Guanylate Cyclase Activity Research Articles

A Phase II study of avenciguat, a novel soluble guanylate cyclase activator, in patients with systemic sclerosis: Study design and rationale of the VITALISScE™ study

A Phase II study of avenciguat, a novel soluble guanylate cyclase activator, in patients with systemic sclerosis: Study design and rationale of the VITALISScE™ study

Real-time imaging of cGMP signaling shows pronounced differences between glomerular endothelial cells and podocytes.

Recent clinical trials of drugs enhancing cyclic guanosine monophosphate (cGMP) signaling for cardiovascular diseases have renewed interest in cGMP biology within the kidney. However, the role of cGMP signaling in glomerular endothelial cells (GECs) and podocytes remains largely unexplored. Using acute kidney slices from mice expressing the FRET-based cGMP biosensor cGi500 in endothelial cells or podocytes enabled real-time visualization of cGMP. Stimulation with atrial natriuretic peptide (ANP) or SNAP (NO donor) and various phosphodiesterase (PDE) inhibitors elevated intracellular cGMP in both cell types. GECs showed a transient cGMP response upon particulate or soluble guanylyl cyclase activation, while the cGMP response in podocytes reached a plateau following ANP administration. Co-stimulation (ANP + SNAP) led to an additive response in GECs. The administration of PDE inhibitors revealed a broader basal PDE activity in GECs dominated by PDE2a. In podocytes, basal PDE activity was mainly restricted to PDE3 and PDE5 activity. Our data demonstrate the existence of both guanylyl cyclase pathways in GECs and podocytes with cell-specific differences in cGMP synthesis and degradation, potentially suggesting new therapeutic options for kidney diseases.

Comparison of sGC activator and sGC stimulator in 5/6 nephrectomized rats on high-salt-diet.

Soluble guanylate cyclase (sGC) stimulators and activators are known to enhance kidney function in various models of chronic kidney disease (CKD) by increasing cyclic guanosine monophosphate (cGMP). Their differential effects on CKD progression, particularly under conditions of oxidative stress, remain unexplored by direct comparative studies. We conducted a side-by-side comparison using 5/6 nephrectomized rats on a high salt diet (5/6Nx+HSD) to evaluate the efficacy of the sGC stimulator BAY 41-8543 and the sGC activator BAY 60-2770 in CKD progression. BAY 41-8543 (1mg/kg; twice daily) and BAY 60-2770 (1mg/kg; once daily) were administered by gavage for 11 weeks. The 5/6Nx+HSD model led to increased plasma creatinine, proteinuria, and blood pressure. Both BAY 41-8543 and BAY 60-2770 significantly reduced systolic and diastolic blood pressure to a similar extent but did not improve renal function parameters. Notably, BAY 60-2770 reduced renal fibrosis, including interstitial fibrosis and glomerulosclerosis, whereas BAY 41-8543 did not. These antifibrotic effects of BAY 60-2770 were independent of blood pressure reduction. Proteomic analysis revealed that BAY 60-2770 corrected the upregulation of 9 proteins associated with apoptosis and fibrosis, including Caspase-3, MKK6 (Mitogen-Activated Protein Kinase Kinase 6), Prdx5 (Peroxiredoxin-5), in the 5/6Nx+HSD group. In contrast, BAY 41-8543 had no significant impact on these proteins. sGC activators were more effective than sGC stimulators in reducing renal fibrosis in 5/6 nephrectomized rats on a high salt diet, and this effect was due to modulation of apoptosis-associated proteins beyond the control of blood pressure.

Soluble Guanylate Cyclase Activators as Potential Novel Pad Therapeutics

Soluble Guanylate Cyclase Activators as Potential Novel Pad Therapeutics

149 - Soluble guanylate cyclase activator, cinaciguat, eliminates radiation-induced senescent cells in late radiation cystitis

149 - Soluble guanylate cyclase activator, cinaciguat, eliminates radiation-induced senescent cells in late radiation cystitis

The Role of Vericiguat in Early Phases of Anterior Myocardial Infarction: A Potential Game-Changer?

