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Drug repurposing is considered a valid approach to accelerate therapeutic solutions for rare diseases. However, it is not as widely applied as it could be, due to several barriers that discourage both industry and academic institutions from pursuing this path. Herein we present the case of an academic multicentre study that considered the repurposing of the old drug guanabenz as a therapeutic strategy in amyotrophic lateral sclerosis. The difficulties encountered are discussed as an example of the barriers that academics involved in this type of study may face. Although further development of the drug for this target population was hampered for several reasons, the study was successful in many ways. Firstly, because the hypothesis tested was confirmed in a sub-population, leading to alternative innovative solutions that are now under clinical investigation. In addition, the study was informative and provided new insights into the disease, which are now giving new impetus to laboratory research. The message from this example is that even a repurposing study with an old product has the potential to generate innovation and interest from industry partners, provided it is based on a sound rationale, the study design is adequate to ensure meaningful results, and the investigators keep the full clinical development picture in mind.

The circadian clock regulates a variety of biological processes that are normally synchronized with the solar day. Disruption of circadian rhythms is associated with health problems. Understanding the signaling mechanisms that couple cell physiology and metabolism to circadian timekeeping will help to develop novel therapeutic strategies. The integrated stress response (ISR) is activated by the cellular stressors to maintain physiological homeostasis by orchestrating mRNA translation. Aberrant ISR has been found in a number of neurological diseases that exhibit disrupted circadian rhythms and sleep. Recent work has started to uncover a critical role for the ISR in regulating the physiology of the circadian clock. Guanabenz (2,6-dichlorobenzylidene aminoguanidine acetate) is an orally bioavailable α2-adrenergic receptor agonist that has been used as an antihypertensive for decades. Recent studies demonstrated that guanabenz can regulate the ISR. Here, we assessed the effects of guanabenz on cellular and behavioral circadian rhythms using a multidisciplinary approach. We found that guanabenz can induce the ISR by increasing eIF2α phosphorylation in cultured fibroblasts as well as in the mouse brain. The hyperphosphorylation of eIF2α by guanabenz is associated with the shortened circadian period in cells and animals and the disruption of behavioral circadian rhythms in mice. Guanabenz administration disrupted circadian oscillations of the clock protein Per1 and Per2 in the mouse suprachiasmatic nucleus, the master pacemaker. These results uncover a significant yet previously unidentified role of guanabenz in regulating circadian rhythms and indicate that exacerbated ISR activation can impair the functions of the brain’s circadian clock by disrupting clock gene expression.

The circadian clock regulates a variety of biological processes that are normally synchronized with the solar day. Disruption of circadian rhythms is associated with health problems. Understanding the signaling mechanisms that couple cell physiology and metabolism to circadian timekeeping will help to develop novel therapeutic strategies. The integrated stress response (ISR) is activated by the cellular stressors to maintain physiological homeostasis by orchestrating mRNA translation. Aberrant ISR has been found in a number of neurological diseases that exhibit disrupted circadian rhythms and sleep. Recent work has started to uncover a critical role for the ISR in regulating the physiology of the circadian clock. Guanabenz (2,6-dichlorobenzylidene aminoguanidine acetate) is an orally bioavailable α2-adrenergic receptor agonist that has been used as an antihypertensive for decades. Recent studies demonstrated that guanabenz can regulate the ISR. Here, we assessed the effects of guanabenz on cellular and behavioral circadian rhythms using a multidisciplinary approach. We found that guanabenz can induce the ISR by increasing eIF2α phosphorylation in cultured fibroblasts as well as in the mouse brain. The hyperphosphorylation of eIF2α by guanabenz is associated with the shortened circadian period in cells and animals and the disruption of behavioral circadian rhythms in mice. Guanabenz administration disrupted circadian oscillations of the clock protein Per1 and Per2 in the mouse suprachiasmatic nucleus, the master pacemaker. These results uncover a significant yet previously unidentified role of guanabenz in regulating circadian rhythms and indicate that exacerbated ISR activation can impair the functions of the brain's circadian clock by disrupting clock gene expression. [ABSTRACT FROM AUTHOR]

Abstract Background We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. Methods HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. Results We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. Conclusions We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes.

