Novel enhancers of guanylyl cyclase-A activity via allosteric modulation

Natriuretic peptide receptor (NPR)-A (also known as NPR-A, NPR1 or guanylyl cyclase-A, GC-A) is an attractive but challenging target to activate with small molecules. GC-A is activated by endogenous atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), and this activation leads to the production of cyclic guanosine monophosphate (cGMP). This system plays an important role in the regulation of cardiovascular and renal homeostasis. However, utilization of this receptor as a drug target has so far been limited to peptides, even though small molecule modulators allow oral administration and longer half-life. We have identified small molecular allosteric enhancers of GC-A, which strengthened ANP or BNP activation in various in vitro and ex vivo systems. These compounds do not mediate their actions through previously described allosteric binding sites or via known mechanisms of action. In addition, their selectivity and activity are dependent on only one amino acid in GC-A. Our findings show that there is a novel allosteric binding site on GC-A, which can be targeted by small molecules that increase the signaling effects of ANP and BNP.

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Discovery of small molecule guanylyl cyclase A receptor positive allosteric modulators

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2109386118 ◽

2021 ◽

Vol 118 (52) ◽

pp. e2109386118

Author(s):

S. Jeson Sangaralingham ◽

Kanupriya Whig ◽

Satyamaheshwar Peddibhotla ◽

R. Jason Kirby ◽

Hampton E. Sessions ◽

...

Keyword(s):

Natriuretic Peptide ◽

Small Molecule ◽

Guanylyl Cyclase ◽

High Throughput Screening ◽

Metabolic Diseases ◽

Ex Vivo ◽

Guanosine Monophosphate ◽

Cardiomyocyte Hypertrophy ◽

Orally Bioavailable

The particulate guanylyl cyclase A receptor (GC-A), via activation by its endogenous ligands atrial natriuretic peptide (ANP) and b-type natriuretic peptide (BNP), possesses beneficial biological properties such as blood pressure regulation, natriuresis, suppression of adverse remodeling, inhibition of the renin-angiotensin-aldosterone system, and favorable metabolic actions through the generation of its second messenger cyclic guanosine monophosphate (cGMP). Thus, the GC-A represents an important molecular therapeutic target for cardiovascular disease and its associated risk factors. However, a small molecule that is orally bioavailable and directly targets the GC-A to potentiate cGMP has yet to be discovered. Here, we performed a cell-based high-throughput screening campaign of the NIH Molecular Libraries Small Molecule Repository, and we successfully identified small molecule GC-A positive allosteric modulator (PAM) scaffolds. Further medicinal chemistry structure–activity relationship efforts of the lead scaffold resulted in the development of a GC-A PAM, MCUF-651, which enhanced ANP-mediated cGMP generation in human cardiac, renal, and fat cells and inhibited cardiomyocyte hypertrophy in vitro. Further, binding analysis confirmed MCUF-651 binds to GC-A and selectively enhances the binding of ANP to GC-A. Moreover, MCUF-651 is orally bioavailable in mice and enhances the ability of endogenous ANP and BNP, found in the plasma of normal subjects and patients with hypertension or heart failure, to generate GC-A–mediated cGMP ex vivo. In this work, we report the discovery and development of an oral, small molecule GC-A PAM that holds great potential as a therapeutic for cardiovascular, renal, and metabolic diseases.

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Abstract 923: Dissociation Between Superoxide Accumulation And Nitroglycerin Induced Tolerance

Circulation ◽

10.1161/circ.116.suppl_16.ii_181-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Vamsi K Addanki ◽

Pei-Suen Tsou ◽

Ho-Leung Fung

Keyword(s):

Nadph Oxidase ◽

Ex Vivo ◽

Dry Weight ◽

Causative Factor ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Protein Accumulation ◽