Anterior myocardial infarction is a critical condition with significant implications for cardiac function and patient prognosis. Despite advancements in reperfusion therapies, optimizing recovery during the early phases of myocardial infarction remains challenging. Anterior myocardial infarction can lead to substantial long-term effects on a patient's health due to extensive damage to the heart muscle, particularly the left ventricle, impacting both quality of life and overall prognosis. Vericiguat, a soluble guanylate cyclase stimulator, has shown promise in heart failure, but its role in early anterior myocardial infarction has not yet been fully explored. By enhancing soluble guanylate cyclase activity, vericiguat may increase cyclic guanosine monophosphate production, leading to vasodilation, inhibition of platelet aggregation, and potential cardioprotective effects. Currently, treatment options for anterior myocardial infarction primarily focus on reperfusion strategies and managing complications. However, there is a critical need for adjunctive therapies that specifically target the pathophysiological changes occurring in the early phases of myocardial infarction. Vericiguat's mechanism of action offers a novel approach to improving vascular function and myocardial health, potentially contributing to innovative treatment strategies that could transform the care and prognosis of patients with anterior myocardial infarction.

Airways Relaxant and Antiasthmatic Activity of Aconitum heterophyllum Wall ex Royle. Roots: A Mechanistic Insight.

Aconitum heterophyllum Wall ex Royle. (Ranunculaceae) is a traditional medicinal herb that has shown extensive pharmacological potential to treat cough, diarrhea, and infectious diseases but no scientific evidence is available to validate its antiasthmatic potential. In this study, we have investigated the tracheal relaxation and antiasthmatic activity of the selected bioactive fraction of A. heterophyllum. Chemical profiling of the most effective fraction obtained via bioassay-guided fractionation was done using LC-MS (Liquid chromatography-mass spectrometry, a technique utilized in the identification, separation, and quantification of known and unknown compounds). Molecular docking analysis of characterized constituents was performed to recognize the binding receptors, followed by an evaluation of the tracheal relaxation ability of active fraction. An acute oral toxicity study of the most effective fraction was done using OECD guidelines 423. Further, the therapeutic efficacy of the fraction was validated in asthma using a guinea pig model of ovalbumin (OVA) induced allergic asthma. The bio-guided activity revealed that hydro-methanolic extract of A. heterophyllum roots (F-1) was the most active fraction. LC-MS analysis of F-1 showed the presence of six major bioactive compounds in F-1. Molecular docking studies revealed strong binding affinities of identified constituents with histaminic receptor (H1) and muscarinic receptor (M3). The ex vivo study demonstrated smooth muscle relaxant activity of F-1 via dysregulating diverse signal transduction pathways viz. histaminic and muscarinic receptors antagonism (non-competitive), stimulation of β2-adrenergic receptor pathway, and soluble guanylyl cyclase activation. The findings of acute oral toxicity studies revealed that F-1 had no toxicity up to the dose of 2000 mg/Kg. The anti-asthmatic therapeutic efficacy of F-1 was further confirmed by the amelioration of respiratory hyperresponsiveness in asthmatic guinea pigs. This is the first evidence-based study showing the antiasthmatic therapeutic potential of the traditionally used herb A. heterophyllum through, computational and animal studies.

Supramolecular complexes of GCAP1: implications for inherited retinal dystrophies

Guanylate Cyclase Activating Protein 1 (GCAP1) is a calcium sensor that regulates the enzymatic activity of retinal Guanylate Cyclase 1 (GC1) in photoreceptors in a Ca2+/Mg2+ dependent manner. While point mutations in GCAP1 have been associated with inherited retinal dystrophies (IRDs), their impact on protein dimerization or on the possible interaction with the potent GC1 inhibitor RD3 (retinal degeneration protein 3) has never been investigated. Here, we integrate exhaustive in silico investigations with biochemical assays to evaluate the effects of the p.(E111V) substitution, associated with a severe form of IRD, on GCAP1 homo- and hetero-dimerization, and demonstrate that wild type (WT) GCAP1 directly interacts with RD3. Although inducing constitutive activation in GC1, the E111V substitution only slightly affects the dimerization of GCAP1. Both WT- and E111V-GCAP1 are predominantly monomeric in the absence of the GC1 target, however E111V-GCAP1 shows a stronger tendency to be monomeric in the Ca2+-bound form, corresponding to GC1 inhibiting state. Reconstitution experiments performed in the co-presence of WT-GCAP1, E111V-GCAP1 and RD3 restored nearly physiological regulation of the GC1 enzymatic activity in terms of cGMP synthesis and Ca2+-sensitivity, suggesting new scenarios for biologics-mediated treatment of GCAP1-associated IRDs.

