unlabelled BAY 58-2667
and subtracted from total binding in every individual assay.
Statistics
Data are presented as means � standard error of the mean
(SEM). GraphPad Prism software version 4.02 (GraphPad Soft-
ware Inc., San Diego, CA, USA) was used for curve fitting and
calculation of EC50 or IC50 values. Ninety-five per cent confi-
dence intervals of EC50 and half maximal inhibitory concen-
tration (IC50) values are given in parentheses. Statistical
comparisons were performed by using the paired Student’s
t-test.
Materials
BAY 58-2667, BAY 41-2272 and HMR 1766 were synthesized
as described (Figure 1; Straub et al., 2001; Stasch et al., 2002b;
Schindler et al., 2006). Tritium labelling of BAY 58-2667 was
performed as described (Shu and Heys, 2000). ODQ was
purchased from Tocris Bioscience (Avonmouth, UK); Zn-
PPIX (zinc-3,18-divinyl-2,7,13,17-tetramethylporphine-8,12-
dipropionic acid), from Sigma. All other chemicals were of
analytical grade and obtained from Sigma.
Results
Inhibition of sGC activity by Zn-PPIX
To validate the hypothesis that HMR 1766 interacts with the
sGC haem pocket as shown for BAY 58-2667, purified recom-
binant haem-free sGC was incubated with concentrations of
BAY 58-2667 or HMR 1766 that activated the enzyme to a
similar extent (Figure 2A). BAY 58-2667 (100 nmol·L-1) acti-
vated the enzyme 69.5-fold (reflecting a specific activity of
10.2 mmol cGMP·mg-1·min-1). At a concentration of 100 mM,
HMR 1766 induced a comparable 72.9-fold activation (to
13.6 mmol cGMP·mg-1·min-1). The addition of increasing con-
centrations of Zn-PPIX resulted in an inhibition of activated
sGC with IC50 values of either 4.8 (2.2–10.2) nmol·L-1 (for BAY
58-2667-activated sGC) or 2.2 (0.9–5.3) nmol·L-1 (for HMR
1766-activated sGC), indicating that both compounds
interacted with the sGC haem pocket.
Competition binding of BAY 58-2667 and HMR 1766
A receptor binding assay using 3H-labelled BAY 58-2667 was
used to further investigate whether BAY 58-2667 and HMR
Molecular protection of sGC
LS Hoffmann et al 3
British Journal of Pharmacology (2009)  ••–••

1766 directly compete for the same binding site (Figure 2B).
Radioactively labelled BAY 58-2667 was incubated with
increasing concentration of unlabelled BAY 58-2667, HMR
1766 or Zn-PPIX. Unlabelled BAY 58-2667 displayed an IC50 of
200 (140.1–281.4) nmol·L-1. Based on a binding constant (KD)
of 13.4 nmol·L-1 (Schmidt et al., 2003), a Ki of 23 nmol·L-1 was
estimated. Zn-PPIX displaced 3H-BAY 58-2667 with an IC50 of
2.9 (1.3–6.3) nmol·L-1. HMR 1766 competed with 3H-BAY
58-2667 only at very high concentrations of �10 mmol·L-1.
Levels of sGC protein in cGMP reporter cells under normal and
haem-oxidizing conditions
sGC degradation was induced by incubating cells for 24 h
with the sGC inhibitor ODQ (Garthwaite et al., 1995; Olesen
et al., 1998; Zhao et al., 2000). Under these conditions, sGC
protein levels decreased by 59.7 and 35.6% for the a1 and b1
sGC subunit respectively (Figure 3). Control experiments in
which cells were incubated with ODQ for only 30 min did not
induce any significant changes in sGC protein levels (data not
shown). Co-incubation with the sGC activator, BAY 58-2667,
prevented the ODQ-induced decrease in sGC protein levels
for both subunits, concentration dependently with a minimal
effective concentration of 10 nmol·L-1 (Figure 3, Table 1). sGC
protein levels of cells treated with BAY 58-2667 alone (i.e.
without haem oxidation by ODQ) remained constant
(Figure 3, Table 1). In contrast, the structurally unrelated sGC
activator HMR 1766 showed no alteration in sGC protein
levels, neither in control nor ODQ-treated reporter cells
(Figure 3). Conversely, exposure of cGMP reporter cells to the
competitive haem pocket antagonist Zn-PPIX led to a slight
reduction of sGC protein levels. However, upon haem oxida-
tion, sGC protein levels in Zn-PPIX-treated cells were higher
than in cells exposed to ODQ alone. As expected, the sGC
stimulator BAY 41-2272 had no effect on sGC protein levels,
neither under control nor under haem-oxidizing conditions.
Effects of haem oxidation on sGC protein levels in ECs
Long-term (24 h, Figure 4) but not short-term (30 min, data
not shown) incubation with ODQ decreased sGC a1 and b1
levels by 57.8 and 46.0% respectively. Unlike the results from
Figure 1 Chemical structures of the sGC activators BAY 58-2667
and HMR 1766, the sGC stimulator BAY 41-2272 and the haem
pocket antagonist zinc protoporphyrin IX (Zn-PPIX). BAY 41-2272,
5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-
pyrimidin-4-ylamine; BAY 58-2667, 4-[((4-carboxybutyl){2-[(4-
phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid; HMR
1766, 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-
(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt; sGC,
soluble guanylyl cyclase; Zn-PPIX, zinc-protoporphyrin IX.
