The Enigma of Norbormide, a Rattus-Selective Toxicant.

Norbormide (NRB) is a Rattus-selective toxicant, which was serendipitously discovered in 1964 and formerly marketed as an eco-friendly rodenticide that was deemed harmless to non-Rattus species. However, due to inconsistent efficacy and the emergence of second-generation anticoagulants, its usage declined, with registration lapsing in 2003. NRBs' lethal action in rats entails irreversible vasoconstriction of peripheral arteries, likely inducing cardiac damage: however, the precise chain of events leading to fatality and the target organs involved remain elusive. This unique contractile effect is exclusive to rat arteries and is induced solely by the endo isomers of NRB, hinting at a specific receptor involvement. Understanding NRB's mechanism of action is crucial for developing species-selective toxicants as alternatives to the broad-spectrum ones currently in use. Recent research efforts have focused on elucidating its cellular mechanisms and sites of action using novel NRB derivatives. The key findings are as follows: NRB selectively opens the rat mitochondrial permeability transition pore, which may be a factor that contributes to its lethal effect; it inhibits rat vascular KATP channels, which potentially controls its Rattus-selective vasoconstricting activity; and it possesses intracellular binding sites in both sensitive and insensitive cells, as revealed by fluorescent derivatives. These studies have led to the development of a prodrug with enhanced pharmacokinetic and toxicological profiles, which is currently undergoing registration as a novel efficacious eco-sustainable Rattus-selective toxicant. The NRB-fluorescent derivatives also show promise as non-toxic probes for intracellular organelle labelling. This review documents in more detail these developments and their implications.

Dynamin-independent CaV1.2 and KCa1.1 channels regulation and vascular tone modulation by the mitochondrial fission inhibitors dynasore and dyngo-4a.

A role for mitochondrial fission in vascular contraction has been proposed based on the vasorelaxant activity of the dynamin (and mitochondrial fission) inhibitors mdivi-1 and dynasore. However, mdivi-1 is capable to inhibit Ba2+ currents through CaV1.2 channels (IBa1.2), stimulate KCa1.1 channel currents (IKCa1.1), and modulate pathways key to the maintenance of vessel active tone in a dynamin-independent manner. Using a multidisciplinary approach, the present study demonstrates that dynasore, like mdivi-1, is a bi-functional vasodilator, blocking IBa1.2 and stimulating IKCa1.1 in rat tail artery myocytes, as well as promoting relaxation of rat aorta rings pre-contracted by either high K+ or phenylephrine. Conversely, its analogue dyngo-4a, though inhibiting mitochondrial fission triggered by phenylephrine and stimulating IKCa1.1, did not affect IBa1.2 but potentiated both high K+- and phenylephrine-induced contractions. Docking and molecular dynamics simulations identified the molecular basis supporting the different activity of dynasore and dyngo-4a at CaV1.2 and KCa1.1 channels. Mito-tempol only partially counteracted the effects of dynasore and dyngo-4a on phenylephrine-induced tone. In conclusion, the present data, along with previous observations (Ahmed et al., 2022) rise caution for the use of dynasore, mdivi-1, and dyngo-4a as tools to investigate the role of mitochondrial fission in vascular contraction: to this end, a selective dynamin inhibitor and/or a different experimental approach are needed.

The drp-1-mediated mitochondrial fission inhibitor mdivi-1 impacts the function of ion channels and pathways underpinning vascular smooth muscle tone.

Mdivi-1 is widely used as a pharmacological tool to inhibit dynamin-related protein-1-mediated mitochondrial fission. Whether this compound may interact directly or indirectly with ion channels or cellular pathways fundamental for the regulation of vascular smooth muscle tone remains unknown. The present study aimed to assess the effect of mdivi-1 on CaV1.2 and KCa1.1 channels, both in vitro and in silico as well as on the mechanical activity of rat aorta rings. Mdivi-1 was an effective CaV1.2 channel blocker, docking in a CaV1.2 channel antagonist binding region, stimulated KCa1.1 channel current, binding to a sensing region common to other stimulators, and possibly inhibited the Rho-kinase pathway. These effects contributed to its vasorelaxant activity observed in rings stimulated with high KCl, phenylephrine, or NaF. Neither structurally different dynamin inhibitors nor a stimulator affected the Ca2+ antagonistic and vasorelaxant activities of the compound. However, mito-tempol reduced its vasorelaxant potency towards phenylephrine. Finally, mdivi-1 antagonized mitochondrial fission triggered by phenylephrine. In conclusion, mdivi-1 is an effective in vitro vasorelaxant agent at concentrations routinely employed to block dynamin-related protein-1. Ion channels and pathways key to the maintenance of vessel active tone are involved in this mechanism. These yet undiscovered off-target effects raise caution for the interpretation of mitochondrial fission signalling.

