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Limited therapeutic efficacy of temozolomide (TMZ) against glioblastomas highlights the importance of exploring new drugs for clinical therapy. Sunitinib, a multitargeted receptor tyrosine kinase inhibitor, is currently being tested as therapy for glioblastomas. Unfortunately, sunitinib still has insufficient activity to cure glioblastomas. Our aim was to determine the molecular mechanisms counteracting sunitinib drug sensitivity and find potential adjuvant drugs for glioblastoma therapy. Through in vitro experiments, transcriptome screening by RNA sequencing, and in silico analyses, we found that sunitinib induced glioma apoptotic death, and downregulated genes were enriched in oncogenic genes of glioblastoma. Meanwhile, sunitinib-upregulated genes were highly associated with the protective autophagy process. Blockade of autophagy significantly enhanced sunitinib's cytotoxicity. Growth arrest and DNA damage-inducible protein (GADD) 34 was identified as a candidate involved in sunitinib-promoted autophagy through activating p38-mitogen-activated protein kinase (MAPK) signaling. Higher GADD34 levels predicted poor survival of glioblastoma patients and induced autophagy formation in desensitizing sunitinib cytotoxicity. Guanabenz, an alpha2-selective adrenergic agonist and GADD34 functional inhibitor, was identified to enhance the efficacy of sunitinib by targeting GADD34-induced protective autophagy in glioblastoma cells, TMZ-resistant cells, hypoxic cultured cells, sphere-forming cells, and colony formation abilities. A better combined treatment effect with sunitinib and guanabenz was also observed by using xenograft mice. Taken together, the sunitinib therapy combined with guanabenz in the inhibition of GADD34-enhanced protective autophagy may provide a new therapeutic strategy for glioblastoma. [ABSTRACT FROM AUTHOR]

Alpha 2 (α 2 -) adrenoceptor agonists, such as clonidine or dexmedetomidine, have been found to inhibit hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels, not only by reducing intracellular cyclic AMP levels but also by directly blocking HCN channels. In this study, we examined the inhibitory effect of guanabenz, a centrally acting α 2 -adrenoceptor agonist with high specificity for α 2A -subtype, on HCN channels in mesencephalic trigeminal nucleus (MTN) neurons which robustly express HCN channels and have been suggested to coexpress α 2A -adrenoceptors. By performing whole-cell patch-clamp recording on MTN neurons in brainstem slices, hyperpolarization-activated inward current (I h) was examined during guanabenz treatment. Guanabenz inhibited I h in a dose-dependent manner, which was likely to be ZD7288-sensitive HCN current as it did not affect barium-sensitive inward rectifying potassium current. Guanabenz not only inhibited I h but also shifted the voltage-dependent activation curve to hyperpolarizing potentials. Interestingly, I h inhibition by guanabenz was not reversed by α 2 -adrenoceptor antagonist atipamezole treatment or by intracellular cyclic AMP perfusion, suggesting that the inhibition may not result from α 2A -adrenoceptor signalling pathway but from direct inhibition of HCN channels. Coherent to our electrophysiological results, single-cell RT-PCR revealed that most MTN neurons lack α 2A -adrenoceptor mRNA. Our study demonstrates that guanabenz can directly inhibit HCN channels in addition to its primary role of activating α 2A -adrenoceptors. [ABSTRACT FROM AUTHOR]

Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N -(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection ( Fraser et al., 2014b ). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R–like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2α in DENV infection using guanabenz, a specific inhibitor of eIF2α dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic. [ABSTRACT FROM AUTHOR]