Molsidomine provides neuroprotection against vincristine-induced peripheral neurotoxicity through soluble guanylyl cyclase activation

Peripheral neurotoxicity is a dose-limiting adverse reaction of primary frontline chemotherapeutic agents, including vincristine. Neuropathy can be so disabling that patients drop out of potentially curative therapy, negatively impacting cancer prognosis. The hallmark of vincristine neurotoxicity is axonopathy, yet its underpinning mechanisms remain uncertain. We developed a comprehensive drug discovery platform to identify neuroprotective agents against vincristine-induced neurotoxicity. Among the hits identified, SIN-1—an active metabolite of molsidomine—prevents vincristine-induced axonopathy in both motor and sensory neurons without compromising vincristine anticancer efficacy. Mechanistically, we found that SIN-1’s neuroprotective effect is mediated by activating soluble guanylyl cyclase. We modeled vincristine-induced peripheral neurotoxicity in rats to determine molsidomine therapeutic potential in vivo. Vincristine administration induced severe nerve damage and mechanical hypersensitivity that were attenuated by concomitant treatment with molsidomine. This study provides evidence of the neuroprotective properties of molsidomine and warrants further investigations of this drug as a therapy for vincristine-induced peripheral neurotoxicity.

Discovery and Optimization of Hsp110 and sGC Dual-Target Regulators for the Treatment of Pulmonary Arterial Hypertension.

Currently, bifunctional agents with vasodilation and ameliorated vascular remodeling effects provide more advantages for the treatment of pulmonary arterial hypertension (PAH). In this study, we first screened the hit 1 with heat shock protein 110 (Hsp110) inhibition effect from our in-house compound library with soluble guanylate cyclase (sGC) activity. Subsequently, a series of novel bisamide derivatives were designed and synthesized as Hsp110/sGC dual-target regulators based on hit 1. Among them, 17i exhibited optimal Hsp110 and sGC molecular activities as well as remarkable cell malignant phenotypes inhibitory and vasodilatory effects in vitro. Moreover, compared to riociguat, 17i showed superior efficacy in attenuating pulmonary vascular remodeling and right ventricular hypertrophy via Hsp110 suppression in hypoxia-induced PAH rat models (i.g.). Notably, our study successfully demonstrated that the simultaneous regulation of Hsp110 and sGC dual targets was a novel and feasible strategy for PAH therapy, providing a promising lead compound for anti-PAH drug discovery.

Activation of NOS-cGMP pathways promotes stress-induced sensitization of behavioral responses in zebrafish

Nitric oxide (NO) is a molecule involved in plasticity across levels and systems. The role of NOergic pathways in stress-induced sensitization (SIS) of behavioral responses, in which a particular stressor triggers a state of hyper-responsiveness to other stressors after an incubation period, was assessed in adult zebrafish. In this model, adult zebrafish acutely exposed to a fear-inducing conspecific alarm substance (CAS) and left undisturbed for an incubation period show increased anxiety-like behavior 24 h after exposure. CAS increased forebrain glutamate immediately after stress and 30 min after stress, an effect that was accompanied by increased nitrite levels immediately after stress, 30 min after stress, 90 min after stress, and 24 h after stress. CAS also increased nitrite levels in the head kidney, where cortisol is produced in zebrafish. CAS-elicited nitrite responses in the forebrain 90 min (but not 30 min) after stress were prevented by a NOS-2 blocker. Blocking NOS-1 30 min after stress prevents SIS; blocking NOS-2 90 min after stress also prevents stress-induced sensitization, as does blocking calcium-activated potassium channels in this latter time window. Stress-induced sensitization is also prevented by blocking guanylate cyclase activation in both time windows, and cGMP-dependent channel activation in the second time window. These results suggest that different NO-related pathways converge at different time windows of the incubation period to induce stress-induced sensitization.

