Background and Purpose
There is a need for effective anti‐COVID‐19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has ...proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID‐19. The purpose of the present study was to find synergistic antiviral combinations for COVID‐19 based on lethal mutagenesis.
Experimental Approach
The effect of combinations of remdesivir and ribavirin on the infectivity of SARS‐CoV‐2 in cell culture has been tested. Viral populations were monitored by ultra‐deep sequencing, and the decrease of infectivity as a result of the treatment was measured.
Key Results
Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS‐CoV‐2, quantified both by CompuSyn (Chou‐Talalay method) and Synergy Finder (ZIP‐score model). In serial passage experiments, virus extinction was readily achieved with remdesivir‐ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi‐species.
Conclusion and Implications
SARS‐CoV‐2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS‐CoV‐2 suppression.
Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important ...drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.
Upon the emergence of SARS-CoV-2 in the human population, it was conjectured that for this coronavirus the dynamic intra-host heterogeneity typical of RNA viruses would be toned down. Nothing of this ...sort is observed. Here we review the main observations on the complexity and diverse composition of SARS-CoV-2 mutant spectra sampled from infected patients, within the framework of quasispecies dynamics. The analyses suggest that the information provided by myriads of genomic sequences within infected individuals may have a predictive value of the genomic sequences that acquire epidemiological relevance. Possibilities to reconcile the presence of broad mutant spectra in the large RNA coronavirus genome with its encoding a 3' to 5' exonuclease proofreading-repair activity are considered. Indeterminations in the behavior of individual viral genomes provide a benefit for the survival of the ensemble. We propose that this concept falls in the domain of "stochastic thinking," a notion that applies also to cellular processes, as a means for biological systems to face unexpected needs.
Fragile X syndrome (FXS), the most common monogenic cause of inherited intellectual disability and autism, is caused by the silencing of the FMR1 gene, leading to the loss of fragile X mental ...retardation protein (FMRP), a synaptically expressed RNA-binding protein regulating translation. The Fmr1 knockout model recapitulates the main traits of the disease. Uncontrolled activity of metabotropic glutamate receptor 5 (mGluR5) and mammalian target of rapamycin (mTOR) signaling seem crucial in the pathology of this disease. The endocannabinoid system (ECS) is a key modulator of synaptic plasticity, cognitive performance, anxiety, nociception and seizure susceptibility, all of which are affected in FXS. The cannabinoid receptors CB1 (CB1R) and CB2 (CB2R) are activated by phospholipid-derived endocannabinoids, and CB1R-driven long-term regulation of synaptic strength, as a consequence of mGluR5 activation, is altered in several brain areas of Fmr1 knockout mice. We found that CB1R blockade in male Fmr1 knockout (Fmr1(-/y)) mice through pharmacological and genetic approaches normalized cognitive impairment, nociceptive desensitization, susceptibility to audiogenic seizures, overactivated mTOR signaling and altered spine morphology, whereas pharmacological blockade of CB2R normalized anxiolytic-like behavior. Some of these traits were also reversed by pharmacological inhibition of mTOR or mGluR5. Thus, blockade of ECS is a potential therapeutic approach to normalize specific alterations in FXS.
Rationale
Endocannabinoids are well poised to regulate crosstalk between energy sensing of hunger and satiety and reward-driven motivation.
Objectives
Here, we aimed to unravel associations between ...plasma endocannabinoids and brain connectivity in homeostatic and reward circuits across hunger and satiety states.
Methods
Fifteen participants (7 females) underwent two counter-balanced resting-state functional magnetic resonance imaging scans, one after overnight fasting and one after consumption of a standardized filling meal (satiety). Before each scan, we drew blood to measure plasma endocannabinoid concentrations (anandamide AEA, anandamide-derived POEA, and 2-arachidonoylglycerol 2-AG), analyzed with liquid chromatography tandem mass spectrometry.
Results
We found that AEA levels were associated with increased connectivity between the lateral hypothalamus and the ventral striatum during satiety. Furthermore, fasting AEA levels correlated with connectivity between the ventral striatum and the anterior cingulate cortex and the insula
.
Conclusions
Altogether, results suggest that peripheral AEA concentrations are sensitive to homeostatic changes and linked to neural communication in reward and salience networks. Findings may have significant implications for understanding normal and abnormal interactions between homeostatic input and reward valuation.