Background: We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. Methods: HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. Results: We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. Conclusions: We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes. [ABSTRACT FROM AUTHOR]

In veterinary medicine, a number of alpha-2 receptor agonists are marketed as sedatives/hypnotics and analgesics, with their principal use being the chemical restraint of large and small animals. Guanabenz (Wytensin®) is an alpha-2 adrenergic receptor agonist marketed for use in humans as an anti-hypertensive agent. Recent reports indicate that guanabenz has been administered to horses in small doses (0.04 mg/kg) for its anti-hypertensive effects. While this offers both benefits of sedation of the horse as well as amelioration of pulmonary hypertension during running exercise and consequent Exercise-Induced Pulmonary Hemorrhage (EIPH), guanabenz is currently proscribed in most racing jurisdictions and its administration to a racing horse can lead to penalties. The Association of Racing Commissioners International (ARCI) lists guanabenz as an ARCI Class 3 agent; Class 3 agents include bronchodilators, anabolic steroids and other drugs with primary effects on the autonomic nervous system, procaine, antihistamines with sedative properties and diuretics and includes amitraz, clonidine, xylazine, detomidine, medetomidine, and romifidine. Guanabenz is unique among alpha-2 agonists in that it differentiates into E- and Z-forms (Fig. 1), with the Z-form lacking hypotensive properties, yet with both E- and Z-forms able to afford relief to cellular stresses related to inflammation or degenerative diseases. The objective of the study was a preliminary description of the pharmacological properties of guanabenz in comparison with clonidine and a number of other alpha-2 agonists. The goal was clinical evaluation of their sedative, analgesic and related activities with the goal of increasing our understanding of the clinical use of such medications and also as a possible prophylaxis for Exercise- Induced Pulmonary Hemorrhage. The clinical study of guanabenz and clonidine was performed in a complete crossover strategy using quantitative markers of sedation, antinociception, heart rate, blood and urine glucose following administration of each compound in five horses. Amitraz, detomidine, medetomidine, romifidine, and xylazine were studied in one horse each. The sedation was quantified by measuring head droop and locomotor activity, while antinociception was measured by Hoof Withdrawal Reflex Latency. Heart rates, urine glucose, urine production and urine specific gravities were also determined by standard clinical chemistry techniques. Guanabenz serum levels and related urinary guanabenz glucuronide levels were determined by established Liquid Chromatography-tandem Mass Spectrometric (LC-MS) methods. In result the clinically effective doses (0.2 mg/kg) of guanabenz produced a rapid and intense sedative effect, with sagging of the lower lip, sunken eyelids, and marked head droop corresponding to plasma guanabenz concentrations that peaked at 120 ng/mL at 2.5 min post-injection (Fig. 2). The initial head height above the ground is considered 100 %, and head heights fell to values ranging 18–40 % with guanabenz, all of which are greater than a 50 % reduction in head height, considered a full clinically useful sedative effect. Despite the intensity of the sedation, all horses remained standing and were able to walk, and the sedation and head droop responses were rapidly reversed by intravenous administration of the alpha-2 receptor antagonist yohimbine, reversals occurring within 10 min of administration. As a pilot investigation this study was extended to six other members of the alpha-2 agonist group, clonidine administered to five horses, and amitraz, detomidine, medetomidine, romifidine, and xylazine to one horse each. Hoof Withdrawal Reflex Latency evaluation demonstrated the considerable analgesic properties of guanabenz, greater than the corresponding potencies among clonidine, detomidine, romifidine, medetomidine and xylazine. Heart rate monitoring showed guanabenz as possessing capacity for prolonged bradycardia, with effects of a single dose lasting for up to 3.5 hr, in contrast with clonidine (1 hr), amitraz (2 hr), detomidine (<1 hr), medetomidine (1 hr), romifidine (2 hr), and xylazine (<1 hr). Peak urine production following guanabenz administration occurred between 1.5 and 3.0 hr after administration (Fig. 6), as indicated by the steeper decline of the urine volume curve during that period. Urine specific gravity dropped to a low of about 1.006 at 2.0 hr after administration and remained at this level for ~1.0 hr. Urine pH remained at 8, and urine protein was negative throughout testing. The other alpha-2 agonists evaluated also caused an increased urine production with a concomitant decrease in specific gravity. The effect of guanabenz had the longest duration on increased urine volume, lasting about 3.0 hr. Xylazine had the shortest diuretic effect, persisting for only about 1.0 hr. Guanabenz along with romifidine and detomidine induced glucosuria whereas other alpha-2 agonists did not. Hyperglycemia and the corresponding glucosuria resulted in a significant diuresis, as shown by the cumulative urine volume. Guanabenz along with amitraz, detomidine and xylazine also produced measurable sedation presenting as reduced locomotor activity (Table 1). While all alpha-2 agonists showed qualitatively similar pharmacological responses, only guanabenz produced an intense and relatively prolonged antinociceptive response. The study is limited by the number of horses examined (five each for guanabenz and clonidine, five for repeat studies that included yohimbine antagonism, and one each for the other agonists). Study design was focused on clinical evaluation of agonist similarities and differences and thus did not specifically generate data for detailed statistical evaluation. In conclusion these studies show that a 100 mg IV dose of guanabenz rapidly induces clinically useful sedation, analgesia and antinociception effects that are more intense and considerably longer-lasting than those produced by other alpha-2 receptor agonists evaluated. Guanabenz also remains detectable in serum up to 8-hours following administration at doses as low as 0.04 mg/kg. In the work reported here, guanabenz administered at 0.2 mg/kg IV showed peak concentrations in serum of 120 ng/ mL at 2.5 min and was detectable for up to 4 hr with its glucuronide metabolite peaking at 120 min post-administration. Although we did not investigate the combination of guanabenz with opioid drugs such as butorphanol for pain management, guanabenz may well be a drug of choice among the other alpha-2 agonists evaluated in this study for administration with opioids for pain management based on maintaining maximum levels of analgesia for longer periods of time. These experiments suggest considerable clinical potential for guanabenz as a sedative and a relatively long-lasting analgesic in equine medicine. Based on these pharmacological properties, guanabenz and related alpha-2 agonists also have considerable potential for clinical use in equine medicine. [ABSTRACT FROM AUTHOR]

