11th International Conference on cGMP

sGc is important for proper endothelial function in vascular beds. Avenciguat (BI 685509) is a novel, potent sGc activator in development for treatment of CKD. Two trials investigated the efficacy and safety of avenciguat in patients with diabetic (NCT04750577) or non-diabetic (NCT04736628) CKD.

In these two Phase II double-blind, placebo-controlled trials of identical design, adults aged ≥18 years with CKD (eGFR ≥20–<90 mL/min/1.73 m², UACR ≥200–<3500 mg/g) were randomised to receive 20 weeks of placebo or avenciguat 1, 2 or 3 mg TID (including a 4-week up-titration period) as adjunct to maximum tolerated ACE inhibitor or ARB. The primary endpoint was change from baseline in UACR in 10-hour urine at Week 20. Safety was monitored throughout both trials. At trial completion, individual patient data were pooled and analysed according to a pre-specified analysis plan.

A total of 500 patients were randomised and received placebo (n=122) or avenciguat 1 mg (n=125),2 mg (n=126) or 3 mg (n=127) TID. Avenciguat reduced UACR in 10-hour urine compared with placebo throughout the treatment period. At Week 20, the placebo-corrected geometric mean percent changes (95% CI) 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. Avenciguat was well tolerated. Hypotension was reported in 33 patients (8.7%) treated with any avenciguat dose and 3 patients (2.5%) receiving placebo.

Avenciguat was effective in lowering albuminuria and was well tolerated in patients with diabetic and non-diabetic CKD.

Background and Aims: Soluble guanylate cyclase (sGC) is a key enzyme in the nitric oxide–cyclic guanosine monophosphate (NO-cGMP) pathway. Oxidative stress impairs NO binding to sGC, preventing the formation of cGMP, contributing to CKD progression and accelerating cardiovascular (CV) disease in patients with CKD. Runcaciguat, a novel sGC activator, potently and selectively activates the hem-free form of sGC under oxidative stress, independently of NO. Activated sGC increases cGMP synthesis, restoring the impaired NO-pathway and potentially preventing CKD progression. This study assessed the efficacy, safety and tolerability of runcaciguat in CKD patients.

Method: The multicentre, randomized, double-blind, placebo-controlled, parallel-group, individual- titration, phase II trial (CONCORD, NCT04507061) enrolled patients aged ≥45 years with CKD (eGFR 25–60 ml/min/1.73 m2, UACR 30–3000 mg/g), ≥3 months stable, maximum tolerated ACEi/ARB treatment and type 2 diabetes and/or hypertension. Patients also had atherosclerotic CV disease and/or heart failure (NYHA class I–II). At 4 weeks after screening, patients were randomized 3:1 to receive runcaciguat od or matching placebo. During the 4-week titration phase, patients were up-titrated weekly from 30 mg to 120 mg, with down-titration acceptable if there were safety issues. Patients received the maximum tolerated dose for ≥4 weeks in the maintenance phase before entering a 30-day safety follow- up. The primary efficacy endpoint was the reduction in UACR from baseline to the average of days 22, 29 and 57 according to a per-protocol analysis for phase 2 studies. The study was separately poweredfor 3 strata: CKD patients with diabetes and SGLT2i co-medication for ≥3 months, CKD patients with diabetes without SGLT2i co-medication, and CKD patients without diabetes.

Results: From September 2020–April 2022, 395 patients from 82 study centres in 13 countries were enrolled and 243 were randomized. In the per protocol set (N=170), 80% were male with a mean ageof 70 years, BMI 30.5 kg/m2, eGFR 41.8 ml/min/1.73 m2 and UACR 220.1 mg/g. Baseline characteristics were largely well balanced between treatment arms and strata, although differences in BMI were observed between the non-diabetic and diabetic strata. An average reduction in UACR of 44% from baseline was observed from day 22 until end of treatment (day 57) in the runcaciguattreatment arm in all strata combined according to a mixed model Bayesian analysis (median 0.565; 10th–90th percentile, 0.526–0.607; >99.9 posterior probability). In contrast, an average UACR reductionof 16% was seen in the placebo arm in all strata combined, but there was no reduction in either diabetic group. Statistically significant reductions in UACR with runcaciguat vs placebo were observed in bothdiabetic strata (>99% posterior probability with 47% and 45% reduction vs placebo), with no significant difference between the strata of patients with diabetes prescribed an SGLT2i or not (p=0.41). Areduction in UACR vs baseline was observed in the runcaciguat arm in all strata combined from day 8 (30 mg od for 7 days). The UACR decreased further during the remainder of the 4-week titration phaseand remained stable at a low level throughout the maintenance phase. After treatment cessation, UACR returned to baseline at the follow-up visit. Up-titration to the highest dose was achieved in ~80% of runcaciguat-treated patients and ~86% of placebo-treated patients. Most treatment-emergent adverse events (TEAEs) were mild or moderate and occurred in 69% and 53% of patients receiving runcaciguat and placebo, respectively. The most common TEAE was peripheral edema in runcaciguat-treated patients (12%) vs nausea in placebo-treated patients (8.5%). Dizziness was the second most frequent TEAE in both groups.

Conclusion: The novel sGC activator runcaciguat significantly reduced UACR vs baseline in patients with CKD on top of ACEi/ARB, with or without concomitant SGLT2i treatment. Runcaciguat was well tolerated with no safety concerns. The results of the CONCORD study suggest that sGC activation may represent a promising approach for the management of patients with CKD with or without type 2 diabetes.