Induced Tolerance

We hypothesize that superoxide (SO) accumulation is not a critical causative factor in inducing nitroglycerin (NTG) tolerance. Using p47phox−/− and gp91−/− mice vs. their respective wild-type (WT) controls, we showed that aorta from mice null of these critical NADPH oxidase subunits exhibited similar vascular tolerance after NTG dosing (20 mg/kg sc, tid for 3 days), as indicated by their ex vivo pEC 50 and cyclic guanosine monophosphate (cGMP, pmol/mg protein) accumulation upon NTG challenge. In vitro aorta SO production (cpm/mg dry weight) was enhanced by NTG incubation both in p47phox null and WT mice (Table 1 ). Pre-exposure of isolated mice aorta to 100 microM NTG for 1 hr resulted in vascular tolerance toward NTG and increased SO accumulation. Oxypurinol (Oxy, 1mM) reduced SO but failed to attenuate vascular tolerance (Table 2 ). In LLC-PK1 cells, pre-exposure to NTG (1 microM for 4 hours) resulted in increased SO accumulation and reduced cGMP response to 3.16 microM NTG vs. vehicle control. Exposure to 1 microM angiotensin II increased SO but did not reduce cGMP response. Taken together, these results indicate that in vivo vascular NTG tolerance in mice does not require the presence of the p47phox and gp91phox subunits of NADPH oxidase, and that increased SO accumulation may be a consequence, rather than a cause, of NTG tolerance. Table 1 Table 2

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Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B

10.1101/191684 ◽

2017 ◽

Cited By ~ 1

Author(s):

Jerid W. Robinson ◽

Jeremy R. Egbert ◽

Julia Davydova ◽

Hannes Schmidt ◽

Laurinda A. Jaffe ◽

...

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Natriuretic Peptide ◽

Guanylyl Cyclase ◽

Growth Factor Receptor ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

List Type ◽

Fibroblast Growth ◽

Reversible Inhibition

AbstractActivating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations of guanylyl cyclase-B (GC-B, also called NPRB or NPR2) cause dwarfism. FGF exposure inhibits GC-B activity in a chondrocyte cell line, but the mechanism of the inactivation is not known. Here, we report that FGF exposure causes dephosphorylation of GC-B in rat chondrosarcoma cells, which correlates with a rapid, potent and reversible inhibition of C-type natriuretic peptide-dependent activation of GC-B. Cells expressing a phosphomimetic mutant of GC-B that cannot be inactivated by dephosphorylation because it contains glutamate substitutions for all known phosphorylation sites showed no decrease in GC-B activity in response to FGF. We conclude that FGF rapidly inactivates GC-B by a reversible dephosphorylation mechanism, which may contribute to the signaling network by which activated FGFR3 causes dwarfism.HighlightsGuanylyl Cyclase-B is expressed in rat chondrosarcoma cellsFGF2 induces a rapid, potent, and reversible inhibition of GC-BFGF2 treatment causes GC-B dephosphorylationFGF2 does not inhibit a dephosphorylation-resistant form of GC-BDephosphorylation is the mechanism of FGF2-dependent inhibition of GC-BAbbreviationscGMPcyclic guanosine monophosphateGCguanylyl cyclaseNPnatriuretic peptidePBSphosphate buffered salineWTwild type

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Phosphodiesterase Type 5 Inhibition Reverts Prostate Fibroblast-to-Myofibroblast Trans-Differentiation

Endocrinology ◽

10.1210/en.2012-1431 ◽

2012 ◽

Vol 153 (11) ◽

pp. 5546-5555 ◽

Cited By ~ 21

Author(s):

Christoph Zenzmaier ◽

Johann Kern ◽

Natalie Sampson ◽

Martin Heitz ◽

Eugen Plas ◽

...

Keyword(s):

Protein Kinase ◽

Ex Vivo ◽

Rho Kinase ◽

Chorioallantoic Membrane ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Protein Levels ◽

