A Role for GABAA Receptor ß3 Subunits in Mediating Harmaline Tremor Suppression by Alcohol: Implications for Essential Tremor Therapy.

Background: Essential tremor patients may find that low alcohol amounts suppress tremor. A candidate mechanism is modulation of α6ß3δ extra-synaptic GABAA receptors, that in vitro respond to non-intoxicating alcohol levels. We previously found that low-dose alcohol reduces harmaline tremor in wild-type mice, but not in littermates lacking δ or α6 subunits. Here we addressed whether low-dose alcohol requires the ß3 subunit for tremor suppression. Methods: We tested whether low-dose alcohol suppresses tremor in cre-negative mice with intact ß3 exon 3 flanked by loxP, and in littermates in which this region was excised by cre expressed under the α6 subunit promotor. Tremor in the harmaline model was measured as a percentage of motion power in the tremor bandwidth divided by overall motion power. Results: Alcohol, 0.500 and 0.575 g/kg, reduced harmaline tremor compared to vehicle-treated controls in floxed ß3 cre- mice, but had no effect on tremor in floxed ß3 cre+ littermates that have ß3 knocked out. This was not due to potential interference of α6 expression by the insertion of the cre gene into the α6 gene since non-floxed ß3 cre+ and cre- littermates exhibited similar tremor suppression by alcohol. Discussion: As α6ß3δ GABAA receptors are sensitive to low-dose alcohol, and cerebellar granule cells express ß3 and are the predominant brain site for α6 and δ expression together, our overall findings suggest alcohol acts to suppress tremor by modulating α6ß3δ GABAA receptors on these cells. Novel drugs that target this receptor may potentially be effective and well-tolerated for essential tremor. Highlights: We previously found with the harmaline essential tremor model that GABAA receptors containing α6 and δ subunits mediate tremor suppression by alcohol. We now show that ß3 subunits in α6-expressing cells, likely cerebellar granule cells, are also required, indicating that alcohol suppresses tremor by modulating α6ß3δ extra-synaptic GABAA receptors.

Resveratrol and 1,25-dihydroxyvitamin D decrease Lingo-1 levels, and improve behavior in harmaline-induced Essential tremor, suggesting potential therapeutic benefits.

Essential tremor (ET) is a neurological disease that impairs motor and cognitive functioning. A variant of the Lingo-1 genetic locus is associated with a heightened ET risk, and increased expression of cerebellar Lingo-1. Lingo-1 has been associated with neurodegenerative processes; however, neuroprotection from ET-associated degeneration can be conferred by the protein Sirt1. Sirt1 activity can be promoted by Resveratrol (Res) and 1,25-dihydroxyvitamin D3 (VitD3), and thus these factors may exert neuroprotective properties through a Sirt1 mechanism. As Res and VitD3 are linked to Sirt1, enhancing Sirt1 could counteract the negative effects of increased Lingo-1. Therefore, we hypothesized that a combination of Res-VitD3 in a harmaline injection model of ET would modulate Sirt1 and Lingo-1 levels. As expected, harmaline exposure (10 mg/kg/every other day; i.p.) impaired motor coordination, enhanced tremors, rearing, and cognitive dysfunction. When Res (5 mg/kg/day; i.p.) and VitD3 (0.1 mg/kg/day; i.p.) were given to adult rats (n = 8 per group) an hour before harmaline, tremor severity, rearing, and memory impairment were reduced. Individual treatment with Res and VitD3 decreased Lingo-1 gene expression levels in qPCR assays. Co-treatment with Res and VitD3 increased and decreased Sirt1 and Lingo-1 gene expression levels, respectively, and in some cases, beneficial effects on behavior were noted, which were not seen when Res or VitD3 were individually applied. Taken together, our study found that Res and VitD3 improved locomotor and cognitive deficits, modulated Sirt1 and Lingo-1. Therefore, we would recommend co-treatment of VitD3 and Res to leverage complementary effects for the management of ET symptoms.

Harmaline induces apoptosis and inhibits migration in A2780 ovarian cancer cells in vitro.