To test the hypothesis of a causal mechanism subtype in HFpEF related to uncoupled NO synthase and apo-sGC, a monocentric, prospective, randomized, standard treatment-controlled, open-label, phase IIa, proof-of-concept clinical trial (REPO-HFPEF IIa) was conducted. 21 HFpEF patients were randomized and exposed for 12 weeks in a 2:1 ratio to the standard of care (SOC) with or without a triple combination of vericiguat, L-citrulline, and folate. Patients were eligible if they were positive for the mechanism-based biomarker plasma NOX5 (≥ 105 ng/ml), apo-sGC-positive (apo-sGC/sGC ratio >1.05), or both. The total count of adverse events (AEs) between treatment groups was 22 in 14 patients in the REPO group and 6 in 7 in the SOC group. The median of peak VO2 at screening for the REPO and SOC groups was 13.1 ml/min/kg (9.4-14.1) and 10.6 ml/min/kg (7.9-12.3), respectively. In a linear mixed regression analysis, the least square mean difference for peak VO2 at visit 4 (end of treatment) was +0.27 (95% CI: -0.74 to 1.27) and -0.23 (95% CI: -1.13 to 0.68) for the REPO and SOC groups, respectively (between treatment p-value=0.340). The effect of treatment on KCCQ, peak VO2, and NT-pro-BNP did not significantly differ across NOX5 and sGC. However, we found a numerical trend towards improvement in KCCQ (+11.9 vs. -13.9, interaction p-value=0.116), peak VO2 (+1.54 vs. +0.45, interaction p-value=0.751), and NT-pro-BNP (-1135.9 pg/ml vs. +658.7 pg/ml, interaction p-value=0.166) in REPO-treated NOX5-positive patients. This differential effect was not found across apo-sGC-positive vs. negative patients: KCCQ (1.2 vs. 11.9, interaction p-value=0.410), peak VO2 (+0.41 vs. 1.91, interaction p-value=0.568), and NT-pro-BNP (-1288.5 pg/ml vs. 298.7 pg/ml, interaction p-value=0.288). Thus, network pharmacology with vericiguat, L-citrulline, and folate in a subgroup of NOX5-positive HFpEF patients is not associated with an increased overall risk of adverse events. There were some numerical signals toward improvement in HF-related quality of life, exercise capacity, and NT-pro-BNP reductions. Further studies with a larger sample size are necessary to validate these findings.

Introduction
Hypertension (HTN) and metabolic syndrome (MetS) are leading and frequently coexisting risk factors for major cardiovascular diseases. The cardiac hormone atrial natriuretic peptide (ANP) plays a key role in blood pressure (BP) and metabolic regulation by activating guanylyl cyclase receptor A and generating cGMP as second messenger.

Objectives
We assessed cardiovascular, metabolic and cGMP properties of MANP, a novel ANP analog, in subjects with HTN and MetS.

Methods
We conducted a double-blind, placebo-controlled trial in 22 patients (17 receiving MANP) with HTN and MetS involving single subcutaneous injection of MANP (2.5 µg/kg) or placebo (NCT03781739).

Results
Compared to baseline, MANP increased plasma cGMP at 30 minutes (delta: 4.8±2.0 pmol/mL, P= 0.02) and 1 hour (delta: 2.9±1.3 pmol/mL, P= 0.03) post-injection. At 6 hours, systolic BP decreased by 5.7±2.9 mmHg (P= 0.06) in the MANP group, whereas no reduction in BP was observed in placebo group. After MANP injection, glucose levels decreased at 2 (delta: -4.7±2.1 mg/mL, P= 0.04) and 4 hours (delta: -13.1±3.9 mg/mL, P= 0.003) compared to baseline while insulin levels remained stable. In the 4 hours post-injection, we also observed a trend of increase in insulin sensitivity index (HOMA2-IS) and decrease in insulin resistance index (HOMA2-IR) in the MANP group, opposite trends were present in placebo group. At 1 hour post-MANP administration, circulating non-esterified fatty acids increased by 108.2±37.4 µM compared to baseline (P= 0.01).

Conclusions
Our findings support favorable cardiovascular and metabolic actions of MANP as a potential therapy for HTN with MetS.

The nitric oxide (NO)/soluble guanylyl cyclase (sGC)signaling pathway in human erythroid cells has been proposed to drive theexpression of γ-globin and control erythropoiesis, but its role in vivo has not yet been demonstrated.We generated erythroid cell-specific eNOS knock-out and knock-in mice, as well as erythroid-specific sGC KO mice, using the loxP/Cre recombinase gene targeting approach. We confirmed the specificity of gene targetingby analyzing erythroid cell-specific DNA recombination, target protein expression, and/or activity using qPCR, western blotting, ELISA, and enzymatic assays.To analyze changes in the hematopoietic phenotype, we analyzed the peripheral blood cell and reticulocyte counts. In the bone marrow and spleen of the mice, we analyzed cellular composition, quantified stage-specific cell differentiation by flow cytometry, and performed a functional colony assay. Moreover, we determined the vascular function, systemic hemodynamics,andNO metabolites.This talk will present the results of these recent and ongoing investigations analyzing the role of the NO/sGC pathway in erythroid cells in mice and their potential significance for human physiology and pathophysiology.

Sepsis is one of the leading causes of death worldwide yet there are limited treatment options for this disease. There has been increasing interest in therapies targeting the microvasculature to improve tissue perfusion and reduce organ damage that often persists despite macrohemodynamic stabilization. CNP is a cardioprotective vasodilator peptide that has been shown to regulate blood pressure, inflammation, and cardiac contractility. Plasma levels of CNP are elevated in patients with sepsis but little is known about the function of this peptide in this syndrome.

LPS inoculation and caecal ligation and puncture (CLP) were performed in mice lacking endothelial-derived CNP (ecCNP ^-/-), cardiomyocyte-derived CNP (cmCNP ^-/-) and natriuretic peptide receptor C (NPR-C ^-/-). A subset of animals were also treated with CNP via mini-pump (0.2mg/kg/day). Cardiovascular hemodynamics, biochemical and inflammatory markers were measured at 24 hr post sepsis induction.

ecCNP ^-/- mice exhibit impaired endothelial function, reduced blood flow and greater expression of inflammatory markers despite exhibiting a higher blood pressure than WT littermates. NPR-C ^-/- mice display similar vascular disturbances to ecCNP ^-/- as well as a greater decline in cardiac function. cmCNP ^-/- mice also present with worse diastolic function following LPS challenge. NPR-C ^-/- mice exhibit more vascular leak in addition to reduced expression of gap and tight junctions. CNP infusion improves blood flow, diastolic function and inflammation.

Our findings indicate endogenous endothelial derived CNP plays a protective role in sepsis by maintaining blood flow, reducing inflammation, preventing oedema and regulating heart function via NPR-C. This peptide may offer a novel therapeutic opportunity in sepsis.

