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In light of the range in presently available observational, reanalysis and model data, we revisit the surface climate response to large tropical volcanic eruptions from the end of the 19th century until present. We focus on the dynamically driven response of the North Atlantic Oscillation (NAO) and the radiative-driven tropical temperature response. Using 10 different reanalysis products and the Hadley Centre Sea Level Pressure observational dataset (HadSLP2) we confirm a positive tendency in the phase of the NAO during boreal winters following large volcanic eruptions, although we conclude that it is not as clear cut as the current literature suggests. While different reanalyses agree well on the sign of the surface volcanic NAO response for individual volcanoes, the spread in the response is often large (∼ 1/2 standard deviation). This inter-reanalysis spread is actually larger for the more recent volcanic eruptions, and in one case does not encompass observations (El Chichón). These are all in the satellite era and therefore assimilate more atmospheric data that may lead to a more complex interaction for the surface response. The phase of the NAO leads to a dynamically driven warm anomaly over northern Europe in winter, which is present in all datasets considered. The general cooling of the surface temperature due to reduced incoming shortwave radiation is therefore disturbed by dynamical impacts. In the tropics, where less dynamically driven influences are present, we confirm a predominant cooling after most but not all eruptions. All datasets agree well on the strength of the tropical response, with the observed and reanalysis response being statistically significant but the modelled response not being significant due to the high variability across models.

Im Gebiet des Helgoland-Riffs (pleistozänes Reliktmaterial) wurden Längs- und Querprofile mit Sedimentechograph und Side-Scan gefahren. Die Profile wurden abgegriffen, abgebohrt und abgedredgt. Einer meist nur geringmächtigen rezenten Deckschicht sind mit einer häufig scharf ausgeprägten Erosionsdiskordanz subrezente und fossile Sedimente untergelagert. Es wurden marine Fazies, Watten- und Brackwasserfazies sowie Torfe, Schmelzwassersande und Pleistozän (Geschiebematerial) angetroffen. Das Untersuchungsgebiet unterliegt z. Z. noch der Erosion. Bei Sturm wird stark umgelagert, grober Sand ist in vielen Fällen wohl als „residual concentration" anzusehen. Aber auch Schlicklagen von mehr als 10 cm Mächtigkeit werden rezent in die Oberflächenschichten eingeschaltet.

Background: Dysregulated pathways in cancer may be hub addicted. Identifying these dysregulated networks for targeting might lead to novel therapeutic options., Objective: Considering the hypothesis that central hubs are associated with increased lethality, identifying key hub targets within central networks could lead to the development of novel drugs with improved efficacy in advanced metastatic solid tumors., Design: Exploring transcriptomic data (22,000 gene products) from the WINTHER trial ( N  = 101 patients with various metastatic cancers), in which both tumor and normal organ-matched tissue were available., Methods: A retrospective in silico analysis of all genes in the transcriptome was conducted to identify genes different in expression between tumor and normal tissues (paired t -test) and to determine their association with survival outcomes using survival analysis (Cox proportional hazard regression algorithm). Based on the biological relevance of the identified genes, hub targets of interest within central networks were then pinpointed. Patients were grouped based on the expression level of these genes ( K -mean clustering), and the association of these groups with survival was examined (Cox proportional hazard regression algorithm, Forest plot, and Kaplan-Meier plot)., Results: We identified four key central hub genes- PLOD3, ARHGAP11A, RNF216 , and CDCA8 , for which high expression in tumor tissue compared to analogous normal tissue had the most significant correlation with worse outcomes. The correlation was independent of tumor or treatment type. The combination of the four genes showed the highest significance and correlation with the poorer outcome: overall survival (hazard ratio (95% confidence interval (CI)) = 10.5 (3.43-31.9) p  = 9.12E-07 log-rank test in a Cox proportional hazard regression model). Findings were validated in independent cohorts., Conclusion: The expression of PLOD3, ARHGAP11A, RNF216 , and CDCA8 constitute, when combined, a prognostic tool, agnostic of tumor type and previous treatments. These genes represent potential targets for intercepting central hub networks in various cancers, offering avenues for novel therapeutic interventions., Competing Interests: V.L., C.B., and F.W. are full-time employees of the Worldwide Innovative Network (WIN) Association—WIN Consortium. WIN Association—WIN Consortium is the owner of the patent family entitled Digital Display. The inventors are V.L. and S.M. E.Ra. receives consulting and is a shareholder of SCOR Health Science, GenoScience Pharma, and Stromacare, is a shareholder of Axoltis, is a Scientific Director of Institut des Vaisseaux et du Sang (IVS), is a member of the Board of Directors of Institut National du Cancer (INCa France). S.M. receives consultancy from WIN Association—WIN Consortium. A.O. receives consulting fees from Roche Israel, MSD Israel, Boehringer Ingelheim, and AstraZeneca. M.D. reports consulting or advisory role: Amgen, AstraZeneca, Bayer, Daiichi Sankyo, GlaxoSmithKline, Ipsen, Merck Serono, MSD, Pierre Fabre, Roche, Servier, Sotio, and Zymeworks. Speakers’ bureau: Amgen, AstraZeneca, Bayer, Merck KgaA, MSD, Pierre Fabre, Pfizer, Roche, Servier, and Terumo. G.B. collaborates in formal clinical trial contracts, IITs, and in joint grants funded by Canadian and Quebec governments with Roche, Merck, Novartis, Astra-Zeneca, Bayer, Esperas, Aurka, Caprion, MRM. U.L. reports advisory board roles at Bayer, Pfizer, and Novartis. Research grants: BMS, Roche, Pfizer, Lilly, Incyte, and GSK. R.L.S. is the Chairman of the WIN Association—WIN Consortium. He declares Research Support: and continues to serve as the PI of the ASCO TAPUR trial. ASCO receives research grants from the following companies in support of this trial: AstraZeneca, Bayer, Boehringer-Ingelheim, Bristol Myers Squibb, Genentech, Lilly, Merck, Pfizer, Seagen, TAIHO. He does not personally receive any compensation from these companies; receives Consulting: he is a consultant to the following companies: Aadi Bioscience*Brylogyx, Cellworks*, Clarified Precision Medicine*, EQRx*, Illumina*, Scandion Oncology* ZephyrAI*, and receive compensation from those designated (*) in the last 2 years. Board Service: He is a member of the Board of Directors of the following companies and receives compensation from each: Clarified Precision Medicine and Leap Therapeutics. R.K. is Chief Medical Officer of the WIN Association—WIN Consortium. She has received research funding from Boehringer Ingelheim, Debiopharm, Foundation Medicine, Genentech, Grifols, Guardant, Incyte, Konica Minolta, MedImmune, Merck Serono, Omniseq, Pfizer, Sequenom, Takeda, and TopAlliance and the NCI; as well as consultant and/or speaker fees and/or advisory board/consultant for Actuate Therapeutics, AstraZeneca, Bicara Therapeutics, Inc., Biological Dynamics, Caris, Datar Cancer Genetics, Daiichi, EISAI, EOM Pharmaceuticals, Iylon, LabCorp, Merck, NeoGenomics, Neomed, Pfizer, Prosperdtx, Regeneron, Roche, TD2/Volastra, Turning Point Therapeutics, X-Biotech; has an equity interest in CureMatch Inc. and IDbyDNA; serves on the Board of CureMatch and CureMetrix, and is a co-founder of CureMatch. I.B.N., A.T.R., J.R., F.C.N., and E.Ru. report no competing interests. All authors have read the policy of conflicts of interests., (© The Author(s), 2024.)