A
B
-11 -10 -9 -8 -7
0
25
50
75
100
BAY 58-2667 (100 nmol·L–1)
HMR 1766 (100 µmol·L–1)
Zn-PPIX (log mol·L–1)
Isolated enzyme
Isolated enzyme
x-
fo
ld
s
ti
m
ul
at
io
n
-11 -10 -9 -8 -7 -6 -5 -4
0
50
100
150 HMR 1766
BAY 58-2667
Zn-PPIX
Concentration (log mol·L–1)
B
in
di
ng
(
%
)
Figure 2 (A) Inhibition of BAY 58-2667 or HMR 1766-induced sGC
activation by Zn-PPIX. Activity was measured by formation of [32P]-
cGMP from [a-32P]-GTP. Isolated sGC was incubated with
100 nmol·L-1 BAY 58-2667 or 100 mmol·L-1 HMR 1766 and increas-
ing concentrations of Zn-PPIX. Data are shown as means � SEM from
five independent experiments performed in duplicate. (B) Compari-
son of the competition binding of BAY 58-2667, HMR 1766 and
Zn-PPIX. Displacement of 100 nmol·L-1 3H-BAY 58-2667 was studied
in a receptor binding assay. BAY 58-2667 showed an average non-
specific binding of 717 dpm and maximal values of 4338 dpm. HMR
1766 had an average non-specific binding of 616 dpm and maximal
values of 4149 dpm. Data are means � SEM from three to five
independent experiments performed in duplicate. BAY 58-2667,
4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)
methyl[benzoic]acid; HMR 1766, 5-chloro-2-(5-chloro-thiophene-2-
sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide
sodium salt; SEM, standard error of the mean; sGC, soluble guanylyl
cyclase; Zn-PPIX, zinc-protoporphyrin IX.
Molecular protection of sGC
4 LS Hoffmann et al
British Journal of Pharmacology (2009)  ••–••

the cGMP reporter cell line, BAY 58-2667 induced a
concentration-dependent increase of the b1 subunit beyond
control even in the absence of ODQ, whereas a1 protein levels
remained unchanged (Figure 4, Table 2). Similar results were
obtained in ODQ-treated ECs, where BAY 58-2667 increased
b1 protein levels beyond the amounts observed in untreated
controls, whereas a1 levels were stabilized at the level of
untreated controls (Table 2, Figure 4). Similar to BAY 58-2667,
exposure of ECs to Zn-PPIX resulted in increased sGC b1
protein levels both under control conditions and upon haem
oxidation beyond the levels of the respective controls. And
even upon haem oxidation, a1 protein levels of Zn-PPIX-
treated cells were higher than in cells treated with only ODQ.
Conversely, neither the NO-independent sGC agonists BAY
41-2272 nor HMR 1766 relevantly increased the levels of
either sGC subunit in ECs under any of the tested conditions.
Figure 3 Effects of 10 mmol·L-1 BAY 58-2667, 10 mmol·L-1 BAY 41-2272, 10 mmol·L-1 HMR 1766 and 5 mmol·L-1 Zn-PPIX on sGC protein levels
under normal and haem-oxidizing conditions in cGMP reporter cells. Haem oxidation was achieved by pre-incubating cells with 10 mmol·L-1
ODQ for 24 h. (A) Representative Western blots of a1 sGC and actin as a loading control (upper panel). Purified rat sGC was used as control.
a1 sGC protein levels as determined by densitometric measurement are shown in the lower panel. (B) Representative Western blots of b1 sGC
and actin as a loading control (upper panel). Purified rat sGC was used as control. b1 sGC protein levels as determined by densitometric
measurement are shown in the lower panel. sGC protein levels are expressed as percentage of the respective control which was set as 100%
(means � SEM of 5–32 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.005: Student’s t-test. BAY 41-2272, 5-cyclopropyl-2-
[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine; BAY 58-2667,4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]
phenethyl}amino)methyl [benzoic] acid; HMR, 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-
benzamide sodium salt; ODQ, 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; sGC, soluble guanylyl cyclase; Zn-PPIX, zinc-protoporphyrin IX.
Molecular protection of sGC
LS Hoffmann et al 5
British Journal of Pharmacology (2009)  ••–••

sGC activity in cGMP reporter cells
For the effects of the haem oxidant ODQ in cGMP reporter
cells to be examined, two approaches were tested, namely a
long-term incubation (24 h) to reduce sGC protein levels, as
well as a short-term incubation (10 min) with ODQ to
oxidize the sGC haem group and inhibit activity without
affecting the enzyme’s protein level. Moreover, long-term
treated cells were additionally short-term treated to test
whether the BAY 58-2667-induced activity of residual sGC
can be further enhanced by acute oxidation. Figure 5 sum-
marizes the results of these protocols on the subsequent sGC
activation by BAY 58-2667, BAY 41-2272, Zn-PPIX or HMR
1766 respectively.
BAY 58-2667 (30 mmol·L-1) activated sGC up to 73-fold with
an EC50 value of 23.3 (13.1–41.3) nmol·L-1. Short-term expo-
sure to ODQ enhanced sGC activation to 129-fold with a
decrease in the EC50 to 9.1 (4.6–17.9) nmol·L-1 (Figure 5A).
Long-term incubation with ODQ resulted in only 78-fold sGC
activation by BAY 58-2667 with a slightly higher EC50 of 15.1
(9.7–23.6) nmol·L-1. When long-term incubated cells were
additionally exposed to a second short-term application of
ODQ, sensitivity to BAY 58-2667 was similar [EC50 9.9 (4.0–
24.5) nmol·L-1, 82-fold activation].
BAY 41-2272 (30 mmol·L-1) stimulated sGC up to 115-fold
with an EC50 of 596 (364.7–973.5) nmol·L-1. Short-term treat-
ment with ODQ (10 min) reduced sGC stimulation to 23-fold
with a corresponding increase in the EC50 to 831 (414.8–1665)
nmol·L-1 (Figure 5B). Long-term haem oxidation resulted in a
further decrease in sGC activity [maximal stimulation 17-fold,
EC50 value of 580 (352.4–953.7) nmol·L-1]. Additional acute
oxidation of long-term treated cells led to a similar EC50 value
of 607 (197.1–1868) nmol·L-1 for BAY 41-2272-induced sGC
activity as in cells that has only long-term treatment. As
expected for a full antagonist, Zn-PPIX had no effect on sGC
activity, neither under haem-oxidizing nor under control
conditions (Figure 5D).
