Guanabenz Research Articles

In silico screening of GMQ-like compounds reveals guanabenz and sephin1 as new allosteric modulators of acid-sensing ion channel 3

Acid-sensing ion channels (ASICs) are voltage-independent cation channels that detect decreases in extracellular pH. Dysregulation of ASICs underpins a number of pathologies. Of particular interest is ASIC3, which is recognised as a key sensor of acid-induced pain and is important in the establishment of pain arising from inflammatory conditions, such as rheumatoid arthritis. Thus, the identification of new ASIC3 modulators and the mechanistic understanding of how these compounds modulate ASIC3 could be important for the development of new strategies to counteract the detrimental effects of dysregulated ASIC3 activity in inflammation. Here, we report the identification of novel ASIC3 modulators based on the ASIC3 agonist, 2-guanidine-4-methylquinazoline (GMQ). Through a GMQ-guided in silico screening of Food and Drug administration (FDA)-approved drugs, 5 compounds were selected and tested for their modulation of rat ASIC3 (rASIC3) using whole-cell patch-clamp electrophysiology. Of the chosen drugs, guanabenz (GBZ), an α2-adrenoceptor agonist, produced similar effects to GMQ on rASIC3, activating the channel at physiological pH (pH 7.4) and potentiating its response to mild acidic (pH 7) stimuli. Sephin1, a GBZ derivative that lacks α2-adrenoceptor activity, has been proposed to act as a selective inhibitor of a regulatory subunit of the stress-induced protein phosphatase 1 (PPP1R15A) with promising therapeutic potential for the treatment of multiple sclerosis. However, we found that like GBZ, sephin1 activates rASIC3 at pH 7.4 and potentiates its response to acidic stimulation (pH 7), i.e. sephin1 is a novel modulator of rASIC3. Furthermore, docking experiments showed that, like GMQ, GBZ and sephin1 likely interact with the nonproton ligand sensor domain of rASIC3. Overall, these data demonstrate the utility of computational analysis for identifying novel ASIC3 modulators, which can be validated with electrophysiological analysis and may lead to the development of better compounds for targeting ASIC3 in the treatment of inflammatory conditions.

Non‐invasive, needle‐free drug delivery for treatment of retinal degeneration on Bardet‐Biedl syndrome

AbstractPurposeTo develop a non‐invasive drug delivery system based on superparamagnetic nanoparticles (NP) for the treatment of retinal degeneration on Bardet‐Biedl syndrome (BBS). The delivery system will be used to deliver two small molecules, valproic acid (VPA) and Guanabenz (GBZ).MethodsA mixture of two NP (one loaded with VPA and other with GBZ) was applied as eyedrops on 14‐days old mice with the use of a magnetic field. The treatment was assessed in both wild type and a mice model for BBS (Bbs12−/−). The effects of the treatment were evaluated 2 weeks post treatment at 1‐month‐old. Retinal structure and function were analysed using electroretinogram recordings (ERG), histology and transmission electron microscopy (TEM).ResultsIn vitro studies showed no important toxic effects in cell cultures for retinal pigmented epithelium (RPE1) and 661W (murine photoreceptor precursor) after treatment with unloaded NP. Scotopic and photopic ERG recordings showed no toxic effect of the NP on photoreceptor (PR) function. TEM analysis indicated the presence of the NP in the PR layer of the retina after eyedrop application. TEM images showed a decrease in Endoplasmic Reticulum (ER) dilatation. The treated group with loaded NP showed an increase in the thickness of the inner nuclear layer, outer nuclear layer, inner PR segment and outer PR segment.ConclusionsThese results highlight the potential for these NP for non‐invasive drug delivery to the retina. Both the presence of the NP and biological effect were shown in our mouse model. The VPA and GBZ combination has already shown decrease of cell death mediated by ER stress in the Bbs12−/− model. This is an unmet clinical need with no curative treatment for retinal ciliopathies. The possibility of delaying PR death would increase the therapeutic window of gene therapy potentially increasing its effectivity.