Inactivating bone-resorbing osteoclasts is a prime therapeutic strategy for the prevention of bone loss in patients with osteopenia and osteoporosis. Synthetic agents such as salubrinal and guanabenz, which attenuate stress to the endoplasmic reticulum, are reported to inhibit development of osteoclasts. However, the mechanism of their inhibitory action on osteoclasts is largely unknown. Using genome-wide expression profiles, we predicted key transcription factors that downregulated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis. Principal component analysis (PCA) predicted a list of transcription factors that were potentially responsible for reversing receptor activator of nuclear factor kappa-B ligand (RANKL)-driven stimulation of osteoclastogenesis. A partial silencing of NFATc1 allowed a selection of transcription factors that were likely to be located upstream of NFATc1. We validated the predicted transcription factors by focusing on two AP-1 transcription factors (c-Fos and JunB) using RAW264.7 pre-osteoclasts as well as primary bone marrow cells. As predicted, their mRNA and protein levels were elevated by RANKL, and the elevation was suppressed by salubrinal and guanabenz. A partial silencing of c-Fos or JunB by RNA interference decreased NFATc1 as well as tartrate-resistant acid phosphatase (TRAP) mRNA. Collectively, a systems-biology approach allows the prediction of a RANKL-salubrinal/guanabenz-NFATc1 regulatory axis, and in vitro assays validate an involvement of AP-1 transcription factors in suppression of osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative disease characterized by the loss of motor neurons in the motor cortex, brain stem and spinal cord. Currently, there is no cure for this lethal disease. Although the mechanism underlying neuronal cell death in ALS remains elusive, growing evidence supports a crucial role of endoplasmic reticulum (ER) stress in the pathogenesis of ALS. Recent reports show that guanabenz, a novel inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, possesses anti-prion properties, attenuates ER stress and reduces paralysis and neurodegeneration in mTDP-43 Caenorhabditis elegans and Danio rerio models of ALS. However, the therapeutic potential of guanabenz for the treatment of ALS has not yet been assessed in a mouse model of ALS. In the present study, guanabenz was administered to a widely used mouse model of ALS expressing copper zinc superoxide dismutase-1 (SOD1) with a glycine to alanine mutation at position 93 (G93A). The results showed that the administration of guanabenz significantly extended the lifespan, delayed the onset of disease symptoms, improved motor performance and attenuated motor neuron loss in female SOD1 G93A mice. Moreover, western blotting results revealed that guanabenz dramatically increased the levels of phosphorylated-eIF2α (P-eIF2α) protein, without affecting total eIF2α protein levels. The results also revealed a significant decrease in the levels of the ER chaperone glucose-regulated protein 78 (BiP/Grp78) and markers of another two ER stress pathways, activating transcription factor 6α (ATF6α) and inositol-requiring enzyme 1 (IRE1). In addition, guanabenz increased the protein levels of anti-apoptotic B cell lymphoma/lewkmia-2 (Bcl-2), and down-regulated the pro-apoptotic protein levels of C/EBP homologous protein (CHOP), Bcl-2-associated X protein (BAX) and cytochrome C in SOD1 G93A mice. Our findings indicate that guanabenz may represent a novel therapeutic candidate for the treatment of ALS, a lethal human disease with an underlying mechanism involving the attenuation of ER stress and mitochondrial stress via prolonging eIF2α phosphorylation. [ABSTRACT FROM AUTHOR]

