DREAM complex inhibitors for the treatment of medical conditions caused by circadian rhythm disorders

Abstract

The present invention relates to an inhibitor of the protein complex dimerization partners, RB-like, E2F and multiple export B class (DREAM complex inhibitor) for use in the treatment of a medical condition caused by a circadian rhythm disorder. The present invention also relates to a DREAM complex inhibitor for use as a sleep mimetic. Furthermore, the present invention also relates to a pharmaceutical composition comprising a DREAM complex inhibitor and one or more pharmaceutically acceptable excipients for use in the treatment of a medical condition caused by a circadian rhythm disorder.

Description

Technical Field

[0001] This invention belongs to the fields of biochemistry and medicine, and more specifically, to the fields of chronobiology and the treatment of medical conditions caused by circadian rhythm disorders.

[0002] This invention relates to an inhibitor (DREAM complex inhibitor) of protein complex dimerized mate bodies, RB-like structures, E2F, and multi-vulval class B, used to treat medical conditions caused by circadian rhythm disorders. The invention also relates to a DREAM complex inhibitor used as a sleep mimic.

[0003] The present invention further relates to a pharmaceutical composition comprising a DREAM complex inhibitor used according to the invention and one or more pharmaceutically acceptable excipients. Background Technology

[0004] As a molecular timing mechanism, the internal circadian clock generates biological rhythms and regulates various physiological processes in mammals (especially humans), such as blood pressure, hormone secretion, sleep-wake cycles, and body temperature. The circadian clock is highly conserved throughout evolution.

[0005] The general mechanism of circadian rhythm disruption involves the scrambled arrangement of the finely coordinated endogenous circadian rhythms, which regulate various physiological and behavioral processes over a period of approximately 24 hours. At the heart of this regulatory system is the suprachiasmatic nucleus (SCN) in the anterior hypothalamus, often referred to as the “master biological clock.” This central pacemaker coordinates and aligns the circadian clocks in different brain regions and surrounding tissues with external synchronizing factors, thereby ensuring the coordinated temporal organization of bodily functions.

[0006] Circadian rhythms are complexly regulated by a combination of endogenous factors (such as genetic and physiological influences) and external and behavioral factors (including exposure to light, activity, and eating schedules). Disruptions in these components can lead to circadian rhythm dysregulation, broadly termed "circadian rhythm disorder."

[0007] A key aspect of circadian rhythm disruption is its two-way relationship with a variety of health consequences. Circadian rhythm disruption not only increases the severity of disease, but many diseases, in turn, can also disrupt circadian rhythms, thus creating an interaction between circadian rhythm regulation and health. This interaction spans multiple tissue levels, from molecular and cellular changes to misalignments between physiological systems and behavioral cycles.

[0008] Measurements of circadian rhythm disturbances include parameters such as circadian rhythm phase (time), alignment between internal-internal or internal-external rhythms, and the period and amplitude of the circadian rhythm. Disruptions in circadian rhythm phase alignment and amplitude are particularly associated with adverse health consequences. These disturbances can occur at a variety of levels, ranging from intrinsic changes at the molecular and cellular levels to misalignments between different tissue levels and with behavioral and environmental cycles.

[0009] The molecular circadian clock is controlled by genes involved in self-regulating feedback loops (e.g., circadian motor output cycle kaput [CLOCK], brain and muscle ARNT-like [BMAL], cycle [PER], rev-erb / nuclear receptor subfamily 1, group D [NR1D], and cryptochrome [CRY]), representing the fundamental mechanism driving circadian rhythms. While SCN rhythms themselves cannot be directly measured in humans, biomarkers such as the timing and amplitude of melatonin onset serve as key indicators of circadian rhythms. Furthermore, disruptions in the sleep-wake rhythm (often accompanied by circadian rhythm dysregulation) significantly contribute to overall health outcomes.

