To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge.

cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

Pulmonary arterial hypertension (PAH) is the most characteristic group of pulmonary hypertensive states. Among other conditions, PAH includes the idiopathic type and forms associated with connective tissue diseases (CTDs), particularly with systemic sclerosis, CREST syndrome, and mixed CTD. Pulmonary hypertension (PH) can be detected by transthoracic Doppler echocardiography. This procedure should be performed routinely once a year in patients with CTD with high risk to develop PAH. The diagnosis of PH needs to be confirmed by right heart catheterization. Recently developed drugs that modulate endothelial function have provided encouraging results in the treatment of PAH with improvement in symptoms, exercise tolerance, and survival.

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Pulmonary Arterial Hypertension

As a chemical transmitter in the mammalian central nervous system, nitric oxide (NO) is still thought a bit of an oddity, yet this role extends back to the beginnings of the evolution of the nervous system, predating many of the more familiar neurotransmitters. During the 20 years since it became known, evidence has accumulated for NO subserving an increasing number of functions in the mammalian central nervous system, as anticipated from the wide distribution of its synthetic and signal transduction machinery within it. This review attempts to probe beneath those functions and consider the cellular and molecular mechanisms through which NO evokes short- and long-term modifications in neural performance. With any transmitter, understanding its receptors is vital for decoding the language of communication. The receptor proteins specialised to detect NO are coupled to cGMP formation and provide an astonishing degree of amplification of even brief, low amplitude NO signals. Emphasis is given to the diverse ways in which NO receptor activation initiates changes in neuronal excitability and synaptic strength by acting at pre- and/or postsynaptic locations. Signalling to non-neuronal cells and an unexpected line of communication between endothelial cells and brain cells are also covered. Viewed from a mechanistic perspective, NO conforms to many of the rules governing more conventional neurotransmission, particularly of the metabotropic type, but stands out as being more economical and versatile, attributes that presumably account for its spectacular evolutionary success.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1460-9568.2008.06285.x

Concepts of neural nitric oxide‐mediated transmission

Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766).

/pdf/no-independent-stimulators-and-activators-of-soluble-au2xwbbp41.pdf

NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential

The contamination of foods and feed with mycotoxins is a commonly known problem. Intense investigations have been conducted to study the occurrence, toxicity, and recently also the prevention and detoxification strategies of mycotoxins in human and animal food chains. Most of the studies have emphasized on "traditional" mycotoxins, such as aflatoxins, ochratoxin A, and trichothecenes. However, one of the most common grain-contaminating genus of fungi, Fusarium spp., is also capable of producing other toxic secondary metabolites - the so-called emerging mycotoxins such as fusaproliferin, beauvericin, enniatins, and moniliformin. So far, only limited data is available on these metabolites. This is not only due to their late recognition but especially the late understanding of their role as mycotoxins. This paper summarizes the existing data on the chemistry, analytical techniques, biosynthesis, production, toxicity, and occurrence data on fusaproliferin, beauvericin, enniatins, and moniliformin. Based on the available studies, attention should be paid to the studies on the distinct significance of these compounds in the human and animal food chains.

Emerging Fusarium -Mycotoxins Fusaproliferin, Beauvericin, Enniatins, And Moniliformin—A Review

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.

https://bpspubs.onlinelibrary.wiley.com/doi/pdf/10.1038/sj.bjp.0704778

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

For receptor binding studies and the elucidation of the mode of action of the potent anthelmintic compound JES 1798 a tritium labeled compound at very high specific activity was necessary. Tritium was introduced by methylation of the N-demethyl precursor JES 2314. The identity of [N-methyl- 3 H]JES 1798 was determined by mass spectrometry. After synthesis and purification, 535 μg [N-methyl- 3 H]JES 1798 were available at a specific activity of 84 Ci/mmol (3.11 TBq/mmol) as determined by mass spectrometry. The total activity was 80 mCi (2.96 GBq). Radiolabeled JES 1798 showed an efficient and specific binding to a membrane fraction from Ascaris suum. Displacement by unlabeled JES 1798 was half-maximal at about 0.72 ± 0.06 μM. Different known enniatins also competed for the [ 3 H]JES 1798-binding in the Ascaris suum membrane preparation. In vitro comparison of JES 1798 with enniatin A, A 1 , B and B 1 or beauvericin revealed that enniatin A showed an anthelmintic activity against Nippostrongylus brasiliensis, Trichinella spiralis and Heterakis spurnosa at a concentration of 5 μg/ml, whereas enniatins A 1 , B and B 1 had an activity at concentrations between 1 and 100 μg/ml. On the other hand beauvericin and JES 1798 exerted their anthelmintic activities at 100 μg/ml and therefore possess minor anthelmintic potency in vitro as compared to the natural occurring enniatins