Lower-limb peripheral artery disease (PAD) is one of the major complications of diabetes mellitus. PAD is associated with poor limb and cardiovascular prognoses, along with a dramatic decrease in life expectancy. Despite major medical advances in the treatment of diabetes, a substantial therapeutic gap remains in the PAD population. Praliciguat is an orally available soluble guanylate cyclase (sGC) stimulator that has been reported both pre-clinically and in early-stage clinical trials to have favorable effects in metabolic and hemodynamic outcomes, suggesting that it may have a potential beneficial effect in PAD. We then evaluated the effect of praliciguat on hind limb ischemia (HLI) recovery in leptin receptor-deficient (Lepr ^db/db). 28 days after surgery. Ischemic foot perfusion and function parameters were better in praliciguat-treated mice than in vehicle controls. Improved ischemic foot perfusion was not associated with either improved traditional cardiovascular risk factors (i.e., weight, glycemia) or increased angiogenesis. However, treatment with praliciguat significantly increased arteriole diameter, decreased ICAM1 expression, and prevented the accumulation of oxidative pro-angiogenic and pro-inflammatory muscle fibers. While investigating the mechanism underlying the beneficial effects of praliciguat therapy, we found that praliciguat significantly downregulates Cxcl12 mRNA expression in cultured myoblasts which in turn may decrease ICAM1 mRNA expression in endothelial cells. Conclusion: Our results demonstrated that praliciguat promotes blood flow recovery in the ischemic muscle of mice with type 2 diabetes, at least in part by increasing arteriole diameter and by downregulating ICAM1 expression.

The natriuretic peptides ANP, BNP and CNP activate transmembrane guanylyl cyclases (GC) that producecyclic GMP (cGMP). We have previously employed targeted FRET-based biosensors to demonstrate that thenatriuretic peptides have differential effects in cardiomyocytes and intact heart, where CNP activates GC-B thatincreases cGMP near troponin I and phospholamban (PLB), enhancing relaxation, while activation of GC-A withANP/BNP modestly increases cGMP only near PLB and do not enhance relaxation, suggesting spatiallyrestricted cGMP signaling from GC-A and GC-B. Using a biosensor targeted to the outer mitochondrialmembrane (OMM), we have found that GC-A and GC-B increase cGMP at the OMM and reduce cardiomyocyteapoptosis. To understand this spatial cGMP signaling from GC-A and GC-B, we combined targeted variants ofthe cGMP scavenger SponGee with our targeted FRET-based cGMP biosensors in cardiac H9c2 cells.Activation of GC-B increased cytosolic cGMP, which was modestly reduced by the lipid raft (Lyn-SponGee),non-raft (SponGee-Kras) or OMM-targeted (OMM-SponGee) cGMP scavengers compared to the untargetedSponGee. At the OMM, cGMP increase from GC-B was reduced in content and kinetics only by the OMMSponGee,while cGMP increase from GC-A was reduced by SponGee-Kras and the untargeted SponGee.Our results indicate that GC-A and GC-B are differentially organized on the plasma membrane and that cGMPreaching the OMM could have different origin. Using cGMP scavengers can therefore be used to deciphersignaling from different areas of the plasma membrane to various subcellular locations in cardiac cells.

Breast cancer has surpassed lung cancer as the leading cause of cancer incidence, with an estimated 2.3 million new cases in 2020, representing approximately 11.7% of all cancer cases worldwide. It remains the most common cause of cancer-associated death among women. Independent of subtype, ~90% of all breast cancer related deaths can be attributed to metastasis, a generally incurable state of disease for which there are few effective therapies. This contextually puts our discovery of a new calcium-dependent signaling pathway, which is elaborated in both cancer cells and within tumor associated macrophages (TAMs), that regulates processes of pathological importance in tumors/metastatic lesions in animal models of advanced breast cancer. Notable was the finding that genetic or pharmacological inhibition of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) activity reduces cancer cell migration/invasion in vitroand metastasis from tumors in vivo. This was determined to be a result of decreased expression of phosphodiesterase-1A (PDE1A), decreased cGMP hydrolysis, and increased activation of protein kinase G (PKG). This ultimately leads to increased phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), interfering with its‘ ability to participate in the actin remodeling activities needed for metastatic progression. Genetic or pharmacological manipulation of PDE1 activity recapitulates these findings. Likely modulating a similar signaling pathway, both CaMKK2 and PDE1 inhibitors also facilitate a favorable polarization of TAMs which in turn increases the number of functionally active cytotoxic T-cells in tumors. Thus, CaMKK2/PDE1 inhibitors represent a novel new class of cancer therapeutics that function by both altering fundamental aspects of cancer cell biology and increasing tumor immunity.

The NO/cGMP signaling cascade is a central regulator of vascular homeostasis. The tumor microvasculature is often abnormal, decreasing immune cell infiltration and the efficacy of anti-cancer therapies. We investigated whether genetic or pharmacological modulation of the NO-sensitive guanylyl cyclase (NO‑GC) affects tumor growth in mouse models of melanoma and breast cancer. Using a combination of immunofluorescence staining and real-time cGMP imaging in tumors of transgenic cGMP sensor mice, we found that the NO/cGMP signaling axis is exclusively present in pericytes of the tumor microvessels. Genetic ablation of NO‑GC in pericytes decreased tumor growth, and administration of a clinically used NO‑GC stimulator increased it. In an in vitro model of human microvessels, pharmacologic NO‑GC inhibition decreased vessel branching and pericyte coverage, while NO‑GC stimulation increased it. Collectively, these results suggest that elevated activity of the NO/cGMP pathway in pericytes may promote tumor growth through unfavorable effects on the tumor microvasculature. Our findings call for studies exploring the possibility of altered tumor growth in cancer patients receiving NO-GC stimulators for cardiovascular indications like heart failure.

Introduction
Glioblastoma is the most aggressive and common malignant primary brain tumor of adolescence. Neuroblastoma is the most common malignant extracranial tumor of infancy. We have already demonstrated the anti-proliferative effect of second messengers cGMP, cAMP, cCMP, cUMP in various cancer cell lines. To expand our research we analyzed that effect in human glioblastoma and neuroblastoma cell lines.

Methods
We used the alamarBlue assay to investigate proliferation in HEK293, U373 (glioblastoma), and SK-N- BE(2) (neuroblastoma) cells. To imitate the functions of cNMPs, acetoxymethyl ester analogues (AMs) were used. Multidrug resistant proteins (MRPs) were inhibited by probenecid and phosphodiesterases (PDEs) by IBMX to alter effects. With qRT-PCR analysis the expression of MRPs, PDEs, soluble adenylyl cyclase (sAC), and soluble guanylyl cyclases (sGC) was analyzed.

Results
All cNMP-AMs showed a time- and concentration-dependent anti-proliferative effect in all three cell lines. Highest reduction of proliferation in HEK293 cells was after treatment with cAMP-AM, cGMP-AM, and cCMP-AM, in U373 cells with cAMP-AM, and cCMP-AM, in SK-N-BE(2) cells with cCMP-AM. Treatment with cNMP-AMs after preincubating with probenecid and IBMX showed an increased effect in HEK293 and SK-N-BE(2) cells after 48 h and in U373 cells after 72 h. MRP4, MRP5 and PDE isoforms are expressed in all three cell lines, the expression of some sGC- isoforms is higher than sAC.