[0010] Lifestyle factors and environmental changes (such as shift work, continuous light exposure, or irregular sleep patterns) lead to circadian rhythm disruption. This disruption is not only a consequence but also a significant contributing factor to a range of health problems, including neurological disorders, mental illnesses, cardiometabolic diseases, and immune disorders. Circadian rhythm disruption and sleep deprivation affect multiple aspects of an organism's health, from cognitive to immune responses. This multifaceted impact can be partly explained by the fact that sleep requires fluid flow and molecular damage clearance in the brain, and that the central nervous system (CNS) mediates excessive biological responses.

[0011] Unbound by existing theories, sleep deprivation and circadian rhythm disruption may affect master regulatory pathways that control fundamental aspects of cellular physiology, thereby modulating the function of multiple cell types. The discovery of this core regulatory mechanism is a key and novel step in developing interventions to restore homeostasis under circadian rhythm dysregulation and sleep deprivation.

[0012] Treatment options for circadian rhythm disorders include lifestyle modifications, light therapy, and pharmacological interventions. Lifestyle modifications require establishing consistent daily routines and optimizing the sleep environment, potentially managing symptoms associated with circadian rhythm disorders. However, the potential drawbacks associated with these treatments must be considered. Achieving lifestyle changes can require significant effort, and their effectiveness may vary from person to person. While light therapy is beneficial, it can cause side effects such as eye strain and headaches.

[0013] Medications provide pharmacological solutions to align sleep patterns with a desired schedule. Melatonin receptor agonists (a type of melatonin medication) are prescribed to regulate melatonin production and treat non-24-hour sleep-wake rhythm disorders. However, they can cause side effects such as dizziness and fatigue. Melatonin supplements are laboratory-prepared forms of the sleep hormone and are recommended for conditions such as delayed sleep-wake disorder. Because they are not regulated by the FDA, the dosage and purity of these supplements may vary between brands, and potential side effects include excessive drowsiness and headaches.

[0014] Caffeine is recommended to prevent daytime sleepiness, but it should be avoided within eight hours of your desired bedtime. Prescription sleep aids such as benzodiazepines and zolpidem can be prescribed to help you fall asleep faster, but they can cause side effects such as muscle weakness and confusion, especially in older adults. Arousal medications such as modafinil and armodafinil help individuals stay awake during shift work, but their effects may be short-lived, and some sleepiness symptoms may persist.

[0015] While these medications provide symptom relief, they typically do not address the underlying causes of cellular and organ dysfunction resulting from circadian rhythm disruption. Furthermore, potential side effects and complications should be carefully considered and discussed with a healthcare professional before initiating any pharmacological intervention.

[0016] In the existing technology, only a few studies have explored the potential mechanisms of circadian rhythm disruption and proposed methods to restore circadian rhythms.

[0017] For example, JP2011195560A describes harmine or harmane alkaloids for improving or restoring circadian rhythms themselves. The methods disclosed herein focus primarily on improving or restoring circadian rhythms themselves and do not mention treating downstream effects or medical conditions caused by circadian rhythm disorders.

[0018] EP4091611A discloses inhibitors for repairing the assembly and / or function of the DREAM complex in subject cells to repair DNA damage. The inhibitors may be selected from the group comprising inhibitory nucleic acids, antibodies, and small molecule inhibitors.

[0019] Abbassi et al. (Pharmacology & Therapeutics, 2015) disclosed the role of DYRK1A and its inhibition in neurodegenerative diseases and cancer. They established a link between DYRK1A inhibition and DREAM complex expression, highlighting its effect on cell proliferation.

[0020] Naert et al. (European Neuropsychopharmacology, 2015) disclosed the use of leucettine 41 as a DYRK1A inhibitor to prevent memory impairment and neurotoxicity induced by oligomeric Aβ25-35 peptide in mice. This study further demonstrates that DYRK1A inhibition (including through other inhibitors such as halamine) has beneficial effects in reducing oxidative stress and memory impairment.

[0021] Existing technologies do not disclose methods specifically targeting the adverse effects caused by circadian rhythm disorders, but primarily focus on reversing or restoring the circadian rhythm itself in various cellular and tissue pathologies unrelated to sleep. Furthermore, no existing technologies provide methods or pathways for treating conditions or disorders directly resulting from circadian rhythm disorders. Additionally, no existing technologies propose using DREAM inhibitors as mimics for restoring sleep.