Ovarian cancer is one of the most prevalent malignancies in women. Harmaline is reported to have powerful anticancer properties. We aimed to investigate the apoptotic and antimetastatic properties of harmaline in A2780 ovarian cancer cells. Cell viability, apoptosis, migration, and invasion were investigated in cells treated with harmaline. Reactive oxygen species (ROS) production, mRNA expression of apoptosis-associated genes, MMP-2, and MMP-9 were measured. Harmaline attenuated the viability of A2780 ovarian cancer cells in a dose- and time-dependent way. Furthermore, compared to NIH/3T3 mouse normal cell line (IC50 = 417 µM), the malignant A2080 cells were more sensitive to harmaline (IC50 = 300 µM after 24 h). Harmaline increased the production of ROS, raised the mRNA expression of p53 and the Bax/Bcl2 ratio. Harmaline also increased the proportion of cells in the late apoptotic and necrotic phases. MMP-2 and MMP-9's mRNA expression, gelatinase activity, and migration of A2780 cells also decreased by harmaline. These findings suggest that harmaline may have the potential to be a therapeutic drug for ovarian cancer by triggering apoptosis and suppressing invasion and migration.

Ayahuasca and Dimethyltryptamine Adverse Events and Toxicity Analysis: A Systematic Thematic Review.

The objective of this paper is to conduct a systematic thematic review of adverse events, safety, and toxicity of traditional ayahuasca plant preparations and its main psychoactive alkaloids (dimethyltryptamine [DMT], harmine, harmaline, and tetrahydroharmine), including discussing clinical considerations (within clinical trials or approved settings). A systematic literature search of preclinical, clinical, epidemiological, and pharmacovigilance data (as well as pertinent reviews and case studies) was conducted for articles using the electronic databases of PubMed and Web of Science (to 6 July 2023) and PsycINFO, ClinicalTrials.gov, and Embase (to 21 September 2022) and included articles in English in peer-reviewed journals. Additionally, reference lists were searched. Due to the breadth of the area covered, we presented the relevant data in a thematic format. Our searches revealed 78 relevant articles. Data showed that ayahuasca or DMT is generally safe; however, some adverse human events have been reported. Animal models using higher doses of ayahuasca have shown abortifacient and teratogenic effects. Isolated harmala alkaloid studies have also revealed evidence of potential toxicity at higher doses, which may increase with co-administration with certain medications. Harmaline revealed the most issues in preclinical models. Nevertheless, animal models involving higher-dose synthetic isolates may not necessarily be able to be extrapolated to human use of therapeutic doses of plant-based extracts. Serious adverse effects are rarely reported within healthy populations, indicating an acceptable safety profile for the traditional use of ayahuasca and DMT in controlled settings. Further randomized, controlled trials with judicious blinding, larger samples, and longer duration are needed.

Molecular docking of bioactive compounds extracted and purified from selected medicinal plant species against covid-19 proteins and in vitro evaluation.

Bioactive compounds are secondary metabolites of plants. They offer diverse pharmacological properties. Peganum harmala is reported to have pharmaceutical effects like insecticidal, antitumor, curing malaria, anti-spasmodic, vasorelaxant, antihistaminic effect. Rosa brunonii has medicinal importance in its flower and fruits effective against different diseases and juice of leaf is reported to be applied externally to cure wounds and cuts. Dryopteris ramosa aqueous leaf extract is used to treat stomach ulcers and stomachaches. Each of these three medicinal plants have been indicated to have anticancer, antiviral, antioxidant, cytotoxic and antifungal effects but efficacy of their bioactive compounds remained unexplored. Study was aimed to explore In-vitro and In-silico anticancer, antiviral, antioxidant, cytotoxic and antifungal effects of bioactive compounds of above three medicinal plants. DPPH and ABTS assay were applied for assessment of antioxidant properties of compounds. Antibacterial properties of compounds were checked by agar well diffusion method. Brine shrimp lethality assay was performed to check cytotoxic effect of compounds. Molecular docking was conducted to investigate the binding efficacy between isolated compounds and targeted proteins. The compound isomangiferrin and tiliroside presented strong antioxidant potential 78.32% (± 0.213) and 77.77% (± 0.211) respectively in DPPH assay while harmaline showed 80.71% (± 0.072) at 200 µg/mL in ABTS assay. The compound harmine, harmaline and PH-HM 17 exhibited highest zone of inhibition 22 mm, 23 mm, 22 mm respectively against Xanthomonas while Irriflophenone-3-C-ß- D-glucopyranoside showed maximum zone of inhibition 34 mm against E. coli. The compound isomangiferrin and vasicine contained strong antibacterial activity 32 mm and 22 mm respectively against S. aureus. The compound mangiferrin, astragalin, tiliroside, quercitin-3-O-rhamnoside showed maximum inhibitory zone 32 mm, 26 mm, 24 mm and 22 mm respectively against Klebsiella pneumoniae. Highest cytotoxic effect was observed by compound tiliroside i.e. 95% with LD50 value 73.59 µg/mL. The compound tiliroside showed the best binding mode of interaction to all targeted proteins presenting maximum hydrophobic interactions and hydrogen bonds. The binding affinity of tiliroside was - 17.9, - 14.9, - 14.6, - 13.8, - 12.8 against different proteins 6VAR, 5C5S, IEA3, 2XV7 and 6LUS respectively. Bioactive compounds are significant natural antioxidants, which could help to prevent the progression of various diseases caused by free radicals. Based on molecular docking we have concluded that phytochemicals can have better anticancer and antiviral potential.