Treatment with increasing concentrations of HMR 1766
resulted in an up to 557-fold activation with an EC50 value of
8.8 (6.6–11.8) mmol·L-1 (Figure 5C). Surprisingly, acute ODQ
did not increase HMR 1766-induced activation, and long-
term ODQ even slightly diminished HMR 1766-induced sGC
activation (Figure 5D), which was also not changed by addi-
tional acute ODQ exposure [EC50 13.5 (6.5–27.9) mmol·L-1].
sGC activity in ECs
In ECs, sGC activity was determined by measuring cGMP
accumulation via radio-immuno assay upon incubating cells
with increasing concentrations of BAY 58-2667, Zn-PPIX,
HMR 1766 or BAY 41-2272 under normal or haem-oxidizing
conditions (Figure 6) respectively. BAY 58-2667 showed a flat
concentration response curve, and the maximal activation
was only eightfold with an EC50 value of 0.3 (0.03–2.3)
mmol·L-1. Haem oxidation potentiated BAY 58-2667-induced
sGC activation up to 134-fold at the highest applied concen-
tration of 10 mmol·L-1, with an EC50 value of 0.2 (0.05–0.7)
mmol·L-1 (Figure 6A).
BAY 41-2272 increased cGMP levels up to 123-fold at the
highest tested concentration of 10 mmol·L-1, which was
reduced to 86-fold after long-term ODQ treatment. The cor-
responding EC50 value was shifted from 737 (326.1–1667) to
946 (411.8–2175) nmol·L-1 respectively (Figure 6B). Incubat-
ing ECs with HMR 1766 lead to a maximal activation of
20-fold, and this was increased to 749-fold upon haem oxi-
dation. EC50 values were 1261 (0.0–28545) and 810 (0.0–419)
mmol·L-1 respectively (Figure 6C).
Activities of mutant b1H105F and b1Y135A/R139A sGCs
The activity of sGC containing either b1H105F or b1Y135A/
R139A was determined in cGMP reporter cells stably trans-
fected with expression vectors encoding for the respective
enzyme mutants. WT, b1H105F and b1Y135A/R139A sGCs
were incubated with increasing concentrations of BAY
58-2667 or HMR 1766 alone or combination with short-term
ODQ treatment (Figure 7). WT sGC was activated up to
17-fold by 30 mmol·L-1 BAY 58-2667, with an EC50 of 0.2
(0.02–1.1) mmol·L-1, and this activation was increased to
64-fold with an EC50 of 2.0 (0.4–9.3) mmol·L-1 by addition of
10 mmol·L-1 ODQ (Figure 7A). Incubation of these WT sGC
expressing cells with HMR 1766 resulted in a maximal acti-
vation of 230-fold by 30 mmol·L-1 HMR 1766, with an EC50 of
Table 1 Effects of increasing concentrations of BAY 58-2667 on sGC protein levels under normal and haem-oxidizing conditions in cGMP
reporter cells stably transfected with WT sGC
cGMP reporter cells a1 sGC b1 sGC
BAY 58-2667 ODQ (10 mmol·L-1) ODQ (10 mmol·L-1)
[mmol·L-1] - + - +
– 100 60 � 4*** 100 36 � 3***
0.01 88 � 7***†† 65 � 5***# 79 � 5***††† 38 � 10***††
0.1 93 � 15††† 76 � 8*** 73 � 10***††† 63 � 13***†††
1 94 � 8***††† 94 � 6***††† 92 � 25††† 88 � 33†††
10 100 � 9††† 95 � 11††† 116 � 10††† 97 � 9†††
Values are expressed as % control (no ODQ or BAY 58-2667) and are means � SEM of 3–32 independent experiments.
***P < 0.005: Student’s t-test (indicated sample vs. control); ††P < 0.01, †††P < 0.005: Student’s t-test (indicated sample vs. ODQ treated control); #P < 0.05: Student’s
t-test (substance treatment vs. substance plus ODQ treatment).
BAY 58-2667, 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid; cGMP, cyclic guanosine monophosphate; ODQ, 1H-(1,2,4)-
oxadiazolo[4,3-a]quinoxalin-1-one; SEM, standard error of the mean; sGC, soluble guanylyl cyclase; WT, wild type.
Molecular protection of sGC
6 LS Hoffmann et al
British Journal of Pharmacology (2009)  ••–••

4.2 (1.6–10.8) mmol·L-1. Co-incubation with ODQ increased
the activation to 328-fold with an EC50 of 1.5 (0.7–3.0)
mmol·L-1 (Figure 7B).