[0022] Therefore, one object of the present invention is to address the unmet need for treatment of medical conditions caused by circadian rhythm disorders, regardless of whether the circadian rhythm itself is restored. Summary of the Invention

[0023] In view of the prior art, the technical problem of the present invention is to provide alternative or improved means for treating medical conditions caused by circadian rhythm disorders.

[0024] Another objective is to provide alternative or improved means of treating medical conditions caused by circadian rhythm disorders, which do not depend on restoring the circadian rhythm itself; and / or means of effectively addressing medical damage caused by circadian rhythm disorders but downstream of the circadian rhythm itself.

[0025] One focus of this invention is to develop interventions with the ability to improve health, even when circadian rhythms and sleep function cannot be restored (whether by choice (social jet lag, shift work) or due to environmental and / or pathological reasons).

[0026] These problems are addressed by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0027] Therefore, the present invention relates to an inhibitor of protein complex dimerization partner, RB-like, E2F and multi-vulval class B (DREAM complex inhibitor) for the treatment of medical conditions caused by circadian rhythm disorders.

[0028] The DREAM complex was first characterized as a core regulator of the activity of circadian rhythm alteration pathways. The pharmacological and gene-inhibiting effects of the DREAM complex alleviated molecular and biological dysfunction caused by circadian rhythm disorders.

[0029] The discovery of this core regulatory mechanism is an important step in developing interventions to restore homeostasis and / or patient health in cases of circadian rhythm disruption and sleep deprivation. In implementation, the restoration of homeostasis and / or patient health occurs independently of circadian rhythm realignment or relief of sleep deprivation. In implementation, the medical effects of DREAM complex inhibitors occur independently of the correction or readjustment of the circadian rhythm itself (and / or downstream of it).

[0030] Circadian rhythms are intrinsic biological cycles with a duration of approximately 24 hours that finely regulate fundamental physiological processes, including eating behavior, sleep-wake cycles, hormone release, and metabolic function. The circadian rhythm system is controlled by the suprachiasmatic nucleus (SCN) in the anterior hypothalamus and is influenced by external stimuli, or "time-measuring factors," such as light exposure. Modern lifestyles, characterized by artificial light, shift work, and continuous 24 / 7 work environments, challenge endogenous circadian rhythms, leading to a variety of health problems. Disruptions to circadian rhythms are associated with adverse health consequences, including cancer, neurodegenerative diseases, cardiovascular problems, and diabetes.

[0031] The primary retinal ganglion cell (SCN), acting as the master circadian rhythm, coordinates circadian rhythm processes by transmitting signals from external stimuli to the peripheral circadian clock. The light-dark detection pathway, mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs), influences melatonin secretion, thereby regulating the sleep-wake cycle. Molecular genetic mechanisms involve a transcription-translational negative feedback loop characterized by key genes such as BMAL1, CLOCK, PER, and CRY. Mutations in these genes can disrupt circadian rhythm function.

[0032] Circadian rhythm sleep disorders (CRSDs) are caused by a misalignment between endogenous biological rhythms and external cues. Intrinsic disorders include sleep phase delay and advance disturbances, irregular sleep-wake rhythm disorders, and non-24-hour sleep-wake disorders. External disorders, such as shift work sleep disorders and jet lag, are caused by a mismatch between the biological clock and an imposed schedule, leading to sleep disturbances and daytime sleepiness.

[0033] Many pharmacological and behavioral interventions for restoring circadian rhythm function and regular sleep patterns are known in the field, such as melatonin supplementation, sleeping pills, and light therapy.