Increase of sympathetic activity has been known to exacerbate osteoporosis through promotion of bone resorption. However, it is largely unknown about involvement of sympathetic activity in exacerbation of periodontitis. In this study, we investigated whether α2-adrenergic receptor (α2-AR) agonist guanabenz which decreases sympathetic activity, attenuates alveolar bone resorption in rats having high sympathetic activity with periodontitis. Volumes of residual alveolar bone and attachment levels in periodontium were examined using micro-computed tomography and hematoxylin-eosin staining, respectively. Furthermore, osteoclast numbers per bone surface and osteoclast surface per bone surface were measured using tartrate-resistant acid phosphatase staining. To examine the suppressive effects of guanabenz on pro-inflammatory cytokines, expression levels of tyrosine hydroxylase (TH), TNF-α, IL1-β, and IL-6 in periodontium were measured using immunohistostaining. Administration of guanabenz attenuated loss of alveolar bone and attachment levels in rats having high sympathetic activity. Furthermore, its administration suppressed osteoclast numbers in rats having high sympathetic activity. TH, TNF-α, IL-1β, and IL-6 positive cells in periodontium in rats treated with guanabenz for 12 weeks, were lower than those in control rats having high sympathetic activity. This study demonstrated administration of α2-AR agonist guanabenz attenuates alveolar bone resorption through decrease of sympathetic activity in rats.

The aim of this study was to determine, in the diet-induced obesity model in rats, the potential of Guanabenz to reduce body weight and ameliorate some metabolic disturbances. Obesity was induced in rats by a high-fat diet. After 10 weeks, rats were treated intraperitoneally with Guanabenz at the two doses: 2 or 5 mg/kg b.w./day, once daily for 25 days. The spontaneous activity of rats was measured for 24 h on the 1st and 24th day of the Guanabenz treatment with a special radio-frequency identification system. Gastric emptying was measured in intragastric phenol red-treated mice by measuring the color of the stomach homogenate 30 min after phenol red administration. Intraperitoneal administration of Guanabenz for 25 days to obese rats resulted in a significant decrease in body weight compared to the baseline values (about 11% at a dose of 5 mg/kg). Both body weight and the amount of adipose tissue in the groups receiving Guanabenz decreased to the levels observed in the control rats fed only standard feed. The anorectic effect occurred in parallel with a reduction in plasma triglyceride levels. We also confirmed the beneficial effect of Guanabenz on plasma glucose level. The present study demonstrates that the administration of Guanabenz strongly inhibits gastric emptying (about 80% at a dose of 5 mg/kg). Guanabenz can successfully and simultaneously attenuate all the disorders and risk factors of metabolic syndrome: hypertension, hyperglycemia, obesity, and dyslipidemia. However, the exact cellular mechanisms of its action require further research. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) pathology is characterized by cognitive impairment, increased acetylcholinesterase (AChE) activity, and impaired neuronal communication. Clinically, AChE inhibitors are being used to treat AD patients; however, these remain unable to prevent the disease progression. Therefore, further development of new therapeutic molecules is required having broad spectrum effects on AD-related various neurodegenerative events. Since repurposing is a quick mode to search the therapeutic molecules; henceforth, this study was conducted to evaluate the anti-Alzheimer activity of drug guanabenz which is already in use for the management of high blood pressure in clinics. The study was performed employing both cellular and rat models of AD along with donepezil as reference drug. Guanabenz treatment in both the experimental models showed significant protection against AD-specific behavioral and pathological indicators like AChE activity, tau phosphorylation, amyloid precursor protein, and memory retention. In conjunction, guanabenz also attenuated the AD-related oxidative stress, impaired mitochondrial functionality (MMP, cytochrome-c translocation, ATP level, and mitochondrial complex I activity), endoplasmic reticulum stress (GRP78, GADD153, cleaved caspase-12), neuronal apoptosis (Bcl-2, Bax, cleaved caspase-3), and DNA fragmentation. In conclusion, findings suggested the panoptic protective effect of guanabenz on disease-related multiple degenerative markers and signaling. Furthermore, clinical trial may shed light and expedite the availability of new therapeutic anti-Alzheimer's molecule for the wellbeing of AD patients. [ABSTRACT FROM AUTHOR]