Conclusions
Efflux and degradation of cNMPs is important. When inhibited with probenecid and IBMX, cNMPs are accumulating and anti-proliferative effect is increased. IBMX alone showed no further decrease of proliferation, therefore MRPs may be more important in the cNMP-metabolism.

Mammalian sGC is a heterodimer composed of α- and β-subunits. The C-terminus of each subunit contains a catalytic domain and the active site is composed of residues from both subunits. The catalytic domains also form a pseudosymmetric site that contains residues known to be involved in nucleotide binding, but lack the complete complement of amino acids required for catalysis. Sequence analysis shows that each subunit also contains well-defined PAS-like domain, and a predicted coiled-coil region. The N-termini of the α- and β-subunits are homologous to the H-NOX ( Heme- Nitric oxide/ OXygen) family of proteins. The N-terminus of β-subunit contains a ferrous heme cofactor that serves a receptor for NO. Additional studies point toward a more complicated role for NO and stimulators in the regulation of activity. Structural and biochemical results have broadened the current molecular view of the regulation of sGC and provide a framework to understand the action of sGC modulators of activity. Activity and structural studies involved with regulation will be discussed. NO also functions as a signaling molecule in Choanoflagellates. Choanoflagellates are a group of free-living unicellular and eukaryotes considered to be the closest living relatives of the animals. The evolutionary history of NO signaling is proving to be informative on function.

Despite widespread recognition of nitric oxide (NO) synthase (NOS) signaling's biological significance, uncertainties persist regarding the chemical nature of NOS-derived bioactivity. Our research reveals that NO-like bioactivity can efficiently be conveyed by mobile NO-ferroheme species, capable of transferring between proteins, partitioning into a hydrophobic phase, and directly activating the sGC– cGMP–PKG pathway without necessitating free NO intermediacy. These NO-ferroheme species, with or without a protein carrier, effectively relax isolated blood vessels and induce hypotension in rodents, particularly potentiated after NOS activity blockade. Notably, NO-ferroheme-induced relaxations remain unaffected by NO scavengers and blood components, suggesting physiological relevance. Thus, NO-ferroheme emerges as a pivotal signaling entity in vascular physiology.

Nitric oxide (NO) regulates vascular function through the activation of soluble guanylate cyclase in vascular smooth muscle and platelets. NO signaling is mediated by paracrine diffusion from endothelial cells to smooth muscle. Many questions relate to how intracellular, paracrine, and endocrine diffusion is possible in hemoglobin/myoglobin-rich environments. This diffusion is constrained by fast reactions of NO with both oxygenated and deoxygenated ferric hemoproteins and by radical-radical reactions with superoxide and hydrogen peroxide. Extensive data now supports the hypothesis that red blood cells support NO-dependent signaling through nitrite reductase reaction pathways of nitrite with deoxygenated hemoglobin and the red blood cell expressed "endothelial" NOS. Diffusion of NO outside of a red blood cell should be limited by scavenging reactions of NO with hemoglobin and multiple scavenging reactions in endothelium and smooth muscle with reactive oxygen species, however, this signaling is observed in preclinical and clinical studies. We propose a potential solution to this paradox based on a recent discovery of a rapid reaction pathway between NO, oxidized heme, and reduced thiols. The reaction of NO with ferric heme in the presence of reduced thiols rapidly generates an NO-ferroheme complex and a thiol radical, with electron transfer from the thiol to the NO-ferric heme complex. This reaction is first-order in NO and produces an NO-ferroheme complex. The NO-ferroheme is stable in red cell membrane and bound to albumin, and promotes potent vasodilation in vivo. Furthermore, this species is not scavenged by hemoglobin or myoglobin. We propose a novel signaling paradigm whereby NO-ferroheme can form via nitrite reduction or red cell eNOS and can transfer a stable NO-ferroheme between membrane, albumin, and smooth muscle, where the NO-ferroheme can activate soluble guanylate cyclase. This new signaling molecule offers a potential explanation for NO signaling in a heme- and reactive oxygen species-rich cellular environment.

Ischemic heart disease is the leading cause of death worldwide. In addition to traditional risk factors as hypertension or hypercholesterolemia, a positive family history increases the risk to suffer from coronary artery disease and myocardial infarction. Among the common variants which are responsible for this observation and which were identified in the past 20 years, several variants are located at gene loci that harbor genes involved in nitric oxide-cGMP signaling. Here, we discuss novel genetic and mechanistic insights regarding the role of cGMP in atherosclerosis. We investigate the role of platelet cGMP in atherosclerosis and possible therapeutic implications. Furthermore, we provide evidence for a role of phosphodiesterase 5A in atherosclerosis based on private mutations and common genetic variation. Finally, we will discuss phenotypic consequences of altered nitric oxide-cGMP signaling in coronary artery disease using phenome-wide analyses.

Cytoglobin is a highly conserved redox sensor heme protein with unknown physiological function. Unexpectedly, genetic deletion of zebrafish cytoglobin ( cygb2) causes cardiac laterality defects showing an abnormal opposite left-right orientation of the heart. In humans, cardiac laterality determination is dependent on motile cilia function. Importantly, primary ciliary dyskinesia (PCD) is associated with low nitric oxide (NO) levels in airway epithelial cells. Using the zebrafish model we found that Cygb2 co-localized with cilia and the NO synthase Nos2b in the embryonic laterality organ and the structure and function of cilia were disrupted in cygb2 mutants. In addition, we detected lower levels of cGMP in mutants. Abnormal ciliary function and organ laterality defects were phenocopied by depletion of nos2b and gucy1a, the canonical NO receptor soluble guanylate cyclase (sGC) homolog in fish, and rescued by exposing cygb2 mutant embryos to an NO donor or an sGC stimulator and with over-expression of nos2b. Consistent with a conserved role in the modulation of ciliary function, cytoglobin knock out in the mouse also impaired airway epithelial ciliary structure and beat frequency. This is the first evidence that cytoglobin is essential for normal developmental stage-specific NO signaling, ciliogenesis and cilia motility, required for the establishment of left-right patterning.