Hepatic improvement within 27 days of avenciguat treatment in Child-Pugh A cirrhosis detected by an oral cholate challenge test.

New methods for measuring hepatic improvement in clinical trials and the clinic are needed. One new method, HepQuant SHUNT, detected dose-dependent improvements in hepatic function and portal physiology in the phase 1b study (NCT03842761) of avenciguat, an activator of soluble guanylyl cyclase that is being developed for the treatment of portal hypertension. Herein, we examined whether HepQuant Duo, an easy-to-administer test version, could similarly detect the effects of avenciguat. Twenty-three patients with Child-Pugh A cirrhosis and liver stiffness >15kPa received either a placebo (n = 5) or a maximum twice-daily avenciguat dose of 1, 2, or 3mg (n = 6 per group) for 28 days. The DuO test was performed at baseline and on days 11 and 27 in each subject. The test involved administering 40mg of d4-cholate orally, measuring d4-cholate concentrations in serum at 20 and 60 minutes, and calculating portal hepatic filtration rate, disease severity index, portal-systemic shunting (SHUNT%), and hepatic reserve (HR%). Avenciguat demonstrated dose-dependent improvement in all test parameters. Changes from baseline in SHUNT% after 27 days' treatment were 0.1 ± 9.0% for placebo, 1.7 ± 5.5% for 1mg twice-daily, -3.2 ± 2.7% for 2mg twice-daily, and -6.1 ± 5.0% for 3mg twice-daily (paired t test for change from baseline p = 0.98, 0.48, 0.04, and 0.03, respectively). The changes detected by HepQuant DuO were similar to those previously observed and reported for HepQuant SHUNT. The results support further study of avenciguat in treating portal hypertension and spotlight the utility of HepQuant DuO in the development of drug therapy for liver disease. HepQuant DuO facilitates the use of function testing to measure hepatic improvement in clinical trials and the clinic.

Soluble guanylate cyclase activator vericiguat prevents anthracycline-mediated cardiotoxicity and sarcopenia through cGMP-NLRP3-cytokine pathway: Potential application in patients with cancer.

e24015 Background: Anthracycline-induced cardiomyopathies and sarcopenia are frequently seen in cancer patients, affecting their overall survival and quality of life; therefore, new cardioprotective strategies are needed in cardioncology. Vericiguat is a new oral guanylate cyclase activator that reduces heart failure hospitalizations or cardiovascular death through improvement of smooth muscle cell relaxation and reduction of myocardial fibrosis and inflammation. In this study, we highlight on the potential cardioprotective properties of vericiguat against anthracycline.mediated cardiotoxicity and sarcopenia. Methods: Human cardiomyocytes (AC-16 cells) and Primary Skeletal Muscle Cells (HSkMC cells) were exposed for 24h h to doxorubicin (DOXO) at 0,3 and 1 µM with or withour a pre-treatment with Vericiguat at 0.1 or 1 or 10 µM for 1h. Mithocondrial cell viability, LDH and Citocrome C release were performed to study cytoprotective properties. TUNEL assay, cGMP and NLRP-3, Myd-88 intracellular levels were quantified through colorimetric and selective ELISA methods. IL-1β, IL-6, IL-8, CXCL-2, TGF-β and IL-18 were also analyzed in cardiac and muscle cell lysates after treatments. Results: Vericiguat exerts a significant cytoprotective and anti-apoptotic effect in cardiac and muscle cell lines during exposure to DOXO. A drastic increase in cGMP expression and reduction in NLRP-3, Myd-88 levels were also seen in Vericiguat-DOXO groups vs DOXO groups ( p < 0.001). Pro-inflammatory and cytotoxic cytokines were also downregulated after Vericiguat treatment during DOXO exposure. Conclusions: Vericiguat significantly reduced cardiotoxic effects and sarcopenia induced by DOXO therapy though enhancement of cGMP levels and reduction of NLRP-3/Myd-88/cytokines pathways. These findings provide a framework for future studies aiming to assess vericiguat for the primary prevention of anthracycline-mediated cardiotoxicity and sarcopenia.