Exploring potooxidative degradation pathways of harmol and harmalol alkaloids in water: effects of pH, excitation sources and atmospheric conditions.

This work explores the photochemical degradation of cationic species of 7-hydroxy-1-methyl-2H-pyrido[3,4-b]indole or harmol (1C) and the corresponding partially hydrogenated derivative 7-hydroxy-1-methyl-3,4-dihydro-2H-pyrido[3,4-b]indole or harmalol (2C) in aqueous solution. UV-visible absorption and fluorescence emission spectroscopy coupled with multivariate data analysis (MCR-ALS and PARAFAC), HPLC and HRESI-MS techniques were used for both quantitative and qualitative analysis. The formation of hydrogen peroxide reactive oxygen species (ROS) was quantified, and the influence of pH, oxygen partial pressure and photoexcitation source on the photochemical degradation of both compounds was assessed. The potential implications on the biosynthesis of ßCs and their biological role in living systems are discussed.

Evaluation of therapeutic role of harmaline: in vitro cytotoxicity targeting nucleic acids.

Use of small molecules as valuable drugs against diseases is still an indefinable purpose due to the lack of in-detail knowledge regarding proper bio-target identification, specificity aspects, mode-mechanism of binding and proper in vitro study. Harmaline, an important beta-carboline alkaloid, shows effective anti-proliferative action against different types of human cancers and is also found to be a nucleic acid targeting natural molecule. This review sought to address the different signal pathways of apoptosis by harmaline in different cancer cell lines and simultaneously to characterize the structure activity aspects of the alkaloid with different motifs of nucleic acid to show its preference, biological efficacy and genotoxicity. The results open up new insights for the design and development of small molecule-based nucleic acid therapeutic agents.

Anticancer Potential of ß-Carboline Alkaloids: An Updated Mechanistic Overview.

his comprehensive review is designed to evaluate the anticancer properties of ß-carbolines derived from medicinal plants, with the ultimate goal of assessing their suitability and potential in cancer treatment, management, and prevention. An exhaustive literature survey was conducted on a wide array of ß-carbolines including, but not limited to, harmaline, harmine, harmicine, harman, harmol, harmalol, pinoline, tetrahydroharmine, tryptoline, cordysinin C, cordysinin D, norharmane, and perlolyrine. Various analytical techniques were employed to identify and screen these compounds, followed by a detailed analysis of their anticancer mechanisms. Natural ß-carbolines such as harmaline and harmine have shown promising inhibitory effects on the growth of cancer cells, as evidenced by multiple in vitro and in vivo studies. Synthetically derived ß-carbolines also displayed noteworthy anticancer, neuroprotective, and cognitive-enhancing effects. The current body of research emphasizes the potential of ß-carbolines as a unique source of bioactive compounds for cancer treatment. The diverse range of ß-carbolines derived from medicinal plants can offer valuable insights into the development of new therapeutic strategies for cancer management and prevention.

Innovative spectrofluorometric method for determination of harmaline and harmine in different matrices.