The identification and characterization of a compound that selectively activates a particular isoform provides unique insight into the molecular differences and functional divergence between the isoforms. This not only allows for a more precise understanding of cellular signalling pathways but also potentially opens up new avenues for targeted therapy of diseases where dysregulated activity of a specific isoform is implicated. In humans, there are two distinct active isoforms of the soluble guanylyl cyclase (sGC): α1/β1 (GC-1) and α2/β1 (GC-2). In the present study we show that runcaciguat is the first isoform-specific sGC activator with a strong preference for GC-1. The activator exhibits antagonistic behaviour towards GC-2, leading to reduced efficacy of the nitric oxide donor DEA/NO. Similar results were generated with the related activator BAY-543. In addition, comparing the sGC activators BAY 60-2770 and BI 703704, a much stronger activation of GC-2 is measurable with BI 703704, whereas the potency of both activators in GC-1 is approximately the same. The classification of the investigated activators into mono- and dicarboxylic acids does not seem to be significant for the observed isoform specificity. It is tempting to hypothesize that the activation of GC-2 may rather increase with the lengthening of the lipophilic tail of the activators. Our laboratory is currently investigating this hypothesis.

cGMP-dependent protein kinase I-α (PKG1α) is a target for pulmonary arterial hypertension due to its role in the regulation of smooth muscle function. While most work has focused on regulation of cGMP turnover, we recently described several small molecule tool compounds which were capable of activating PKG1α via a cGMP independent pathway. Selected molecules were crystallized in the presence of PKG1α and were found to bind to an allosteric site proximal to the low-affinity nucleotide binding domain. These molecules act to displace the switch helix and cause activation of PKG1α representing a new mechanism for the activation and control of this critical therapeutic path. The described structures are vital to understanding the function and control of this key regulatory pathway.

Introduction: cGMP-dependent protein kinase I (PKG I) can be activated by its non-canonical activator cAMP. However, cAMP most likely pushes PKG I in a distinct different, yet potentially physiologically relevant conformation.

Results: Binding affinities of cAMP and cGMP to PKG I were determined using surface Plasmon resonance (SPR) and different conformations of PKG Iα were measured by size exclusion chromatography in the presence and absence of cyclic nucleotides, divalent metal ions and nucleotides. The novel switchSENSE technology (Dynamic Biosensors, Munich) can be utilized to measure reversible conformational changes of PKG I directly. The technology has two modes of operation: In fluorescence proximity sensing (static) mode, changes in the proximity of a fluorophore are detected, subsequently rate constants and affinities can be calculated. In dynamic response mode, the lag-values change depending on the hydrodynamic diameter of the protein attached to the DNA nanolevers. These conformational changes can be induced by the binding of cyclic nucleotides or other effectors to the protein kinase. Both, static and dynamic mode demonstrate dose dependent conformational changes in PKG I in response to cGMP and cAMP. These differences have an impact on kinase activation (activation constant for PKG Iβ with VASPtide as substrate 200 nM for cGMP and in the μM range for cAMP). Interestingly, the maximal activity of the cAMP activated PKG I is significantly lower compared to cGMP-activated protein. Our data provide mechanistic insight into the crosstalk between cGMP- and cAMP- dependent protein kinase signaling pathways.

Red blood cells (RBCs) regulate cardiovascular function via a mechanism involving nitric oxide-like bioactivity, but the signaling and the identity of any mediator released by RBCs have remained unknown. We have investigated whether RBCs exposed to hypoxia mediates cardioprotection during ischemia-reperfusion and explored the signaling involved. Administration of hypoxic RBCs or the extracellular supernatant from mouse RBCs exposed to hypoxia to isolated hearts subjected to ischemia-reperfusion improved post-ischemic cardiac function and reduced infarct size. This cardioprotective effect was abolished by blocking sGC in the RBCs or when exposing RBCs from sGC knockout mice to hypoxia, suggesting that RBC sGC is required for the protective effect. Exposure of RBCs to hypoxia resulted in increased extracellular levels of cGMP, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on cGMP transport, sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice and humans to increase nitric oxide bioactivity further enhanced the cardioprotective effect of RBCs. Pharmacological stimulation of RBC sGC mimicked the cardioprotective effect of hypoxia and reversed the negative effect of RBCs from patients with type 2 diabetes on post-ischemic cardiac function via a mechanism involving release of cGMP and activation of cardiac protein kinase G. It is concluded that RBCs generate and export cGMP as a response to hypoxia mediating cardioprotection via a paracrine effect. This effect can be further augmented by pharmacological stimulation of RBC sGC suggesting a therapeutic target in ischemic heart disease.

cGMP is critically involved in the regulation of multiple intracellular events in cardiomyocytes which are crucial for both normal function and disease such as heart failure. Phosphodiesterases (PDEs), as the enzymes which degrade cyclic nucleotides can be well targeted therapeutically and have been in focus of investigation because they can actively shape subcellular cGMP nanodomains linked to functional responses. While lots of studies focused on PDE5 and PDE9 as typical cGMP degrading PDEs, the role of dual-specific PDE1 and PDE3 in terms of cGMP regulation has been mostly ignored. In our recent studies, we have looked at cGMP hydrolysis by PDE1 and PDE3 and how its regulates cardiomyocyte contractility and hypertrophy via subcellular nanodomains using live cell imaging. We found that PDE1 inhibition amplifies cGMP pools generated by the NO-dependent but not the particulate guanylyl cyclase. Furthermore, it improves cardiomyocyte relaxation when combined with NO-donors or natriuretic peptides. PDE3, which is often referred to as cGMP inhibited PDE, contributes substantially to cGMP hydrolysis in these cells und undergoes posttranslational regulation upon ischemic injury which could impact the development of cardiac hypertrophy and the effects of cGMP elevating drugs.

Although NO-induced cGMP has a long-known function in the vascular system, its function in the heart is less well established. In cardiac slices of healthy mice, NO-induced cGMP enters cardiac myocytes through gap junctions -as shown using a FRET-based cGMP indicator specifically expressed in cardiac myocytes- and amplifies β-receptor-induced cAMP by inhibiting PDE3.To identify the source of NO-induced cGMP, mice devoid of NO-sensitive guanylyl cyclase 1 (NO-GC1) specifically in Tcf21-expressing fibroblasts were generated. In acute slices of these mice, NO-GC stimulators did no longer enhance isoprenaline-stimulated cAMP in cardiac myocytes and did not enhance isoprenaline-induced phospholamban phosphorylation.To investigate whether the function of NO-induced cGMP is altered under pathophysiological condi-tions, Angiotensin II-treated mice with cardiac hypertrophy were analysed. In these mice, expression of PDE2 (cGMP-stimulated phosphodiesterase) was increased and PDE2 gained a stronger impact on NO-induced cGMP as analysed by the FRET-based cGMP indicator cGi-500. Accordingly, NO-induced cGMP was shown to decrease cAMP and phospholamban phosphorylation. Interestingly, basal cAMP levels as well as phospholamban phosphorylation were increased compared to untreated mice.In sum, Tcf21-expressing cardiac fibroblasts were identified as source of NO-induced cGMP in cardiac myocytes. Cardiac hypertrophy reversed the effect of NO-induced cGMP on cAMP in myocytes. Instead of increasing cAMP and phospholamban phosphorylation via PDE3 as observed under control conditi¬ons, NO-induced cGMP decreased cAMP and phospholamban phosphorylation via PDE2 in Angio¬tensin II-induced hypertrophy.

