Inhibition of salt inducible kinases reduces rhythmic HIV-1 replication and reactivation from latency.

Human immunodeficiency virus type 1 (HIV-1) causes a major burden on global health, and eradication of latent virus infection is one of the biggest challenges in the field. The circadian clock is an endogenous timing system that oscillates with a ~24 h period regulating multiple physiological processes and cellular functions, and we recently reported that the cell intrinsic clock regulates rhythmic HIV-1 replication. Salt inducible kinases (SIK) contribute to circadian regulatory networks, however, there is limited evidence for SIKs regulating HIV-1 infection. Here, we show that pharmacological inhibition of SIKs perturbed the cellular clock and reduced rhythmic HIV-1 replication in circadian synchronised cells. Further, SIK inhibitors or genetic silencing of Sik expression inhibited viral replication in primary cells and in a latency model, respectively. Overall, this study demonstrates a role for salt inducible kinases in regulating HIV-1 replication and latency reactivation, which can provide innovative routes to better understand and target latent HIV-1 infection.

Molecular components of the circadian clock regulate HIV-1 replication.

Human immunodeficiency virus 1 (HIV-1) causes major health burdens worldwide and still lacks curative therapies and vaccines. Circadian rhythms are endogenous daily oscillations that coordinate an organism's response to its environment and invading pathogens. Peripheral viral loads of HIV-1 infected patients show diurnal variation; however, the underlying mechanisms remain unknown. Here, we demonstrate a role for the cell-intrinsic clock to regulate rhythmic HIV-1 replication in circadian-synchronized systems. Silencing the circadian activator Bmal1 abolishes this phenotype, and we observe BMAL1 binding to the HIV-1 promoter. Importantly, we show differential binding of the nuclear receptors REV-ERB and ROR to the HIV-long terminal repeat at different circadian times, demonstrating a dynamic interplay in time-of-day regulation of HIV-1 transcription. Bioinformatic analysis shows circadian regulation of host factors that control HIV-1 replication, providing an additional mechanism for rhythmic viral replication. This study increases our understanding of the circadian regulation of HIV-1, which can ultimately inform new therapies.

The multiple roles of salt-inducible kinases in regulating physiology.

Salt-inducible kinases (SIKs), which comprise a family of three homologous serine-threonine kinases, were first described for their role in sodium sensing but have since been shown to regulate multiple aspects of physiology. These kinases are activated or deactivated in response to extracellular signals that are cell surface receptor mediated and go on to phosphorylate multiple targets including the transcription cofactors CRTC1-3 and the class IIa histone deacetylases (HDACs). Thus, the SIK family conveys signals about the cellular environment to reprogram transcriptional and posttranscriptional processes in response. In this manner, SIKs have been shown to regulate metabolic responses to feeding/fasting, cell division and oncogenesis, inflammation, immune responses, and most recently, sleep and circadian rhythms. Sleep and circadian rhythms are master regulators of physiology and are exquisitely sensitive to regulation by environmental light and physiological signals such as the need for sleep. Salt-inducible kinases have been shown to be central to the molecular regulation of both these processes. Here, we summarize the molecular mechanisms by which SIKs control these different domains of physiology and highlight where there is mechanistic overlap with sleep/circadian rhythm control.

Sleep and circadian rhythm disruption alters the lung transcriptome to predispose to viral infection.

Sleep and circadian rhythm disruption (SCRD), as encountered during shift work, increases the risk of respiratory viral infection including SARS-CoV-2. However, the mechanism(s) underpinning higher rates of respiratory viral infection following SCRD remain poorly characterized. To address this, we investigated the effects of acute sleep deprivation on the mouse lung transcriptome. Here we show that sleep deprivation profoundly alters the transcriptional landscape of the lung, causing the suppression of both innate and adaptive immune systems, disrupting the circadian clock, and activating genes implicated in SARS-CoV-2 replication, thereby generating a lung environment that could promote viral infection and associated disease pathogenesis. Our study provides a mechanistic explanation of how SCRD increases the risk of respiratory viral infections including SARS-CoV-2 and highlights possible therapeutic avenues for the prevention and treatment of respiratory viral infection.