Effect of Nitrosyl Iron Complex with 3,4-Dichlorothiophenolyls on the Level of Cyclic Nucleotide In Vitro.

The effect of a promising NO donor, a binuclear nitrosyl iron complex (NIC) with 3,4-dichlorothiophenolyls [Fe2(SC6H3Cl2)2(NO)4], on the adenylate cyclase and soluble guanylate cyclase enzymatic systems was studied. In in vitro experiments, this complex increased the concentration of important secondary messengers, such as cAMP and cGMP. An increase of their level by 2.4 and 4.5 times, respectively, was detected at NIC concentration of 0.1 mM. The ligand of the complex, 3,4-dichlorothiophenol, produced a less pronounced effect on adenylate cyclase. It was shown that the effect of this complex on the activity of soluble guanylate cyclase was comparable to the effect of anionic nitrosyl complex with thiosulfate ligands that exhibits vasodilating and cardioprotective properties.

Effect of Avenciguat on Albuminuria in Patients with CKD: Two Randomized Placebo-Controlled Trials.

Avenciguat is a novel, potent soluble guanylate cyclase activator in development for CKD. Two trials investigated avenciguat in diabetic (NCT04750577) and non-diabetic (NCT04736628) CKD. A prespecified pooled analysis of two randomized, double-blind, placebo-controlled trials of identical design. Adults with CKD (eGFR ≥20 and <90 mL/min/1.73 m 2 , urine albumin-to-creatinine ratio [UACR] ≥200 and <3500 mg/g) were randomized to 20 weeks of placebo or avenciguat 1, 2, or 3 mg three times daily (TID; adjunctive to angiotensin-converting-enzyme inhibitor or angiotensin receptor blocker. Primary endpoint: change from baseline in UACR in 10-hour urine at week 20, analyzed per protocol. Secondary endpoint: UACR change from baseline in first morning void urine at week 20. Safety was monitored throughout. Overall 500 patients (mean age 62 years [SD 13]; mean eGFR 44 mL/min/1.73 m 2 [SD 18] and median 10-hour UACR 719 [interquartile range 379-1285] mg/g ) received placebo (n=122) or avenciguat 1 mg (n=125), 2 mg (n=126), or 3 mg (n=127) TID. All 243 patients in study one and 27/261 patients in study two had diabetes. Avenciguat 1, 2, and 3 mg TID reduced UACR in 10-hour and first morning void urine versus placebo throughout the treatment period. At week 20, placebo-corrected geometric mean changes (95% confidence interval) from baseline in UACR in 10-hour urine with avenciguat 1, 2, and 3 mg TID were -15.5% (-26.4, -3.0), -13.2% (-24.6, -0.1), and -21.5% (-31.7, -9.8), respectively, analyzed per protocol. Corresponding changes in first morning void urine were -19.4% (-30.0, -7.3), -15.5% (-26.9, -2.5), and -23.4% (-33.5, -11.8), respectively. Avenciguat was well tolerated, overall frequency of adverse events was low and similar to placebo. The number of patients who discontinued study drug due to adverse events with avenciguat 1, 2, and 3 mg TID were 5 (4%), 11 (9%), and 11 (9%), respectively, compared with 4 (3%) in the placebo group. Avenciguat lowered albuminuria, and was well tolerated in patients with CKD.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of BI 685509, a soluble guanylyl cyclase activator, in healthy volunteers: Results from two randomized controlled trials

