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1.  Insights into BAY 60-2770 activation and S-nitrosylation-dependent desensitization of soluble guanylyl cyclase via crystal structures of homologous Nostoc H-NOX domain complexes 
Biochemistry  2013;52(20):10.1021/bi301657w.
The soluble guanylyl cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC’s salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveals that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization.
PMCID: PMC3775905  PMID: 23614626
The journal of sexual medicine  2012;10(3):704-718.
Soluble guanylate cyclase (sGC) is the receptor for nitric oxide (NO) and in pathophysiologic conditions where NO formation or bioavailability is impaired, erectile dysfunction (ED) occurs.
The aim of this study was to investigate erectile responses to the sGC stimulator BAY 41-8543 in physiologic and pathophysiologic conditions.
Increases in intracavernosal pressure (ICP) in response to intracavernosal (ic) injections of BAY 41-8543 were investigated in the anesthetized rat.
Main Outcome Measures
Increases in ICP/MAP in response to ic injections of BAY 41-8543 and the interaction of BAY 41-8543 with exogenous and endogenously released NO were investigated and the effect of the sGC stimulator on cavernosal nerve injury was assessed. The mechanism of the increase in ICP/MAP in response to ic injection of acetylcholine was investigated.
The ic injections of BAY 41-8543 increased ICP/MAP and the duration of the response. BAY 41-8543 was less potent than SNP and ic injections of BAY 41-8543 and SNP produced a larger response than the algebraic sum of responses to either agent alone. Simultaneous ic injection of BAY 41-8543 and cavernosal nerve stimulation produced a greater response than either intervention alone. Atropine and cavernosal nerve crush injury decreased the response to nerve stimulation and ic injection of BAY 41-8543 restored the response.
These data show that BAY 41-8543 has significant erectile activity and can synergize with exogenous and endogenously released NO. This study shows that atropine and nerve crush attenuate the response to cavernosal nerve stimulation and that BAY 41-8543 can restore the response. The results with atropine, L-NAME and hexamethonium indicate that the response to ic injection of acetylcholine is mediated by muscarinic receptors and the release of NO with no significant role for nicotinic receptors. These results suggest that BAY 41-8543 would be useful in the treatment of ED.
PMCID: PMC3594361  PMID: 22989320
5.  The selective rho-kinase inhibitor Azaindole-1 has long lasting erectile activity in the rat 
Urology  2013;81(2):465.e7-465.e14.
To investigate the effects of the selective Rho-kinase (ROCK) inhibitor azaindole-1 on erectile function under physiologic and pathophysiologic conditions in the rat.
The effect of intracavernosal (i.c.) injections of azaindole-1 on change in ICP, ICP/MAP, AUC, and response duration were investigated in the anesthetized rat under control conditions and when NANC neurotransmission and cholinergic function or sGC were inhibited or after cavernosal nerve crush injury.
The i.c. injections of azaindole-1 produced dose-related increases in ICP/MAP and AUC that were long lasting at the highest doses studied when compared with the prototypical ROCK-inhibitor fasudil. Erectile responses were not altered by 7-NI and atropine in doses that reduced the response to cavernosal nerve stimulation by 86%, indicating that they were independent of NO release by cavernosal nerves or activation of muscarinic receptors in the corpora cavernosa. Erectile responses to azaindole-1 were not altered by the sGC inhibitor ODQ in a dose that attenuated responses to the NO donor SNP indicating that they were independent of an action on sGC. The erectile response to ic injections of azaindole-1 or Y-27632 which was reported to be NO/cGMP- dependent were not attenuated after cavernosal nerve crush injury.
The present studies indicate azaindole-1 has long lasting erectile activity that is independent of NO release, muscarinic receptor, or sGC activation or the integrity of the cavernosal nerves.
PMCID: PMC3564057  PMID: 23374844
Azaindole-1; selective Rho-kinase inhibitor; erectile dysfunction; oxidative stress; impaired cavernosal nerve function
7.  NO-independent stimulation or activation of soluble guanylyl cyclase during early reperfusion limits infarct size 
Cardiovascular Research  2013;101(2):220-228.
Guanylyl cyclase-cyclic guanosine monophosphate signalling plays an important role in endogenous cardioprotective signalling. The aim was to assess the potential of direct pharmacological activation and stimulation of soluble guanylyl cyclase, targeting different redox states of the enzyme, to limit myocardial necrosis during early reperfusion.