The regulation of circadian entrainment in mice by the adenosine the A 2A /A 1 receptor antagonist CT1500.

Circadian entrainment in mice relies primarily on photic cues that trigger the transcription of the core clock genes Period1/2 in the suprachiasmatic nucleus (SCN), thus aligning the phase of the clock with the dawn/dusk cycle. It has been shown previously that this pathway is directly regulated by adenosine signalling and that adenosine A2A/A1 receptor antagonists can both enhance photic entrainment and phase shift circadian rhythms of wheel-running behaviour in mice. In this study, we tested the ability of CT1500, a clinically safe adenosine A2A/A1 receptor antagonist to effect circadian entrainment. We show that CT1500 lengthens circadian period in SCN ex vivo preparations. Furthermore, we show in vivo that a single dose of CT1500 enhances re-entrainment to a shifted light dark cycle in a dose-dependent manner in mice and also phase shifts the circadian clock under constant dark with a clear time-of-day related pattern. The phase response curve shows CT1500 causes phase advances during the day and phase delays at dusk. Finally, we show that daily timed administration of CT1500 can entrain the circadian clock to a 24 h rhythm in free-running mice. Collectively, these data support the use of CT1500 in the treatment of disorders of circadian entrainment.

Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection.

We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-µL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8-94.7%) and specificity of 92.4% (95% CI 83.2-97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.

Adenosine integrates light and sleep signalling for the regulation of circadian timing in mice.

The accumulation of adenosine is strongly correlated with the need for sleep and the detection of sleep pressure is antagonised by caffeine. Caffeine also affects the circadian timing system directly and independently of sleep physiology, but how caffeine mediates these effects upon the circadian clock is unclear. Here we identify an adenosine-based regulatory mechanism that allows sleep and circadian processes to interact for the optimisation of sleep/wake timing in mice. Adenosine encodes sleep history and this signal modulates circadian entrainment by light. Pharmacological and genetic approaches demonstrate that adenosine acts upon the circadian clockwork via adenosine A1/A2A receptor signalling through the activation of the Ca2+ -ERK-AP-1 and CREB/CRTC1-CRE pathways to regulate the clock genes Per1 and Per2. We show that these signalling pathways converge upon and inhibit the same pathways activated by light. Thus, circadian entrainment by light is systematically modulated on a daily basis by sleep history. These findings contribute to our understanding of how adenosine integrates signalling from both light and sleep to regulate circadian timing in mice.

Patient fibroblast circadian rhythms predict lithium sensitivity in bipolar disorder.

Bipolar disorder is a chronic neuropsychiatric condition associated with mood instability, where patients present significant sleep and circadian rhythm abnormalities. Currently, the pathophysiology of bipolar disorder remains elusive, but treatment with lithium continues as the benchmark pharmacotherapy, functioning as a potent mood stabilizer in most, but not all patients. Lithium is well documented to induce period lengthening and amplitude enhancement of the circadian clock. Based on this, we sought to investigate whether lithium differentially impacts circadian rhythms in bipolar patient cell lines and crucially if lithium's effect on the clock is fundamental to its mood-stabilizing effects. We analyzed the circadian rhythms of bipolar patient-derived fibroblasts (n = 39) and their responses to lithium and three further chronomodulators. Here we show, relative to controls (n = 23), patients exhibited a wider distribution of circadian period (p < 0.05), and that patients with longer periods were medicated with a wider range of drugs, suggesting lower effectiveness of lithium. In agreement, patient fibroblasts with longer periods displayed muted circadian responses to lithium as well as to other chronomodulators that phenocopy lithium. These results show that lithium differentially impacts the circadian system in a patient-specific manner and its effect is dependent on the patient's circadian phenotype. We also found that lithium-induced behavioral changes in mice were phenocopied by modulation of the circadian system with drugs that target the clock, and that a dysfunctional clock ablates this response. Thus, chronomodulatory compounds offer a promising route to a novel treatment paradigm. These findings, upon larger-scale validation, could facilitate the implementation of a personalized approach for mood stabilization.

TRESK is a key regulator of nocturnal suprachiasmatic nucleus dynamics and light adaptive responses.