[0034] The natural hormone melatonin is known to promote adequate sleep duration in cases of sleep disorders and jet lag. Hypnotics target different molecules; key examples include benzodiazepine receptor agonists (BZRAs) (eszopiclone, zaleplon, and zolpidem), benzodiazepine hypnotics (eszolam, flurazepam, hydroxychloroquine, triazolam, and quazepam), dual appetite receptor antagonists (DORAs) (daridorexant, lemborexant, and suvorexant), histamine receptor antagonists (doxepine), and melatonin receptor agonists (rameltein). While effective in inducing sleep, most of these compounds cause side effects that can impair quality of life (drowsiness, dizziness, headache, diarrhea, and addiction). Recently, light therapy, consisting of repeated and limited exposure to bright light (with the same light intensity and duration) at fixed times throughout the day, has been proposed as an effective non-pharmacological intervention for circadian rhythm sleep disorders, insomnia, and sleep disturbances in patients with Alzheimer's disease and dementia. However, this approach remains controversial.

[0035] In general, all existing solutions focus on inducing and / or maintaining sleep by restoring circadian rhythms. However, circadian rhythm restoration is not always feasible and contrasts with circadian rhythm readjustment due to the inherent challenges of adjusting to potentially persistent lifestyle or behavioral changes. For example, such limitations can be caused by a complete lack or persistent disruption of circadian rhythm components (e.g., attributed to genetic mutations) or the presence of irreversible circadian rhythm disruptive stimuli (such as prolonged sleep deprivation).

[0036] In situations where restoring defective circadian rhythm components and / or removing interfering stimuli prove ineffective, the present invention aims to restore and / or improve the health of target peripheral cells, even in cases where circadian rhythms are not restored, are reversible, or are permanently disrupted.

[0037] Therefore, one object of the present invention is to separate sleep and / or sleep disorders from their physiological (i.e., health or medical) consequences in the body. Another object of the present invention is to provide means for addressing medical conditions or injuries associated with and / or caused by circadian rhythm disorders, without relying on the necessity of restoring the circadian rhythm itself.

[0038] All current interventions for sleep deprivation typically rely on sleep restoration, but this is not entirely feasible in all cases. For example, this approach often fails in patients affected by severe brain dysfunction, such as dementia. Moreover, even if night shift workers sleep during the day with the help of regular sleep medication, they still suffer from the health consequences of circadian rhythm disruption. Furthermore, all existing pharmacological interventions, including melatonin, induce side effects that impair overall health, cognitive function, and even involve addiction.

[0039] In this invention, the inventors discovered that circadian rhythm disruption is associated with a number of negative health effects through the activity of a chromatin remodeling complex called DREAM (Dimerization partner, RB-like, E2F and multi-vulval class B).

[0040] Subsequently, as illustrated in the examples below, genetic inactivation of the DREAM complex restored the health of animals affected by persistent disruption of their circadian rhythms. The same rescue was observed after exposure to a DREAM inhibitor.

[0041] There is no indication in the art of an effective intervention equivalent to inhibiting the DREAM complex, which rapidly salvages an organism's health in persistent circadian rhythm dysfunction, acting downstream of the circadian clock. Therefore, the findings of this invention are unexpected, and the DREAM inhibitors of this invention exhibit unexpected and beneficial effects on health recovery in organisms with impaired circadian rhythms, independent of or downstream of circadian clock restoration.

[0042] Therefore, this invention also proposes a novel clinical situation superior to previously used similar molecules, such as halamine or other DREAM complex inhibitors. The discovery and observation of DREAM complex inhibition, independent of restoring the circadian rhythm, leads to the resolution of medical symptoms and underlying pathologies in organisms with disrupted biological clocks, demonstrating a novel technical effect previously unidentified in the art. From this novel technical effect arises a novel clinical situation in which such DREAM complex inhibitors can be administered to different patient populations at different doses or in different dosing regimens, as previously known from earlier studies of halamine or other DREAM complex inhibitors. The identification of novel bio / technological effects based on this invention also enables the treatment of novel patient groups and the achievement of novel therapeutic effects in said groups, thereby defining novel medical treatments.

[0043] In this implementation, inhibition of the DREAM complex rapidly salvages the organism's health in persistent circadian rhythm dysfunction.