Abstract: Increased central sympathetic outflow secondary to afferent sympathetic excitation has been implicated in the hypertensive effect of the immunosuppressant drug cyclosporine (CSA). The present study investigated the roles of central α2-adrenoceptors and I1-imidazoline receptors in modulating the hypertensive action of CSA. The blood pressure (BP) response to CSA in conscious rats was assessed in the absence and presence of peripherally or centrally acting sympatholytic drugs. Also, the effect of selective pharmacologic blockade of α2 or I1 receptors by yohimbine and efaroxan, respectively, on the pressor response to CSA was evaluated. CSA (20 mg/kg i.v.) produced a rapid increase in BP that peaked (25±4 mm Hg) after approximately 4 min and continued for the 45 min study duration. Ganglionic (hexamethonium 20 mg/kg) or α1-adrenoceptor (prazosin 1 mg/kg) blockade reduced the pressor effect of CSA. Pressor responses to phenylephrine (α1-adrenoceptor agonist) were not affected by CSA, thereby eliminating a possible role for alterations of vascular α1-adrenoceptor responsiveness in CSA hypertension. CSA hypertension was attenuated in rats pretreated intravenously with drugs that reduce central sympathetic tone including clonidine (mixed α2/I1-receptor agonist, 30 μg/kg) or moxonidine (selective I1-receptor agonist, 100 μg/kg) in contrast to no effect for guanabenz (selective α2-receptor agonist, 30 μg/kg). Intracisternal (i.c.) administration of moxonidine also reduced CSA hypertension. Selective blockade of central I1 (efaroxan, 0.15 μg/rat, i.c.) but not α2 (yohimbine, 25 μg/5 μl/rat, i.c.) receptors abolished the hypertensive response to CSA. Together, these findings highlight that CSA elicits its hypertensive effect via disruption of central sympathoinhibitory pathways which include I1-imidazoline receptors. [Copyright &y& Elsevier]

Abstract: In cystic fibrosis respiratory epithelial cells, the absence or dysfunction of the chloride channel CFTR (Cystic Fibrosis Transmembrane conductance Regulator) results in reduced chloride ion transport. In contrast, Ca2+-stimulated Cl− secretion is intact in cystic fibrosis airway epithelia. One possible target for drug discovery aiming at treating cystic fibrosis is to correct the ionic imbalance through stimulation of alternative ionic pathways that may compensate the failure of epithelial Cl− conductance. Here, using a simple high-throughput screening assay to search for Cl− channels modulators in the cystic fibrosis nasal epithelial cell line JME-CF15, the compound guanabenz (Wytensin®), an α2-selective adrenergic agonist was found positive. Using iodide effluxes and electrophysiological recordings, we showed that guanabenz-activated (EC50 =831 nM) a DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) sensitive and Ca2+ dependent Cl− channel (CaCC). Guanabenz activated a linear Cl− channel with unitary single-channel conductance of 8 pS. Recording calcium signals in CF15 cells showed that guanabenz increased the intracellular Ca2+ concentration stimulating an influx of Ca2+. In the absence of extracellular Ca2+, the guanabenz effects on Ca2+ influx and activation of CaCC were both abolished. These data demonstrate that guanabenz activates Ca2+-dependent Cl− channels via a Ca2+ influx in human cystic fibrosis airway epithelial cells. [Copyright &y& Elsevier]

The activation of α2-adrenoceptors has been reported to impair memory functions in both rats and humans. The α2-adrenoceptor subtype responsible for this detrimental effect is still unknown. The effect of the α2-agonists clonidine and guanabenz on memory processes, in dependence to the time of administration, was evaluated in the mouse passive avoidance test. Clonidine (0.02–0.2 mg kg-1 i.p.) and guanabenz (0.1–0.3 mg kg-1 i.p.) induced amnesia in a dose-dependent manner. From time–course experiments emerged that the impairment of memory function was detectable only when clonidine and guanabenz were administered 60 min before or immediately after the training test, respectively. This detrimental effect was prevented by pretreatment with the α2-antagonist yohimbine (1–3 mg kg-1 i.p.) and by the α2A-antagonist BRL-44408 (0.3–1 mg kg-1 i.p.). By contrast, the α2B,C antagonists ARC-239 (10 mg kg-1 i.p.) and prazosin (1 mg kg-1 i.p.) did not revert the amnesia induced by both clonidine and guanabenz. At the highest effective doses, clonidine and guanabenz were devoid of behavioral side-effects as well as maintained unaltered the motor coordination, as revealed by the rota-rod test. Furthermore, none of the compounds used modified the spontaneous motility as indicated by the Animex apparatus. These results indicate that clonidine and guanabenz impaired memory processes in a mouse passive avoidance paradigm through the selective activation of the α2A-adrenoceptor subtype. [Copyright &y& Elsevier]