The suprachiasmatic nucleus (SCN) is a complex structure dependent upon multiple mechanisms to ensure rhythmic electrical activity that varies between day and night, to determine circadian adaptation and behaviours. SCN neurons are exposed to glutamate from multiple sources including from the retino-hypothalamic tract and from astrocytes. However, the mechanism preventing inappropriate post-synaptic glutamatergic effects is unexplored and unknown. Unexpectedly we discovered that TRESK, a calcium regulated two-pore potassium channel, plays a crucial role in this system. We propose that glutamate activates TRESK through NMDA and AMPA mediated calcium influx and calcineurin activation to then oppose further membrane depolarisation and rising intracellular calcium. Hence, in the absence of TRESK, glutamatergic activity is unregulated leading to membrane depolarisation, increased nocturnal SCN firing, inverted basal calcium levels and impaired sensitivity in light induced phase delays. Our data reveals TRESK plays an essential part in SCN regulatory mechanisms and light induced adaptive behaviours.

Effects of the putative lithium mimetic ebselen on pilocarpine-induced neural activity.

Lithium, commonly used to treat bipolar disorder, potentiates the ability of the muscarinic agonist pilocarpine to induce seizures in rodents. As this potentiation by lithium is reversed by the administration of myo-inositol, the potentiation may be mediated by inhibition of inositol monophosphatase (IMPase), a known target of lithium. Recently, we demonstrated that ebselen is a 'lithium mimetic' in regard to behaviours in both mice and man. Ebselen inhibits IMPase in vitro and lowers myo-inositol in vivo in the brains of mice and men, making ebselen the only known inhibitor of IMPase, other than lithium, that penetrates the blood-brain barrier. Our objective was to determine the effects of ebselen on sensitization to pilocarpine-induced seizures and neural activity. We administered ebselen at different doses and time intervals to mice, followed by injection of a sub-seizure dose of pilocarpine. We assessed seizure and neural activity by a subjective seizure rating scale, by monitoring tremors, and by induction of the immediate early gene c-fos. In contrast to lithium, ebselen did not potentiate the ability of pilocarpine to induce seizures. Unexpectedly, ebselen inhibited pilocarpine-induced tremor as well as pilocarpine-induced increases in c-fos mRNA levels. Both lithium and ebselen inhibit a common target, IMPase, but only lithium potentiates pilocarpine-induced seizures, consistent with their polypharmacology at diverse molecular targets. We conclude that ebselen does not potentiate pilocarpine-induced seizures and instead, reduces pilocarpine-mediated neural activation. This lack of potentiation of muscarinic sensitization may be one reason for the lack of side-effects observed with ebselen treatment clinically.

Sleep-Wake Disturbance Related to Ocular Disease: A Systematic Review of Phase-Shifting Pharmaceutical Therapies.

PURPOSE: Light input, via the eyes, is essential for regulating circadian rhythms. Eye diseases can cause disruption of vital biological rhythms. Of totally blind people, 87% report sleep problems. There are no UK guidelines for visual disturbance-related circadian rhythm disruption. Our objective was to systematically review the literature to determine the effectiveness of pharmacological agents on the sleep quality of patients with sleep disturbance related to ocular disease. METHODS: We searched CENTRAL, MEDLINE, EMBASE, PsycINFO, and CINAHL alongside protocol registries and citation searches. We assessed the risk of bias using the Cochrane Risk of Bias Assessment Tool and assessed the strength of overall evidence using GRADE criteria. RESULTS: Four studies (n=116) met the inclusion criteria. Low-quality evidence showed that melatonin can cause entrainment (1 study), increases in total sleep time (all 3 studies), and reduction in sleep latency (1 study). Low-to-moderate quality evidence showed tasimelteon causes a significant improvement in entrainment, midpoint of sleep timing, lower-quartile of night-time sleep, and upper-quartile of daytime sleep. CONCLUSIONS: Results should be treated with caution as the melatonin studies had risks of bias due to inadequate reporting of randomization and masking procedures. The tasimelteon trial had a risk of reporting bias due to changing the outcomes after enrolling participants. Despite the paucity of trials, melatonin and tasimelteon may cause entrainment and improve subjective sleep measures with limited side effects. TRANSLATIONAL RELEVANCE: Given the relative cost melatonin may be a viable choice for treatment of circadian rhythm sleep disorders in the blind and warrants further research.