Nitrobenzoxadiazole derivatives of the rat selective toxicant norbormide as fluorescent probes for live cell imaging.

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats, but relatively harmless to other rodents and mammals. As a vasoactive agent, NRB induces a species-specific vasocontractile effect that is restricted to the peripheral arteries of the rat. Despite the precise mechanisms behind this phenomenon having yet to be fully clarified, it is postulated that the molecular target of NRB could be located within the plasma membrane of rat peripheral artery myocytes (e.g. rat caudal artery myocytes). As such, the primary objective of this study was to develop a fluorescently labelled derivative of NRB to investigate its subcellular distribution/localization in both NRB-sensitive (freshly isolated rat caudal artery myocytes, FIRCAMs) and NRB-insensitive (human hepatic stellate, LX2) cells. Of the examples prepared, lead structure endo-NRB-NBD-bPA subsequently demonstrated retention of the parent toxicant's pharmacological profile (in terms of its ability to induce both a vasocontractile response in rat caudal artery rings in vitro, and a lethal end-point in rats in vivo). Endo-NRB-NBD-bPA was also shown to be significantly less permeable (an integral feature in the design of fluorescent probes targeting cell-surface receptors) to both LX2 cells and FIRCAMs. Disappointingly, no fluorescence could be observed on the plasma membrane of FIRCAMs stained with endo-NRB-NBD-bPA.

Norbormide-Based Probes and Their Application for Mitochondrial Imaging in Drosophila Melanogaster.

Fluorescent live imaging on Drosophila melanogaster is a microscopy technique in rapid expansion. The growing number of probes available to detect cellular components and the relatively easy genetic manipulation of fruit fly make this model one of the most used for in vivo analysis of several physiological and/or pathological processes. Here we describe the chemical synthesis of two norbormide-derived BODIPY-conjugated fluorescent probes (NRBMC009 and NRBZLW0047). Moreover, we describe the larval dissection method, and subsequent live imaging acquisition. Both probes are able to label mitochondria in different Drosophila larval tissues, which allows for the characterization of mitochondrial morphological alterations by using a simple and quick method that avoids the fixation artefacts that often occur in immunofluorescence studies.

Ritanserin blocks CaV1.2 channels in rat artery smooth muscles: electrophysiological, functional, and computational studies.

CaV1.2 channel blockers or 5-HT2 receptor antagonists constitute effective therapy for Raynaud's syndrome. A functional link between the inhibition of 5-HT2 receptors and CaV1.2 channel blockade in arterial smooth muscles has been hypothesized. Therefore, the effects of ritanserin, a nonselective 5-HT2 receptor antagonist, on vascular CaV1.2 channels were investigated through electrophysiological, functional, and computational studies. Ritanserin blocked CaV1.2 channel currents (ICa1.2) in a concentration-dependent manner (Kr = 3.61 µM); ICa1.2 inhibition was antagonized by Bay K 8644 and partially reverted upon washout. Conversely, the ritanserin analog ketanserin (100 µM) inhibited ICa1.2 by ~50%. Ritanserin concentration-dependently shifted the voltage dependence of the steady-state inactivation curve to more negative potentials (Ki = 1.58 µM) without affecting the slope of inactivation and the activation curve, and decreased ICa1.2 progressively during repetitive (1 Hz) step depolarizations (use-dependent block). The addition of ritanserin caused the contraction of single myocytes not yet dialyzed with the conventional method. Furthermore, in depolarized rings, ritanserin, and to a lesser extent, ketanserin, caused a concentration-dependent relaxation, which was antagonized by Bay K 8644. Ritanserin and ketanserin were docked at a region of the CaV1.2 α1C subunit nearby that of Bay K 8644; however, only ritanserin and Bay K 8644 formed a hydrogen bond with key residue Tyr-1489. In conclusion, ritanserin caused in vitro vasodilation, accomplished through the blockade of CaV1.2 channels, which was achieved preferentially in the inactivated and/or resting state of the channel. This novel activity encourages the development of ritanserin derivatives for their potential use in the treatment of Raynaud's syndrome.

Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon.

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.

The Selective Rat Toxicant Norbormide Blocks KATP Channels in Smooth Muscle Cells But Not in Insulin-Secreting Cells.

Norbormide is a toxicant selective for rats to which it induces a widespread vasoconstriction. In a recent paper, we hypothesized a role of ATP-sensitive potassium (KATP) channels in norbormide-induced vasoconstriction. The current study was undertaken to verify this hypothesis by comparing the effects of norbormide with those of glibenclamide, a known KATP channel blocker. The whole-cell patch-clamp method was used to record KATP currents in myocytes freshly isolated from the rat and mouse caudal artery and from the rat gastric fundus, as well as in insulin-secreting pancreatic beta cells (INS-1 cells). Smooth muscle contractile function was assessed on either rat caudal artery rings or gastric fundus strips. Molecular modeling and docking simulation to KATP channel proteins were investigated in silico. Both norbormide (a racemic mixture of endo and exo isomers) and glibenclamide inhibited KATP currents in rat and mouse caudal artery myocytes, as well as in gastric fundus smooth muscle cells. In rat INS-1 cells, only glibenclamide blocked KATP channels, whereas norbormide was ineffective. The inhibitory effect of norbormide in rat caudal artery myocytes was not stereo-specific as both the endo isomers (active as vasoconstrictor) and the exo isomers (inactive as vasoconstrictor) had similar inhibitory activity. In rat caudal artery rings, norbormide-induced contraction was partially reverted by the KATP channel opener pinacidil. Computational approaches indicated the SUR subunit of KATP channels as the binding site for norbormide. KATP channel inhibition may play a role in norbormide-induced vasoconstriction, but does not explain the species selectivity, tissue selectivity, and stereoselectivity of its constricting activity. The lack of effect in INS-1 cells suggests a potential selectivity of norbormide for smooth muscle KATP channels.

Live applications of norbormide-based fluorescent probes in Drosophila melanogaster.

In this study we investigated the performance of two norbormide (NRB)-derived fluorescent probes, NRBMC009 (green) and NRBZLW0047 (red), on dissected, living larvae of Drosophila, to verify their potential application in live cell imaging confocal microscopy. To this end, larval tissues were exposed to NRB probes alone or in combination with other commercial dyes or GFP-tagged protein markers. Both probes were rapidly internalized by most tissues (except the central nervous system) allowing each organ in the microscope field to be readily distinguished at low magnification. At the cellular level, the probes showed a very similar distribution (except for fat bodies), defined by loss of signal in the nucleus and plasma membrane, and a preferential localization to endoplasmic reticulum (ER) and mitochondria. They also recognized ER and mitochondrial phenotypes in the skeletal muscles of fruit fly models that had loss of function mutations in the atlastin and mitofusin genes, suggesting NRBMC009 and NRBZLW0047 as potentially useful screening tools for characterizing ER and mitochondria morphological alterations. Feeding of larvae and adult Drosophilae with the NRB-derived dyes led to staining of the gut and its epithelial cells, revealing a potential role in food intake assays. In addition, when flies were exposed to either dye over their entire life cycle no apparent functional or morphological abnormalities were detected. Rapid internalization, a bright signal, a compatibility with other available fluorescent probes and GFP-tagged protein markers, and a lack of toxicity make NRBZLW0047 and, particularly, NRBMC009 highly performing fluorescent probes for live cell microscopy studies and food intake assays in Drosophila.

Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging.

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12. Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells.

Manipulation of Mitochondria Dynamics Reveals Separate Roles for Form and Function in Mitochondria Distribution.