Background and Purpose: First-generation soluble guanylate cyclase (sGC) stimulators have shown clinical benefit in pulmonary hypertension (riociguat) and chronic heart failure (vericiguat). However, given the broad therapeutic opportunities for sGC stimulators, tailored molecules for distinct indications are required., Experimental Approach: We report the high-throughput screening (HTS)-based discovery of a second generation of sGC stimulators from a novel imidazo[1,2-a]pyridine lead series. An intense medicinal chemistry programme resulted in the discovery of the sGC stimulator BAY 1165747 (BAY-747). The pharmacokinetic profile of BAY-747 was determined in different species, and it was broadly characterized in pharmacological model systems relevant for vasodilatation and hypertension., Key Results: BAY-747 is a highly potent sGC stimulator in vitro. In addition, BAY-747 showed an excellent pharmacokinetic profile with long half-life and low peak-to-trough ratio. BAY-747 was investigated in experimental in vivo models of malignant and resistant hypertension (rHT). In spontaneously hypertensive (SH) rats, BAY-747 caused a dose-related and long-lasting decrease in mean arterial blood pressure (MAP). Oral treatment over 12 days resulted in a persistent decrease. BAY-747 provided additional benefit when dosed on top of losartan, amlodipine or spironolactone and even on top of triple combinations of frequently used antihypertensive drugs. In a new canine model of rHT, BAY-747 caused a dose-related and long-lasting (>6 h) MAP decrease., Conclusion and Implications: BAY-747 is a potent, orally available sGC stimulator. BAY-747 shows long-acting pharmacodynamic effects with a very low peak-to-trough ratio. BAY-747 could be a treatment alternative for patients with hypertension, especially those not responding to standard-of-care therapy., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Herein, we describe the identification, chemical optimization, and preclinical characterization of novel soluble guanylate cyclase (sGC) stimulators. Given the very broad therapeutic opportunities for sGC stimulators, new tailored molecules for distinct indications with specific pharmacokinetics, tissue distribution, and physicochemical properties will be required in the future. Here, we report the ultrahigh-throughput (uHTS)-based discovery of a new class of sGC stimulators from an imidazo[1,2- a ]pyridine lead series. Through the extensive and staggered optimization of the initial screening hit, liabilities such as potency, metabolic stability, permeation, and solubility could be substantially improved in parallel. These efforts resulted ultimately in the discovery of the new sGC stimulators 22 and 28 . It turned out that BAY 1165747 (BAY-747, 28 ) could be an ideal treatment alternative for patients with hypertension, especially those not responding to standard anti-hypertensive therapy (resistant hypertension). BAY-747 ( 28 ) demonstrated sustained hemodynamic effects up to 24 h in phase 1 studies.

After acute myocardial infarction, early reperfusion is the most effective strategy for reducing cardiac damage and improving clinical outcome. However, restoring blood flow to the ischemic myocardium can paradoxically induce injury by itself (reperfusion injury), with microvascular dysfunction being one contributing factor. α 2B adrenergic receptors have been hypothesized to be involved in this process. To assess α 2B -related pharmacology, we identified a novel α 2B antagonist by HTS. The HTS hit showed limited α 2A selectivity as well as low solubility and was optimized toward BAY-6096, a potent, selective, and highly water-soluble α 2B antagonist. Key aspects of the optimization were the introduction of a permanently charged pyridinium moiety to achieve very good aqueous solubility and the inversion of an amide to prevent genotoxicity. BAY-6096 dose-dependently reduced blood pressure increases in rats induced by an α 2B agonist, demonstrating the role of α 2B receptors in vascular constriction in rats.

Background: The current model of clinical drug development in oncology displays major limitations due to a high attrition rate in patient enrollment in early phase trials and a high failure rate of drugs in phase III studies., Objective: Integrating transcriptomics for selection of patients has the potential to achieve enhanced speed and efficacy of precision oncology trials for any targeted therapies or immunotherapies., Methods: Relative gene expression level in the metastasis and normal organ-matched tissues from the WINTHER database was used to estimate in silico the potential clinical benefit of specific treatments in a variety of metastatic solid tumors., Results: As example, high mRNA expression in tumor tissue compared to analogous normal tissue of c-MET and its ligand HGF correlated in silico with shorter overall survival (OS; p  < 0.0001) and may constitute an independent prognostic marker for outcome of patients with metastatic solid tumors, suggesting a strategy to identify patients most likely to benefit from MET-targeted treatments. The prognostic value of gene expression of several immune therapy targets (PD-L1, CTLA4, TIM3, TIGIT, LAG3, TLR4) was investigated in non-small-cell lung cancers and colorectal cancers (CRCs) and may be useful to optimize the development of their inhibitors, and opening new avenues such as use of anti-TLR4 in treatment of patients with metastatic CRC., Conclusion: This in silico approach is expected to dramatically decrease the attrition of patient enrollment and to simultaneously increase the speed and detection of early signs of efficacy. The model may significantly contribute to lower toxicities. Altogether, our model aims to overcome the limits of current approaches., Competing Interests: − Dr. Vladimir Lazar, Catherine Bresson, and Fanny Wunder are full-time employees of Worldwide Innovative Network (WIN) Association – WIN Consortium. − Shai Magidi receives consultancy fees from Worldwide Innovative Network (WIN) Association – WIN Consortium. − Worldwide Innovative Network (WIN) Association – WIN Consortium is the owner of the patent family entitled Digital Display. The inventors are Dr. Vladimir Lazar and Shai Magidi. − Dr. Baolin Zhang disclaimer: The views expressed in this manuscript are those of the author and do not represent the views or policies of the U.S. FDA. − Pr. Christophe Le Tourneau has participated in advisory boards from Celgene, AstraZeneca, MSD, BMS, Merck Serono, Nanobiotix, Rakuten, Seatlle Genetics, and Roche. − Pr. Eric Raymond : SCOR Health Science – consulting and shareholder; GenoScience Pharma – consulting and shareholder, Axoltis – shareholder, Stromacare – consulting and shareholderInstitut des Vaisseaux et du Sang (IVS) – Scientific Director, Institut National du Cancer (INCa France) – Member of the Board of Directors. − Pr. Michel Ducreux reports consulting or advisory role: Amgen, AstraZeneca, Bayer, Daiichi Sankyo, Merck Serono, MSD, Pierre Fabre, Roche, Servier. Speakers’ bureau: AstraZeneca, Bayer, Roche, Terumo Amir Onn receives consulting fees from: Roche Israel, MSD Israel, Boehringer Ingelheim, and AstraZeneca. − Enriqueta Felip reports consulting or advisory role: Abbvie, Amgen, AstraZeneca, Bayer, Blue Print Medicines, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, GSK, Janssen, Merck, MSD, Novartis, Pfizer, Puma Biotechnology, Roche, Sanofi Genzyme, Takeda; Speakers’ bureau or expert testimony: AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, Medscape, MSD, Novartis, Peervoice, Pfizer, Prime Oncology, Roche, Springer, Takeda, Touchime, CME Outfitters; Research grant or funding: Grant for Oncology Innovation (GOI), Fundación Merck Salud; Other: Grífols (independent member of the board). − Dr. Josep Tabernero declares scientific consultancy role for Array Biopharma, AstraZeneca, Bayer, BeiGene, Boehringer Ingelheim, Chugai, Genentech, Inc., Genmab A/S, Halozyme, Imugene Limited, Inflection Biosciences Limited, Ipsen, Kura Oncology, Lilly, MSD, Menarini, Merck Serono, Merrimack, Merus, Molecular Partners, Novartis, Peptomyc, Pfizer, Pharmacyclics, ProteoDesign SL, Rafael Pharmaceuticals, F. Hoffmann-La Roche Ltd, Sanofi, SeaGen, Seattle Genetics, Servier, Symphogen, Taiho, VCN Biosciences, Biocartis, Foundation Medicine, HalioDX SAS and Roche Diagnostics. − Gerald Batist collaborates in formal clinical trial contracts, IITs and in joint grants funded by Canadian and Quebec governments with Roche, Merck, Novartis, AstraZeneca, Bayer, Esperas, Aurka, Caprion, MRM. − Razelle Kurzrock is Chief Medical Officer of the Worldwide Innovative Network (WIN) Association – WIN Consortium. She has received research funding from Genentech, Merck Serono, Pfizer, Boehringer Ingelheim, TopAlliance, Takeda, Incyte, Debiopharm, Medimmune, Sequenom, Foundation Medicine, Konica Minolta, Grifols, Omniseq, and Guardant, as well as consultant and/or speaker fees and/or advisory board for X-Biotech, Neomed, Pfizer, Actuate Therapeutics, Roche, Turning Point Therapeutics, TD2/Volastra, Bicara Therapeutics, Inc., has an equity interest in IDbyDNA and CureMatch Inc, serves on the Board of CureMatch and CureMetrix, and is a co-founder of CureMatch. − Richard L. Schilsky is the Chairman of Worldwide Innovative Network (WIN) Association – WIN Consortium. He declares Research Support: continues to serve as the PI of the ASCO TAPUR trial. ASCO receives research grants from the following companies in support of this trial: AstraZeneca, Bayer, Boehringer-Ingelheim, Bristol Myers Squibb, Genentech, Lilly, Merck, Pfizer, Seagen. He does not personally receive any compensation from these companies; receives Consulting: he is consultant to the following companies: Brylogyx, Cellworks*, Clarified Precision Medicine*, EQRx*, Illumina*, Scandion Oncology* and receive compensation from those designated (*). − Dr. Baolin Zhang, Dr. Jacques Raynaud and Pr. Eitan Rubin, declare no potential conflicts of interest., (© The Author(s), 2023.)