Guanabenz Attenuates Acetaminophen-Induced Liver Toxicity and Synergizes Analgesia in Mice.

Endoplasmic reticulum (ER) stress has been shown to be involved in the hepatotoxicity of acetaminophen (APAP). Guanabenz (GA), a widely known antihypertensive drug, is reported to exhibit an anti-ER stress effect. In this study, we investigated the potential of GA as an antidote against APAP-induced hepatotoxicity. The underlying biochemical mechanisms for the hepatoprotective effect of GA were explored. Here we found that treatment of mice with GA (10 mg/kg) before APAP overdose dramatically prevented APAP-induced liver enzyme elevation and resultant toxicity in mice, as indicated by suppression of elevated serum alanine aminotransferase (ALT) levels and liver histological analysis. Importantly, delayed administration of GA within 6 h after APAP overdose also showed an almost equivalent protective effect against APAP liver toxicity. Mechanistically, several pathways are involved in the protective effect of GA against APAP-induced live toxicity, including attenuation of ER stress and oxidative stress, increased levels of nontoxic phase I and II metabolites of APAP, decrease in the formation of toxic N-acetyl-p-benzoquinone imine (NAPQI), and its subsequent protein binding. Importantly, combination of GA with APAP exhibited synergistic interaction in the latter's analgesic activity, while sparing its antipyretic action. These findings provide the preclinical evidence of GA as a promising antidote for treatment of APAP-induced liver toxicity and raise a possibility of its combination with APAP in clinical settings.

PromISR-6, a Guanabenz Analogue, Improves Cellular Survival in an Experimental Model of Huntington's Disease.

Guanabenz (GBZ), an α2-adrenergic agonist, demonstrated off-target effects that restored protein homeostasis and ameliorated pathobiology in experimental models of neurodegenerative disease. However, GBZ did not directly activate the integrated stress response (ISR), and its proposed mode of action remains controversial. Utilizing an iterative in silico screen of over 10,000 GBZ analogues, we analyzed 432 representative compounds for cytotoxicity in Wild-type, PPP1R15A-/-, and PPP1R15B-/- mouse embryonic fibroblasts. Nine compounds clustering into three functional groups were studied in detail using cell biological and biochemical assays. Our studies demonstrated that PromISR-6 is a potent GBZ analogue that selectively activated ISR, eliciting sustained eIF2α phosphorylation. ISRIB, an ISR inhibitor, counteracted PromISR-6-mediated translational inhibition and reduction in intracellular mutant Huntingtin aggregates. Reduced protein synthesis combined with PromISR-6-stimulated autophagic clearance made PromISR-6 the most efficacious GBZ analogue to reduce Huntingtin aggregates and promote survival in a cellular model of Huntington's disease.

Guanabenz Prevents d-Galactosamine/Lipopolysaccharide-Induced Liver Damage and Mortality.

Multi-organ failure in response to uncontrolled microbial infection is characterized by low blood pressure accompanied by a systemic over-inflammation state, caused by massive pro-inflammatory cytokines release and liver damage. Recently, the integrated stress response (ISR), characterized by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, was involved with controlling apoptosis in stressed hepatocytes and associated with poor survival to endotoxin challenge. Lipopolysaccharide (LPS) alone is able to induce the ISR in hepatocytes and can trigger massive liver damage along with tumor necrosis factor-alpha (TNF-α) expression. Consequently, drugs interfering with eIF2α phosphorylation may represent potential candidates for the treatment of such pathologies. We, therefore, used Guanabenz (GBZ), a small compound with enhancing eIF2α phosphorylation activity to evaluate its effect on bacterial LPS sensing and endotoxemia. GBZ is confirmed here to have an anti-inflammatory activity by increasing in vitro interleukin-10 (IL-10) production by LPS-stimulated dendritic cells. We further show that in the d-galactosamine (d-galN)/LPS-dependent lethality model, intraperitoneal injection of GBZ promoted mice survival, prevented liver damage, increased IL-10 levels, and inhibited TNF-α production. GBZ and its derivatives could therefore represent an interesting pharmacological solution to control systemic inflammation and associated acute liver failure.