Mitochondria shape is controlled by membrane fusion and fission mediated by mitofusins, Opa1, and Drp1, whereas mitochondrial motility relies on microtubule motors. These processes govern mitochondria subcellular distribution, whose defects are emphasized in neurons because of their polarized structure. We have studied how perturbation of the fusion/fission balance affects mitochondria distribution in Drosophila axons. Knockdown of Marf or Opa1 resulted in progressive loss of distal mitochondria and in a distinct oxidative phosphorylation and membrane potential deficit. Downregulation of Drp1 rescued the lethality and bioenergetic defect caused by neuronal Marf RNAi, but induced only a modest restoration of axonal mitochondria distribution. Surprisingly, Drp1 knockdown rescued fragmentation and fully restored aberrant distribution of axonal mitochondria produced by Opa1 RNAi; however, Drp1 knockdown did not improve viability or mitochondria function. Our data show that proper morphology is critical for proper axonal mitochondria distribution independent of bioenergetic efficiency. The health of neurons largely depends on mitochondria function, but does not depend on shape or distribution.

Preliminary studies of berberine and its semi-synthetic derivatives as a promising class of multi-target anti-parkinson agents.

Parkinson's disease (PD) is a neurodegenerative disorder bearing motor and nonmotor symptoms. The treatment today is symptomatical rather than preventive or curative and this leaves the field open for the search of both novel molecular targets and drug candidates. Interference with α-synuclein fibrillation, monoamine oxidase (MAO) inhibition, modulation of adenosine receptors and the inhibition of specific phosphodiesterase (PDE) isoforms are some of the currently pursued strategies. We synthesised and studied some semi-synthetic berberine derivatives using a set of in silico tools. We evaluated their drug-likeness and tested the compounds against a set of target proteins involved in the onset or progression of PD, with a particular attention to MAO-B. Preliminary in vitro assay on MAO-B confirmed our in silico predictions.

Cav1.2 channel current block by the PKA inhibitor H-89 in rat tail artery myocytes via a PKA-independent mechanism: Electrophysiological, functional, and molecular docking studies.

To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca2+ current through Cav1.2 channels [ICa1.2], we have documented a marked capacity of the isoquinoline H-89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the ICa1.2 inhibitory activity of H-89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in-depth analysis of H-89 vascular effects on both ICa1.2 and contractility was undertaken by performing whole-cell patch-clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H-89 inhibited ICa1.2 with a pIC50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6-22 amide or enhanced by the PKA stimulators 6-Bnz-cAMP and 8-Br-cAMP. Inhibition of ICa1.2 by H-89 appeared almost irreversible upon washout, was charge carrier- and voltage-dependent, and antagonised by the Cav1.2 channel agonist (S)-(-)-Bay K 8644. H-89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage-dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration-dependent manner. H-89 docked at the α1C subunit in a pocket region close to that of (S)-(-)-Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr-1489. Finally, both high K+- and (S)-(-)-Bay K 8644-induced contractions of rings were fully reverted by H-89. In conclusion, these results indicate that H-89 inhibited vascular ICa1.2 and, consequently, the contractile function through a PKA-independent mechanism. Therefore, caution is recommended when interpreting experiments where H-89 is used to inhibit vascular smooth muscle PKA.

Isoflavones from Maclura pomifera: structural elucidation and in silico evaluation of their interaction with PDE5.

While osajin and pomiferin are known for their anticancer, antibacterial and antidiabetic properties, scandenone and auriculasin have been proposed as anti-inflammatory and antinociceptive agents. Curiously, these two couples of molecules are, from a chemical point of view, structural isomers which can all be extracted from Maclura pomifera. Although previous works described, separately, the isolation in reasonable amounts of the sole osajin/pomiferin couple or of scandenone/auriculasin, we report the extraction and characterization using direct spectral and chromatographical comparison of the four compounds. 2D NMR allowed to unambiguously assign the correct structures to the isomers. The compounds were screened in silico against PDE5 and their interaction pattern with the protein was compared with that of icarisid II, a natural PDE5 inhibitor.

An NBD Derivative of the Selective Rat Toxicant Norbormide as a New Probe for Living Cell Imaging.