Strong evidence suggests that endoplasmic reticulum stress plays a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) through altered regulation of proteostasis. Robust preclinical findings demonstrated that guanabenz selectively inhibits endoplasmic reticulum stress-induced eIF2α-phosphatase, allowing misfolded protein clearance, reduces neuronal death and prolongs survival in in vitro and in vivo models. However, its safety and efficacy in patients with ALS are unknown. To address these issues, we conducted a multicentre, randomized, double-blind trial with a futility design. Patients with ALS who had displayed an onset of symptoms within the previous 18 months were randomly assigned in a 1:1:1:1 ratio to receive 64 mg, 32 mg or 16 mg of guanabenz or placebo daily for 6 months as an add-on therapy to riluzole. The purpose of the placebo group blinding was to determine safety but not efficacy. The primary outcome was the proportion of patients progressing to higher stages of disease within 6 months as measured using the ALS Milano-Torino staging system, compared with a historical cohort of 200 patients with ALS. The secondary outcomes were the rate of decline in the total revised ALS functional rating scale score, slow vital capacity change, time to death, tracheotomy or permanent ventilation and serum light neurofilament level at 6 months. The primary assessment of efficacy was performed using intention-to-treat analysis. The treatment arms using 64 mg and 32 mg guanabenz, both alone and combined, reached the primary hypothesis of non-futility, with the proportions of patients who progressed to higher stages of disease at 6 months being significantly lower than that expected under the hypothesis of non-futility and a significantly lower difference in the median rate of change in the total revised ALS functional rating scale score. This effect was driven by patients with bulbar onset, none of whom (0/18) progressed to a higher stage of disease at 6 months compared with those on 16 mg guanabenz (4/8; 50%), the historical cohort alone (21/49; 43%; P = 0.001) or plus placebo (25/60; 42%; P = 0.001). The proportion of patients who experienced at least one adverse event was higher in any guanabenz arm than in the placebo arm, with higher dosing arms having a significantly higher proportion of drug-related side effects and the 64 mg arm a significantly higher drop-out rate. The number of serious adverse events did not significantly differ between the guanabenz arms and the placebo. Our findings indicate that a larger trial with a molecule targeting the unfolded protein response pathway without the alpha-2 adrenergic related side-effect profile of guanabenz is warranted. [ABSTRACT FROM AUTHOR]

Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative conditions that share a characteristic feature of degeneration of the longest axons within the corticospinal tract, which leads to progressive spasticity and weakness of the lower limbs. Mutations of over 70 genes produce defects in various biological pathways: axonal transport, lipid metabolism, endoplasmic reticulum (ER) shaping, mitochondrial function, and endosomal trafficking. HSPs suffer from an adequate therapeutic plan. Currently the treatments foreseen for patients affected by this pathology are physiotherapy, to maintain the outgoing tone, and muscle relaxant therapies for spasticity. Very few clinical studies have been conducted, and it’s urgent to implement preclinical animal studies devoted to pharmacological test and screening, to expand the rose of compounds potentially attractive for clinical trials. Small animal models, such as Drosophila melanogaster and zebrafish, have been generated, analyzed, and used as preclinical model for screening of compounds and their effects. In this work, we briefly described the role of HSP-linked proteins in the organization of ER endomembrane system and in the regulation of ER homeostasis and stress as a common pathological mechanism for these HSP forms. We then focused our attention on the pharmacodynamic and pharmacokinetic features of some recently identified molecules with antioxidant property, such as salubrinal, guanabenz, N-acetyl cysteine, methylene blue, rapamycin, and naringenin, and on their potential use in future clinical studies. Expanding the models and the pharmacological screening for HSP disease is necessary to give an opportunity to patients and clinicians to test new molecules.

Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative conditions that share a characteristic feature of degeneration of the longest axons within the corticospinal tract, which leads to progressive spasticity and weakness of the lower limbs. Mutations of over 70 genes produce defects in various biological pathways: axonal transport, lipid metabolism, endoplasmic reticulum (ER) shaping, mitochondrial function, and endosomal trafficking. HSPs suffer from an adequate therapeutic plan. Currently the treatments foreseen for patients affected by this pathology are physiotherapy, to maintain the outgoing tone, and muscle relaxant therapies for spasticity. Very few clinical studies have been conducted, and it's urgent to implement preclinical animal studies devoted to pharmacological test and screening, to expand the rose of compounds potentially attractive for clinical trials. Small animal models, such as Drosophila melanogaster and zebrafish, have been generated, analyzed, and used as preclinical model for screening of compounds and their effects. In this work, we briefly described the role of HSP-linked proteins in the organization of ER endomembrane system and in the regulation of ER homeostasis and stress as a common pathological mechanism for these HSP forms. We then focused our attention on the pharmacodynamic and pharmacokinetic features of some recently identified molecules with antioxidant property, such as salubrinal, guanabenz, N-acetyl cysteine, methylene blue, rapamycin, and naringenin, and on their potential use in future clinical studies. Expanding the models and the pharmacological screening for HSP disease is necessary to give an opportunity to patients and clinicians to test new molecules. [ABSTRACT FROM AUTHOR]

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]

A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.

Drug repurposing is considered a valid approach to accelerate therapeutic solutions for rare diseases. However, it is not as widely applied as it could be, due to several barriers that discourage both industry and academic institutions from pursuing this path. Herein we present the case of an academic multicentre study that considered the repurposing of the old drug guanabenz as a therapeutic strategy in amyotrophic lateral sclerosis. The difficulties encountered are discussed as an example of the barriers that academics involved in this type of study may face. Although further development of the drug for this target population was hampered for several reasons, the study was successful in many ways. Firstly, because the hypothesis tested was confirmed in a sub-population, leading to alternative innovative solutions that are now under clinical investigation. In addition, the study was informative and provided new insights into the disease, which are now giving new impetus to laboratory research. The message from this example is that even a repurposing study with an old product has the potential to generate innovation and interest from industry partners, provided it is based on a sound rationale, the study design is adequate to ensure meaningful results, and the investigators keep the full clinical development picture in mind., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ambrosini, Dalla Bella, Ravasi, Melazzini and Lauria.)

Polyglutamine expansion diseases are a group of hereditary neurodegenerative disorders that develop when a CAG repeat in the causative genes is unstably expanded above a certain threshold. The expansion of trinucleotide CAG repeats causes hereditary adult-onset neurodegenerative disorders, such as Huntington's disease, dentatorubral–pallidoluysian atrophy, spinobulbar muscular atrophy and multiple forms of spinocerebellar ataxia (SCA). The most common dominantly inherited SCA is the type 3 (SCA3), also known as Machado–Joseph disease (MJD), which is an autosomal dominant, progressive neurological disorder. The gene causatively associated with MJD is ATXN3. Recent studies have shown that this gene modulates endoplasmic reticulum (ER) stress. We generated transgenic Caenorhabditis elegans strains expressing human ATXN3 genes in motoneurons, and animals expressing mutant ATXN3-CAG89 alleles showed decreased lifespan, impaired movement, and rates of neurodegeneration greater than wild-type ATXN3-CAG10 controls. We tested three neuroprotective compounds (Methylene Blue, guanabenz and salubrinal) believed to modulate ER stress and observed that these molecules rescued ATXN3-CAG89 phenotypes. Furthermore, these compounds required specific branches of the ER unfolded protein response (UPRER), reduced global ER and oxidative stress, and polyglutamine aggregation. We introduce new C. elegans models for MJD based on the expression of full-length ATXN3 in a limited number of neurons. Using these models, we discovered that chemical modulation of the UPRER reduced neurodegeneration and warrants investigation in mammalian models of MJD.