Structure-activity relationship study around guanabenz identifies two derivatives retaining antiprion activity but having lost α2-adrenergic receptor agonistic activity.

Guanabenz (GA) is an orally active α2-adrenergic agonist that has been used for many years for the treatment of hypertension. We recently described that GA is also active against both yeast and mammalian prions in an α2-adrenergic receptor-independent manner. These data suggest that this side-activity of GA could be explored for the treatment of prion-based diseases and other amyloid-based disorders. In this perspective, the potent antihypertensive activity of GA happens to be an annoying side-effect that could limit its use. In order to get rid of GA agonist activity at α2-adrenergic receptors, we performed a structure-activity relationship study around GA based on changes of the chlorine positions on the benzene moiety and then on the modifications of the guanidine group. Hence, we identified the two derivatives 6 and 7 that still possess a potent antiprion activity but were totally devoid of any agonist activity at α2-adrenergic receptors. Similarly to GA, 6 and 7 were also able to inhibit the protein folding activity of the ribosome (PFAR) which has been suggested to be involved in prion appearance/maintenance. Therefore, these two GA derivatives are worth being considered as drug candidates.

Inhibitors of eIF2α Dephosphorylation Slow Replication and Stabilize Latency in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that permanently infects warm-blooded vertebrates through its ability to convert into a latent tissue cyst form. The latent form (bradyzoite) can reinitiate a life-threatening acute infection if host immunity wanes, most commonly in AIDS or organ transplant patients. We have previously shown that bradyzoite development is accompanied by phosphorylation of the parasite eukaryotic initiation factor 2 alpha subunit (eIF2α), which dampens global protein synthesis and reprograms gene expression. In this study, we analyzed the activities of two specific inhibitors of eIF2α dephosphorylation, salubrinal (SAL) and guanabenz (GA). We establish that these drugs are able to inhibit the dephosphorylation of Toxoplasma eIF2α. Our results show that SAL and GA reduce tachyzoite replication in vitro and in vivo. Furthermore, both drugs induce bradyzoite formation and inhibit the reactivation of latent bradyzoites in vitro. To address whether the antiparasitic activities of SAL and GA involve host eIF2α phosphorylation, we infected mutant mouse embryonic fibroblast (MEF) cells incapable of phosphorylating eIF2α, which had no impact on the efficacies of SAL and GA against Toxoplasma infection. Our findings suggest that SAL and GA may serve as potential new drugs for the treatment of acute and chronic toxoplasmosis.

Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding

The ribosome, the protein synthesis machinery of the cell, has also been implicated in protein folding. This activity resides within the domain V of the main RNA component of the large subunit of the ribosome. It has been shown that two antiprion drugs 6-aminophenanthridine (6AP) and Guanabenz (GA) bind to the ribosomal RNA and inhibit specifically the protein folding activity of the ribosome. Here, we have characterized with biochemical experiments, the mode of inhibition of these two drugs using ribosomes or ribosomal components active in protein folding (referred to as ’ribosomal folding modulators’ or RFMs) from both bacteria Escherichia coli and yeast Saccharomyces cerevisiae, and human carbonic anhydrase (HCA) as a sample protein. Our results indicate that 6AP and GA inhibit the protein folding activity of the ribosome by competition with the unfolded protein for binding to the ribosome. As a result, the yield of the refolded protein decreases, but the rate of its refolding remains unaffected. Further, 6AP- and GA mediated inhibition of RFM mediated refolding can be reversed by the addition of RFMs in excess. We also demonstrate with delayed addition of the ribosome and the antiprion drugs that there is a short time-span in the range of seconds within which the ribosome interacts with the unfolded protein. Thus we conclude that the protein folding activity of the ribosome is conserved from bacteria to eukaryotes and most likely the substrate for RFMs is an early refolding state of the target protein.