Harmaline and harmine are naturally occurring closely related ß-carboline alkaloids found in Peganum and Banisteriopsis plants. They have historical significance in traditional practices due to their potential psychoactive and therapeutic properties. Herein, a highly sensitive spectrofluorometric method was developed for the quantifying of harmaline and harmine in diverse matrices, including pure forms, seed samples, and spiked plasma. The procedures lie in addressing the challenge of overlapping fluorescence spectra exhibited by harmaline and harmine through the incorporation of hydroxypropyl-ß-cyclodextrin, altering their chemical properties and fluorescence characteristics. Synchronous fluorescence measurements coupled with first derivative mathematical technique make it possible to distinguish between the harmaline and harmine at 419 and 456 nm, respectively. The method effectiveness is demonstrated through spectral analysis, optimization of the measurement conditions, adopting validation parameters and application to the pure form, seed samples and spiked human plasma. This methodology facilitates accurate determination of these alkaloids over the concentration range of 10─200 ng/mL. Thus, the developed approach provides a robust mean for the precise determination of harmaline and harmine, contributing to analytical chemistry's ongoing efforts to address complex challenges in quantification across diverse matrices.

Search for Novel Therapies for Essential Tremor Based on Positive Modulation of α6-Containing GABAA Receptors.

Background: Prior work using GABAA receptor subunit knockouts and the harmaline model has indicated that low-dose alcohol, gaboxadol, and ganaxolone suppress tremor via α6ßδ GABAA receptors. This suggests that drugs specifically enhancing the action of α6ßδ or α6ßγ2 GABAA receptors, both predominantly expressed on cerebellar granule cells, would be effective against tremor. We thus examined three drugs described by in vitro studies as selective α6ßδ (ketamine) or α6ßγ2 (Compound 6, flumazenil) receptor modulators. Methods: In the first step of evaluation, the maximal dose was sought at which 6/6 mice pass straight wire testing, a sensitive test for psychomotor impairment. Only non-impairing doses were used to evaluate for anti-tremor efficacy in the harmaline model, which was assessed in wildtype and α6 subunit knockout littermates. Results: Ketamine, in maximally tolerated doses of 2.0 and 3.5 mg/kg had minimal effect on harmaline tremor in both genotypes. Compound 6, at well-tolerated doses of 1-10 mg/kg, effectively suppressed tremor in both genotypes. Flumazenil suppressed tremor in wildtype mice at doses (0.015-0.05 mg/kg) far lower than those causing straight wire impairment, and did not suppress tremor in α6 knockout mice. Discussion: Modulators of α6ßδ and α6ßγ2 GABAA receptors warrant attention for novel therapies as they are anticipated to be effective and well-tolerated. Ketamine likely failed to attain α6ßδ-active levels. Compound 6 is an attractive candidate, but further study is needed to clarify its mechanism of action. The flumazenil results provide proof of principle that targeting α6ßγ2 receptors represents a worthy strategy for developing essential tremor therapies. Highlights: We tested for harmaline tremor suppression drugs previously described as in vitro α6ßδ or α6ßγ2 GABAA receptor-selective modulators. Well-tolerated flumazenil doses suppressed tremor in α6-wildtype but not α6-knockout mice. Compound 6 and ketamine failed to display this profile, likely from off-target effects. Selective α6 modulators hold promise as tremor therapy.

Harmaline toxicity on dorsal striatal neurons and its role in tremor.

Harmaline is one of the ß-carboline derivative compounds that is widely distributed in the food chain and human tissues. Harmine, a dehydrogenated form of harmaline, appeared to have a higher concentration in the brain, and appeared to be elevated in essential tremor (ET) and Parkinson's disease. Exogenous harmaline exposure in high concentration has myriad consequences, including inducing tremor, and causing neurodegeneration of Purkinje cells in the cerebellum. Harmaline-induced tremor is an established animal model for human ET, but its underlying mechanism is still controversial. One hypothesis posits that the inferior olive-cerebellum pathway is involved, and CaV3.1 T-type Ca2+ channel is a critical target of action. However, accumulating evidence indicates that tremor can be generated without disturbing T-type channels. This implies that additional neural circuits or molecular targets are involved. Using in vitro slice Ca2+-imaging and patch clamping, we demonstrated that harmaline reduced intracellular Ca2+ and suppressed depolarization-induced spiking activity of medium spiny striatal neurons (MSN), and this effect of harmaline can be partially attenuated by sulpiride (5 µM). In addition, the frequencies of spontaneous excitatory post-synaptic currents (sEPSCs) on MSNs were also significantly attenuated. Furthermore, the induced tremor in C57BL/6 J mice by harmaline injections (i.p. 12.5-18 mg/kg) was also shown to be attenuated by sulpiride (20 mg/kg). This series of experiments suggests that the dorsal striatum is a site of harmaline toxic action and might contribute to tremor generation. The findings also provide evidence that D2 signaling might be a part of the mechanism underlying essential tremor.