ABSTRACT Toxoplasma gondii is an intracellular parasite that has infected one-third of humans. The infection is permanent because the replicative form (tachyzoite) converts into a latent tissue cyst form (bradyzoite) that evades host immunity and is impervious to current drugs. The continued presence of these parasitic cysts hinders treatment and leads to chronic infection that has been linked to behavioral changes in rodents and neurological disease in humans. How these behavioral changes occur, and whether they are due to parasite manipulation or the host response to infection, remains an outstanding question. We previously showed that guanabenz possesses antiparasitic activity; here, we show that guanabenz reproducibly lowers brain cyst burden up to 80% in chronically infected male and female BALB/cJ mice when given intraperitoneally but not when administered by gavage or in food. Regardless of the administration route, guanabenz reverses Toxoplasma-induced hyperactivity in latently infected mice. In contrast, guanabenz increases cyst burden when given to chronically infected C57BL/6J mice yet still reverses Toxoplasma-induced hyperactivity. Examination of the brains from chronically infected BALB/cJ and C57BL/6J mice shows that guanabenz decreases inflammation and perivascular cuffing in each strain. Our study establishes a robust model for cyst reduction in BALB/cJ mice and shows for the first time that it is possible to reverse a key behavioral change associated with latent toxoplasmosis. The rescue from parasite-induced hyperactivity correlates with a decrease in neuroinflammation rather than reduced cyst counts, suggesting that some behavioral changes arise from host responses to infection. IMPORTANCE Toxoplasma gondii is a common parasite of animals, including up to one-third of humans. The single-celled parasite persists within hosts for the duration of their life as tissue cysts, giving rise to chronic infection. Latent toxoplasmosis is correlated with neurological dysfunction in humans and results in dramatic behavioral changes in rodents. When infected, mice and rats adapt behaviors that make them more likely to be devoured by cats, the only host that supports the sexual stage of the parasite. In this study, we establish a new mouse model of tissue cyst depletion using a drug called guanabenz and show that it is possible to reverse a key behavior change back to normal in infected animals. We also show that the mechanism appears to have nothing to do with parasite cyst burden but rather the degree of neuroinflammation produced by chronic infection.

Abstract—The body's response to stress is mediated by the interaction of catecholamines with adrenergic α- and β-receptors. It has been established that, in contrast to α1- and β-adrenoceptors, α2-adrenoceptors are involved in feedback loops of the simpatoadrenal system, which controls the release of catecholamines (norepinephrine and epinephrine) to save energy resources and the function of peripheral organs under an overload of catecholamines. Violations of the feedback mechanisms lead to various pathologies, including hypertrophic heart remodeling, followed by the development of heart failure. We previously found the expression of α2-adrenoreceptors in the plasma membrane of cardiomyocytes, where they participate in the regulation of the intracellular level of Ca2+. We suggested that these receptors are able to locally control the response of the heart muscle to stress in addition to the traditional mechanism. We have shown in this study that sarcolemmal α2-adrenoreceptors attenuate the positive inotropic effect of adrenergic stimulation in papillary muscles by controlling the level of free Ca2+ in cytosol. The ability of α2-adrenoreceptor agonists to suppress intracellular spontaneous Ca2+ oscillations increases the efficiency of systolic function of papillary muscles and reduces the likelihood of dangerous ventricular arrhythmia. Thus, α2-adrenoreceptors in the sarcolemmal membrane of cardiomyocytes have their own protective potential, which can prevent pathological remodeling of the heart muscle under chronic stress. [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]

α2-Adrenoceptors (α2-AR) found in the cardiomyocyte's sarcolemma represent a very important negative feedback for control of myocardial contractility by endogenous catecholamines. Earlier, we showed that the endogenous neurotransmitter agmatine in micromolar concentrations via α2-AR activates the nitric oxide (NO) synthesis, enhancing the Ca2+ pumping into sarcoplasmic reticulum (SR). In the millimolar doses it inhibits Ca2+ sequestration by SR Ca2+ ATPase (SERCA), acting through the first type of imidazoline receptors. Here, we study the functional activity of agmatine, as well as a specific α2-agonist, guanabenz, in respect to spontaneous Ca2+-transients in SHR cardiomyocytes of the early age (2–2.5 months), and adulthood animals (8–9 months). α2-mediated cardioprotective effect was almost twofold decreased in SHR cardiac cells compared to normotensive rats of the corresponding age, despite the fact that both α2A- and α2B-AR protein levels were significantly increased in SHR cardiomyocytes. NO-mediated facilitation of SERCA activity is substantially reduced in SHR cardiomyocytes vs. normotensive rats. These data suggest that the SHR phenotype starting from early age shows signs of the impaired sarcolemmal α2-AR signaling, which can aggravate the development of this cardiovascular pathology. • α2AR signaling is impaired in SHR cardiomyocytes starting from 2 months old. • α2AR-mediated NO synthesis is substantially decreased in SHR vs. normotensive rats. • NO-mediated facilitation of SERCA is practically abrogated in SHR cardiomyocytes. • Protein levels of α2A-AR and α2B-AR subtypes are heightened in SHR cardiomyocytes. • Impaired α2AR-signaling may relative to the deterioration of myocardial functions in SHR. [ABSTRACT FROM AUTHOR]