[0044] In this implementation, the inhibition of the DREAM complex acts downstream of the circadian rhythm clock.

[0045] In this implementation, inhibition of the DREAM complex acts downstream of the circadian rhythm clock and separates circadian rhythm disturbances from their negative health effects.

[0046] In some implementations, the inhibitor is a DYRK1A inhibitor, also known as an inhibitor of "bispecific tyrosine phosphorylation-regulated kinase 1A" (DYRK1A inhibitor).

[0047] With the emergence of DYRK1A as a therapeutic target, a variety of inhibitors have been identified. In practice, DYRK1A inhibitors can be classified into three types: ATP-competitive (Type I), partially binding non-ATP-competitive (Type II), and specifically pocket-binding non-ATP-competitive (Type III). Natural products such as halamine and synthetic compounds containing indazole, azaindole, or benzothiazole moieties have shown activity as DYRK1A inhibitors. Clinical trials, such as those involving epigallocatechin-3-gallate (EGCG) (a polyphenolic component derived from green tea), highlight the potential therapeutic applications of DYRK1A inhibitors in addressing diseases associated with DYRK1A dysregulation.

[0048] It is worth noting that DYRK1A inhibitors include, but are not limited to, halamine, leucettine-L41, 5-IT, GNF4877, and epigallocatechin gallate (EGCG). Other promising compounds such as PST-001, Silmitasertib, Lorecivivint, and FRTX-02 are at various stages of clinical development.

[0049] In this implementation, the DYRK1A inhibitor is a non-selective ATP-competitive DYRK1A inhibitor.

[0050] In this embodiment, the DYRK1A inhibitor is halamine, leucettine-L41, 5-IT, GNF4877 and / or epigallocatechin gallate (EGCG).

[0051] In this implementation, the DYRK1A inhibitor is PST-001, Silmitasertib, Lorecivivint, and / or FRTX-02.

[0052] In some embodiments, the inhibitor is a β-carboline alkaloid, preferably a harmalaalkaloid, or a pharmacologically acceptable salt or ester thereof.

[0053] In this implementation, the inhibitor is halamine or a pharmacologically acceptable salt or ester thereof.

[0054] Halmine, an alkaloid used in this invention, is a DREAM complex inhibitor used to inactivate DREAM in cases of circadian rhythm disruption. It is one of the traditional therapies known to humans for a long time. Its recent therapeutic benefits include depression, Parkinson's disease, and Alzheimer's disease, with side effects only detectable at very high doses. Unlike conventional sleep medications, in cases of circadian rhythm disruption, Halmine's inhibition of DREAM acts directly on downstream molecular targets mediating health loss. In this way, health damage is rapidly salvaged, even if circadian rhythm behavior itself remains disrupted (e.g., in shift workers and dementia patients).

[0055] What is truly surprising is that Halmine inhibits DREAM without requiring the restoration of synergistic circadian rhythms to mitigate associated health losses. This is a unique feature absent in any existing treatment. Furthermore, all current sleep medications indirectly regulate health by using sleep restoration as a mandatory intermediate step. In contrast, inhibiting DREAM (e.g., through Halmine) acts directly on downstream molecular targets causing health losses, resulting in a faster positive effect without requiring a reset of the circadian rhythm itself. To the inventors' knowledge, no side effects comparable to those of conventional sleep medications have been reported with Halmine.

[0056] In this implementation, the inhibitor is an inhibitor of Dyrk (INDY) or a pharmacologically acceptable salt or ester thereof.

[0057] In an embodiment, the subunits of the DREAM complex are suppressed, wherein the suppressed subunits of the DREAM complex are LIN9, LIN54 and / or RBBP4 / RBBP7.

[0058] In one implementation, the repressed subunit of the DREAM complex is LIN9.

[0059] In one implementation, the repressed subunit of the DREAM complex is LIN54.

[0060] In one embodiment, the repressed subunit of the DREAM complex is lin-53 / RBBP4 / RBBP7.