This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of BI 685509 after oral single rising doses (SRDs) or multiple rising doses (MRDs) in healthy volunteers. In the SRD trial (NCT02694354; February 29, 2016), within each of the three dose groups (DGs), six subjects received BI 685509 (1.0, 2.5, or 5.0 mg) and two received placebo (N = 24). In the MRD trial (NCT03116906; April 17, 2017), within each of the five DGs, nine subjects received BI 685509 (uptitrated to 1 mg once daily [qd; DG1], 2.5 mg twice daily [DG2], 5.0 mg qd [DG3]; 3.0 mg three times daily [tid; DG4] or 4.0 mg tid [DG5]) and three received placebo, for 14–17 days (N = 60). In the SRD trial, 7/24 subjects (29.2%) had ≥ 1 adverse event (AE), most frequently orthostatic dysregulation (n = 4). In the MRD trial, 26/45 subjects (57.8%) receiving BI 685509 had ≥ 1 AE, most frequently orthostatic dysregulation and fatigue (each n = 12). Tolerance development led to a marked decrease in orthostatic dysregulation events (DG3). BI 685509 was rapidly absorbed after oral administration, and exposure increased in a dose-proportional manner after single doses. Multiple dosing resulted in near–dose-proportional increase in exposure and limited accumulation. BI 685509 pharmacokinetics appeared linear with time; steady state occurred 3–5 days after each multiple-dosing period. Increased plasma cyclic guanosine monophosphate and decreased blood pressure followed by a compensatory increase in heart rate indicated target engagement. BI 685509 was generally well tolerated; orthostatic dysregulation may be appropriately countered by careful uptitration.

Primary Infection and Supernatants Generated by Pseudomonas aeruginosa ExoY Have Lasting Effects on Cyclic Nucleotide Signaling in Pulmonary Endothelial Cells

Rationale: Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of hospital-acquire pneumonia. This gram-negative bacterium has an arsenal of virulence factors including the type three secretion system (TTSS), which encodes for an injection type needle and exotoxins. One of the four exotoxins, ExoY, is a promiscuous nucleotidylyl cyclase that utilizes F-actin as a cofactor to generate both canonical and non-canonical cyclic nucleotides (cAMP, cGMP and cCMP and cUMP respectively). While it is well known that activation of endogenous adenylyl and guanylyl cyclase leads to intracellular increases in cAMP and cGMP and release into the extracellular space, whether ExoY generated cyclic nucleotides are also effluxed out of the cell into the extracellular space is not known. In addition, it is unclear whether presence of the bacteria themselves affects endogenous cAMP signaling. Further, infection with P. aeruginosa expressing ExoY generates virulent cytotoxic amyloids from lung endothelium; however, whether these cytotoxic amyloids disrupt agonist-induced endogenous cAMP generation is unknown. Thus, we tested the hypothesis that during primary infection, cAMP and cGMP are effluxed into the extracellular space, while the primary infection itself and sterile supernatants from the primary infection attenuate isoproterenol induce increases in cAMP in pulmonary microvascular endothelial cells (PMVECs). Methods: During primary infection, PMVECs were inoculated with PBS (vehicle control) or 3 different strains of P. aeruginosa at an moi 20:1. Strains ExoY+ and ExoYK81M have a fully functional TTSS and express either catalytically active or inactive ExoY, respectively, while a third strain, ExoYTM, is unable to translocate ExoY into eukaryotic cells. During the last 15 minutes of the 4-hour infection, cells were stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. In separate experiments, supernatants from a primary infection were generated using the same three strains of bacteria at moi 20:1 and 4 hours after inoculation, the supernatants collected and sterilized using a 0.2 μm filter. Sterilized supernatants were added to naïve PMVECs and during the last 15 minutes of the 4-hour inoculation, cells stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. Results: Cyclic AMP and cGMP were elevated in both the lysate and media of PMVECs following 4-hour infection with ExoY+ and the total isoproterenol and rolipram-stimulated cAMP response was attenuated by the presence of bacteria, ExoYK81M and ExoYTM, and further blunted with ExoY+ infection. Further, inoculation with ExoYK81M and ExoYTM supernatants attenuated the total isoproterenol and rolipram-stimulated cAMP response, which was further blunted by ExoY+ supernatants. Conclusions: Overall, we demonstrate PMVECs release a large cAMP and cGMP pool into the extracellular space and both primary infection and supernatants derived from the primary infection attenuate agonist induced elevations in cAMP. HL121513, HL66299. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1100: Soluble Guanylate Cyclase Activators As Potential Novel Pad Therapeutics