The post-receptorial mechanism of the amnesic action of the α2-agonists clonidine and guanabenz was investigated in the mouse passive avoidance test. Animals were i.c.v. injected with pertussis toxin (PTX) or with antisense oligonucleotides, complementary to the sequence of the α-subunit mRNA of Gi1, Gi2, Gi3, Go1 and Go2 proteins. The administration of PTX (0.25 μg per mouse i.c.v.) reversed the amnesia induced by both α2-agonists. Similarly, anti-Giα1 (6.25–12.5 μg per mouse i.c.v.), anti-Giα3 (3.12–12.5 μg per mouse i.c.v.), anti-Goα1 (12.5–25 μg per mouse i.c.v.) antagonised the detrimental effect induced by clonidine and guanabenz. By contrast, pretreatment with anti-Giα2 (3.12–25 μg per mouse i.c.v.) and anti-Goα2 (12.5–25 μg per mouse i.c.v.) never modified the impairment of memory processes induced by the α2-agonists. At the highest effective doses, none of the compounds used impaired motor coordination (rota rod test), nor modified spontaneous motility and inspection activity, (hole board test). These results indicate the involvement of Gi1, Gi3, and Go1, but not Gi2 and Go2, protein subtypes in the transduction mechanism responsible for the induction of amnesia by clonidine and guanabenz. [Copyright &y& Elsevier]

Procyclidine and ethopropazine, widely used as anti-parkinsonian agents because of their anti-cholinergic action, are also known to have NMDA antagonist properties. Unlike other NMDA antagonists, these agents—because of their anti-cholinergic action—are devoid of neurotoxic side effects. In the present study, we used a sciatic nerve ligation model that produces a hyperalgesic (neuropathic pain) state in adult rats to evaluate the ability of procyclidine or ethopropazine, either alone or in combination with an α2 adrenergic agonist, to ameliorate neuropathic pain. We found that both procyclidine and ethopropazine alleviated thermal hyperalgesia in a dose dependent manner; when a marginally effective dose of these agents was combined with an ineffective dose of an α2 adrenergic agonist (clonidine or guanabenz), the combination therapy provided effective and long-lasting relief from neuropathic pain. In addition, the combination therapy was free from neurotoxic or behavioral side effects, and hyperactivity, a side effect associated with procyclidine monotherapy, was counteracted by clonidine. [Copyright &y& Elsevier]

Moxonidine is a centrally-active imidazoline compound with preferential affinity for imidazoline receptors (IR) over α2-adrenoceptors (α2AR). Clinically, moxonidine has proven advantageous for treating hypertension over pure α2-adrenergic agonists (i.e., guanabenz) due to its lowered incidence of sedative side effects. The present experiments reveal divergent behavioral effects of low doses of moxonidine and guanabenz in C57Bl/6 mice in an exploratory arena. Low-dose moxonidine (0.05 mg kg−1 i.p.) elicited an increase in novel object contacts (+36%) and more movement into central space (+56%; P<0.01) compared to saline-injected controls; whereas guanabenz induced only dose-responsive sedative-like behaviors in the same paradigm. Yet, the two agonists were indistinguishable in terms of blood pressure changes over a similar dose range (0.025–0.1 mg kg−1 i.p.) in consciously free-moving mice (Δ mean±S.E.M.=−12.3±3.2 mm Hg for moxonidine versus −13.5±1.9 mm Hg for guanabenz). As expected of α2AR involvement, the sedative-like effects of guanabenz were completely blocked by pretreatment with the non-imidazoline α2AR-antagonist, SKF86466 (0.5 or 1.0 mg kg−1 i.p.). However, the pro-exploratory effects of low doses of moxonidine (0.05 or 0.1 mg kg−1) were not antagonized by SKF86466. These results suggest that moxonidine acts preferentially through a non-adrenergic mechanism, possibly IR-mediated, to elicit pro-exploratory behavior. [Copyright &y& Elsevier]