[0061] While the small-molecule DYRK1A inhibitor INDY is well-known in the art for its ability to inhibit the DREAM complex, its use in treating medical conditions associated with and / or induced by circadian rhythm disorders is not mentioned. Notably, EP4091611A1 teaches the inhibition of DREAM complex assembly and / or function to repair DNA damage in subject cells. EP4091611A1 discloses inhibitors such as halamine and INDY. However, compared to this invention, it explicitly emphasizes the use of such DREAM inhibitors for targeting the lins-52, lins-35, eFL-1, and dpll-1 subunits of the DREAM complex, demonstrating significant differences in the specific subunits affected within the DREAM complex.

[0062] There is no indication in the art that inhibiting the subunits LIN9, LIN54, and / or lin-53 / RBBP4 / RBBP7, alone or in combination, will improve health, despite persistent circadian rhythm disruption. The therapy according to the invention is neither intended to restore circadian rhythms nor is it a direct treatment for sleep disorders. The primary objective of the invention is to address the medical conditions associated with sleep deprivation and its health consequences.

[0063] Therefore, technicians should not expect that inhibiting the DREAM complex with a DYRK1A inhibitor will affect subunits within the complex other than those mentioned in EP4091611A1. Furthermore, such inhibition should not be anticipated to provide an alternative or ameliorative approach for addressing medical conditions caused by circadian rhythm disorders.

[0064] In implementation, the inhibitor comprises an affinity agent that binds to the DREAM complex or its subunits, an antibody or an antigen-binding fragment thereof, or is composed of such an antibody, or comprises an antisense or interfering nucleic acid molecule that targets the DREAM complex or its subunits, such as short interfering RNA (siRNA).

[0065] In a preferred embodiment, the inhibition of the DREAM complex is transient. In this embodiment, transient inhibition includes pharmacological activity as a DREAM inhibitor after a certain period of time following administration, and preferably is discontinuous, for example, at certain times throughout a 24-hour period, where the DREAM inhibition is not significant or is at a low or negligible level.

[0066] The DREAM complex can be inhibited by various methods known to those skilled in the art, such as small molecule disruption of interactions, antibody blocking of specific binding sites, and nucleic acid molecules preventing the synthesis of proteins within the complex.

[0067] In one embodiment, a DREAM complex inhibitor is a small molecule, affinity reagent, antibody, or antigen-binding fragment thereof that targets the DREAM complex and / or one or more subunits of the DREAM complex, resulting in a reduction or inhibition of DREAM complex function.

[0068] In one embodiment, a DREAM complex inhibitor is a nucleic acid molecule capable of reducing the expression or function of the DREAM complex. For example, antisense oligonucleotides or interfering nucleic acid substances can be used to target (reduce) the expression of one or more subunits of the DREAM complex. Those skilled in the art know nucleic acid-based methods for reducing the expression of DREAM complex subunits.

[0069] In other embodiments, the DREAM complex can be inhibited by targeted protein degradation (TPD), for example by a targeted protein degradation chimera (PROTAC) molecule. As known to those skilled in the art, a targeted protein degradation chimera (PROTAC) is a heterobifunctional molecule consisting of two active domains and a linker, capable of removing specific unwanted proteins (see Luh et al., Angewandte Chemie International Edition. 59 (36): 15448–15466). PROTACs typically consist of two covalently linked protein-binding molecules: one capable of binding to an E3 ubiquitin ligase, and the other capable of binding to the target protein intended for degradation. The E3 ligase is recruited to the target protein, leading to ubiquitination and subsequent degradation via the proteasome. By using this method, efficient degradation of one or more DREAM complex subunits can be achieved, thereby enabling DREAM complex inhibition according to the invention.

[0070] In some implementations, the medical condition is induced by and / or includes loss of tissue function, oxidative stress, and / or impaired cellular homeostasis.