Igf Binding ◽

Phosphodiesterase Type

Abstract Phosphodiesterase type 5 (PDE5) inhibitors have been demonstrated to improve lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). Because BPH is primarily driven by fibroblast-to-myofibroblast trans-differentiation, this study aimed to evaluate the potential of the PDE5 inhibitor vardenafil to inhibit and reverse trans-differentation of primary human prostatic stromal cells (PrSC). Vardenafil, sodium nitroprusside, lentiviral-delivered short hairpin RNA-mediated PDE5 knockdown, sodium orthovanadate, and inhibitors of MAPK kinase, protein kinase G, Ras homolog family member (Rho) A, RhoA/Rho kinase, phosphatidylinositol 3 kinase and protein kinase B (AKT) were applied to PrSC treated with basic fibroblast growth factor (fibroblasts) or TGFβ1 (myofibroblasts) in vitro, in chicken chorioallantoic membrane xenografts in vivo, and to prostatic organoids ex vivo. Fibroblast-to-myofibroblast trans-differentiation was monitored by smooth muscle cell actin and IGF binding protein 3 mRNA and protein levels. Vardenafil significantly attenuated TGFβ1-induced PrSC trans-differentiation in vitro and in chorioallantoic membrane xenografts. Enhancement of nitric oxide/cyclic guanosine monophosphate signaling by vardenafil, sodium nitroprusside, or PDE5 knockdown reduced smooth muscle cell actin and IGF binding protein 3 mRNA and protein levels and restored fibroblast-like morphology in trans-differentiated myofibroblast. This reversal of trans-differentiation was not affected by MAPK kinase, protein kinase G, RhoA, or RhoA/Rho kinase inhibition, but vardenafil attenuated phospho-AKT levels in myofibroblasts. Consistently, phosphatidylinositol 3 kinase or AKT inhibition induced reversal of trans-differentiation, whereas the tyrosine phosphatase inhibitor sodium orthovanadate abrogated the effect of vardenafil. Treatment of prostatic organoids with vardenafil ex vivo reduced expression of myofibroblast markers, indicating reverse remodeling of stroma towards a desired higher fibroblast/myofibroblast ratio. Thus, enhancement of the nitric oxide/cyclic guanosine monophosphate signaling pathway by vardenafil attenuates and reverts fibroblast-to-myofibroblast trans-differentiation, hypothesizing that BPH patients might benefit from long-term therapy with PDE5 inhibitors.

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Cyclic Guanosine Monophosphate Modulates Locomotor Acceleration Induced by Nitric Oxide but not Serotonin in Clione limacina Central Pattern Generator Swim Interneurons

Integrative Organismal Biology ◽

10.1093/iob/obaa045 ◽

2020 ◽

Author(s):

Thomas J Pirtle ◽

Richard A Satterlie

Keyword(s):

Nitric Oxide ◽

Central Pattern Generator ◽

Guanylyl Cyclase ◽

Signal Transduction Pathway ◽

Soluble Guanylyl Cyclase ◽

Cyclic Guanosine Monophosphate ◽

Pattern Generator ◽

Guanosine Monophosphate ◽

Cellular Changes ◽

Clione Limacina

Abstract Typically, the marine mollusk, Clione limacina, exhibits a slow, hovering locomotor gait to maintain its position in the water column. However, the animal exhibits behaviorally relevant locomotor swim acceleration during escape response and feeding behavior. Both nitric oxide and serotonin mediate this behavioral swim acceleration. In this study, we examine the role that the second messenger, cGMP, plays in mediating nitric oxide and serotonin-induced swim acceleration. We observed that the application of an analog of cGMP or an activator of soluble guanylyl cyclase increased fictive locomotor speed recorded from Pd-7 interneurons of the animal’s locomotor central pattern generator. Moreover, inhibition of soluble guanylyl cyclase decreased fictive locomotor speed. These results suggest that basal levels of cGMP are important for slow swimming and that increased production of cGMP mediates swim acceleration in Clione. Because nitric oxide has its effect through cGMP signaling and because we show herein that cGMP produces cellular changes in Clione swim interneurons that are consistent with cellular changes produced by serotonin application, we hypothesize that both nitric oxide and serotonin function via a common signal transduction pathway that involves cGMP. Our results show that cGMP mediates nitric oxide-induced but not serotonin-induced swim acceleration in Clione.