Methods and results
Rat isolated hearts were subjected to reversible left coronary artery occlusion (ischaemia-reperfusion) and infarct size was assessed by the tetrazolium staining technique. Administration during early reperfusion of BAY 41-2272, an NO-independent, haem-dependent stimulator of soluble guanylyl cyclase targeting the reduced state, or BAY 60-2770, an NO-independent, haem-independent activator targeting the oxidized state, significantly limited infarct size. Inhibition of NO synthesis did not abrogate this protection, but exogenous perfusion of NO with BAY 41-2272 produced a synergistic effect. The haem site oxidiser, ODQ abrogated the protection afforded by BAY 41-2272 but potentiated the protection afforded by BAY 60-2770. Targeting both the reduced and oxidized forms of sGC together did not afford additive protection.
Targeting either reduced or oxidized forms of sGC during early reperfusion affords cardioprotection, providing support for the concept that direct sGC manipulation at reperfusion has therapeutic potential for the management of acute myocardial infarction.
PMCID: PMC3896250  PMID: 24259501
Ischaemia-reperfusion; cGMP; NO; sGC
12.  Effects of Different Pulmonary Vasodilators on Arterial Saturation in a Model of Pulmonary Hypertension 
PLoS ONE  2013;8(8):e73502.
Approved therapies for pulmonary arterial hypertension can induce oxygen desaturation when administered to patients with secondary forms of pulmonary hypertension (PH), probably due to an increase in ventilation/perfusion mismatch. Thus, so far these treatments have largely failed in secondary forms of PH.
We established an animal model of heterogeneous lung ventilation to evaluate the desaturation potential of mechanistically distinct vasoactive drugs launched or currently in clinical development for the treatment of PH. Single-lung ventilation was induced in five groups (N = 6) of anesthetized minipigs (7 weeks, 4 to 5 kg BW), and their hemodynamic parameters were monitored before and after intravenous injection of control (vehicle only), endothelin antagonist (bosentan; 0.3, 1, 3, 10 mg/kg), phosphodiesterase type 5 inhibitor (sildenafil; 3, 10, 30, 100 µg/kg), and soluble guanylate cyclase stimulators (BAY 41–8543 and riociguat; 1, 3, 10, 30 µg/kg). Cumulative doses were administered before successive unilateral ventilation cycles. The doses were chosen to achieve equal effect on blood pressure by the different pharmacologic principles.
Single-lung ventilation resulted in transient increases in mean pulmonary artery pressure (mPAP) and desaturation. In contrast to control, all drugs dose-dependently decreased hypoxic mPAP (a positive treatment effect) and increased area under the arterial hemoglobin saturation curve (unwanted desaturation effect). Riociguat and bosentan reduced hypoxic mPAP to the greatest extent, while the soluble guanylate cyclase stimulators riociguat and BAY 41–8543 lowered arterial oxygen saturation of hemoglobin the least.
Future investigations will be required to confirm these findings in clinical settings.
PMCID: PMC3756006  PMID: 24015306
13.  Kynurenine is a novel endothelium-derived relaxing factor produced during inflammation 
Nature medicine  2010;16(3):279-285.
Control of blood vessel tone is central to vascular homeostasis. Here, we show that metabolism of tryptophan to kynurenine by indoleamine 2,3-dioxygenase (IDO) expressed in endothelial cells contributes to arterial vessel relaxation and the control of blood pressure. Infection of mice with malarial parasites (Plasmodium berghei), and experimental induction of endotoxemia, caused endothelial expression of IDO, resulting in decreased plasma tryptophan, increased kynurenine, and hypotension. Pharmacological inhibition of IDO increased blood pressure in systemically inflamed mice, but not in mice deficient for IDO or interferon-γ, which is required for IDO induction. Tryptophan dilated pre-constricted porcine coronary arteries only if active IDO and an intact endothelium were both present. Kynurenine dose-dependently decreased blood pressure in spontaneously hypertensive rats, inhibited contraction of arteries, and relaxed pre-constricted rings endothelium-independently. Arterial relaxation by kynurenine was mediated by activation of the adenylate and soluble guanylate cyclase pathways.
PMCID: PMC3556275  PMID: 20190767
14.  The Soluble Guanylyl Cyclase Activator Bay 58-2667 Selectively Limits Cardiomyocyte Hypertrophy 
PLoS ONE  2012;7(11):e44481.