Incubation of cGMP reporter cells expressing the haem-free
sGC mutant b1H105F with BAY 58-2667 resulted in a maximal
activation of 45-fold, which was only slightly increased to
55-fold by addition of ODQ [determined EC50 were 11.6 (3.2–
41.8) nmol·L-1 and 7.6 (1.8–31.2) nmol·L-1 respectively
(Figure 7C)]. The highest concentration of HMR 1766
(30 mmol·L-1) activated b1H105F cells up to 95-fold [EC50 0.8
(0.5–1.1) mmol·L-1] and 100-fold [EC50 0.8 (0.4–1.8) mmol·L-1]
by the addition of ODQ (Figure 7D). The double mutant
b1Y135A/R139A was activated neither by BAY 58-2667 nor by
BAY 58-2667 in combination with ODQ (Figure 7E) and only
Figure 4 Effects of 10 mmol·L-1 BAY 58-2667, 10 mmol·L-1 BAY 41-2272, 10 mmol·L-1 HMR 1766 and 5 mmol·L-1 Zn-PPIX on sGC protein levels
under normal and haem-oxidizing conditions in endothelial cells. Haem oxidation was achieved by incubation of the cells with 10 mmol·L-1
ODQ for 24 h. (A) Representative Western blots of a1 sGC and actin as a loading control (upper panel). Purified rat sGC was used as control.
a1 sGC protein levels as determined by densitometric measurement are shown in the lower panel. (B) Representative Western blots of b1 sGC
and actin as a loading control (upper panel). Purified rat sGC was used as control. b1 sGC protein levels as determined by densitometric
measurement are shown in the lower panel. sGC protein levels are expressed as percentage of the respective control which was set as 100%
(means � SEM of 5–29 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.005: Student’s t-test. BAY 41-2272, 5-cyclopropyl-2-[1-
(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine; BAY 58-2667, 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]
phenethyl}amino)methyl[benzoic]acid; HMR 1766, 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-
phenyl)-benzamide sodium salt; ODQ, 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; sGC, soluble guanylyl cyclase; Zn-PPIX, zinc-
protoporphyrin IX.
Molecular protection of sGC
LS Hoffmann et al 7
British Journal of Pharmacology (2009)  ••–••

Table 2 Effects of increasing concentrations of BAY 58-2667 on sGC protein levels under normal and haem-oxidizing conditions
in endothelial cells
ECs a1 sGC b1 sGC
BAY 58-2667 ODQ (10 mmol·L-1) ODQ (10 mmol·L-1)
[mmol·L-1] - + - +
– 100 58 � 4*** 100 46 � 3***
0.01 108 � 3***††† 60 � 8***### 106 � 5***††† 53 � 5***###
0.1 99 � 11†† 64 � 15*** 111 � 11**††† 74 � 9***†††#
1 100 � 9††† 92 � 9**†† 138 � 13***††† 129 � 17***†††
10 103 � 9††† 122 � 12***††† 162 � 24***††† 163 � 22***†††
Values are expressed as % control (no ODQ or BAY 58-2667) and are means � SEM of 4–29 independent experiments.
**P < 0.01, ***P < 0.005: Student’s t-test (indicated sample vs. control); ††P < 0.01, †††P < 0.005: Student’s t-test (indicated sample vs. ODQ treated control); #P
< 0.05, ###P < 0.005: Student’s t-test (substance treatment vs. substance plus ODQ treatment).
BAY 58-2667, 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid; cGMP, cyclic guanosine monophosphate; EC, endothelial
cell; ODQ, 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; SEM, standard error of the mean; sGC, soluble guanylyl cyclase.
BA
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cGMP reporter cells
0
0
50
100
150
-9 -8 -7 -6 -5 -4
Short-term ODQ
Control
Long-term ODQ
Long-term and short-term ODQ
BAY 58-2667 (log mol·L–1)
x-
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50
100
150
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Long-term ODQ
Long-term and short-term ODQ
BAY 41-2272 (log mol·L–1)
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0
0
250
500
750
-9 -8 -7 -6 -5 -4
Control
Short-term ODQ
Long-term ODQ
Long-term and short-term ODQ
HMR 1766 (log mol·L–1)
x-
fo
ld
s
tim
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0
0.0
2.5
5.0
7.5
10.0
-9 -8 -7 -6 -5 -4
Control
Short-term ODQ
Long-term ODQ
Zn-PPIX (log mol·L–1)
x-
fo
ld
s
tim
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Figure 5 Concentration response curves of wildtype sGC in cGMP reporter cells incubated with increasing concentrations of BAY 58-2667 (A),
BAY 41-2272 (B), HMR 1766 (C) or Zn-PPIX (D) alone, in combination with 10 mmol·L-1 ODQ for 10 min, after 24 h pretreatment with
10 mmol·L-1 ODQ and pretreatment with additional treatment with 10 mmol·L-1 ODQ for 10 min. Data are means � SEM from 7–19
independent experiments performed in quadruplicate. sGC activation is represented as x-fold stimulation compared with non-stimulated
control. Following basal activities were measured: (A) 10 min BAY 58-2667 1091 RLUs; 10 min BAY 58-2667 + ODQ 867 RLUs; 24 h
ODQ/10 min BAY 58-2667 1441 RLUs; 24 h ODQ/10 min BAY 58-2667 + ODQ 866 RLUs. (B) 10 min BAY 41-2272 980 RLUs; 10 min BAY
41-2272 + ODQ 1800 RLUs; 24 h ODQ/10 min BAY 41-2272 1069 RLUs; 24 h ODQ/10 min BAY 41-2272 + ODQ 584 RLUs. (C) 10 min HMR
1766 1528 RLUs; 10 min HMR 1766 + ODQ 1380 RLUs; 24 h ODQ/10 min HMR 1766 1411 RLUs; 24 h ODQ/10 min HMR 1766 + ODQ 856
RLUs. (D) 10 min Zn-PPIX 1130 RLUs; 10 min Zn-PPIX + ODQ 1131 RLUs; 24 h ODQ/10 min Zn-PPIX 1750 RLUs. BAY 41-2272, 5-cyclopropyl-
2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine; BAY 58-2667, 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]
phenethyl}amino)methyl[benzoic]acid; cGMP, cyclic guanosine monophosphate; HMR 1766, 5-chloro-2-(5-chloro-thiophene-2-
sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt; ODQ, 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; RLU,
relative light unit; SEM, standard error of the mean; sGC, soluble guanylyl cyclase; Zn-PPIX, zinc-protoporphyrin IX.