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ANP and BNP Exert Anti-Inflammatory Action via NPR-1/cGMP Axis by Interfering with Canonical, Non-Canonical, and Alternative Routes of Inflammasome Activation in Human THP1 Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22010024 ◽

2020 ◽

Vol 22 (1) ◽

pp. 24

Author(s):

Letizia Mezzasoma ◽

Vincenzo Nicola Talesa ◽

Rita Romani ◽

Ilaria Bellezza

Keyword(s):

Natriuretic Peptide ◽

Specific Inhibitor ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Caspase 8 ◽

Inflammasome Activation ◽

Intracellular Cgmp ◽

Dependent Protein ◽

Anti Inflammatory ◽

Alternative Routes

Dysregulated inflammasome activation and interleukin (IL)-1β production are associated with several inflammatory disorders. Three different routes can lead to inflammasome activation: a canonical two-step, a non-canonical Caspase-4/5- and Gasdermin D-dependent, and an alternative Caspase-8-mediated pathway. Natriuretic Peptides (NPs), Atrial Natriuretic Peptide (ANP) and B-type Natriuretic Peptide (BNP), binding to Natriuretic Peptide Receptor-1 (NPR-1), signal by increasing cGMP (cyclic guanosine monophosphate) levels that, in turn, stimulate cGMP-dependent protein kinase-I (PKG-I). We previously demonstrated that, by counteracting inflammasome activation, NPs inhibit IL-1β secretion. Here we aimed to decipher the molecular mechanism underlying NPs effects on THP-1 cells stimulated with lipopolysaccharide (LPS) + ATP. Involvement of cGMP and PKG-I were assessed pre-treating THP-1 cells with the membrane-permeable analogue, 8-Br-cGMP, and the specific inhibitor KT-5823, respectively. We found that NPs, by activating NPR-1/cGMP/PKG-I axis, lead to phosphorylation of NLRP3 at Ser295 and to inflammasome platform disassembly. Moreover, by increasing intracellular cGMP levels and activating phosphodiesterases, NPs interfere with both Gasdermin D and Caspase-8 cleavage, indicating that they disturb non-canonical and alternative routes of inflammasome activation. These results showed that ANP and BNP anti-inflammatory and immunomodulatory actions may involve the inhibition of all the known routes of inflammasome activation. Thus, NPs might be proposed for the treatment of the plethora of diseases caused by a dysregulated inflammasome activation.

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Release of cyclic guanosine monophosphate evaluated as a diagnostic tool in cardiac diseases

Clinical Chemistry ◽

10.1093/clinchem/37.2.186 ◽

1991 ◽

Vol 37 (2) ◽

pp. 186-190 ◽

Cited By ~ 39

Author(s):

Karl-P Vorderwinkler ◽

Eilka Artner-Dworzak ◽

Gab Jakob ◽

Johanne Mair ◽

Franz Diensti ◽

...

Keyword(s):

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Cardiac Diseases ◽

Blood Samples ◽

Cutoff Value ◽

Intracellular Cgmp ◽

Glycerol Trinitrate ◽

Clinical Measurements ◽

And Storage

Abstract Concentrations of atrial natriuretic peptide (ANP) are increased in plasma of patients with impaired cardiac and renal function. The second messenger of ANP, cyclic guanosine monophosphate (cGMP), is released into the plasma specifically upon stimulation of cells with ANP. Although nitrates can also activate intracellular cGMP synthesis, we detected no increase in plasma cGMP concentrations after infusions of glycerol trinitrate. Because immunoreactive ANP is highly susceptible to degradation and nonspecific influences in blood samples, determinations of ANP require immediate centrifugation and storage of plasma at -20 degrees C. In contrast, we found that cGMP is stable for five days in vitro in blood samples containing EDTA. In 147 healthy blood donors, the upper cutoff value for plasma cGMP was 6.60 nmol/L, not significantly different (P greater than 0.05) from that for 222 patients with disorders other than cardiovascular and renal. In 69 patients with manifest congestive heart failure (NYHA stages II-IV), 65 had increased cGMP values. Using the above cutoff value for cGMP gave diagnostic sensitivity of 94.2% and specificity of 93.7%. Plasma cGMP may thus provide an alternative for routine clinical measurements of ANP in cardiac diseases in the absence of renal disorders.

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