Aim: Vanishing White Matter (VWM) is a devastating leucoencephalopathy without effective treatment options. Patients have mutations in the EIF2B1-5 genes, encoding the five subunits of eIF2B, a guanine exchange factor that is an important regulator of protein translation. We recently developed mouse models for VWM that replicate the human disease. To study disease improvement after treatment in these mice, it is essential to have sensitive biomarkers related to disease stage. The Bergmann glia of the cerebellum, an astrocytic subpopulation, translocate into the molecular layer in symptomatic VWM mice and patients. This study looked at the prospects of using Bergmann glia pathology as an objective disease marker for VWM. Methods: We defined a new quantitative measurement of Bergmann glia pathology in the cerebellum of VWM mice and patients. To test the sensitivity of this new marker for improvement, VWM mutant mice received long-term treatment with Guanabenz, an FDAapproved anti-hypertensive agent affecting eIF2B activity. Results: Bergmann glia translocation was significantly higher in symptomatic VWM mice and VWM patients than in controls and worsened over the disease course. Both Bergmann glia pathology and cerebellar myelin pathology improved with Guanabenz treatment in mice, showing that Bergmann glia translocation is a sensitive measurement for improvement. Conclusions: Bergmann glia translocation can be used to objectively assess effects of treatment in VWM mice. Future treatment strategies involving compounds regulating eIF2 phosphorylation might benefit VWM patients. [ABSTRACT FROM AUTHOR]

Reduced function of parkin appears to be a central pathogenic event in Parkinson disease (PD). Increasing parkin levels enhances survival in models of PD-related neuronal death and is a promising therapeutic objective. Previously, we demonstrated that the transcription factor ATF4 promotes survival in response to PD-mimetic stressors by maintaining parkin levels. ATF4 translation is up-regulated by phosphorylation of the translation initiation factor eIF2α. The small molecule guanabenz enhances eIF2α phosphorylation by blocking the function of GADD34, a regulatory protein that promotes eIF2α dephosphorylation. We tested the hypothesis that guanabenz, by inhibiting GADD34 and consequently increasing eIF2α phosphorylation and elevating ATF4, would improve survival in models of PD by up-regulating parkin. We found that GADD34 is strongly induced by 6-OHDA, and that GADD34 localization is dramatically altered in dopaminergic substantia nigra neurons in PD cases. We further demonstrated that guanabenz attenuates 6-hydroxydopamine (6-OHDA) induced cell death of differentiated PC12 cells and primary ventral midbrain dopaminergic neurons in culture, and of dopaminergic neurons in the substantia nigra of mice. In culture models, guanabenz also increases eIF2α phosphorylation and ATF4 and parkin levels in response to 6-OHDA. Furthermore, if either ATF4 or parkin is silenced, then the protective effect of guanabenz is lost. We also found similar results in a distinct model of neuronal death: primary cultures of cortical neurons treated with the topoisomerase I inhibitor camptothecin, in which guanabenz limited camptothecin-induced neuronal death in an ATF4- and parkin-dependent manner. In summary, our data suggest that guanabenz and other GADD34 inhibitors could be used as therapeutic agents to boost parkin levels and thereby slow neurodegeneration in PD and other neurodegenerative conditions. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

Chronic toxoplasmosis which is positively correlated with many neuropsychiatric problems has no curative treatment till now; due to the resistant tissue cysts especially in the brain. In search of an effective treatment, guanabenz-loaded polyethylene glycol poly lactic-co-glycolic acid (PEG-PLGA) nanoparticles was evaluated against chronic experimental toxoplasmosis. For this purpose, each mouse was infected with 10 cysts of Toxoplasma gondii (ME 49 strain). Treated mice received either guanabenz alone (5 mg/kg/day) in subgroup IIa or guanabenz-loaded nanoparticles by full dose in subgroup IIb or guanabenz-loaded nanoparticles by the half dose (2.5 mg/kg/day) in subgroup IIc. Subgroup Ie was treated by pyrimethamine and sulfadiazine. The treatment started on day 25 post-infection for 19 successive days. Then Parasitological, histopathological, immunohistochemical, immunological and ultrastructural morphological studies were performed. The results showed that: subgroup IIb showed the highest statistically significant reduction in the neuroinflammation and brain tissue cysts (77%) with a significant higher efficacy in comparison with pyrimethamine and sulfadiazine and showed the highest level of IFN-γ, while the lowest level was in subgroup IIa. All group II mice showed similar changes of depression and compression of the wall of the cyst. This is marked in subgroup IIb with release of crescent shaped bradyzoite outside the cyst. PEG-PLGA nanoparticles had no toxic effect on the liver or the kidney of the mice. It could be concluded that guanabenz-loaded PEG-PLGA nanoparticles could be promising and safe for treatment of chronic toxoplasmosis. Chronic toxoplasmosis which is positively correlated with many neuropsychiatric problems has no curative treatment till now; due to the resistant tissue cysts. Guanabenzloaded polyethylene glycol poly lacticco-glycolic acid (PEG-PLGA) nanoparticles caused significant brain tissue cysts reduction (77%) in comparison with pyrimethamine and sulfadiazine combination therapy. [Display omitted] • Guanabenz repurposing as an anti- Toxoplasma drug with higher efficacy when incorporated with PEG-PLGA nanoparticles. • Guanabenz-loaded nanoparticles achieved highly significant Toxoplasma brain cyst reduction with reduced neuroinflammation. • Guanabenz-loaded nanoparticles caused marked compression of the cyst wall with release of crescent shaped bradyzoite. • Guanabenz-loaded nanoparticles had an immune stimulant effect by increasing mean interferon gamma level. • PEG-PLGA nanoparticles had no toxic effect on the liver or the kidney. [ABSTRACT FROM AUTHOR]