Molecular protection of sGC
8 LS Hoffmann et al
British Journal of Pharmacology (2009)  ••–••

slightly activated by HMR 1766 (5-fold) or HMR 1766 and
ODQ (6-fold; Figure 7F).
sGC protein levels of b1H105F sGC and b1Y135A/R139A sGC
Incubation of the WT sGC expressing cell line with ODQ for
24 h decreased sGC protein levels by 49% (a1) and 61% (b1)
(Figure 8). BAY 58-2667 was able to rescue sGC a1 protein to
control levels, whereas sGC b1 was increased beyond control.
This observation was not affected by additional ODQ. In the
cell line expressing the haem-free mutant b1H105F, oxidation
did not result in reduced protein levels of both subunits. The
addition of BAY 58-2667 both under control and haem-
oxidizing conditions strongly increased b1H105F protein levels
with weaker effects on a1 sGC. In cells expressing haem-free
b1Y135A/R139A, sGC protein levels remained unchanged both
under control and haem-oxidizing conditions, and BAY
58-2667 had no effect on the protein levels of either subunit
(Figure 8).
Discussion
The NO-cGMP pathway plays a key role in the cardiovascular
system, and its impairment is associated with different car-
diovascular diseases. Recent findings suggest that increased
levels of oxidative stress as observed under pathophysiologi-
cal conditions can lead to oxidation or even loss of the sGC
haem group, rendering the enzyme insensitive to NO and
prone to ubiquitin-mediated degradation (Stasch et al., 2006;
Meurer et al., 2007; Xia et al., 2007). These results might
explain at least partially the observed reduction of sGC
protein levels in different animal models of cardiovascular
diseases (Ruetten et al., 1999; Kagota et al., 2001; Melichar
et al., 2004).
Occupation of the sGC haem pocket with high affinity
metallo-porphyrins or compounds resembling the spatial
structure and charge distribution of haem such as BAY
58-2667 is able to prevent the oxidation-induced degradation
of sGC (Stasch et al., 2006; Meurer et al., 2007). In parallel,
Schindler et al., (2006) identified another haem-independent
sGC activator that, although structurally unrelated, shares
some characteristics with BAY 58-2667 such as the activation
of haem-free sGC. The first evidence suggested an interaction
of HMR 1766 with the haem pocket, as described for metallo-
porphyrins and BAY 58-2667. However, whether this com-
pound binds to the sGC haem site and if it is able to prevent
the oxidation-induced sGC degradation as shown for Zn-PPIX
and BAY 58-2667 have not been investigated yet. To clarify
BA
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Endothelial cells
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200
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Control
BAY 58-2667 (log mol·L–1)
x-
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50
100
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200
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Control
BAY 41-2272 (log mol·L–1)
x-
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250
500
750
1000
-9 -8 -7 -6 -5 -4
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Long-term ODQ
HMR 1766 (log mol·L–1)
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0.0
2.5
5.0
7.5
10.0
-9 -8 -7 -6 -5 -4
Control
Long-term ODQ
Zn-PPIX (log mol·L–1)
x-
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Figure 6 Effects of BAY 58-2667, BAY 41-2272, HMR 1766 and Zn-PPIX on sGC activity under normal and haem-oxidizing conditions.
Endothelial cells were treated with different concentrations of BAY 58-2667 (A), HMR 1766 (C), Zn-PPIX (D) for 30 min or BAY 41-2272 (B)
for 15 min with or without 24 h ODQ (10 mmol·L-1) pretreatment. sGC activity was determined by measurement of cGMP accumulation via
radio-immunoassay. Data are expressed as x-fold stimulation of control (means � SEM of 8–14 independent experiments). The basal cGMP
contents was as follows: (in fmol cGMP per well): (A) BAY 58-2667 409; BAY 58-2667 + ODQ 24 h 19; (B) BAY 41-2272 484; BAY 41-2272
+ ODQ 24 h 26; (C) HMR 1766 241; HMR 1766 + ODQ 24 h 16; (D) Zn-PPIX 408; Zn-PPIX + ODQ 24 h 19. BAY 41-2272, 5-cyclopropyl-
2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine; BAY 58-2667, 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)
oxy]phenethyl}amino)methyl[benzoic]acid; cGMP, cyclic guanosine monophosphate; HMR 1766, 5-chloro-2-(5-chloro-thiophene-2-
sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt; ODQ, 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; sGC,
soluble guanylyl cyclase; Zn-PPIX, zinc-protoporphyrin IX.
Molecular protection of sGC
LS Hoffmann et al 9
British Journal of Pharmacology (2009)  ••–••

this open question and to shed light on the general mecha-
nism of sGC stabilization, the present study investigated the
molecular requirements of sGC activation and stabilization
using the sGC activators, BAY 58-2667 and HMR 1766, and
the haem pocket antagonist, Zn-PPIX, on sGC activity and
protein levels in two cell systems expressing WT and mutant
sGC.