Oxidative stress and mitochondrial dysfunction are important aspects of pathogenesis, particularly in the brain, which is highly dependent on oxygen, and the protection of astrocytes is essential for neuroprotection. In this context, imidazoline drugs have been reported to be neuroprotective. Our recent study showed that imidazoline drugs, including guanabenz, inhibit the naphthazarin-induced oxidative cytotoxicity associated with lysosomal destabilization. We now report on a study into the protective effects of rilmenidine and AGN 192403, which have affinity for imidazoline-1 receptors, on the cytotoxicity induced by naphthazarin and inhibitors of mitochondrial respiration in astrocytes. Cytotoxicity was measured grossly by LDH release and by measuring changes in lysosomal membrane stability and features of mitochondrial membrane permeabilization. Naphthazarin-induced cytotoxicity was evidenced by the ordered development of lysosomal acridine orange relocation, decrease in mitochondrial potential, cytochrome c release, and caspase-9 activation, and was inhibited by guanabenz, rilmenidine, and AGN 192403. Antimycin A and rotenone induced mitochondrial dysfunction primarily, and their cytotoxicities were inhibited only by AGN 192403. Rilmenidine and guanabenz may have a lysosomal stabilizing effect, which underlies their protective effects. AGN 192403 might affect the mitochondrial cell death cascades, and had a novel protective effect on the cytotoxicity associated with mitochondrial dysfunction. [Copyright &y& Elsevier]

Oxidative stress is a primary pathogenesis in the brain, which is particularly vulnerable to oxidative stress. Maintenance of astrocyte functions under oxidative stress is essential to prevent neuronal injuries and to recover neuronal functions in various pathologic conditions. Imidazoline drugs have affinities for imidazoline receptors, which are highly distributed in the brain, and have been shown to be neuroprotective. This study presented the protective effects of several imidazoline drugs against oxidative cytotoxicity, in primary cultures of astrocytes. Imidazoline drugs, such as idazoxan, guanabenz, guanfacine, BU224, and RS-45041-190, showed protective effects against naphthazarin-induced oxidative cytotoxicity, as evidenced by LDH release and Hoechst 33342/propidium iodide staining. The imidazoline drugs stabilized lysosomes and inhibited naphthazarin-induced lysosomal destabilization, as evidenced by acridine orange relocation. Guanabenz inhibited, the leakage of lysosomal cathepsin D to cytosol, the decreased mitochondrial potential, and the release of mitochondrial cytochrome c, which were induced by naphthazarin. The lysosomal destabilization by oxidative stress and other apoptotic signals and subsequent cathepsin D leakage to the cytosol can induce apoptotic changes of mitochondria and eventually cell death. Therefore, lysosomal stabilization by imidazoline drugs may be ascribed to their protective effects against oxidative cytotoxicity. [Copyright &y& Elsevier]

• We present further evidence that guanabenz significantly reduces the number of latent Toxoplasma gondii cysts in BALB/c mice. • Brain cyst counts rebound after cessation of guanabenz treatment without causing symptoms of acute toxoplasmosis. • We tested whether the re-expansion of cysts could be impeded by pyrimethamine during or after guanabenz treatment. Toxoplasma gondii is a protozoan parasite that causes opportunistic infection in immunocompromised individuals. The parasite forms latent tissue cysts that are refractory to current treatments and give rise to life-threatening reactivated infection following immune suppression. Previously, we showed that guanabenz sharply reduces brain cyst count in BALB/c mice harboring latent toxoplasmosis; however, whether cyst count would change once drug treatment stopped was not addressed. In the present study, we observed a rebound in brain cysts following the discontinuation of guanabenz or a guanabenz-pyrimethamine combination therapy. The re-expansion of brain cysts was not accompanied by symptoms of acute toxoplasmosis. We also tested whether the rebound in cyst counts could be ameliorated by administering pyrimethamine during or after guanabenz treatment. [ABSTRACT FROM AUTHOR]