Despite advances in the treatment of heart failure in recent years, options for patients are still limited and the disease is associated with considerable morbidity and mortality. Modulating cyclic guanosine monophosphate levels within the natriuretic peptide signaling pathway by inhibiting PDE9A has been associated with beneficial effects in preclinical heart failure models. We herein report the identification of BAY-7081, a potent, selective, and orally bioavailable PDE9A inhibitor with very good aqueous solubility starting from a high-throughput screening hit. Key aspect of the optimization was a switch in metabolism of our lead structures from glucuronidation to oxidation. The switch proved being essential for the identification of compounds with improved pharmacokinetic profiles. By studying a tool compound in a transverse aortic constriction mouse model, we were able to substantiate the relevance of PDE9A inhibition in heart diseases.

Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr -/- mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. I mp or ta ntly, p ha rm ac ol ogical sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr -/- mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD., Competing Interests: Competing interests H.S. has received personal fees from MSD Sharp & Dohme, Amgen, Bayer Vital GmbH, Boehringer Ingelheim, Daiichi Sankyo, Novartis, Servier, Brahms, Bristol Myers Squibb, Medtronic, Sanofi Aventis, Synlab, Pfizer and Vifor T as well as grants and personal fees from AstraZeneca that are unrelated to the submitted work. H.S. and T.K. are named inventors on a patent application for the prevention of restenosis after angioplasty and stent implantation (patent applicants: Klinikum rechts der Isar, German Heart Centre Munich; inventors: T. Kessler, A. Kastrati, H. Schunkert; application no. PCT/EP2021/053116; status: pending), which is unrelated to the submitted work. T.K. received lecture fees from Bayer HealthCare Pharmaceuticals. L.D., F.W. and P.S. are full-time employees of Bayer HealthCare Pharmaceuticals. The other authors declare no competing interests.

Background: SARS-CoV-2 (COVID-19) elicits a T-cell antigen-mediated immune response of variable efficacy. To understand this variability, we explored transcriptomic expression of angiotensin-converting enzyme 2 ( ACE2 , the SARS-CoV-2 receptor) and of immunoregulatory genes in normal lung tissues from patients with non-small cell lung cancer (NSCLC)., Methods: This study used the transcriptomic and the clinical data for NSCLC patients generated during the CHEMORES study [ n  = 123 primary resected (early-stage) NSCLC] and the WINTHER clinical trial ( n  = 32 metastatic NSCLC)., Results: We identified patient subgroups with high and low ACE2 expression ( p  = 1.55 × 10 -19 ) in normal lung tissue, presumed to be at higher and lower risk, respectively, of developing severe COVID-19 should they become infected. ACE2 transcript expression in normal lung tissues (but not in tumor tissue) of patients with NSCLC was higher in individuals with more advanced disease. High- ACE2 expressors had significantly higher levels of CD8+ cytotoxic T lymphocytes and natural killer cells but with presumably impaired function by high Thymocyte Selection-Associated High Mobility Group Box Protein TOX ( TOX ) expression. In addition, immune checkpoint-related molecules - PD-L1, CTLA-4, PD-1 , and TIGIT - are more highly expressed in normal (but not tumor) lung tissues; these molecules might dampen immune response to either viruses or cancer. Importantly, however, high inducible T-cell co-stimulator ( ICOS ), which can amplify immune and cytokine reactivity, significantly correlated with high ACE2 expression in univariable analysis of normal lung (but not lung tumor tissue)., Conclusions: We report a normal lung immune-tolerant state that may explain a potential comorbidity risk between two diseases - NSCLC and susceptibility to COVID-19 pneumonia. Further, a NSCLC patient subgroup has normal lung tissue expressing high ACE2 and high ICOS transcripts, the latter potentially promoting a hyperimmune response, and possibly leading to severe COVID-19 pulmonary compromise., (© The Author(s), 2022.)

Background: Oxidative stress associated with severe cardiopulmonary diseases leads to impairment in the nitric oxide/soluble guanylate cyclase signaling pathway, shifting native soluble guanylate cyclase toward heme-free apo-soluble guanylate cyclase. Here we describe a new inhaled soluble guanylate cyclase activator to target apo-soluble guanylate cyclase and outline its therapeutic potential., Methods: We aimed to generate a novel soluble guanylate cyclase activator, specifically designed for local inhaled application in the lung. We report the discovery and in vitro and in vivo characterization of the soluble guanylate cyclase activator mosliciguat (BAY 1237592)., Results: Mosliciguat specifically activates apo-soluble guanylate cyclase leading to improved cardiopulmonary circulation. Lung-selective effects, e.g., reduced pulmonary artery pressure without reduced systemic artery pressure, were seen after inhaled but not after intravenous administration in a thromboxane-induced pulmonary hypertension minipig model. These effects were observed over a broad dose range with a long duration of action and were further enhanced under experimental oxidative stress conditions. In a unilateral broncho-occlusion minipig model, inhaled mosliciguat decreased pulmonary arterial pressure without ventilation/perfusion mismatch. With respect to airway resistance, mosliciguat showed additional beneficial bronchodilatory effects in an acetylcholine-induced rat model., Conclusion: Inhaled mosliciguat may overcome treatment limitations in patients with pulmonary hypertension by improving pulmonary circulation and airway resistance without systemic exposure or ventilation/perfusion mismatch. Mosliciguat has the potential to become a new therapeutic paradigm, exhibiting a unique mode of action and route of application, and is currently under clinical development in phase Ib for pulmonary hypertension., (© 2022. The Author(s).)