Mapping Pharmacologically Evoked Neurovascular Activation and Its Suppression in a Rat Model of Tremor Using Functional Ultrasound: A Feasibility Study.

Functional ultrasound (fUS), an emerging hemodynamic-based functional neuroimaging technique, is especially suited to probe brain activity and primarily used in animal models. Increasing use of pharmacological models for essential tremor extends new research to the utilization of fUS imaging in such models. Harmaline-induced tremor is an easily provoked model for the development of new therapies for essential tremor (ET). Furthermore, harmaline-induced tremor can be suppressed by the same classic medications used for essential tremor, which leads to the utilization of this model for preclinical testing. However, changes in local cerebral activities under the effect of tremorgenic doses of harmaline have not been completely investigated. In this study, we explored the feasibility of fUS imaging for visualization of cerebral activation and deactivation associated with harmaline-induced tremor and tremor-suppressing effects of propranolol. The spatial resolution of fUS using a high frame rate imaging enabled us to visualize time-locked and site-specific changes in cerebral blood flow associated with harmaline-evoked tremor. Intraperitoneal administration of harmaline generated significant neural activity changes in the primary motor cortex and ventrolateral thalamus (VL Thal) regions during tremor and then gradually returned to baseline level as tremor subsided with time. To the best of our knowledge, this is the first functional ultrasound study to show the neurovascular activation of harmaline-induced tremor and the therapeutic suppression in a rat model. Thus, fUS can be considered a noninvasive imaging method for studying neuronal activities involved in the ET model and its treatment.

Alcohol and Ganaxolone Suppress Tremor via Extra-Synaptic GABAA Receptors in the Harmaline Model of Essential Tremor.

Background: A long-standing question is why essential tremor often responds to non-intoxicating amounts of alcohol. Blood flow imaging and high-density electroencephalography have indicated that alcohol acts on tremor within the cerebellum. As extra-synaptic δ-subunit-containing GABAA receptors are sensitive to low alcohol levels, we wondered whether these receptors mediate alcohol's anti-tremor effect and, moreover, whether the δ-associated GABAA receptor α6 subunit, found abundantly in the cerebellum, is required. Methods: We tested the hypotheses that low-dose alcohol will suppress harmaline-induced tremor in wild-type mice, but not in littermates lacking GABAA receptor δ subunits, nor in littermates lacking α6 subunits. As the neurosteroid ganaxolone also activates extra-synaptic GABAA receptors, we similarly assessed this compound. The harmaline mouse model of essential tremor was utilized to generate tremor, measured as a percentage of motion power in the tremor bandwidth (9-16 Hz) divided by background motion power at 0.25-32 Hz. Results: Ethanol, 0.500 and 0.575 g/kg, and ganaxolone, 7 and 10 mg/kg, doses that do not impair performance in a sensitive psychomotor task, reduced harmaline tremor compared to vehicle-treated controls in wild-type mice but failed to suppress tremor in littermates lacking the δ or the α6 GABAA receptor subunit. Discussion: As cerebellar granule cells are the predominant brain site intensely expressing GABAA receptors containing both α6 and δ subunits, these findings suggest that this is where alcohol acts to suppress tremor. It is anticipated that medications designed specifically to target α6ßδ-containing GABAA receptors may be effective and well-tolerated for treating essential tremor. Highlights: How does alcohol temporarily ameliorate essential tremor? This study with a mouse model found that two specific kinds of GABA receptor subunits were needed for alcohol to work. As receptors with both these subunits are found mainly in cerebellum, this work suggests this is where alcohol acts to suppress tremor.

Harmaline exerts potentially anti-cancer effects on U-87 human malignant glioblastoma cells in vitro.