In acute myocardial infarction (AMI), cytokines such as TNFα impair coronary endothelial barrier functions. This allows the transmigration of neutrophils, which attract monocytes and macrophages for the clearance of dead cells. Inflammation after ischemia is necessary, but too much inflammation is deleterious. In general, increased endothelial cAMP and/or cGMP levels are enhanced, whereas decreased cAMP/cGMP levels weaken the barrier. Such decreases can be evoked by phosphodiesterases (PDEs). Our project aims to characterize the role of the cyclic GMP-stimulated dual esterase PDE2 in the regulation of endothelial barrier functions and myocardial immune cell infiltration after ischemia.

In cultured human coronary microvascular endothelial cells (ECs), TNFα increased PDE2 expression together with augmented expression of proinflammatory adhesion proteins such as VCAM-1. Pharmacological inhibition of PDE2 enhanced endothelial cGMP/cAMP levels and attenuated thrombin-induced barrier dysfunction as well as TNFα-induced VCAM-1 expression. To dissect the pathophysiological relevance, we generated a novel genetic mouse model with conditional, endothelial-restricted PDE2 deletion. Immunoblotting and PDE2 activity assays demonstrated efficiency and selectivity. Such EC PDE2 KO mice exhibited no noticeable phenotypic changes. Under resting conditions, their arterial blood pressure and coronary EC barrier were unaltered. Experimental AMI was associated with significant increases in cardiac PDE2 expression. In control mice, this was concomitant to marked myocardial infiltration by neutrophils, monocytes/macrophages, and dendritic cells. Notably, in EC PDE2 KO mice, myocardial inflammation after ischemia was significantly reduced.

Understanding the role of PDE2 in the regulation of endothelial barrier functions may unravel targets for novel therapies of AMI.

The complex nature of polygenic diseases encompasses a broad range of underlying pathologies arising from heterogeneous molecular mechanisms and exhibiting a diversity of disease phenotypes. This complexity can be formalized using molecular interaction networks (e.g., the protein-protein interaction network) and analyzed using the basic tenets of graph theory. Among the features of these network that can be discerned are included subnetworks that are specific to a particular disease (i.e., a disease module), unique mechanisms or pathways that govern the disease phenotype, and the identification of potential drug targets that exist within or near the disease module. In this presentation, I will discuss the computational approaches used to identify disease modules and drug targets, including approaches to drug repositioning or repurposing; experimental approaches to characterizing the dynamic interactions of drugs and their targets in real time in single cells; and efficient single-cell strategies for assessing combinations of drugs. These principles will be illustrated with examples from specific diseases, and their implications for cell signaling pathways, including cGMP signaling, considered. The strategies presented will ultimately contribute to and guide the ongoing evolution of precision medicine.

Natriuretic peptides and their receptors have been implicated in a broad range of physiological processes, regulating blood pressure, cardiac hypertrophy and fibrosis, fat metabolism, and long bone growth. They mediate their action through the modulation of intracellular levels of cGMP and cAMP. Here we report a completely novel function for natriuretic peptide signaling in the control of cell fate in the embryonic ectoderm. Using a combination of morpholino-based knockdowns, pharmacological inhibitors and rescue assays in Xenopus embryos we show that natriuretic peptide receptor 3 (Npr3), natriuretic peptide receptor 1 (Npr1) and the natriuretic peptide Nppc cooperate in the regulation of neural crest and cranial placode formation, two cell populations that make a major contribution to the craniofacial skeleton and paired sensory organs in vertebrates. Npr3 plays an especially pivotal role in this process by differentially regulating these two developmental programs. Our findings suggest that Npr3 has a dual function in the embryonic ectoderm, it (i) reduces ligand-mediated activation of Npr1 to generate cGMP levels compatible with neural crestformation, and (ii) modulates cAMP levels via inhibition of adenylyl cyclase to promote cranial placode fate. Therefore the intracellular modulation of these two-second messengers participates in the segregation of these cell populations during embryogenesis.

The natriuretic peptides (NPs) signal through their membrane receptors NPRA (GC-A; for ANP and BNP) and NPRB (GC-B; for CNP), which generate cGMP. A third receptor, NPRC, is a negative regulator of NP signaling: it binds all three NPs and internalizes/degrades them. The relative levels of NPRA or NPRB to NPRC thus dictates the signaling strength of cGMP produced. In recent studies we explored the possibility that another means by which NPRC might dampen signaling through NPRA and NPRB is through the formation of receptor heterodimers. The consequence of such an event would similarly be a decrease in cGMP production. We show evidence that such heterodimers do form and that the blunting of cGMP signaling may also include the internalization of these heterodimers. Other events that seem to contribute to the net signaling strength of NPs is a regulated decrease in ‘inhibitory’ components of the NP signaling system. In these studies, we have focused on adipose tissue, an important ‘organ’ that is a pivotal regulator of whole-body metabolic homeostasis. ANP and BNP stimulate adipocyte lipolysis as well as a process of net energy expenditure in brown and ‘beige’ adipocytes. We show that two important ‘negative’ regulators of NP signaling in adipocytes – NPRC and phosphodiesterase-9 (PDE9) – are transcriptionally down-regulated during catecholamine/β-adrenergic receptor stimulation of lipolysis and brown/beige adipocyte thermogenesis. In concert with elevated ANP production and release from the heart, these events appear to coordinately foster NP signaling strength in the adipocyte to protect from fat mass gain and metabolic disease.