Background: The Worldwide Innovative Network (WIN) Consortium has developed the Simplified Interventional Mapping System (SIMS) to better define the cancer molecular milieu based on genomics/transcriptomics from tumor and analogous normal tissue biopsies. SPRING is the first trial to assess a SIMS-based tri-therapy regimen in advanced non-small cell lung cancer (NSCLC)., Methods: Patients with advanced NSCLC (no EGFR, ALK, or ROS1 alterations; PD-L1 unrestricted; ≤2 prior therapy lines) received avelumab, axitinib, and palbociclib (3 + 3 dose escalation design)., Results: Fifteen patients were treated (five centers, four countries): six at each of dose levels 1 (DL1) and DL2; three at DL3. The most common ≥Grade 3 adverse events were neutropenia, hypertension, and fatigue. The recommended Phase II dose (RP2D) was DL1: avelumab 10 mg/kg IV q2weeks, axitinib 3 mg po bid, and palbociclib 75 mg po daily (7 days off/21 days on). Four patients (27%) achieved a partial response (PR) (progression-free survival [PFS]: 14, 24, 25 and 144+ weeks), including two after progression on pembrolizumab. Four patients attained stable disease (SD) that lasted ≥24 weeks: 24, 27, 29, and 64 weeks. At DL1 (RP2D), four of six patients (66%) achieved stable disease (SD) ≥6 months/PR (2 each). Responders included patients with no detectable PD-L1 expression and low tumor mutational burden., Conclusions: Overall, eight of 15 patients (53%) achieved clinical benefit (SD ≥ 24 weeks/PR) on the avelumab, axitinib, and palbociclib combination. This triplet showed antitumor activity in NSCLC, including in tumors post-pembrolizumab progression, and was active at the RP2D, which was well tolerated. NCT03386929 clinicaltrial.gov., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Autotaxin (ATX) plays an important role in (patho-)physiological lysophosphatidic acid (LPA) signaling. Here we describe the establishment of novel cell-based ATX assay formats. ATX-mediated LPA generation is detected by using a stable LPA receptor reporter cell line. In a first assay variant, ATX-mediated LPA generation is started in the absence of cells and the reaction mix is transferred to the reporter cells after stopping the reaction (two-tube assay). In a second assay variant, ATX is added to the reporter cells expressing the known autotaxin binding partners integrin β1, integrin β3 and the LPA receptor 1. LPA generation is started in the presence of cells and is detected in real-time (one-tube assay). Structurally diverse ATX inhibitors with different binding modes were characterized in both cell-based assay variants and were also tested in the well-established biochemical choline release assay. ATX inhibitors displayed similar potencies, regardless if the assay was performed in the absence or presence of cells, and comparable results were obtained in all three assay formats. In summary, our novel cell-based ATX assay formats are well-suited for sensitive detection of enzyme activity as well as for the characterization of ATX inhibitors in the presence and absence of cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Herein we describe the discovery, mode of action, and preclinical characterization of the soluble guanylate cyclase (sGC) activator runcaciguat. The sGC enzyme, via the formation of cyclic guanosine monophoshphate, is a key regulator of body and tissue homeostasis. sGC activators with their unique mode of action are activating the oxidized and heme-free and therefore NO-unresponsive form of sGC, which is formed under oxidative stress. The first generation of sGC activators like cinaciguat or ataciguat exhibited limitations and were discontinued. We overcame limitations of first-generation sGC activators and identified a new chemical class via high-throughput screening. The investigation of the structure-activity relationship allowed to improve potency and multiple solubility, permeability, metabolism, and drug-drug interactions parameters. This program resulted in the discovery of the oral sGC activator runcaciguat (compound 45 , BAY 1101042). Runcaciguat is currently investigated in clinical phase 2 studies for the treatment of patients with chronic kidney disease and nonproliferative diabetic retinopathy.

The expanding targeted therapy landscape requires combinatorial biomarkers for patient stratification and treatment selection. This requires simultaneous exploration of multiple genes of relevant networks to account for the complexity of mechanisms that govern drug sensitivity and predict clinical outcomes. We present the algorithm, Digital Display Precision Predictor (DDPP), aiming to identify transcriptomic predictors of treatment outcome. For example, 17 and 13 key genes were derived from the literature by their association with MTOR and angiogenesis pathways, respectively, and their expression in tumor versus normal tissues was associated with the progression-free survival (PFS) of patients treated with everolimus or axitinib (respectively) using DDPP. A specific eight-gene set best correlated with PFS in six patients treated with everolimus: AKT2, TSC1, FKB-12, TSC2, RPTOR, RHEB, PIK3CA, and PIK3CB (r = 0.99, p = 5.67E-05). A two-gene set best correlated with PFS in five patients treated with axitinib: KIT and KITLG (r = 0.99, p = 4.68E-04). Leave-one-out experiments demonstrated significant concordance between observed and DDPP-predicted PFS (r = 0.9, p = 0.015) for patients treated with everolimus. Notwithstanding the small cohort and pending further prospective validation, the prototype of DDPP offers the potential to transform patients' treatment selection with a tumor- and treatment-agnostic predictor of outcomes (duration of PFS).

Precision medicine focuses on DNA abnormalities, but not all tumors have tractable genomic alterations. The WINTHER trial ( NCT01856296 ) navigated patients to therapy on the basis of fresh biopsy-derived DNA sequencing (arm A; 236 gene panel) or RNA expression (arm B; comparing tumor to normal). The clinical management committee (investigators from five countries) recommended therapies, prioritizing genomic matches; physicians determined the therapy given. Matching scores were calculated post-hoc for each patient, according to drugs received: for DNA, the number of alterations matched divided by the total alteration number; for RNA, expression-matched drug ranks. Overall, 303 patients consented; 107 (35%; 69 in arm A and 38 in arm B) were evaluable for therapy. The median number of previous therapies was three. The most common diagnoses were colon, head and neck, and lung cancers. Among the 107 patients, the rate of stable disease ≥6 months and partial or complete response was 26.2% (arm A: 23.2%; arm B: 31.6% (P = 0.37)). The patient proportion with WINTHER versus previous therapy progression-free survival ratio of >1.5 was 22.4%, which did not meet the pre-specified primary end point. Fewer previous therapies, better performance status and higher matching score correlated with longer progression-free survival (all P < 0.05, multivariate). Our study shows that genomic and transcriptomic profiling are both useful for improving therapy recommendations and patient outcome, and expands personalized cancer treatment.

Corin (atrial natriuretic peptide-converting enzyme, EC 3.4.21) is a transmembrane serine protease expressed in cardiomyocytes. Corin exerts its cardioprotective effects via the proteolytic cleavage and activation of pro-atrial natriuretic peptide (pro-ANP) to ANP. We recently described an ANP reporter cell line stably expressing the ANP receptor, a cGMP-dependent cation channel used as a real-time cGMP biosensor, and the Ca 2+ -sensitive photoprotein aequorin. Here, we describe the generation of a novel reporter cell line expressing the calcium biosensor GCaMP6 instead of aequorin. In contrast to the luminescence-based assay, ANP stimulation of our novel GCaMP6 reporter cell resulted in stable, long-lasting fluorescence signals. Using this novel reporter system, we were able to detect pro-ANP to ANP conversion by purified, soluble wildtype corin (solCorin), but not the active site mutant solCorin(S985A), resulting in left-shifted concentration-response curves. Furthermore, cellular pro-ANPase activity could be detected on HEK 293 cells after transient expression of wildtype corin. In contrast, corin activity was not detected after transfection with the inactive corin(S985A) variant. In supernatants from cardiomyocyte-derived HL-1 cells pro-ANP to ANP conversion could also be detected, while in HL-1 corin knockout cells no conversion was observed. These findings underline the role of corin as the pro-ANP convertase. Our novel fluorescence-based ANP reporter cell line is well-suited for the sensitive detection of corin activity, and may be used for the identification and characterization of novel corin modulators., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cyclic nucleotides cAMP and cGMP are central second messengers in cardiac cells and critical regulators of cardiac physiology as well as pathophysiology. Consequently, subcellular compartmentalization allows for spatiotemporal control of cAMP/cGMP metabolism and subsequent regulation of their respective effector kinases PKA or PKG is most important for cardiac function in health and disease. While acute cAMP-mediated signalling is a mandatory prerequisite for the physiological fight-or-flight response, sustained activation of this pathway may lead to the progression of heart failure. In contrast, acute as well as sustained cGMP-mediated signalling can foster beneficial features, e.g. anti-hypertrophic and vasodilatory effects. These two signalling pathways seem to be intuitively counteracting and there is increasing evidence for a functionally relevant crosstalk between cAMP and cGMP signalling pathways on the level of cyclic nucleotide hydrolysing phosphodiesterases (PDEs). Among this diverse group of enzymes, PDE2 may fulfill a unique integrator role. Equipped with dual substrate specificity for cAMP as well as for cGMP, it is the only cAMP hydrolysing PDE, which is allosterically activated by cGMP. Recent studies have revealed strongly remodelled cAMP/cGMP microdomains and subcellular concentration profiles in different cardiac pathologies, leading to a putatively enhanced involvement of PDE2 in cAMP/cGMP breakdown and crosstalk compared to the other cardiac PDEs. This review sums up the current knowledge about molecular properties and regulation of PDE2 and explains the complex signalling network encompassing PDE2 in order to better understand the functional role of PDE2 in distinct cell types in cardiac health and disease. Moreover, this review gives an outlook in which way PDE2 may serve as a therapeutic target to treat cardiac disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The first-in-class soluble guanylate cyclase (sGC) stimulator riociguat was recently introduced as a novel treatment option for pulmonary hypertension. Despite its outstanding pharmacological profile, application of riociguat in other cardiovascular indications is limited by its short half-life, necessitating a three times daily dosing regimen. In our efforts to further optimize the compound class, we have uncovered interesting structure-activity relationships and were able to decrease oxidative metabolism significantly. These studies resulting in the discovery of once daily sGC stimulator vericiguat (compound 24, BAY 1021189), currently in phase 3 trials for chronic heart failure, are now reported.