BACKGROUND: Harmaline is a ß-carboline alkaloid that can be extracted from the seeds of Peganum harmala. Harmaline has been shown to exhibit a potent cytotoxic effect against tumor cells. In this study, the anti-glioblastoma activity of harmaline was investigated in vitro. METHODS AND RESULTS: Cell viability, apoptosis, and cell cycle arrest were assessed in U-87 cells treated with harmaline at different doses. Reactive oxygen species (ROS) generation and the mRNA expression of apoptosis-associated genes were assessed. The anti-metastatic effect of harmaline on U-87 cells was evaluated by gelatin zymography assay where matrix metalloproteinase [MMP]-2/-9 enzymatic activity was measured, and the scratch assay was used to assess migratory responses. Flow cytometry demonstrated that harmaline could suppress the proliferation and induce sub-G1 cell cycle arrest and apoptotic cell death in glioblastoma cells. Harmaline treatment was also associated with an upregulation of the cell cycle-related genes, p21 and p53, and pro-apoptotic Bax, as well as the induction of ROS. The zymography assay indicated that the essential steps of metastasis were potently suppressed by harmaline through inhibiting the expression of MMP-2 and - 9. In addition, the migration of U-87 cells was significantly reduced after harmaline treatment. CONCLUSION: Our data suggest a basis for further research of harmaline which has potential cytotoxic activities in glioblastoma cells; inducing cell cycle arrest and apoptosis, repression of migration, possibly invasion, and metastasis.

GABA-A Alpha 2/3 but Not Alpha 1 Receptor Subunit Ligand Inhibits Harmaline and Pimozide-Induced Tremor in Rats.

Treatment of tremors, such as in essential tremor (ET) and Parkinson's disease (PD) is mostly ineffective. Exact tremor pathomechanisms are unknown and relevant animal models are missing. GABA-A receptor is a target for tremorolytic medications, but current non-selective drugs produce side effects and have safety liabilities. The aim of this study was a search for GABA-A subunit-specific tremorolytics using different tremor-generating mechanisms. Two selective positive allosteric modulators (PAMs) were tested. Zolpidem, targeting GABA-A α1, was not effective in models of harmaline-induced ET, pimozide- or tetrabenazine-induced tremulous jaw movements (TJMs), while the novel GABA-A α2/3 selective MP-III-024 significantly reduced both the harmaline-induced ET tremor and pimozide-induced TJMs. While zolpidem decreased the locomotor activity of the rats, MP-III-024 produced small increases. These results provide important new clues into tremor suppression mechanisms initiated by the enhancement of GABA-driven inhibition in pathways controlled by α2/3 but not α1 containing GABA-A receptors. Tremor suppression by MP-III-024 provides a compelling reason to consider selective PAMs targeting α2/3-containing GABA-A receptors as novel therapeutic drug targets for ET and PD-associated tremor. The possibility of the improved tolerability and safety of this mechanism over non-selective GABA potentiation provides an additional rationale to further pursue the selective α2/3 hypothesis.

Akkermansia muciniphila protects mice against an emerging tick-borne viral pathogen.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by a phlebovirus in the Bunyaviridae family. Infection can result in systemic inflammatory response syndrome with a high fatality rate, and there are currently no treatments or vaccines available. The microbiota has been implicated in host susceptibility to systemic viral infection and disease outcomes, but whether the gut microbiota is implicated in severe fever with thrombocytopenia syndrome virus (SFTSV) infection is unknown. Here, we analysed faecal and serum samples from patients with SFTS using 16S ribosomal RNA-sequencing and untargeted metabolomics, respectively. We found that the gut commensal Akkermansia muciniphila increased in relative abundance over the course of infection and was reduced in samples from deceased patients. Using germ-free or oral antibiotic-treated mice, we found that A. muciniphila produces the ß-carboline alkaloid harmaline, which protects against SFTSV infection by suppressing NF-κB-mediated systemic inflammation. Harmaline indirectly modulated the virus-induced inflammatory response by specifically enhancing bile acid-CoA: amino acid N-acyltransferase expression in hepatic cells to increase conjugated primary bile acids, glycochenodeoxycholic acid and taurochenodeoxycholic acid. These bile acids induced transmembrane G-protein coupled receptor-5-dependent anti-inflammatory responses. These results indicate the probiotic potential of A. muciniphila in mitigating SFTSV infection.