Norbormide (NRB) is a unique compound that acts directly on rat vascular myocytes to trigger a contractile process, through an as yet unknown mechanism, which results in the selective contraction of rat peripheral arteries. To gain insight into the mechanisms involved in NRB rat-selective activity, we investigated the subcellular distribution of NRB-AF12, a nitrobenzoxadiazole (NBD)-derivative of NRB, in living NRB-sensitive and NRB-insensitive cells. In both cell types, NRB-AF12 localized to the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and endosomes; however, in NRB-sensitive cells, the fluorescence also extended to the plasma membrane. NRB-AF12 was rapidly internalized into the cells, could easily be washed out and then reloaded back into the same cells, all with a high degree of reproducibility. Cells exposed for 24 h to NRB-AF12 did not show apparent signs of toxicity, even at concentrations of the dye (10 µM) much higher than those required for fluorescence labeling (500 ηM). The distribution pattern of NRB-AF12 fluorescence was near identical to that of ER-Tracker® (Er-Tr), a fluorescent derivative of glibenclamide, a known KATP channel blocker. Displacement tests did not demonstrate, but at the same time did not rule out the possibility of a common target for ER-Tr, NRB-AF12, NRB, and glibenclamide. On the basis of these results we hypothesize a common target site for NRB-AF12 and ER-Tr, and a similar target profile for NRB and glibenclamide, and propose NRB-AF12 as an alternative fluorescence probe to ER-Tracker. Furthermore, NRB-based fluorescence derivatives could be designed to selectively label single cellular structures.

An intracellular adrenomedullin system reduces IL-6 release via a NF-kB-mediated, cAMP-independent transcriptional mechanism in rat thymic epithelial cells.

Thymic epithelial cells (TECs) play a key role in the regulation of central immune tolerance by expressing autoantigens and eliminating self-reactive T cells. In a previous paper we reported that adrenomedullin (ADM) and its co-receptor protein RAMP2 are located intracellularly in newborn human thymic epithelial cells (TECs). This work has two main aims: (1) to examine the cellular localization of ADM and its receptor in TECs of adult Wistar rats to validate this animal model for the study of the ADM system and its function(s) in thymus; (2) to investigate the potential modulating effect of ADM on the NF-kB pathway, which is involved through the production of cytokines such as IL-6, in the maturation of T-lymphocytes and immunological tolerance. Our results show that, similarly to human newborn TECs, ADM is localized to the cytoplasm of adult rat TECs, and RAMP2 is expressed in the nucleus but not in the plasma membrane. Pretreatment of TECs for 4h with ADM significantly reduced lipopolysaccharide (LPS)-induced release of IL-6 (P<0.001) and expression of the p65 subunit of NF-kB, while doubled the expression of IkBα (P<0.001), the physiological inhibitor of NF-kB nuclear translocation. These effects were not mediated by activation of the cAMP pathway, a signalling cascade that is rapidly activated by ADM in cells that express plasma membrane RAMP2, but were the consequence of a reduction in the transcription of p65 (P<0.001) and an increase in the transcription of IkBα (P<0.05). On the basis of these findings we propose that in rat TECs ADM reduces IL-6 secretion by modulating NF-kB genes transcription through an interaction with a receptor localized to the nucleus. This may partly explain the protective effects of ADM in autoimmune diseases and points to the ADM system of TECs as a novel potential target for immunomodulating drugs.

Fatty Acid-Derived Pro-Toxicants of the Rat Selective Toxicant Norbormide.

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, as an acute vasoactive, NRB has a rapid onset of action which makes it relatively unpalatable to rats, often leading to sublethal uptake and accompanying bait shyness. A series of NRB-derived pro-toxicants (3a - i, 4a - i, and 5a - i) were prepared in an effort to 'mask' this acute response and improve both palatability and efficacy. Their synthesis, in vitro biological evaluation (vasocontractile response in rat vasculature, stability in selected rat media) and palatability/efficacy in Sprague-Dawley, wild Norway, and wild ship rats is described. Most notably, pro-toxicant 3d was revealed to be free of all pre-cleavage vasoconstrictory activity in rat caudal artery and was subsequently demonstrated to release NRB in the presence of rat blood, liver, and pancreatic enzymes. Moreover, it consistently displayed a high level of acceptance by rats in a two-choice bait-palatability and efficacy trial, with accompanying high mortality. On this evidence, fatty acid ester prodrugs would appear to offer a promising platform for the further development of NRB-derived toxicants with enhanced palatability and efficacy profiles.

New Therapeutic Applications of Phosphodiesterase 5 Inhibitors (PDE5-Is).