Rationale: Phosphodiesterase 2 is a dual substrate esterase, which has the unique property to be stimulated by cGMP, but primarily hydrolyzes cAMP. Myocardial phosphodiesterase 2 is upregulated in human heart failure, but its role in the heart is unknown., Objective: To explore the role of phosphodiesterase 2 in cardiac function, propensity to arrhythmia, and myocardial infarction., Methods and Results: Pharmacological inhibition of phosphodiesterase 2 (BAY 60-7550, BAY) led to a significant positive chronotropic effect on top of maximal β-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated β-adrenoceptor-mediated inotropy and chronotropy. Conversely, ECG telemetry in heart-specific phosphodiesterase 2-transgenic (TG) mice showed a marked reduction in resting and in maximal heart rate, whereas cardiac output was completely preserved because of greater cardiac contraction. This well-tolerated phenotype persisted in elderly TG with no indications of cardiac pathology or premature death. During arrhythmia provocation induced by catecholamine injections, TG animals were resistant to triggered ventricular arrhythmias. Accordingly, Ca 2+ -spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca 2+ leakage and lower basal phosphorylation levels of Ca 2+ -cycling proteins including ryanodine receptor type 2. Moreover, TG demonstrated improved cardiac function after myocardial infarction., Conclusions: Endogenous phosphodiesterase 2 contributes to heart rate regulation. Greater phosphodiesterase 2 abundance protects against arrhythmias and improves contraction force after severe ischemic insult. Activating myocardial phosphodiesterase 2 may, thus, represent a novel intracellular antiadrenergic therapeutic strategy protecting the heart from arrhythmia and contractile dysfunction., (© 2016 American Heart Association, Inc.)

The apelin ligand receptor system is an important target to develop treatment strategies for cardiovascular diseases. Although apelin exhibits strong inotropic effects, its pharmaceutical application is limited because no agonist with suitable properties is available. On the one hand, peptide ligands are too instable, and on the other hand, small-molecule agonists show only low potency. This study describes the development of apelin (APJ) receptor agonists with not only high activity but also metabolic stability. Several strategies including capping of termini, insertion of unnatural amino acids, cyclization, and lipidation were analyzed. Peptide activity was tested using a Ca 2+ -mobilization assay and the degradation of selected analogues was analyzed in rat plasma. The best results were obtained by N-terminal lipidation of a 13-mer apelin derivative. This analogue displayed a half-life of 29 h in rat plasma, compared with 0.025 h for the wild-type peptide. Furthermore, in vivo pharmacokinetics revealed a clearance of 0.049 L h -1  kg -1 and a half-life of 0.36 h. In summary, amino acid substitution and fatty acid modification resulted in a potent and 1000-fold more stable peptide that exhibits high pharmaceutical potential., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The meiotic cell cycle of mammalian oocytes in preovulatory follicles is held in prophase arrest by diffusion of cGMP from the surrounding granulosa cells into the oocyte. Luteinizing hormone (LH) then releases meiotic arrest by lowering cGMP in the granulosa cells. The LH-induced reduction of cGMP is caused in part by a decrease in guanylyl cyclase activity, but the observation that the cGMP phosphodiesterase PDE5 is phosphorylated during LH signaling suggests that an increase in PDE5 activity could also contribute. To investigate this idea, we measured cGMP-hydrolytic activity in rat ovarian follicles. Basal activity was due primarily to PDE1A and PDE5, and LH increased PDE5 activity. The increase in PDE5 activity was accompanied by phosphorylation of PDE5 at serine 92, a protein kinase A/G consensus site. Both the phosphorylation and the increase in activity were promoted by elevating cAMP and opposed by inhibiting protein kinase A, supporting the hypothesis that LH activates PDE5 by stimulating its phosphorylation by protein kinase A. Inhibition of PDE5 activity partially suppressed LH-induced meiotic resumption as indicated by nuclear envelope breakdown, but inhibition of both PDE5 and PDE1 activities was needed to completely inhibit this response. These results show that activities of both PDE5 and PDE1 contribute to the LH-induced resumption of meiosis in rat oocytes, and that phosphorylation and activation of PDE5 is a regulatory mechanism., (© 2016 by the Society for the Study of Reproduction, Inc.)

In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte., (© 2014. Published by The Company of Biologists Ltd.)

Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As light-regulated actuators, photoreceptors underpin optogenetics, which denotes the noninvasive, reversible, and spatiotemporally precise perturbation by light of living cells and organisms. Of particular versatility, naturally occurring photoactivated adenylate cyclases promote the synthesis of the second messenger cAMP under blue light. Here, we have engineered a light-activated phosphodiesterase (LAPD) with complementary light sensitivity and catalytic activity by recombining the photosensor module of Deinococcus radiodurans bacterial phytochrome with the effector module of Homo sapiens phosphodiesterase 2A. Upon red-light absorption, LAPD up-regulates hydrolysis of cAMP and cGMP by up to sixfold, whereas far-red light can be used to down-regulate activity. LAPD also mediates light-activated cAMP and cGMP hydrolysis in eukaryotic cell cultures and in zebrafish embryos; crucially, the biliverdin chromophore of LAPD is available endogenously and does not need to be provided exogenously. LAPD thus establishes a new optogenetic modality that permits light control over diverse cAMP/cGMP-mediated physiological processes. Because red light penetrates tissue more deeply than light of shorter wavelengths, LAPD appears particularly attractive for studies in living organisms.

We report here the generation and pharmacological characterization of two novel PDE 4B1 and PDE 4D3 reporter cell lines. Intracellular cAMP levels are monitored in these cells by a cAMP-sensitive biosensor. We used the recombinant PDE 4B1 and PDE 4D3 reporter cell lines to characterize the cellular effects of various competitive and allosteric PDE 4 inhibitors. In addition, we compared the cellular activity of these PDE 4 inhibitors with the in vitro inhibition of full-length PDE 4D3 and a truncated enzyme comprising the PDE 4D3 catalytic domain. Two different groups of PDE 4 inhibitors could be identified. The first group, including competitive inhibitors like roflumilast, cilomilast and piclamilast, shows similar in vitro activity on full-length and truncated PDE 4D3 and comparably low cellular activity. The second group, including the allosteric inhibitors PMNPQ, D159153, and D159404, shows much better inhibition of full-length versus truncated PDE 4D3. In addition, these compounds show high cellular activity. Our data obtained with the prototype PDE 4 inhibitor rolipram show that rolipram has properties intermediate between the two groups. The results imply that these novel PDE 4 reporter cell lines are well-suited for the characterization of the cellular activity of PDE 4 inhibitors and may also support a better understanding of the complex PDE 4 pharmacology.