Tools for the study of ribosome‐borne protein folding activity

In addition to its role in protein synthesis, which involves a peptidyl transferase activity, the ribosome has also been described to be able to assist protein folding, at least in vitro, as presented in a Research Highlight (Das, et al., Biotechnol. J. 2008). This in vitro-described ribosome-borne protein folding activity (RPFA) is yet poorly characterized in vivo, in part because of the lack of tools to study its biological significance. There is substantial evidence documenting RPFA in vitro, and an assay intended to detect this activity in vivo has been set up in bacteria, but this assay is indirect. In this review, we describe the different tools and tests currently available to study RPFA. We put a special emphasis on the various available inhibitors of this activity and in particular, we discuss the use of 6-aminophenanthridine (6AP) and guanabenz (GA), two antiprion drugs that were very recently shown to specifically inhibit RPFA in vitro without any significant effect on the activity of the ribosome in protein synthesis. Therefore, these drugs should allow determining the potential biological role of RPFA. Importantly, the biological activity of 6AP and GA suggest a possible involvement of RPFA in human proteinopathies.

Antihypertensive Drug Guanabenz Is Active In Vivo against both Yeast and Mammalian Prions

BackgroundPrion-based diseases are incurable transmissible neurodegenerative disorders affecting animals and humans.Methodology/Principal FindingsHere we report the discovery of the in vivo antiprion activity of Guanabenz (GA), an agonist of α2-adrenergic receptors routinely used in human medicine as an antihypertensive drug. We isolated GA in a screen for drugs active in vivo against two different yeast prions using a previously described yeast-based two steps assay. GA was then shown to promote ovine PrPSc clearance in a cell-based assay. These effects are very specific as evidenced by the lack of activity of some GA analogues that we generated. GA antiprion activity does not involve its agonist activity on α2-adrenergic receptors as other chemically close anti-hypertensive agents possessing related mechanism of action were found inactive against prions. Finally, GA showed activity in a transgenic mouse-based in vivo assay for ovine prion propagation, prolonging slightly but significantly the survival of treated animals.Conclusion/SignificanceGA thus adds to the short list of compounds active in vivo in animal models for the treatment of prion-based diseases. Because it has been administrated for many years to treat hypertension on a daily basis, without major side-effects, our results suggest that it could be evaluated in human as a potential treatment for prion-based diseases.

Enhanced Sympathoexcitatory and Pressor Responses to Chronic Central Sodium Loading in Dahl Salt-Sensitive Vs. -Resistent Rats

P46 Increased responsiveness to increases in CSF Na + by high salt intake may contribute to salt-sensitive hypertension in Dahl S rats. To test this hypothesis, Dahl S and R rats received a chronic icv infusion of artificial CSF (aCSF) or aCSF containing 0.8 M Na + (5 μl/hr) for 14 days, using osmotic minipumps. On day 14 in conscious rats, CSF was sampled and BP, HR and renal sympathetic nerve activity (RSNA) were recorded at rest and in response to air stress, icv α 2 -adrenoceptor agonist guanabenz (50 μg), icv ouabain (0.6 μg), and iv phenylephrine and nitropusside to estimate baroreflex function. Data are mean±SEM (n=8-13). *p + caused more severe hypertension, larger responses to air stress and guanabenz, and more blunting of the arterial baroreflex gain in S vs R rats. These findings suggest that enhanced neuronal responsiveness to CSF Na + is one of the mechanisms contributing to salt-sensitive hypertension in Dahl S rats.

Effect of NG-monomethyl-L-arginine on arcade arterioles of rat spinotrapezius muscles