BACKGROUND: Phosphodiesterase 5 inhibitors (PDE5-Is) sildenafil, vardenafil, tadalafil and the recently approved avanafil represent the first-line choice for both on-demand and chronic treatment of erectile dysfunction (ED). In addition to this, sildenafil and tadalafil, have also been approved for the treatment of pulmonary arterial hypertension. Due to its expression and localization in many tissues, PDE5 and its regulation has been reported to be involved in several other diseases. OBJECTIVE: We aim to provide an updated overview of the emerging therapeutic applications of PDE5-Is besides ED, taking into account the latest ongoing research reports. METHODS: We searched online databases (Pubmed, Reaxys, Scopus) to lay the bases for an accurate, quality criteria-based literature update. We focused our attention on most recent research reports, in particular when supported by pre-clinical and clinical data. RESULTS: The regulation of PDE5 may influence pathological conditions such as, among the others, heart failure, cystic fibrosis, cancer, CNS-related diseases, diabetes and dysfunctions affecting male urinary/reproductive system. CONCLUSION: Sildenafil, vardenafil, tadalafil and the other chemical entities considered PDE5-Is showed overall positive results and significant improvements in the studied disease, thus some discordant results, in particular when comparing pre-clinical and clinical data, have to be pointed out, suggesting that further insights are needed especially to assess the exact molecular pathway underlying.

Semi-synthetic derivatives of natural isoflavones from Maclura pomifera as a novel class of PDE-5A inhibitors.

Natural (iso)flavonoids have been recently reported to inhibit cyclic nucleotide phosphodiesterases (PDEs) and induce vasorelaxation, albeit the results described in the literature are discordant. The cGMP-selective isoform PDE-5A, in particular, represents the target of sildenafil and its analogues in the treatment of erectile dysfunction (ED) and pulmonary hypertension by promoting relaxation in vascular smooth muscle through the activation of the NO/cGMP pathway. We undertook this study to verify if osajin and pomiferin, two natural prenylated isoflavones and major constituents of Maclura pomifera extracts previously investigated for their anticancer, antibacterial and antidiabetic properties, show inhibitory activity on PDE-5A. These two isoflavones were isolated from the plant extracts and then synthetically modified to obtain a set of semi-synthetic derivatives with slight and focused modifications on the natural scaffold. The compounds were at first screened against PDE-5A in vitro and, based on the encouraging results, further tested for their relaxant effect on isolated rat artery rings. Computational docking studies were also carried out to explore the mode of interaction with the target protein. The obtained data were compared to the behaviour of the well-known PDE-5A inhibitor sildenafil. Our results demonstrate that semi-synthetic derivatives of osajin and pomiferin show an inhibitory effect on the isolated enzyme that, for some of the compounds, is accompanied by a vasorelaxant activity. Based on our findings, we propose the here described isoflavones as potential lead compounds for the development, starting from natural scaffolds, of a new class of PDE-5A inhibitors with vasorelaxant properties.

NAD(+)-dependent SIRT1 deactivation has a key role on ischemia-reperfusion-induced apoptosis.

Ischemia-reperfusion (IR) leads to severe organ injury and dysfunction. Sirtuins (SIRTs) are a family of histone deacetylases (HDACs) that require nicotinamide adenine dinucleotide (NAD(+)) for the deacetylation reaction. SIRTs play a major role in counteracting cellular stress and apoptosis. This study aimed to investigate the mechanisms of heart protection against apoptosis by SIRTs and the molecular pathways involved in SIRTs regulation and function in a rat model of IR injury. Hearts of male Wistar-Kyoto rats were subjected to 30-min ischemia followed by reperfusion up to 6h. IR increased cardiomyocyte apoptosis; the cleavage of caspase 3, induced a transient upregulation of SIRT1 and downregulation of SIRT6 expression, but decreased SIRT1 activity and reduced NAD(+) content. IR also increased forkhead box protein O1 (FoxO1) expression and FoxO1 binding to SIRT1 promoter region. Resveratrol restored SIRT1 activity and NAD(+) level by an AMPK-dependent mechanism, reduced cardiomyocyte apoptosis, and attenuated caspase 3 cleavage via heat shock factor-1 deacetylation and heat shock protein (HSP) expression upregulation. Our data show new potential molecular mechanisms of up and downstream regulation of SIRT1 in IR. The interplay among FoxO1, SIRT1, NAD(+), AMPK, HSP, and SIRT6 depicts a complex molecular network that protects the heart from apoptosis during IR and may be susceptible to therapeutic interventions.