Many physiological and pathophysiological processes are regulated by cAMP. Different therapies directly or indirectly influence the cellular concentration of this second messenger. A wide variety of receptors either activates or inhibits adenylate cyclases in order to induce proper physiological responses. A key event in this signalling system is the direct and dynamic interaction of Gαi1 subunits with adenylate cyclases. We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) and monitored receptor-stimulated interactions between Gαi1 and AC5 in single intact cells with high temporal resolution. We observed that FRET between Gαi1 and AC5 developed at much lower concentration of agonist compared with the overall Gi-protein activity resulting in a left-shift of the concentration-response curve by approximately one order of magnitude. Furthermore, Gi1-protein-mediated attenuation of AC5-dependent increases in cAMP occurred at comparable low concentrations of agonist. On analysing the dynamics we found the dissociation of the Gαi1 subunits and AC5 to occur significantly slower than the G-protein deactivation and to be insensitive to RGS4 (regulator of G-protein signalling type 4) expression. This led us to the conclusion that AC5, by binding active Gαi1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

Receptor guanylyl cyclases are implicated in a growing number of pathophysiologies and, therefore, represent an important target class for drug development. We report here the generation and pharmacological characterization of three particulate guanylyl cyclase (pGC) reporter cell lines. Plasmid constructs encoding the natriuretic peptide receptors GC-A and GC-B, and the heat-stable enterotoxin receptor GC-C, were stably transfected in a parental reporter cell line expressing a cyclic nucleotide-gated (CNG) cation channel, acting as the biosensor for intracellular cGMP. In our reporter cell lines pGC activity can be monitored in living cells in real-time . By using different natural as well as synthetic receptor ligands of the natriuretic and guanylin peptide families, we show that our reporter assay monitors pGC activity with very high sensitivity. In contrast to previous findings, we could detect significant stimulation of GC-A and GC-B by each of the natriuretic peptides ANP, BNP and CNP. In addition, the clearance receptor ligand Cys-ANF(4-18) and the ANP receptor antagonist Arg-ANF(6-18) were characterized as partial GC-A agonists. The results imply that our novel pGC reporter cell lines are well suited for the characterization of receptor pharmacology and may be used for natural ligand characterization of guanylyl cyclase orphan receptors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 min, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2h, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In the female reproductive tract, mammalian sperm undergo a regulated sequence of prefusion changes that "prime" sperm for fertilization. Among the least understood of these complex processes are the molecular mechanisms that underlie sperm guidance by environmental chemical cues. A "hard-wired" Ca(2+) signaling strategy that orchestrates specific motility patterns according to given functional requirements is an emerging concept for regulation of sperm swimming behavior. The molecular players involved, the spatiotemporal characteristics of such motility-associated Ca(2+) dynamics, and the relation between a distinct Ca(2+) signaling pattern and a behavioral sperm phenotype, however, remain largely unclear. Here, we report the functional characterization of two human sperm chemoreceptors. Using complementary molecular, physiological, and behavioral approaches, we comparatively describe sperm Ca(2+) responses to specific agonists of these novel receptors and bourgeonal, a known sperm chemoattractant. We further show that individual receptor activation induces specific Ca(2+) signaling patterns with unique spatiotemporal dynamics. These distinct Ca(2+) dynamics are correlated to a set of stimulus-specific stereotyped behavioral responses that could play vital roles during various stages of prefusion sperm-egg chemical communication.

Background and Purpose: Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory and cardiovascular disorders. Their actions are mediated by CysLT(1) and CysLT(2) receptors. Here we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy) benzoic acid (HAMI3379), the first potent and selective CysLT(2) receptor antagonist., Experimental Approach: Pharmacological characterization of HAMI3379 was performed using stably transfected CysLT(1) and CysLT(2) receptor cell lines, and isolated, Langendorff-perfused, guinea pig hearts., Key Results: In a CysLT(2) receptor reporter cell line, HAMI3379 antagonized leukotriene D(4)- (LTD(4)-) and leukotriene C(4)- (LTC(4)-) induced intracellular calcium mobilization with IC(50) values of 3.8 nM and 4.4 nM respectively. In contrast, HAMI3379 exhibited very low potency on a recombinant CysLT(1) receptor cell line (IC(50) > 10 000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT receptor cell lines. In binding studies using membranes from the CysLT(2) and CysLT(1) receptor cell lines, HAMI3379 inhibited [(3)H]-LTD(4) binding with IC(50) values of 38 nM and >10 000 nM respectively. In isolated Langendorff-perfused guinea pig hearts HAMI3379 concentration-dependently inhibited and reversed the LTC(4)-induced perfusion pressure increase and contractility decrease. The selective CysLT(1) receptor antagonist zafirlukast was found to be inactive in this experimental setting., Conclusions and Implications: HAMI3379 was identified as a potent and selective CysLT(2) receptor antagonist, which was devoid of CysLT receptor agonism. Using this compound, we showed that the cardiac effects of CysLTs are predominantly mediated by the CysLT(2) receptor.

Conventional drug design embraces the "one gene, one drug, one disease" philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology.

AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An ANP- and nitric oxide (NO)/soluble guanylate cyclase (sGC)-induced insertion of AQP2 into the plasma membrane is reported from different models. However, functional data on the insertion of AQP2 is missing. We used primary cultured inner medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein-quenching measurements, and immunofluorescence and Western blotting to analyze the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression, and water permeability. While AVP led to acceleration in osmotically induced swelling, ANP had no effect. However, in AVP-pretreated cells ANP significantly decreased the kinetics of cell swelling. This effect was mimicked by 8-bromo-cGMP and blunted by PKG inhibition. Stimulation of the NO/sGC pathway or direct activation of sGC with BAY 58-2667 had similar effects to ANP. In cells treated with AVP, AQP2 was predominantly localized in the plasma membrane, and after additional incubation with ANP AQP2 was mostly localized in the cytosol, indicating an increased retrieval of AQP2 from the plasma membrane by ANP. Western blot analysis showed that ANP was able to reduce AVP-induced phosphorylation of AQP2 at position S256. In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a decreased localization of AQP2 in the plasma membrane which is mediated by cGMP and PKG.

Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impairments of the NO-sGC signaling pathway have been implicated in the pathogenesis of cardiovascular and other diseases. Direct stimulation of sGC represents a promising therapeutic strategy particularly for the treatment of pulmonary hypertension (PH), a disabling disease associated with a poor prognosis. Previous sGC stimulators such as the pyrazolopyridines BAY 41-2272 and BAY 41-8543 demonstrated beneficial effects in experimental models of PH, but were associated with unfavorable drug metabolism and pharmacokinetic (DMPK) properties. Herein we disclose an extended SAR exploration of this compound class to address these issues. Our efforts led to the identification of the potent sGC stimulator riociguat, which exhibits an improved DMPK profile and exerts strong effects on pulmonary hemodynamics and exercise capacity in patients with PH. Riociguat is currently being investigated in phase III clinical trials for the oral treatment of PH.