The effect of the specific inhibitor of nitric oxide (NO) formation NG-monomethyl-L-arginine (L-NMMA) on resting arteriolar diameter and on actions of both endothelium-dependent and -independent vasoactive substances was investigated using intravital microscopy in rats. The spinotrapezius muscle of anaesthetized normotensive rats was suspended in a Krebs-Henseleit bath containing tetrodotoxin (3 x 10(-7) M), indomethacin (2.8 x 10(-5) M), and propranolol (10(-6) M) to block sympathetic nerve conduction, prostacyclin formation, and beta-adrenergic receptors, respectively. Acetylcholine (ACh), nitroprusside (NP), norepinephrine (NE), phenylephrine (PE), and guanabenz (GB) were topically applied before and after superfusion of L-NMMA (10(-5) to 10(-4) M). Superfusion of L-NMMA reduced arteriolar diameter and caused dose-dependent increases in arteriolar tone. The onset of action of L-NMMA was nearly immediate. L-NMMA inhibited vasodilator responses to the endothelium-dependent vasodilator ACh but not to the endothelium-independent NP. NE induced dose-related vasoconstriction that was significantly potentiated by L-NMMA. These effects were partially reversed by addition of L-arginine (10(-3) M). Potentiation of vasoconstriction elicited by NE was still observed after inhibition of alpha 1-adrenoceptors with prazosin, but potentiation was abolished by the alpha 2-adrenoceptor antagonist yohimbine. L-NMMA potentiated arteriolar vasoconstriction elicited by the alpha 2-adrenergic receptor agonist GB but not by the alpha 1-adrenoceptor agonist PE. These findings with L-NMMA suggest that resting diameter of arterioles is modulated by endogenous NO biosynthesis and that endothelium-dependent vasodilators act through the formation of endogenous NO to exert their action in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

O-059 - Influence of guanabenz(GB) on dextran-induced paw edema and histamine release from isolated peritoneal mast cells in rats

O-059 - Influence of guanabenz(GB) on dextran-induced paw edema and histamine release from isolated peritoneal mast cells in rats

Different pharmacological interaction of clonidine and guanabenz with antidepressive drugs.

A study was made of the effect of several antidepressive drugs with different mechanisms of action on cardiovascular response induced by clonidine (CLO) or guanabenz (GUA) in anaesthetized normotensive rats. The acute pretreatment (1 mg.kg-1) with desipramine (DMI), maprotiline (MAP) and mianserin (MIA) reduced the hypotensive and bradycardiac responses induced by CLO (10 micrograms.kg-1), while only MIA prevented these central actions when elicited by GUA (30 micrograms.kg-1). Only chronic treatment with DMI reduced the hypotensive effect of CLO, but none of long-term antidepressant administrations decreased the fall in blood pressure induced by GUA. The long-term administration of DMI, MAP and MIA abolished the bradycardia from both agonists, CLO and GUA. The different pharmacological interaction of CLO and GUA with (acute or chronic) antidepressive treatments could be explained by a structural difference between the two agonists, and also by differences in localization and/or subtype of the alpha-adrenoceptors involved in their central cardiovascular effects.

β-adrenergic activity and conformation of the antihypertensive specific α 2-agonist drug, guanabenz

In recent research a new series of specific drugs, one of which is guanabenz (GBZ, 2,6(di-chlorobenzyliden)-aminoguanidine)† has been introduced into the clinical treatment of centrally mediated hypertension. Guanabenz (GBZ) is considered to be among the most specific α 2-adrenergic agonists, acting similarly to clonidine by decreasing the sympathetic outflow from the brain to the peripheral circulatory system. In the present report we show that GBZ displays a significant affinity for β-adrenoceptors. In displacement studies of the iodinated β-antagonist [ 125I]cyanopindolol (CYP) from turkey erythrocyte membranes, the dissociation constant of GBZ was 3.8 μM. Inhibition of the (−) epinephrine induced adenylate cyclase activity by GBZ is competitive, with an apparent dissociation constant of 30 μM. A similar value was obtained by studies of GBZ's effect on the (−)epinephrine-induced [ 3H]cAMP accumulation in intact turkey erythrocytes. In view of its unexpected affinity for β-adrenoceptors, we examined the three-dimensional structure of crystalline GBZ. In these studies substantial differences between clonidine and GBZ were observed, despite their strong structural resemblance. These dissimilarities (angle of rotation φ = 39.7° as compared to 76° in clonidine, and the rotational restriction of clonidine as compared to the greater mobility in rotation of GBZ) could explain the difference of specificity between these two compounds.