Cardiovascular diseases remain the leading cause of death in industrialized countries. Central to this statistic is the lack of treatments to effectively prevent or reverse the severe pathologic remodeling that characterizes a failing heart. Ageing is a major risk factor for the development of heart hypertrophy and fibrosis. Natriuretic peptides, via their cGMP-synthesizing guanylyl cyclase receptors, GC-A (for ANP and BNP) and GC-B (for CNP), exert protective cardiovascular and metabolic actions. The role of their shared receptor-C (NPR-C), lacking the GC domain, is less clear. Depending on the cell type and biological context, NPR-C seems to either internalize the NPs or mediate their effects. Endothelial cells (ECs) express all three receptors for NPs. To dissect the impact of endothelial NPR-C on cardiovascular ageing, we generated mice with EC-restricted deletion (KO).

In control mice, ageing was associated with increased arterial blood pressure and progressive cardiac fibrosis and hypertrophy. EC NPR-C KO mice showed reduced clearance of ANP from the circulation and stabilized ANP and CNP plasma levels. Despite this, throughout various ages their blood pressure was not different from control littermates. Furthermore, cardiac interstitial collagen contents were unchanged. However, aging-related cardiac enlargement and cardiomyocyte hypertrophy were markedly blunted and left ventricular systolic and diastolic functions were preserved. This was linked to augmented cardiac cGMP levels and enhanced PKGI-dependent stimulation of antihypertrophic and lusitropic pathways in cardiomyocytes.

Chronic inhibition of the NPR-C-mediated endothelial clearance of NPs improves their protective cGMP-mediated cardiac effects, attenuating ageing-related heart hypertrophy and dysfunction despite elevated blood pressure.

Supported by the Deutsche Forschungsgemeinschaft (DFG KU 1037/14-1 and SFB 1525)

The heart extends beyond its fundamental role in circulating blood, doubling as an endocrine organ through the secretion of cardiac natriuretic peptides. These peptides orchestrate blood pressure regulation via the natriuretic peptide receptor-1 (NPR1), known alternatively as NPR-A, ANPR, or GC-A. They achieve this by binding to the receptor's extracellular domain, subsequently activating its intracellular guanylyl cyclase domain to generate the second messenger, cyclic guanosine monophosphate (cGMP). However, the intricate architecture and the domain interactions of full-length GC-A have remained obscure. This investigation employs cryo-electron microscopy, functional analyses, and molecular dynamics simulations to explore the structure of full-length human GC-A in both the absence and presence of atrial natriuretic peptide (ANP). Our results reveal the sophisticated architecture and regulatory mechanisms of this single-pass transmembrane receptor guanylyl cyclase, shedding light on its functional dynamics and unveiling potential therapeutic avenues.

Natriuretic peptides are important regulators in the cardiovascular and renal system with pleiotropic effects. Atrial (ANP) and brain natriuretic peptide (BNP) activate the natriuretic peptide receptor A (NPR-A), causing production of cyclic guanosine monophosphate (cGMP). Various designer natriuretic peptides have been developed and investigated for treatment of heart failure and hypertension, but their major limitations are that they have short half-life and are not available for oral administration. Our novel approach is to find small molecular drugs to help and treat patients with cardiovascular diseases (CVDs), through targeting NPR-A. We have identified small molecular allosteric enhancers of NPR-A, which increase the efficacy and potency of BNP and ANP in their ability to activate NPR‐A and generate cGMP. These compounds were characterized as NPR-A-selective allosteric enhancers, and their activity is dependent on one unique amino acid in NPR-A. Finally, a pre-study in an animal model was performed to test the formulation and assure the lead compound is well tolerated by the animals.

The C-natriuretic peptide (CNP) analog vosoritide recently became the first drug approved for the treatment of achondroplasia in children, clearly a significant milestone in the history of this rare disease. However, the dosing regimen is very burdensome for the young patients, requiring daily subcutaneous injections for potentially more than a decade. At ProLynx, using our half-life extension technology we have developed a long-acting CNP analog conjugate that demonstrates equal or greater efficacy to vosoritide while at the same time requiring less frequent, more convenient dosing. Our half-life extension technology utilizes β-eliminative linkers to attach a drug to pre-fabricated tetra-polyethylene glycol hydrogel microspheres (tetra-PEG microspheres). When injected subcutaneously, these microspheres act as a stationary depot where the chemistry of the β-eliminative linkers allows for controlled release of the attached drug, which translates directly to control of the plasma half-life. Two microsphere conjugates were prepared with release rates designed to allow once-weekly and once-monthly administration using a highly active, deamidation resistant CNP analog, [Gln ^6,14]CNP-38. In vitro studies in mice demonstrated that [Gln ^6,14]CNP-38 was slowly released from the conjugates into the systemic circulation with biphasic elimination pharmacokinetics and terminal half-lives of ∼200 h and ∼600 h. In addition, studies in mice using weekly to monthly dosing regimens demonstrated growth comparable to or exceeding daily injections of vosoritide. Further studies in cynomolgus monkeys showed a similarly extended terminal half-live of ~600 h. Simulations of the human pharmacokinetics indicate that plasma levels of [Gln ^6,14]CNP-38 could be maintained within the required therapeutic window for monthly dosing intervals, significantly reducing the patient burden from ~30 injections per month to one.

Patients with myocardial infarction (MI) and heart failure (HF) often develop life-threatening arrhythmia. In the diseased heart, arrhythmia are forced by the imbalance between cyclic nucleotide pathways displaying a detrimental overactive cAMP- and a suppressed beneficial cGMP-signaling. Phosphodiesterase 2 (PDE2) is a keynode enzyme that connects both pathways via its unique property of cGMP-mediated activation to increase cAMP hydrolysis in cardiomyocytes (CM). PDE2 expression is upregulated in HF. Thus, the cGMP-induced PDE2 activation by either natriuretic peptides (NP) or vericiguat (VE) stimulating particulate or soluble guanylate cyclases might represent a new antiarrhythmic therapy.

First, we demonstrated that the pharmacological inhibition or the CM-specific genetic deletion of PDE2 increased arrhythmia development after ischemia reperfusion injury in ex-vivo perfused mouse hearts. Importantly, the cGMP-mediated PDE2 stimulation with CNP or its approved drug analogue vosoritide (VO) reduced arrhythmia in WT hearts, while the PDE2 inhibitor BAY60-7550 prevent the observed antiarrhythmic effect.

Second, we studied the underlying mechanisms on cellular level. In isolated CM, the β-adrenergic stimulation with isoprenaline (ISO) enhanced pro-arrhythmic triggers, like the I _CaL, Ca ²⁺-sparks (CaSp) and -waves (SCW). Interestingly, the simultaneous incubation with CNP or VO significantly reduced the number of CaSp, SCW and I _CaL amplitude. Of note, VE also decreased ISO-induced arrhythmogenic triggers in CM. Selective PDE2 inhibition or knockout clearly prevented the beneficial effects of cGMP-generating stimulators. Finally, we verified the antiarrhythmic effects of cGMP-induced PDE2 activation in CM and ex-vivo perfused hearts from diseased mice with HF, showing evidence for a novel therapeutic strategy.