We report here the generation and pharmacological characterization of a phosphodiesterase 2A (PDE2A) reporter cell line. Human PDE2A was stably transfected in a parental cell line expressing the atrial natriuretic peptide (ANP) receptor and the cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the biosensor for intracellular cGMP. In this reporter cell line, cGMP levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel. By using different PDE inhibitors, we could show that our PDE2A reporter assay specifically monitors PDE2A inhibition with high sensitivity. In the absence of ANP stimulation, the PDE2A selective inhibitors EHNA, BAY 60-7550 and PDP did not increase basal luminescence levels in this experimental setting. However, in combination with ANP, these inhibitors stimulated luminescence signals and induced leftward shifts of ANP concentration-response curves. Similar results were obtained when using IBMX, trequinsin and dipyridamole, which inhibit PDE2A nonselectively with lower potency. PDP, the most potent PDE2A inhibitor known to date, was found to exhibit much lower cellular activity as anticipated from its biochemical PDE2A inhibitory activity. By cellular uptake and transport studies we could show that PDP's cell permeability is low and that the compound is a substrate for an efflux transporter. Other PDE inhibitors including vinpocetine, milrinone, rolipram, sildenafil, zaprinast, BRL 50481 and BAY 73-6691 did not stimulate luminescence signals on our PDE2A reporter cell line. The results imply that this novel PDE2A reporter assay provides an efficient, high throughput means for the identification and characterization of PDE2A inhibitors.

Functional cell-based assays have gained increasing importance for microplate-based high throughput screening (HTS). The use of high-density microplates, most prominently 1536-well plates, and miniaturized assay formats allow screening of comprehensive compound collections with more than 1 million compounds at ultra-high throughput, i.e. in excess of 100,000 samples per day. uHTS operations with numerous campaigns per year should generally support this throughput at all different steps of the process, including the underlying compound logistics, the (automated) testing of the corporate compound collection in the bioassay, and the subsequent follow-up studies for hit confirmation and characterization. A growing number of reports document the general feasibility of cell-based uHTS in microliter volumes. In addition, full automation with integrated robotic systems allows the realization of also complex assay protocols with multiple liquid handling and signal detection steps. For this review, cell-based assays are categorized based on the kinetics of the cellular response to be quantified in the test and the readout method employed. Thus, assays measuring fast cellular responses with high temporal resolution, e.g., receptor mediated calcium signals or changes in membrane potential, are at one end of this spectrum, while tests quantifying cellular transcriptional responses mark the opposite end. Trends for cell-based uHTS assays developed at Bayer-Schering Pharma are, first, to incorporate assay integral reference signals allowing the experimental differentiation of target hits from non-specifically acting compounds, and second, to make use of kinetic, real-time readouts providing additional information on the mode-of-action of test compounds.

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) receptors and their respective ligands play important roles in cardiovascular (patho-)physiology. Functional expression of ADM and CGRP receptors requires the presence of the calcitonin receptor-like receptor (CRLR) together with receptor-activity-modifying proteins (RAMPs). We have characterized the expression patterns of CRLR and RAMP1 to RAMP3 in human cardiovascular-related tissues by quantitative polymerase chain reaction. We could identify high expression levels of CRLR, RAMP1, and RAMP2 in human heart and various blood vessels. RAMP3 expression in these tissues, however, was detectable at significantly lower levels. In addition, we describe here a novel, aequorin luminescence-based G protein-coupled receptor reporter assay that enables the real-time detection of receptor activation in living cells. In the assay system, intracellular cAMP levels are monitored with high sensitivity by using a modified, heteromultimeric cyclic nucleotide-gated channel mediating calcium influx. G(q)-coupled receptor activation is detected via aequorin luminescence stimulated by calcium release from intracellular stores. Using this novel reporter assay, we established and characterized stable ADM1 and CGRP1 receptor cell lines. The peptide ligands ADM, CGRP1, and CGRP2 were characterized as potent agonists at their respective receptors. In contrast, intermedin acted as a weak agonist on both receptors and showed only partial agonism on the ADM1 receptor. Agonist activities were effectively antagonized by the receptor antagonists ADM(22-52) and CGRP(8-37). Various vasoactive ADM fragments were also characterized but showed no activity on the ADM1 receptor cell line. In addition, luminescence signal kinetics after activation of G(s)- and G(q)-coupled receptors were found to be markedly different.

Unlabelled: Liver cirrhosis is a chronic disease with high mortality rate and need for effective pharmacological intervention. The fibrotic remodelling of liver tissue is crucially dependent on hepatic stellate cell activation. Activation of hepatic stellate cells is reduced by an increase in cyclic guanosine monophosphate (cGMP). Stable cGMP analogues also reduce the contractile response of hepatic stellate cells. However, cGMP production is downregulated in the cirrhotic liver due to the reduced activity of the endothelial nitric oxide synthase., Objective: Here we report that the novel activator of soluble guanylate cyclase (sGC), BAY 60-2770 (4-({(4-carboxybutyl) [2- (5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl]methoxy}phenyl)ethyl] amino}methyl)benzoic acid), which increases the activity of sGC in a nitric oxide-independent manner, attenuates liver fibrosis in two rat models., Methods: The compound was studied in the pig serum model and the carbon tetrachloride model. Fibrosis was assessed by estimating the increase in fibrous collagen by micromorphometry of histological sections stained with Sirius Red/Fast Green and by measuring total hepatic collagen., Results: BAY 60-2770, on a recombinant sGC reporter cell line, stimulated the luminescence signals with an EC50 value of 5.4 +/- 1.2 nmol/L. In the presence of [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 pmol/L) the EC50 was shifted to 0.39 +/- 0.11 nmol/L. In both fibrosis models, once daily oral administration of BAY 60-2770 concomittantly with the fibrotic stimulus prevented 60-75% of fibrosis, the lowest effective dose being 0.1 mg/kg in the pig serum model and 0.3 mg/kg in the carbon tetrachloride model. The treatment was well tolerated by all animals. The doses used were devoid of any significant influence on systemic blood pressure., Conclusion: Nitric oxide-independent activation of sGC might be an innovative therapeutic approach for the treatment of liver fibrosis of necro-inflammatory and immunological origin.

Nitric oxide (NO) plays an important role in protection against the onset and progression of various cardiovascular disorders. Therefore, the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based, and highly sensitive reporter assay for NO generated by endothelial nitric oxide synthase (eNOS). In a coculture system, NO production is indirectly monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx mediated by the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor. Using this NO reporter assay, we performed a fully automated high-throughput screening campaign for stimulators of NO synthesis. The coculture system reflects most aspects of the natural NO/cGMP pathway, namely, Ca(2+)-dependent and Ca(2+)-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The NO reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP signaling pathway.

We report here the in vitro characterization of 1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one (BAY 73-6691), the first potent and selective inhibitor of phosphodiesterase 9 (PDE9), which is currently under preclinical development for the treatment of Alzheimer's disease. This compound selectively inhibits human (IC50 = 55 nM) and murine (IC50 = 100 nM) PDE9 activity in vitro and shows only moderate activity against other cyclic nucleotide-specific phosphodiesterases. We also report the generation and characterization of a stably transfected PDE9 Chinese hamster ovary cell line, additionally expressing soluble guanylate cyclase (sGC), the olfactory cyclic nucleotide-gated cation channel CNGA2 and the photoprotein aequorin. In this cell line, intracellular cGMP levels can be monitored in real-time via aequorin luminescence induced by Ca2+ influx through CNGA2, acting as the intracellular cGMP sensor. This simple and sensitive assay system was used for the characterization of the cellular activity of the new PDE9 inhibitor. BAY 73-6691 alone did not significantly increase basal cGMP levels in this experimental setting. However, in combination with submaximal stimulating concentrations of the sGC activator 4-[((4-carboxybutyl)[2-[(4-phenethyl-benzyl)oxy]phenethyl]amino)methyl] benzoic acid (BAY 58-2667), the compound induced concentration-dependent luminescence signals and intracellular cGMP accumulation. The PDE9 inhibitor significantly potentiated the cGMP signals generated by sGC activating compounds such as BAY 58-2667 or 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and induced leftward shifts of the corresponding concentration-response curves. Using our newly generated PDE9 reporter cell line, we could show that BAY 73-6691 is able to efficiently penetrate cells and to inhibit intracellular PDE9 activity.