Although evidence now suggests cGMP is a negative regulator of cardiac hypertrophy, the direct consequences of the soluble guanylyl cyclase (sGC) activator BAY 58-2667 on cardiac remodeling, independent of changes in hemodynamic load, has not been investigated. In the present study, we tested the hypothesis that the NO•-independent sGC activator BAY 58-2667 inhibits cardiomyocyte hypertrophy in vitro. Concomitant impact of BAY 58-2667 on cardiac fibroblast proliferation, and insights into potential mechanisms of action, were also sought. Results were compared to the sGC stimulator BAY 41-2272.
Neonatal rat cardiomyocytes were incubated with endothelin-1 (ET1, 60nmol/L) in the presence and absence of BAY 41-2272 and BAY 58-2667 (0.01–0.3 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. The impact of both sGC ligands on basal and stimulated cardiac fibroblast proliferation in vitro was also determined.
We now demonstrate that BAY 58-2667 (0.01–0.3 µmol/L) elicited concentration-dependent antihypertrophic actions, inhibiting ET1-mediated increases in cardiomyocyte 2D area and de novo protein synthesis, as well as suppressing ET1-induced cardiomyocyte superoxide generation. This was accompanied by potent increases in cardiomyocyte cGMP accumulation and activity of its downstream signal, vasodilator-stimulated phosphoprotein (VASP), without elevating cardiomyocyte cAMP. In contrast, submicromolar concentrations of BAY 58-2667 had no effect on basal or stimulated cardiac fibroblast proliferation. Indeed, only at concentrations ≥10 µmol/L was inhibition of cardiac fibrosis seen in vitro. The effects of BAY 58-2667 in both cell types were mimicked by BAY 41-2272.
Our results demonstrate that BAY 58-2667 elicits protective, cardiomyocyte-selective effects in vitro. These actions are associated with sGC activation and are evident in the absence of confounding hemodynamic factors, at low (submicromolar) concentrations. Thus this distinctive sGC ligand may potentially represent an alternative therapeutic approach for limiting myocardial hypertrophy.
PMCID: PMC3492396  PMID: 23144773
15.  The Soluble Guanylate Cyclase Stimulator Riociguat Ameliorates Pulmonary Hypertension Induced by Hypoxia and SU5416 in Rats 
PLoS ONE  2012;7(8):e43433.
The nitric oxide (NO)–soluble guanylate cyclase (sGC)–cyclic guanosine monophosphate (cGMP) signal-transduction pathway is impaired in many cardiovascular diseases, including pulmonary arterial hypertension (PAH). Riociguat (BAY 63–2521) is a stimulator of sGC that works both in synergy with and independently of NO to increase levels of cGMP. The aims of this study were to investigate the role of NO–sGC–cGMP signaling in a model of severe PAH and to evaluate the effects of sGC stimulation by riociguat and PDE5 inhibition by sildenafil on pulmonary hemodynamics and vascular remodeling in severe experimental PAH.
Methods and Results
Severe angioproliferative PAH was induced in rats by combined exposure to the vascular endothelial growth factor receptor antagonist SU5416 and hypoxia (SUHx). Twenty-one days thereafter rats were randomized to receive either riociguat (10 mg/kg/day), sildenafil (50 mg/kg/day) or vehicle by oral gavage, for 14 days until the day of the terminal hemodynamic measurements. Administration of riociguat or sildenafil significantly decreased right ventricular systolic pressure (RVSP). Riociguat significantly decreased RV hypertrophy (RVH) (0.55±0.02, p<0.05), increased cardiac output (60.8±.8 mL/minute, p<0.05) and decreased total pulmonary resistance (4.03±0.3 mmHg min−1 ml−1 100 g BW, p<0.05), compared with sildenafil and vehicle. Both compounds significantly decreased the RV collagen content and improved RV function, but the effects of riociguat on tricuspid annular plane systolic excursion and RV myocardial performance were significantly better than those of sildenafil (p<0.05). The proportion of occluded arteries was significantly lower in animals receiving riociguat than in those receiving vehicle (p<0.05); furthermore, the neointima/media ratio was significantly lower in those receiving riociguat than in those receiving sildenafil or vehicle (p<0.05).
Riociguat and sildenafil significantly reduced RVSP and RVH, and improved RV function compared with vehicle. Riociguat had a greater effect on hemodynamics and RVH than sildenafil.
PMCID: PMC3422306  PMID: 22912874
16.  Soluble Guanylate Cyclase as an Emerging Therapeutic Target in Cardiopulmonary Disease 
Circulation  2011;123(20):2263-2273.
PMCID: PMC3103045  PMID: 21606405
cardiovascular diseases; hypertension; pulmonary; soluble guanylate cyclase; nitric oxide; riociguat

Results 1-25 (52)