Introduction: Peripheral arterial disease (PAD) is major cause of limb loss in the United States with limited treatment modalities. Understanding the genetic etiology of PAD may provide rationale for novel therapeutic strategies. Here, we report a novel association between PAD and a likely endothelial cell specific cis-regulatory element of Nitric Oxide Synthase 3 ( NOS3 ). We hypothesize that targeting of the nitric oxide (NO)-cyclic GMP (cGMP) axis with Riociguat, an FDA-approved soluble guanylate cyclase (sGC) activator, will improve perfusion and exercise capacity in a murine PAD model. Methods: We meta-analyzed genetic associations with PAD using summary statistics from European participants in the UK Biobank (UKB), Copenhagen Hospital Biobank - Cardiovascular Disease Cohort and Danish Blood Donor Study (CHB-CVDC/DBDS), and the Million Veteran Program (MVP). Human NOS3 deficiency was modeled using Enos +/- mice with C57/B6 controls. We induced hindlimb ischemia (HLI) via double ligation of the femoral artery as described previously. Riociguat (3mg/kg/day) was administered 4 days prior and 28 days after HLI induction. We measured exercise capacity by placing animals on a treadmill with increasing speed and incline. We measured hindlimb perfusion using laser doppler perfusion imaging and assessed relative perfusion of the ischemic limb to the contralateral control limb. Results: We identified novel association of the minor allele of rs3918226, which lies within the promoter of NOS3, with PAD (OR: 1.06; 95% CI: 1.04-1.09; P = 1.3 x 10-9). Induction of hindlimb ischemia in Enos +/- animals resulted in impaired exercise capacity compared to wild-type controls. In Riociguat-treated Enos +/- mice, we observed a significant increase in distance traveled during exercise testing at 14 days which was sustained for the duration of the study period (276 vs 156 meters, p&lt;0.05). We did not find a significant improvement in hindlimb perfusion over the course of the study period. Conclusion: Here, we identify a novel genetic association with PAD and demonstrate that in a genetic model of PAD we can improve exercise capacity by targeting the NO-cGMP axis with Riociguat. Future studies will assess the clinical utility of sGC activators like Riociguat in humans.

NMR Structure of Retinal Guanylate Cyclase Activating Protein 5 (GCAP5) with R22A Mutation That Abolishes Dimerization and Enhances Cyclase Activation.

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with ∼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

The sGCa Vericiguat Exhibit Cardioprotective and Anti-Sarcopenic Effects through NLRP-3 Pathways: Potential Benefits for Anthracycline-Treated Cancer Patients.

Anthracycline-induced cardiomyopathies and sarcopenia are frequently seen in cancer patients, affecting their overall survival and quality of life; therefore, new cardioprotective and anti-sarcopenic strategies are needed. Vericiguat is a new oral guanylate cyclase activator that reduces heart failure hospitalizations or cardiovascular death. This study highlighted the potential cardioprotective and anti-sarcopenic properties of vericiguat during anthracycline therapy. Human cardiomyocytes and primary skeletal muscle cells were exposed to doxorubicin (DOXO) with or without a pre-treatment with vericiguat. Mitochondrial cell viability, LDH, and Cytochrome C release were performed to study cytoprotective properties. Intracellular Ca++ content, TUNEL assay, cGMP, NLRP-3, Myd-88, and cytokine intracellular levels were quantified through colorimetric and selective ELISA methods. Vericiguat exerts significant cytoprotective and anti-apoptotic effects during exposure to doxorubicin. A drastic increase in cGMP expression and reduction in NLRP-3, MyD-88 levels were also seen in Vericiguat-DOXO groups vs. DOXO groups (p < 0.001) in both cardiomyocytes and human muscle cells. GCa vericiguat reduces cytokines and chemokines involved in heart failure and sarcopenia. The findings that emerged from this study could provide the rationale for further preclinical and clinical investigations aimed at reducing anthracycline cardiotoxicity and sarcopenia in cancer patients.