[0071] As illustrated in the example below, RNAi treatment of the PER homolog lin-42 in *C. elegans* induced persistent but moderate circadian rhythm disruption, serving as a mimicry of lifestyle-induced circadian rhythm irregularities. This intervention was also applied to a *C. elegans* model of protein homeostasis stress and accelerated aging, followed by proteomics and molecular and functional assays. Furthermore, transcriptomic analysis was performed in mice exhibiting altered circadian rhythm behaviors and in PER1-knockdown human cells. These assays confirmed the link between circadian rhythm disruption and impaired homeostasis, particularly under additional cellular stress. Excessive conserved and universally expressed molecular targets altered due to circadian rhythm distortion were identified, consistent with various health losses associated with circadian rhythm impairment.

[0072] In this implementation, the patient suffers from sleep deprivation.

[0073] In this implementation, the treatment is independent of the restoration of the circadian rhythm, and the treatment and associated biological effects preferably occur downstream of the circadian rhythm.

[0074] Sleep deprivation and circadian rhythm disruption are common occurrences in the modern world. It is well documented that a persistent lack of coordinated circadian behavior / sleep leads to a range of health impairments, from immune deficiencies to cognitive decline. While sleep disorders can often be treated with specialized medications that actively promote sleep, this is not always the case. Factors such as insensitivity, cognitive side effects, or addiction can prevent individuals from becoming dependent on regular sleeping pills. Furthermore, individuals may avoid sleep for extended periods due to occupational reasons (shift work and similar professions).

[0075] In mammals, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) coordinates peripheral oscillators throughout the body. Clock genes, including BMAL1, CLOCK, NPAS2, CRY1 / 2, and PER1-3, form self-regulating feedback loops that drive circadian rhythm oscillations. Posttranscriptional modifications, such as phosphorylation of casein kinase family members, play a role in clock accuracy. Auxiliary loops involve Rev-erbα, Rorα, DEC1, DEC2, and D-site albumin promoter-binding proteins. Thus, sleep regulation involves both the circadian pacemaker and homeostatic sleep drives. The SCN influences sleep duration through neuronal projections. For example, so-called Process C determines sleep duration, while Process S constructs sleep drives. Mutations in clock genes are associated with sleep disorders, and polymorphisms in the PER gene are associated with diurnal bias. Mutations in the CLOCK:BMAL1 complex, casein kinase I, and clock genes affect the length of the circadian rhythm cycle, linking circadian rhythms to sleep regulation.

[0076] Conversely, prolonged wakefulness affects the expression of clock genes in the cerebral cortex, upregulating genes Per1 and Per2. Independent of the time of day, homeostatic sleep drives develop an increased demand for sleep in response to prolonged wakefulness. Sleep deprivation affects the firing rate of SCN neurons, and the expression of clock genes in the cerebral cortex is influenced by prolonged wakefulness. The bidirectional relationship between circadian rhythms and sleep regulation involves complex interactions in which clock genes influence sleep patterns. Conversely, sleep disturbances (such as sleep deprivation) can affect the expression and function of clock genes.

[0077] Therefore, this intervention acts on the downstream molecular effects of circadian rhythm disruption, thereby bypassing sleep regulation and acting on its downstream molecular targets to improve health, even if sleep itself is not restored.

[0078] In some implementations, the medical condition to be treated includes acute medical injuries associated with sleep deprivation and circadian rhythm disruption.

[0079] In some implementations, acute medical injury is one or more of the following: fatigue, drowsiness, cognitive impairment, brain fog, memory impairment, gastrointestinal discomfort, cardiovascular injury, changes in blood pressure, decreased immunity, mood changes, depression, anxiety, and / or paranoia.

[0080] Sleep disorders can lead to a number of health consequences. Short-term effects in healthy individuals can include increased stress sensitivity, physical problems, reduced quality of life, mood disturbances, emotional disorders, and cognitive deficits. In children and adolescents, sleep disorders negatively impact psychosocial health, academic performance, and risky behavior. Adults experience psychosocial problems such as mood disturbances, emotional disorders, and cognitive deficits. Sleep disorders alter cognition and performance, affecting attention, executive function, memory, decision-making, and judgment. In individuals with underlying medical conditions, sleep disorders reduce quality of life (QoL), affecting patients with chronic kidney disease or liver transplant conditions. Therefore, the DREAM inhibitors of this invention are suitable for the treatment and / or prevention of such conditions caused by circadian rhythm disruption and / or sleep deprivation.