Cyclic nucleotide-gated (CNG) channels transduce chemical signals into electrical signals in sensory receptors and neurons and are essential for vision and smell. Although they are members of the voltage-gated ion channel superfamily, CNG channels are insensitive to membrane potential and are instead activated by intracellular cGMP or cAMP. To better understand how CNG channels work as molecular machines, we have determined high-resolution structures of cGMP-activated eukaryotic (nematode and human) CNG channels in various conditions and states. I will briefly display these structures and illustrate how they provide unprecedented mechanistic insights into CNG channel properties and mechanisms. I will in particular showcase and dissect the structures of the cGMP-activated human cone photoreceptor CNG channel, which is composed of CNGA3 and CNGB3 subunits. We have captured this channel in closed, transition, pre-open and open states in detergent or lipid nanodisc, all with fully bound cGMP. The pre-open and open states are obtained only in the lipid nanodisc, suggesting a critical role of lipids in tuning the energetic landscape of CNGA3/CNGB3 activation. The different states exhibit subunit-unique, incremental and distinct conformational rearrangements that originate in the cyclic nucleotide-binding domain (which is 50-60 Å away from the activation gate located in the central cavity), propagate through the coupling C-linker/gating ring to the transmembrane domain, and gradually open the cavity gate in S6. Our study illustrates a spatial conformational-change wave of allosteric gating of a vertebrate CNG channel by its natural ligand and provides an expanded framework for studying CNG functions and channelopathy.

Optogenetic manipulation of cells or organisms became successful in neuroscience, especially with the introduction of the light-gated ion channel Channelrhodopsin-2 as an easily applicable tool. The optogenetic toolbox was enriched with the application of earlier discovered and engineered photoreceptors, or newly discovered photoreceptors from nature. The application field was expanded from neuroscience to other fields like cardiovascular research etc. Recently, an optogenetic approach was also clinically applied for partial recovery of visual function in a blind patient.

Cyclic nucleotides, specifically cAMP and cGMP, play pivotal roles as ubiquitous second messengers, governing a myriad of biological processes. The dysregulation of cyclic nucleotide signaling is implicated in various diseases, rendering it a crucial focus in pharmaceutical research. The application of optogenetics to manipulate cAMP was initially demonstrated by expressing the photoactivated adenylyl cyclase from the unicellular alga Euglena gracilis (EuPAC) in Drosophila melanogaster. Subsequently, the discovery of a much smaller PAC from the soil bacterium Beggiatoa (bPAC) has greatly advanced the field, providing an efficient tool for intracellular cAMP manipulation in genetically targeted cells. The bPAC was mutated into a photoactivated guanylyl cyclase (GC) for optogenetic manipulation of cGMP but showed low efficacy.

Later, a natural light-gated guanylyl cyclase, Cyclop, was characterized from the fungus Blastocladiella emersonii, and determined to be the first enzyme rhodopsin with 8 transmembrane helices. In addition, new enzyme rhodopsin with light-gated PDE activity was discovered from a protist Salpingoeca rosetta. I present recent progress with light-gated GCs and PDEs for optogenetic manipulating cGMP.

Acute and chronic pain, although common, are often poorly treated by existing therapies. Accordingly, there is a substantial unmet medical need for safe and efficacious treatments. Recent studies revealed that cyclic nucleotide phosphodiesterases (PDE) contribute to the processing of pain, and that PDE might be targets for pain therapy. Here we found that the PDE10A isoform is expressed in pain processing neurons in dorsal root ganglia (DRG) and the spinal cord in mice. Incubation of DRG neuron cultures with a selective PDE10A inhibitor increased the cGMP levels in enzyme immunoassays. Real-time FRET-based cGMP imaging confirmed increased cGMP signals in DRG neurons after PDE10A inhibition. Notably, PDE10A inhibitor treatment inhibited the pain behavior in mice in models of acute nociceptive pain and persistent inflammatory pain without the development of antinociceptive tolerance. Together, our data support the idea that PDE10A is a suitable target for development of efficacious analgesic drugs.

Malaria, caused by protozoan parasites of the Plasmodium genus, killed over 600,000 people and infected another 250 million in 2023. The toll from malaria is likely to increase due to the spread of drugresistancein the parasite population and insecticide-resistance in the mosquito vectors. Progress towards controlling malaria requires new drugs will novel mechanisms of action. One promising drug target is the P. falciparum cGMP-dependent protein kinase (PfPKG), the major effector of cGMP signaling in the parasite. It is essential for viability at several steps of the parasite’s complex life cycle. Its genetic or pharmacological inhibition blocks invasion and exit of parasites from host hepatocytes and erythrocytes, making it an attractive target for achieving pre-exposure prophylaxis against malaria. Towards this end, we defined the structure-activity relationship of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. Cellular acitivity of compounds was determined against two parasite stages. Their specificity was evaluated using transgenic parasites expressing PfPKG carrying a substituted "gatekeeper" residue that occludes the compound binding site. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.

Haem, a fundamental prosthetic group, mediates oxygen transport and metabolism in proteins while exerting signalling functions in haemolytic disorders through its pro-oxidant, pro-inflammatory, and cytotoxic effects when unbound. In addition to its established functions, haem plays a crucial role in regulating haem-responsive proteins such as apo-soluble guanylate cyclase (apo-sGC) and the REV-ERB nuclear receptors, both of which are targets for clinical drug development. Through a comprehensive series of in vitro assays involving apo-sGC, REV-ERB, and four other haem-sensing proteins—Slo1/BK potassium channel, cystathionin-β-synthase, Gis1 transcription factor, and hNaV1.5 sodium channel—we discovered unexpected interactions between apo-sGC activators, traditionally developed for cardiovascular and pulmonary conditions, and REV-ERB, along with most of the tested haem-responsive proteins. Heme concentration dynamics were unaffected. Conversely, REV-ERB ligands, designed initially to address sleep disorders and metabolic syndromes, influenced apo-sGC activity. This investigation significantly broadens our understanding of the pharmacological implications of haem and haem mimetics, highlighting the importance of integrating haem-sensing protein assays into the pharmacological profiling of a class of haem mimetic pharmacophores such as apo-sGC activators and REV-ERB modulators to mitigate potential off-target effects and adverse reactions.