To validate a putative interaction of HMR 1766 with the
sGC haem pocket, sGC activity and competition binding
assays were performed with BAY 58-2667, HMR 1766 and
Zn-PPIX. Activity assays with purified sGC showed unequivo-
cally that the high-affinity metallo-porphyrin is able to
inhibit BAY 58-2667 and HMR 1766-induced sGC activation,
suggesting an interaction of HMR 1766 with the haem-
binding site as shown for BAY 58-2667 (Schmidt et al., 2004;
Schindler et al., 2006; Stasch et al., 2006). The competition
binding assays performed here support this view, as unla-
belled BAY 58-2667 and Zn-PPIX displaced 3H-BAY 58-2667
cGMP reporter cells
A WT
0
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E Y135A/R139A F Y135A/R139A
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5
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10
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Control
Short-term ODQ
BAY 58-2667 (log mol·L–1)
x-
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0
0
3
5
8
10
-9 -8 -7 -6 -5 -4
Control
Short-term ODQ
HMR 1766 (log mol·L–1)
x-
fo
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Figure 7 Concentration response curves of cGMP reporter cells stably transfected with WT (A, B) a1/b1H105F sGC (C, D) or a1/b1Y135A/
R139A sGC (E, F) incubated with increasing concentrations of BAY 58-2667 (A, C ,E) or HMR 1766 (B, D, F) alone or in combination with
short-term ODQ (10 mmol·L-1) treatment. Data are means � SEM from four to eight independent experiments performed in duplicate. sGC
activation is represented as x-fold stimulation compared with non-stimulated control. Following basal activities from which x-fold stimulation
was calculated were measured: (A) BAY 58-2667 2986 RLUs; BAY 58-2667 + ODQ 10 min 3078 RLUs; (B) HMR 1766 2957 RLUs; HMR 1766
+ ODQ 10 min 2654 RLUs; (C) BAY 58-2667 16796 RLUs; BAY 58-2667 + ODQ 10 min 18266 RLUs; (D) HMR 1766 16624 RLUs; HMR 1766
+ ODQ 10 min 19589 RLUs; (E) BAY 58-2667 3626 RLUs, BAY 58-2667 + ODQ 10 min 3822 RLUs; (F) HMR 1766 3494 RLUs; HMR 1766 +
ODQ 10 min 3498 RLUs. BAY 58-2667, 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid; cGMP, cyclic
guanosine monophosphate; HMR 1766, 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-
benzamide sodium salt; ODQ, 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; RLU, relative light unit; sGC, soluble guanylyl cyclase; WT,
wildtype.
Molecular protection of sGC
10 LS Hoffmann et al
British Journal of Pharmacology (2009)  ••–••

from the enzyme. The results obtained with HMR 1766 were
less clear; the relative low affinity of this compound pre-
vented full displacement of radioactively labelled BAY
58-2667 from the enzyme. Nevertheless, at high micromolar
concentrations, HMR 1766 reduced 3H-BAY 58-2667 binding
to 67%, suggesting that HMR 1766, BAY 58-2667 and Zn-PPIX
bind to the same or at least partially overlapping binding
sites. The concentration of HMR 1766 used for this study is
higher than the concentration used by Schindler et al. but was
chosen to achieve similar sGC activation by HMR 1766 and
BAY 58-2667. This difference in sGC sensitivity might be due
to the facts that we used recombinant rat sGC expressed in
and purified from a baculovirus/Sf9 insect cell system, instead
of native bovine sGC. Heterologous expression in insect cells
might result in an sGC that lacks certain post-translational
modifications such as phosphorylation, which might affect
sGC activity (Meurer et al., 2005).
Using Western blot analysis, the effect of these com-
pounds on sGC protein levels upon haem oxidation by the
sGC inhibitor, ODQ, was established. Long-term treatment
of ECs and cGMP reporter cells with ODQ resulted in dra-
matically reduced protein levels of both sGC subunits, as
observed for other primary cells (Stasch et al., 2006). This
oxidation-induced degradation becomes prominent when
the incubation time exceeds 2 h, whereas short-term incu-
bations had no effect on sGC protein levels (Stasch et al.,
2006). BAY 58-2667 and Zn-PPIX prevented this oxidation-
induced decrease. In agreement with previous findings
(Stasch et al., 2006), protein levels remained unchanged in
cells treated with the sGC stimulator BAY 41-2272, suggest-
ing that signalling events downstream of cGMP are not
likely to be involved in sGC stabilization. Our results
obtained with HMR 1766 were very surprising. Despite the
fact that both BAY 58-2667 and HMR 1766 appear to
Figure 8 Effects of BAY 58-2667 on protein levels under normal and oxidative conditions of WT, a1/b1H105F sGC and a1/b1Y135A/R139A
sGC. cGMP reporter cells were incubated with 10 mmol·L-1 ODQ or 10 mmol·L-1 BAY 58-2667 alone or combined for 24 h as indicated. (A)
Representative Western blots of a1 sGC and actin as a loading control (upper panel). Purified rat sGC was used as control. a1 sGC protein levels
as determined by densitometric measurement are shown in the lower panel. (B) Representative Western blots of b1 sGC and actin as a loading
control (upper panel). Purified rat sGC was used as control. b1 sGC protein levels as determined by densitometric measurement are shown in
the lower panel. sGC protein levels are normalized to the respective control, which was set as 100% (means � SEM of three to
eight independent experiments). *P < 0.05, **P < 0.01, ***P < 0.005: Student’s t-test. BAY 58-2667, 4-[((4-carboxybutyl){2-[(4-
phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid; ODQ, 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; SEM, standard error or the
mean; sGC, soluble guanylyl cyclase; WT, wildtype.
Molecular protection of sGC
LS Hoffmann et al 11
British Journal of Pharmacology (2009)  ••–••

interact with the sGC haem pocket, HMR 1766 did not
show any protective effect on sGC protein levels.
The differences in protein levels under normal and haem-
oxidizing conditions reflected the observed changes in sGC
activity. Short-term incubation with 10 mmol·L-1 ODQ has
been shown to potentiate BAY 58-2667-induced sGC activa-
tion at most, indicating that the majority of cellular sGC is
converted into the oxidized/haem-free state, which can be
activated by BAY 58-2667. As no impact on sGC protein levels
has been reported for short-term ODQ incubations, the
maximal BAY 58-2667-induced sGC activation upon short-
term ODQ treatment compared with the combined long- and
short-term ODQ incubation should reflect the decrease in sGC
protein levels. Under this condition, a reduction of maximal
sGC activation by BAY 58-2667 of 69% (129 to 82-fold activa-
tion) was observed, matching the observed reduction in sGC
protein levels of 60% for a1 sGC and 36% of b1 sGC.