Salubrinal and guanabenz are two known inhibitors of dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α), and they suppress osteoclastogenesis through downregulating nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master molecule of osteoclastogenesis. The mechanism of NFATc1 suppression is not well understood. Using genome-wide microarray analysis, we investigated molecular regulators of osteoclastogenesis, in particular, in response to salubrinal and guanabenz. We identified two sets of genes: a set of genes that were upregulated by receptor activator of nuclear factor kappa-B ligand (RANKL) and downregulated by salubrinal and guanabenz; and the other set of genes that were downregulated by RANKL and upregulated by salubrinal and guanabenz. The microarray and qPCR result revealed that a zinc finger protein, FYVE domain containing 21 (Zfyve21), as well as DNA-damage-inducible transcript 4 (Ddit4), were suppressed by RANKL and upregulated by salubrinal and guanabenz. A partial silencing of Zfyve21 or Ddit4 attenuated salubrinal- and guanabenz-driven suppression of NFATc1. Collectively, this study demonstrates that Zfyve21 and Ddit4 are two inhibitors of osteoclastogenesis. We expect that they may potentially serve as novel targets for preventing bone loss from skeletal diseases such as osteoporosis. [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]

Integrated stress responses (ISR) may lead to cell death and tissue degeneration via eukaryotic translation initiation factor 2 α (eIF2α)-mediated signaling. Alleviating ISR by modulating eIF2α phosphorylation can reduce the symptoms associated with various diseases. Guanabenz is known to elevate the phosphorylation level of eIF2α and reduce pro-inflammatory responses. However, the mechanism of its action is not well understood. In this study, we investigated the signaling pathway through which guanabenz induces anti-inflammatory effects in immune cells, in particular macrophages. Genome-wide mRNA profiling followed by principal component analysis predicted that colony stimulating factor 2 (Csf2, or GM-CSF as granulocyte macrophage colony stimulating factor) is involved in the responses to guanabenz. A partial silencing of Csf2 or eIF2α by RNA interference revealed that Interleukin-6 (IL6), Csf2, and Cyclooxygenase-2 (Cox2) are downregulated by guanabenz-driven phosphorylation of eIF2α. Although expression of IL1β and Tumor Necrosis Factor-α (TNFα) was suppressed by guanabenz, their downregulation was not directly mediated by eIF2α signaling. Collectively, the result herein indicates that anti-inflammatory effects by guanabenz are mediated by not only eIF2α-dependent but also eIF2α-independent signaling. [ABSTRACT FROM AUTHOR]

Supraphysiological concentrations (SPCs) of triiodo-L-thyronine (T3) have been used in the treatment of a number of nonviral diseases. However, the signaling mechanisms that regulate the function of T3 at these concentrations and their role in modulating cellular stress pathways and antiviral responses are unknown. Here, we have investigated the effects of SPCs of T3 on integrated stress response (ISR) signaling pathways and the replication of vesicular stomatitis virus (VSV). T3 amplified Poly IC-induced activation of RNA-dependent protein kinase, induced phosphorylation of eIF2α, stress granule (SG) formation, IRE1α phosphorylation, XBP1 splicing, and the expression of stress markers. T3 inhibited VSV replication by modulating SG formation and the expression of stress response markers. ISR activator guanabenz also inhibited VSV replication and amplified T3-induced anti-VSV response. To summarize, we have uncovered novel functions of T3 at SPCs as an activator of ISR signaling pathways and an inhibitor of VSV replication. This study offers a proof of principle of the concept that ISR activating agents like SPC of T3 and guanabenz can be potential antiviral agents. [ABSTRACT FROM AUTHOR]