The concept of a mild mutagen was coined to describe a minor mutagenic activity exhibited by some nucleoside analogues that potentiated their efficacy as antiretroviral agents. In the present study, ...we report the mild mutagen activity of sofosbuvir (SOF) for hepatitis C virus (HCV). Serial passages of HCV in human hepatoma cells, in the presence of SOF at a concentration well below its cytotoxic concentration 50 (CC
) led to pre-extinction populations whose mutant spectra exhibited a significant increase of C→U transitions, relative to populations passaged in the absence of SOF. This was reflected in an increase in several diversity indices that were used to characterize viral quasispecies. The mild mutagenic activity of SOF was largely absent when it was tested with isogenic HCV populations that displayed high replicative fitness. Thus, SOF can act as a mild mutagen for HCV, depending on HCV fitness. Possible mechanisms by which the SOF mutagenic activity may contribute to its antiviral efficacy are discussed.
Williams–Beuren syndrome (WBS) is a rare genetic multisystemic disorder characterized by mild-to-moderate intellectual disability and hypersocial phenotype, while the most life-threatening features ...are cardiovascular abnormalities. Nowadays, there are no pharmacological treatments to directly ameliorate the main traits of WBS. The endocannabinoid system (ECS), given its relevance for both cognitive and cardiovascular function, could be a potential druggable target in this syndrome. We analyzed the components of the ECS in the complete deletion (CD) mouse model of WBS and assessed the impact of its pharmacological modulation in key phenotypes relevant for WBS. CD mice showed the characteristic hypersociable phenotype with no preference for social novelty and poor short-term object-recognition performance. Brain cannabinoid type-1 receptor (CB1R) in CD male mice showed alterations in density and coupling with no detectable change in main endocannabinoids. Endocannabinoid signaling modulation with subchronic (10 days) JZL184, a selective inhibitor of monoacylglycerol lipase, specifically normalized the social and cognitive phenotype of CD mice. Notably, JZL184 treatment improved cardiovascular function and restored gene expression patterns in cardiac tissue. These results reveal the modulation of the ECS as a promising novel therapeutic approach to improve key phenotypic alterations in WBS.
Williams-Beuren syndrome (WBS) is a rare disorder that causes hyper-social behavior, intellectual disability, memory problems, and life-threatening overgrowth of the heart. Behavioral therapies can help improve the cognitive and social aspects of the syndrome and surgery is sometimes used to treat the effects on the heart, although often with limited success. However, there are currently no medications available to treat WBS.
The endocannabinoid system – which consists of cannabis-like chemical messengers that bind to specific cannabinoid receptor proteins – has been shown to influence cognitive and social behaviors, as well as certain functions of the heart. This has led scientists to suspect that the endocannabinoid system may play a role in WBS, and drugs modifying this network of chemical messengers could help treat the rare condition.
To investigate, Navarro-Romero, Galera-López et al. studied mice which had the same genetic deletion found in patients with WBS. Similar to humans, the male mice displayed hyper-social behaviors, had memory deficits and enlarged hearts. Navarro-Romero, Galera-López et al. found that these mutant mice also had differences in the function of the receptor protein cannabinoid type-1 (CB1).
The genetically modified mice were then treated with an experimental drug called JZL184 that blocks the breakdown of endocannabinoids which bind to the CB1 receptor
.
This normalized the number and function of receptors in the brains of the WBS mice, and reduced their social and memory symptoms. The treatment also restored the animals’ heart cells to a more normal size, improved the function of their heart tissue, and led to lower blood pressure. Further experiments revealed that the drug caused the mutant mice to activate many genes in their heart muscle cells to the same level as normal, healthy mice
.
These findings suggest that JZL184 or other drugs targeting the endocannabinoid system may help ease the symptoms associated with WBS. More studies are needed to test the drug’s effectiveness in humans with this syndrome. Furthermore, the dramatic effect JZL184 has on the heart suggests that it might also help treat high blood pressure or conditions that cause the overgrowth of heart cells.
We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of ...SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.
We report a quantification of the decrease of effectiveness of antiviral agents directed to hepatitis C virus, when the agents are added during an ongoing infection in cell culture vs. when they are ...added at the beginning of the infection. Major determinants of the decrease of inhibitory activity are the time post-infection of inhibitor administration and viral replicative fitness. The efficacy decrease has been documented with antiviral assays involving the combination of the direct-acting antiviral agents, daclatasvir and sofosbuvir, and with the combination of the lethal mutagens, favipiravir and ribavirin. The results suggest that strict antiviral effectiveness assays in preclinical trials may involve the use of high fitness viral populations and the delayed administration of the agents, relative to infection onset.
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