As shown in Figure 3, both BAY 41-2272 and HMR 1766
were unable to prevent oxidation-induced degradation of
sGC. Therefore, we expected similar results for BAY 41-2272-
or HMR 1766-induced sGC activity.
BAY 41-2272 activated sGC 115-fold under control condi-
tions (Figure 5B). Combination of short- and long-term treat-
ment reduced sGC activation to 7%. This is even lower than
protein levels in ODQ and BAY 41-2272-treated cells, which
were decreased by 57% (a1 sGC) and 43% (b1 sGC).
But, in contrast to BAY 41-2272, HMR 1766-induced sGC
activity was unchanged or only slightly diminished compared
with normal conditions. This discrepancy might be due to
technical limitations, as the strong activation of sGC at high
concentrations of HMR 1766 resulted in RLUs at the limit of
detection. Although the addition of ODQ slightly increases
the amount of RLUs, the expected HMR 1766 plus ODQ-
induced maximum activation can presumably not be mea-
sured. Again, the highest concentration of HMR 1766 chosen
for sGC activity measurements was higher than the concen-
trations used by Schindler et al. (2006). The differences in
efficacy might be explained by the use of different cell lines or
primary cells.
Comparison of Figures 5A and 6A shows that ECs were not
stimulated to the same extend as cGMP reporter cells under
control conditions. This might be due to the different cell
types. In ECs, compared with cGMP reporter cells, the low
BAY 58-2667-induced stimulation might reflect a small pool
of naturally oxidized/haem-free sGC. On the other hand, this
would argue for a bigger pool of haem-free/oxidized sGC in
cGMP reporter cells compared with ECs. Mingone et al. (2006)
showed that the levels of haem precursors (e.g. 5-amino-
laevulinic acid) directly impact on haem synthesis and, as a
result, in the relative amount of NO-sensitive, haem-
containing sGC. As the cGMP reporter cells express sGC at
very high levels, it might be possible that the native cellular
haem synthesis is not able to match the needs of this artifi-
cially high expression, resulting in increased relative amounts
of BAY 58-2667-sensitive, haem-free enzyme. Further studies
applying haemin or 5-amino-laevulinic-acid might be able to
shed light on this question. Wolin (2009) has shown that the
haem precursor PPIX accumulates in vascular tissue incubated
with 5-amino-laevulinic acid and thereby stimulates sGC and
induces pulmonary artery relaxation.
In ECs, a dramatic increase in sGC activator-induced acti-
vation of sGC following long-term treatment with ODQ was
observed. The haem-free state of sGC is preferentially targeted
by sGC activators (Stasch et al., 2006; Roy et al., 2008). More-
over, haem loss in only a small proportion of sGC pool results
in a dramatic increase of BAY 58-2667-induced sGC activity
(Roy et al., 2008). In contrast, only a small decrease of BAY
41-2272-induced activation of ODQ pretreated sGC was
observed, which might suggest a receptor reserve.
Protein levels of BAY 41-2272 and ODQ-treated ECs and
cGMP reporter cells decreased by about 50% and reflected the
measured sGC activity. In ECs, BAY 41-2272-induced activity
was lowered by about 25%, and the corresponding EC50 was
increased to about 25%. In cGMP reporter cells, sGC activity
was diminished to an even greater extent.
In summary, BAY 58-2667 and Zn-PPIX but not BAY
41-2272 prevented sGC from oxidation-induced degradation.
The measured sGC activity reflected the observed changes in
protein levels. Importantly, the different sGC activators
showed an unexpectedly different profile with respect to sGC
protein and activity levels. Both haem-independent sGC acti-
vators, BAY 58-2667 and HMR 1766, induced sGC activation
under normal and haem-oxidizing conditions, but only BAY
58-2667 was able to prevent oxidation-induced enzyme deg-
radation. This molecular characteristic might be explained by
different levels of haem mimicry and overlapping but not
identical binding sites.
The sGC stabilizing ligands, BAY 58-2667 and Zn-PPIX,
have the same chemical motif as haem and bind to the haem
site with much higher affinity than the native prosthetic
group. Furthermore, mutation analyses and structural models
showed that both ligands interact with the haem-binding
residues Y135 and R139 of the b1 subunit (Schmidt et al.,
2004).
To investigate the interaction of the two haem-independent
sGC activators, BAY 58-2667 and HMR 1766, the activation
patterns of these compounds have been recorded with differ-
ent sGC mutations that are known to effect haem-binding.
Mutation of the axial haem ligand b1H105F has been shown
to cause the expression of haem-free enzyme (Foerster et al.,
1996), although this mutation did not preclude subsequent
reconstitution of the enzyme with porphyrins (Schmidt et al.,
2004). In contrast, the soluble mutant b1Y135A/R139A, which
lacks the essential haem-binding residues, cannot be recon-
stituted with PPIX (Schmidt et al., 2004). Furthermore, Y135
and R139 were also identified as binding sites for BAY 58-2667
as the double mutant is no longer activated by BAY 58-2667
(Schmidt et al., 2004).
This double mutant a1/b1Y135A/R139A was used to deter-
mine whether both BAY 58-2667 and HMR 1766 activate sGC
by binding to these residues within the haem binding motif
(Schmidt et al., 2004).
When measuring sGC activity in cGMP reporter cells
expressing these haem-free sGC variants, HMR 1766 and BAY
58-2667 apparently did not bind to the same residues.