Antiviral activities of plant-derived indole and ß-carboline alkaloids against human and avian influenza viruses.

The persistent evolution of drug-resistant influenza strains represents a global concern. The innovation of new treatment approaches through drug screening strategies and investigating the antiviral potential of bioactive natural-based chemicals may address the issue. Herein, we screened the anti-influenza efficacy of some biologically active indole and ß-carboline (ßC) indole alkaloids against two different influenza A viruses (IAV) with varied host range ranges; seasonal influenza A/Egypt/NRC098/2019(H1N1) and avian influenza A/chicken/Egypt/N12640A/2016(H5N1). All compounds were first assessed for their half-maximal cytotoxic concentration (CC50) in MDCK cells and half-maximal inhibitory concentrations (IC50) against influenza A/H5N1. Intriguingly, Strychnine sulfate, Harmalol, Harmane, and Harmaline showed robust anti-H5N1 activities with IC50 values of 11.85, 0.02, 0.023, and 3.42 µg/ml, respectively, as compared to zanamivir and amantadine as control drugs (IC50 = 0.079 µg/ml and 17.59 µg/ml, respectively). The efficacy of the predefined phytochemicals was further confirmed against influenza A/H1N1 and they displayed potent anti-H1N1 activities compared to reference drugs. Based on SI values, the highly promising compounds were then evaluated for antiviral efficacy through plaque reduction assay and consistently they revealed high viral inhibition percentages at non-toxic concentrations. By studying the modes of antiviral action, Harmane and Harmalol could suppress viral infection via interfering mainly with the viral replication of the influenza A/H5N1 virus, whilst Harmaline exhibited a viricidal effect against the influenza A/H5N1 virus. Whereas, Strychnine sulfate elucidated its anti-influenza potency by interfering with viral adsorption into MDCK cells. Consistently, chemoinformatic studies showed that all studied phytochemicals illustrated HB formations with essential peptide cleft through the NH of indole moiety. Among active alkaloids, harmalol displayed the best lipophilicity metrics including ligand efficiency (LE) and ligand lipophilic efficiency (LLE) for both viruses. Compounds geometry and their ability to participate in HB formation are very crucial.

Cerebellar α6GABAA Receptors as a Therapeutic Target for Essential Tremor: Proof-of-Concept Study with Ethanol and Pyrazoloquinolinones.

Ethanol has been shown to suppress essential tremor (ET) in patients at low-to-moderate doses, but its mechanism(s) of action remain unknown. One of the ET hypotheses attributes the ET tremorgenesis to the over-activated firing of inferior olivary neurons, causing synchronic rhythmic firings of cerebellar Purkinje cells. Purkinje cells, however, also receive excitatory inputs from granule cells where the α6 subunit-containing GABAA receptors (α6GABAARs) are abundantly expressed. Since ethanol is a positive allosteric modulator (PAM) of α6GABAARs, such action may mediate its anti-tremor effect. Employing the harmaline-induced ET model in male ICR mice, we evaluated the possible anti-tremor effects of ethanol and α6GABAAR-selective pyrazoloquinolinone PAMs. The burrowing activity, an indicator of well-being in rodents, was measured concurrently. Ethanol significantly and dose-dependently attenuated action tremor at non-sedative doses (0.4-2.4 g/kg, i.p.). Propranolol and α6GABAAR-selective pyrazoloquinolinones also significantly suppressed tremor activity. Neither ethanol nor propranolol, but only pyrazoloquinolinones, restored burrowing activity in harmaline-treated mice. Importantly, intra-cerebellar micro-injection of furosemide (an α6GABAAR antagonist) had a trend of blocking the effect of pyrazoloquinolinone Compound 6 or ethanol on harmaline-induced tremor. In addition, the anti-tremor effects of Compound 6 and ethanol were synergistic. These results suggest that low doses of ethanol and α6GABAAR-selective PAMs can attenuate action tremor, at least partially by modulating cerebellar α6GABAARs. Thus, α6GABAARs are potential therapeutic targets for ET, and α6GABAAR-selective PAMs may be a potential mono- or add-on therapy.