Synthesis of a radiolabeled enniatin cyclodepsipeptide [3H‐methyl]JES 1798

For receptor binding studies and the elucidation of the mode of action of the potent anthelmintic compound PF1022A a tritium labeled compound with very high specific activity was necessary. Tritium was introduced into the compound by methylation of the [bis-N-demethyl]precursor of PF1022A (PF1022-219). The identity of [bis-N-methyl- 3 H]PF1022A was determined by LC/MS. After synthesis and purification, 88.9 μg [bis-N-methyl- 3 H]PF1022A were available showing a specific activity of 162 Ci/mmol (5,99 TBq/mmol) determined by mass spectrometry. The total activity was 15 mCi (555 MBq). Radiolabeled PF1022A showed an efficient and specific binding to a membrane fraction from Ascaris suum. Displacement by unlabeled PF1022A was half-maximal at about 40 nM. At 100-fold higher concentrations the biologically much less effective optical antipodean (PF1022-001) competed for maximal 40% of the [ 3 H]PF1022A-binding in the Ascaris suum membrane preparation. In-vitro comparison of PF1022A with its optical antipodean revealed a more than 100-fold higher anthelmintic activity of PF1022A against Heterakis spumova, Nippostrongylus brasiliensis and Trichinella spiralis.

Synthesis of a radiolabeled cyclodepsipeptide [3H-methyl]PF1022A

Nexavar™, Sorafenib tosylate (BAY 43-9006 tosylate) is a potent small molecule Raf kinase inhibitor for the treatment of hyperproliferative disorders such as cancer. Both radiolabeled and stable isotope labeled compounds were required for drug absorption, distribution, metabolism and excretion (ADME) and quantitative mass spectrometry bio-analytical studies. Nexavar™ labeled with carbon-14 in the carboxamide group was prepared in two steps in an overall radiochemical yield of 42% starting from 4-chloro-N-methyl-2-pyridine-[14C]carboxamide. The [2H3,15N] version of Nexavar™ was prepared in 75% yield based on 4-chloro-N-[2H3]methyl-2-pyridine-[15N]carboxamide. The pyridine N-oxide metabolite labeled with carbon-14 as well as with deuterium and nitrogen-15 and was synthesized by oxidation in yields of 59% and 87%, respectively. Starting from [2H2, 13C]formaldehyde the N-hydroxymethyl metabolite was labeled with carbon-13 and deuterium in one step in a 45% overall yield. Copyright © 2006 John Wiley & Sons, Ltd.

Syntheses of [2H3, 15N], [14C]Nexavar™ and its labeled metabolites

Tritium was introduced into the new orally active, selective phosphodiesterase type V (PDE V) inhibitor vardenafil (Levitra®), by reduction of a suitable amide precursor with freshly prepared lithium aluminum tritide. A specific activity of 52.7 Ci/mmol (1.95 TBq/mmol) was achieved. In order to overcome the usual technical difficulties during the preparation of complex tritides a new and easy labeling technique which has considerable potential for various tritia-tion procedures, was developed. Copyright © 2003 John Wiley & Sons, Ltd.

Ulrich Pleiss

Papers

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

Synthesis of a radiolabeled enniatin cyclodepsipeptide [3H‐methyl]JES 1798

Synthesis of a radiolabeled cyclodepsipeptide [3H-methyl]PF1022A

Syntheses of [2H3, 15N], [14C]Nexavar™ and its labeled metabolites

Synthesis of [3H] vardenafil, Levitra®, using a new labeling technique