Although BAY 58-2667 is able to strongly activate the haem-
free sGC mutant H105F, it was not able to induce any activa-
tion of the double mutant a1/b1Y135A/R139A, indicating that
these residues are crucially important for BAY 58-2667
binding, as shown earlier (Schmidt et al., 2004). In contrast,
Molecular protection of sGC
12 LS Hoffmann et al
British Journal of Pharmacology (2009)  ••–••

HMR 1766 still activates the double mutant although its acti-
vation is diminished compared with WT sGC. These data
clearly suggest that Y135 and R139 do not affect binding of
HMR 1766 in the same way or extent as they affect binding of
BAY 58-2667 or haem. Zhou et al. (2008) used docking simu-
lations based on the sGC structure of Nostoc sp to identify
putative regions through which HMR 1766 interacts with
sGC. Contrary to our findings in living cells, they postulated
Y135 and R139 as binding partners of HMR 1766, suggesting
a BAY 58-2667-like binding mode.
Considering the results from the receptor binding assay,
which showed that HMR 1766 competes with BAY 58-2667
only at high concentrations whereas it can be readily replaced
by low amounts of Zn-PPIX, it becomes evident that BAY
58-2667 bears noticeably more resemblance to haem. In con-
trast, HMR 1766 seems to interact with different residues from
those interacting with BAY 58-2667, although their binding
sites might overlap at least partially.
Basing on these findings, we hypothesized that the differ-
ent binding modes to the haem pocket might be responsible
for the differences between HMR 1766 and BAY 58-2667
with respect to protection of sGC from oxidation-induced
degradation. When using the same sGC mutants described
above in Western blots, it became apparent that the protec-
tive function of BAY 58-2667 is also mediated by the haem-
like occupation of the haem. This protection can only be
observed for WT and b1H105F sGC, which are activated by
BAY 58-2667, but not for b1Y135A/R139A sGC, which
neither is activated by BAY 58-2667 nor can be reconstituted
with PPIX (Schmidt et al., 2004). HMR 1766 was not used in
this set of experiments because of its lack of protective or
stabilizing properties, which was already demonstrated in
WT-sGC-expressing cells, and HMR 1766-induced activity in
Y135A/R139A sGC-expressing cells showed its different
binding mode.
Another difference becomes obvious by comparing a1 and
b1 sGC levels: Oxidation-induced degradation of the a1
subunit is not prevented by BAY 58-2667 and Zn-PPIX to the
same extent as observed for the b1 subunit. Based on our data
with b1H105F sGC, it is more likely that the b subunit is
predominantly affected by ODQ. Incubation with ODQ does
not lead to a significant decrease in sGC protein levels in cells
expressing b1H105F, as observed in cells expressing WT sGC.
In addition, the BAY 58-2667 activation profile of b1H105F
expressing cells resembles the pattern of BAY 58-2667-
induced sGC activity in ODQ treated WT cells. In agreement
with Stasch et al. (2006), we assume that the physiological
turnover of a1 sGC is not affected due to the lack of haem
binding, and, thus, any changes that are mediated via the
haem binding site do not apply to a1 sGC directly. The
changes we observed for a1 sGC protein levels may rather be
based on counter regulatory mechanisms as described for the
a1 and a2 knockout mice (Mergia et al., 2006). Here, deletion
of a subunits results in a concomitant decrease of b1 protein
levels. Moreover, Friebe et al. (2007) demonstrated that b1
knockout mice lack the a1 subunit. We suppose that the
observed changes underlie a similar mechanism of counter
regulation, which results in decreased a1 protein levels when
b1 subunits are depleted due to oxidation. However, instability
of single a1 subunits cannot be an explanation for the
Figure 9 Model of the role of sGC’s haem group and sGC targeting compounds in protection of sGC. Under haem-oxidizing conditions such
as oxidative stress, binding of the sGC activator BAY 58-2667 stabilizes sGC and thereby protects sGC from degradation. In contrast, binding
of the sGC activator HMR 1766 or the sGC stimulator BAY 41-2272 cannot stabilize sGC and therefore sGC like haem-free sGC is ubiquitinated
and degraded. BAY 41-2272, 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine; BAY 58-2667, 4-[((4-
carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid; sGC, soluble guanylyl cyclase; HMR 1766, 5-chloro-2-(5-
chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt.
Molecular protection of sGC
LS Hoffmann et al 13
British Journal of Pharmacology (2009)  ••–••

decrease in a1 sGC protein levels, as homodimers can form
and are stable (Zabel et al., 1999).
In summary, our results show that BAY 58-2667 and
Zn-PPIX but neither BAY 41-2272 nor HMR 1766 prevent
oxidation-induced degradation of sGC and decreased sGC
activity. BAY 58-2667’s protective effect depends on high-
affinity binding to the haem-binding pocket in a manner that
reassembles the native prosthetic group including the inter-
action with the haem binding motif Y-x-S-x-R. HMR 1766
lacks these properties, making it a distinct class of sGC acti-
vators. The results of this study are summarized in Figure 9. To
our knowledge, BAY 58-2667 is thus the first pharmacological
enzyme ligand, which, in addition to activating, also stabi-
lizes its own target. As cardiovascular diseases are associated
with increased levels of oxidative stress, it can be expected
that the relative amount of haem-oxidized/haem-free sGC is
increased under pathophysiological conditions. This view is
in agreement to results obtained in a clinical trial with
patients suffering from acute decompensated heart failure,
which suggest the presence of a pool of haem-free/oxidized
sGC in humans (Schmidt et al., 2009). This imbalance would
be translated into decreased sGC protein levels due to accel-
erated degradation of the oxidation-impaired enzyme. The
sGC stabilizing features of BAY 58-2667 described here might
help to overcome this imbalance by preventing sGC from
degradation and thus improving cardiovascular disease.
Further pharmacological and clinical studies with sGC activa-
tors will provide more information on the in vivo efficacy and
effects in the treatment of cardiovascular diseases.
Conflict of interest
LS Hoffmann, S Schaefer, Y Keim and JP Stasch are fulltime
employees of Bayer HealthCare.
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