Soluble guanylate cyclase, a heterodimer consisting of an alpha- and a heme-containing beta-subunit, is the major receptor for the biological messenger nitric oxide (NO) and is involved in various signal transduction pathways. The heme moiety of the enzyme is bound between the axial heme ligand histidine(105) and the recently identified counterparts of the heme propionic acids, tyrosine(135) and arginine(139). The latter residues together with an invariant serine(137) form the unique heme binding motif Y-x-S-x-R. In this work, we show that replacement of the serine(137) with alanine destabilizes the binding of the heme moiety and impairs NO-mediated soluble guanylate cyclase activation.

We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.

Soluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO- and heme-independent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between heme-containing and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the beta(1)-subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity.

1. Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (alpha/ss) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. 2. Through a high-throughput screen we identified BAY 58-2667, an amino dicarboxylic acid which potently activates sGC in an NO-independent manner. In contrast to NO, YC-1 and BAY 41-2272, the sGC stimulators described recently, BAY 58-2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem deficient. 3. Binding studies with radiolabelled BAY 58-2667 show a high affinity site on the enzyme. 4. Using photoaffinity labelling studies we identified the amino acids 371 (alpha-subunit) and 231 - 310 (ss-subunit) as target regions for BAY 58-2667. 5. sGC activation by BAY 58-2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not influenced by nitrate tolerance. 6. BAY 58-2667 shows a potent antihypertensive effect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic effects of BAY 58-2667 and GTN are very similar on the arterial and venous system. 7. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.

Tissue expression and distribution of the high-conductance Ca(2+)-activated K+ channel Slo was investigated in rat brain by immunocytochemistry, in situ hybridization, and radioligand binding using the novel high-affinity (Kd 22 pM) ligand [3H]iberiotoxin-D19C ([3H]IbTX-D19C), which is an analog of the selective maxi-K peptidyl blocker IbTX. A sequence-directed antibody directed against Slo revealed the expression of a 125 kDa polypeptide in rat brain by Western blotting and precipitated the specifically bound [3H]IbTX-D19C in solubilized brain membranes. Slo immunoreactivity was highly concentrated in terminal areas of prominent fiber tracts: the substantia nigra pars reticulata, globus pallidus, olfactory system, interpeduncular nucleus, hippocampal formation including mossy fibers and perforant path terminals, medial forebrain bundle and pyramidal tract, as well as cerebellar Purkinje cells. In situ hybridization indicated high levels of Slo mRNA in the neocortex, olfactory system, habenula, striatum, granule and pyramidal cell layer of the hippocampus, and Purkinje cells. The distribution of Slo protein was confirmed in microdissected brain areas by Western blotting and radioligand-binding studies. The latter studies also established the pharmacological profile of neuronal Slo channels. The expression pattern of Slo is consistent with its targeting into a presynaptic compartment, which implies an important role in neural transmission.

A novel potassium channel beta-subunit (Kv beta 3) was cloned from rat brain being the third member of a Kv beta subunit gene family. It is a protein of 403 amino acid residues with a 68% amino acid sequence homology to Kv beta 1.1. Kv beta 3 is primarily expressed in rat brain having a distribution distinct to those of Kv beta 1.1 and Kv beta 2. This subunit also has a long N-terminal structure and induces inactivation in N-terminal deleted Kv1.4 but not in other members of the Kv1 channel family. Similarly to Kv beta 1.1, the Kv beta 3-induced inactivation is regulated by the intracellular redox potential.

A large variety of potassium channels is involved in regulating integration and transmission of electrical signals in the nervous system. Different types of neurons, therefore, require specific patterns of potassium channel subunits expression and specific regulation of subunit coassembly into heteromultimeric channels, as well as subunit-specific sorting and segregation. This was investigated by studying in detail the expression of six different alpha-subunits of voltage-gated potassium channels in the rat hippocampus, cerebellum, olfactory bulb and spinal cord, combining in situ hybridization and immunocytochemistry. Specific polyclonal antibodies were prepared for five alpha-subunits (Kv1.1, Kv1.2, Kv1.3 Kv1.4, Kv1.6) of the Shaker-related subfamily of rat Kv channels, which encode delayed-rectifier type and rapidly inactivating A-type potassium channels. Their distribution was compared to that of an A-type potassium channel (Kv3.4), belonging to the Shaw-related subfamily of rat Kv channels. Our results show that these Kv channel alpha-subunits are differentially expressed in rat brain neurons. We did not observe in various neurons a stereotypical distribution of Kv channel alpha-subunits to dendritic and axonal compartments, but a complex differential subcellular subunit distribution. The different Kv channel subunits are targeted either to presynaptic or to postsynaptic domains, depending on neuronal cell type. Thus, distinct combinations of Kv1 alpha-subunits are co-localized in different neurons. The implications of these findings are that both differential expression and assembly as well as subcellular targeting of Kv channel alpha-subunits may contribute to Kv channel diversity and thereby to presynaptic and postsynaptic membrane excitability.

We have cloned a mammalian (rat) homologue of Drosophila ether á go-go (eag) cDNA, which encodes a distinct type of voltage activated potassium (K) channel. The derived Drosophila and rat eag polypeptides share > 670 amino acids, with a sequence identity of 61%, exhibiting a high degree of similarity at the N-terminus, the hydrophobic core including the pore forming P region and a potential cyclic nucleotide binding site. Rat eag mRNA is specifically expressed in the central nervous system. In the Xenopus oocyte expression system rat eag mRNA gives rise to voltage activated K channels which have distinct properties in comparison with Drosophila eag channels and other voltage activated K channels. Thus, the rat eag channel further extends the known diversity of K channels. Most notably, the kinetics of rat eag channel activation depend strongly on holding membrane potential. Hyperpolarization slows down the kinetics of activation; conversely depolarization accelerates the kinetics of activation. This novel K channel property may have important implications in neural signal transduction allowing neurons to tune their repolarizing properties in response to membrane hyperpolarization.

Structural and functional diversity of voltage-gated Kv1-type potassium channels in rat brain is enhanced by the association of two different types of subunits, the membrane-bound, poreforming alpha-subunits and a peripheral beta-subunit. We have cloned a beta-subunit (Kv beta 1) that is specifically expressed in the rat nervous system. Association of Kv beta 1 with alpha-subunits confers rapid A-type inactivation on non-inactivating Kv1 channels (delayed rectifiers) in expression systems in vitro. This effect is mediated by an inactivating ball domain in the Kv beta 1 amino terminus.

Previously, we characterized a Shaker-related family of voltage-gated potassium channels (RCK) in rat brain. Now, we describe a second family of voltage-gated potassium channels in the rat nervous system. This family is related to the Drosophila Shaw gene and has been dubbed Raw. In contrast to the RCK potassium channel family the Raw family utilizes extensive alternative splicing for expressing potassium channel subunits with variant C-termini. These alternative C-termini do not appear to influence the electrophysiological and pharmacological properties as studied in the Xenopus oocyte expression system. In situ hybridizations to sections of rat brain indicate that members of the Raw family are expressed in distinct areas of the central nervous system. Probably, Raw channels are expressed predominantly as homomultimers. Immunocytochemical experiments with antibodies against Raw3 and RCK4 proteins which form two distinct A-type potassium channels indicate that in hippocampus the two channels are expressed both in different neurons and in the same ones. In general, properties of Raw potassium channels appeared to be similar to RCK channels. However, Raw outward currents, in contrast to RCK currents, exhibit an intense rectification at test potentials higher than +20 to +40 mV. RCK and Raw channel subunits did not measurably coassemble into RCK/Raw heteromultimers after coinjecting RCK and Raw cRNA into Xenopus oocytes. These results suggest that members of the RCK and the Raw potassium channel families express potassium channels which form independent outward current systems. Combining the results of in situ hybridizations, immunocytochemical staining and expression of the cloned potassium channels in Xenopus oocytes demonstrates that unrestrained mixing of potassium channel subunits to form hybrid channels does not occur in the rat central nervous system. A single neuron is able to express multiple, independently assembled potassium channels.