The genetics of circadian rhythms, sleep and health.

Circadian rhythms are 24-h rhythms in physiology and behaviour generated by molecular clocks, which serve to coordinate internal time with the external world. The circadian system is a master regulator of nearly all physiology and its disruption has major consequences on health. Sleep and circadian rhythm disruption (SCRD) is a ubiquitous feature in today's 24/7 society, and studies on shift-workers have shown that SCRD can lead not only to cognitive impairment, but also metabolic syndrome and psychiatric illness including depression (1,2). Mouse models of clock mutants recapitulate these deficits, implicating mechanistic and causal links between SCRD and disease pathophysiology (3-5). Importantly, treating clock disruption reverses and attenuates these adverse health states in animal models (6,7), thus establishing the circadian system as a novel therapeutic target. Significantly, circadian and clock-controlled gene mutations have recently been identified by Genome-Wide Association Studies (GWAS) in the aetiology of sleep, mental health and metabolic disorders. This review will focus upon the genetics of circadian rhythms in sleep and health.

Circadian rhythms and metabolism: from the brain to the gut and back again.

This paper focuses on the relationship between the circadian system and glucose metabolism. Research across the translational spectrum confirms the importance of the circadian system for glucose metabolism and offers promising clues as to when and why these systems go awry. In particular, basic research has started to clarify the molecular and genetic mechanisms through which the circadian system regulates metabolism. The study of human behavior, especially in the context of psychiatric disorders, such as bipolar disorder and major depression, forces us to see how inextricably linked mental health and metabolic health are. We also emphasize the remarkable opportunities for advancing circadian science through big data and advanced analytics. Advances in circadian research have translated into environmental and pharmacological interventions with tremendous therapeutic potential.

Effects of the potential lithium-mimetic, ebselen, on impulsivity and emotional processing.

RATIONALE: Lithium remains the most effective treatment for bipolar disorder and also has important effects to lower suicidal behaviour, a property that may be linked to its ability to diminish impulsive, aggressive behaviour. The antioxidant drug, ebselen, has been proposed as a possible lithium-mimetic based on its ability in animals to inhibit inositol monophosphatase (IMPase), an action which it shares with lithium. OBJECTIVES: The aim of the study was to determine whether treatment with ebselen altered emotional processing and diminished measures of risk-taking behaviour. METHODS: We studied 20 healthy participants who were tested on two occasions receiving either ebselen (3600 mg over 24 h) or identical placebo in a double-blind, randomized, cross-over design. Three hours after the final dose of ebselen/placebo, participants completed the Cambridge Gambling Task (CGT) and a task that required the detection of emotional facial expressions (facial emotion recognition task (FERT)). RESULTS: On the CGT, relative to placebo, ebselen reduced delay aversion while on the FERT, it increased the recognition of positive vs negative facial expressions. CONCLUSIONS: The study suggests that at the dosage used, ebselen can decrease impulsivity and produce a positive bias in emotional processing. These findings have implications for the possible use of ebselen in the disorders characterized by impulsive behaviour and dysphoric mood.

Oxford Lithium Trial (OxLith) of the early affective, cognitive, neural and biochemical effects of lithium carbonate in bipolar disorder: study protocol for a randomised controlled trial.

BACKGROUND: Despite lithium's being the most effective drug for bipolar disorder and in clinical use for decades, we still know very little about its early effects relevant to its mode of action. METHODS/DESIGN: The Oxford Lithium Trial is a double-blind, randomised, placebo-controlled study of 6-week lithium treatment in participants with bipolar disorder and mood instability. Its aim is to identify early clinical, neurocognitive and biological effects. Participants (n = 40) will undergo an intensive battery of multi-modal investigations, including remote monitoring of mood, activity and physiology, as well as cognitive testing, fMRI and magnetoencephalography, together with biochemical and gene expression measurements to assess renal, inflammatory and circadian effects. DISCUSSION: The findings derived from this trial may be of value in predicting subsequent therapeutic response or side effects, not only relevant to the use of lithium but also providing a potential signature to help in more rapid evaluation of novel mood stabilisers. In this respect, OxLith is a step towards the development of a valid experimental medicine model for bipolar disorder. TRIAL REGISTRATION: ISRCTN91624955 . Registered on 22 January 2015.

Effects of the potential lithium-mimetic, ebselen, on brain neurochemistry: a magnetic resonance spectroscopy study at 7 tesla.

RATIONALE: Lithium is an effective treatment for bipolar disorder, but safety issues complicate its clinical use. The antioxidant drug, ebselen, may be a possible lithium-mimetic based on its ability to inhibit inositol monophosphatase (IMPase), an action which it shares with lithium. OBJECTIVES: Our primary aim was to determine whether ebselen lowered levels of inositol in the human brain. We also assessed the effect of ebselen on other brain neurometabolites, including glutathione, glutamate, glutamine, and glutamate + glutamine (Glx) METHODS: Twenty healthy volunteers were tested on two occasions receiving either ebselen (3600 mg over 24 h) or identical placebo in a double-blind, random-order, crossover design. Two hours after the final dose of ebselen/placebo, participants underwent proton magnetic resonance spectroscopy ((1)H MRS) at 7 tesla (T) with voxels placed in the anterior cingulate and occipital cortex. Neurometabolite levels were calculated using an unsuppressed water signal as a reference and corrected for individual cerebrospinal fluid content in the voxel. RESULTS: Ebselen produced no effect on neurometabolite levels in the occipital cortex. In the anterior cingulate cortex, ebselen lowered concentrations of inositol (p = 0.028, Cohen's d = 0.60) as well as those of glutathione (p = 0.033, d = 0.58), glutamine (p = 0.024, d = 0.62), glutamate (p = 0.01, d = 0.73), and Glx (p = 0.001, d = 1.0). CONCLUSIONS: The study suggests that ebselen produces a functional inhibition of IMPase in the human brain. The effect of ebselen to lower glutamate is consistent with its reported ability to inhibit the enzyme, glutaminase. Ebselen may have potential as a repurposed treatment for bipolar disorder.

Effect of the Putative Lithium Mimetic Ebselen on Brain Myo-Inositol, Sleep, and Emotional Processing in Humans.

Lithium remains the gold standard in treating bipolar disorder but has unwanted toxicity and side effects. We previously reported that ebselen inhibits inositol monophosphatase (IMPase) and exhibits lithium-like effects in animal models through lowering of inositol. Ebselen has been tested in clinical trials for other disorders, enabling us to determine for the first time the effect of a blood-brain barrier-penetrant IMPase inhibitor on human central nervous system (CNS) function. We now report that in a double-blind, placebo-controlled trial with healthy participants, acute oral ebselen reduced brain myo-inositol in the anterior cingulate cortex, consistent with CNS target engagement. Ebselen decreased slow-wave sleep and affected emotional processing by increasing recognition of some emotions, decreasing latency time in the acoustic startle paradigm, and decreasing the reinforcement of rewarding stimuli. In summary, ebselen affects the phosphoinositide cycle and has CNS effects on surrogate markers that may be relevant to the treatment of bipolar disorder that can be tested in future clinical trials.

Scaffold hopping with virtual screening from IP3 to a drug-like partial agonist of the inositol trisphosphate receptor.

Inositol 1,4,5-trisphosphate (IP3 ) is a universal signalling molecule that releases calcium from stores within cells by activating the IP3 receptor. Although chemical tools that modulate the IP3 receptor exist, none is ideal due to trade offs between potency, selectivity and cell permeability, and their chemical properties make them challenging starting points for optimisation. Therefore, to find new leads, we used virtual screening to scaffold hop from IP3 by using the program ROCS to perform a 3D ligand-based screen of the ZINC database of purchasable compounds. We then used the program FRED to dock the top-ranking hits into the IP3 binding pocket of the receptor. We tested the 12 highest-scoring hits in a calcium-release bioassay and identified SI-9 as a partial agonist. SI-9 competed with [(3) H]IP3 binding, and reduced histamine-induced calcium signalling in HeLa cells. SI-9 has a novel 2D scaffold that represents a tractable lead for designing improved IP3 receptor modulators.