[0081] In another implementation, medical conditions include chronic medical conditions associated with sleep deprivation and circadian rhythm disorders.

[0082] In the implementation of the invention, the chronic medical conditions are neurological conditions, mental conditions, cardiovascular conditions, metabolic conditions, allergic conditions, immune conditions, gastrointestinal conditions, rheumatic conditions, proliferative conditions (cancer), microbiome conditions, multi-organ conditions, and / or pulmonary conditions.

[0083] Circadian rhythm disturbances significantly affect neurological disorders, influencing their development, presentation, and severity. Disorders such as cerebrovascular diseases, epilepsy, migraines, multiple sclerosis, and various neurodevelopmental disorders are associated with circadian rhythm disturbances. In neurodevelopmental disorders such as autism spectrum disorder (ASD), dysregulated cortisol rhythms and low-amplitude melatonin rhythms have been observed. Single nucleotide polymorphisms (SNPs), particularly in the melatonin receptor, may contribute to ASD. Smith-Magilli syndrome (SMS) involves the deletion of RAI1, a gene associated with melatonin regulation. Treatment strategies for SMS include administration of β1-adrenergic antagonists and melatonin supplementation. In neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD), circadian rhythm disturbances predict disease progression. Melatonin and light therapy have shown potential to control circadian rhythm-related symptoms in PD and AD. Strategies targeting circadian rhythm function may slow age-related cognitive decline in AD.

[0084] Circadian rhythm disruption is prevalent in mental illnesses, particularly schizophrenia and mood disorders such as depression, bipolar disorder, and seasonal affective disorder (SAD). SAD, characterized by depression during periods of reduced daylight, demonstrates the impact of circadian rhythm dysregulation on depressive symptoms. "Circadian rhythm depression" is a term coined to describe mood disorders that exhibit rhythmicity, seasonality, and treatment response influenced by circadian rhythm factors. Human studies have linked altered expression of circadian clock genes and genetic polymorphisms within these genes to mood disorders. Earlier sleep midpoints are associated with a lower risk of depression.

[0085] Circadian rhythm dysregulation, where environmental or behavioral rhythms deviate from the core or peripheral biological clock, increases the risk of cardiovascular disease (CVD). Shift work is associated with myocardial infarction (MI) and stroke, possibly induced by hypertension and inflammation. In a mouse stroke model, prolonged shift work upregulates inflammatory mediators, exacerbating stroke severity. Night shift workers face an increased CVD risk due to IL-6-mediated inflammation induced by circadian rhythm disruption, manifested as carotid intima-media thickening. Dysregulation alters blood pressure regulation, thereby affecting the cardioprotective nocturnal drop in blood pressure. Cardiac arrhythmias exhibit diurnal variations, influenced by circadian rhythm dysregulation and non-24-hour light / dark cycles.

[0086] The complex link between circadian rhythms and metabolism is crucial for optimal energy utilization. Disruptions to the circadian rhythm significantly impact metabolic health, leading to impaired glucose tolerance, insulin resistance, and an increased risk of diabetes and obesity. Disruptions to core circadian rhythm genes, social jet lag, and sleep pattern irregularities contribute to metabolic damage. The timing of food intake, light exposure, and personalized interventions, such as time-restricted eating, play key roles in mitigating these risks. Obesity is associated with CLOCK gene variants, and shift workers face an increased risk of metabolic disorders.

[0087] The circadian rhythm is associated with immune function, influencing health and susceptibility to infection. In the context of COVID-19, circadian rhythm dysregulation caused by shift work has been associated with an increased risk of SARS-CoV-2 infection. The effects of the circadian rhythm system extend to asthma, where gene mutations affecting the biological clock lead to defects in the epithelial barrier, potentially increasing asthma susceptibility. This dysregulation, exacerbated by factors such as hypoxia, may increase the risk of allergic reactions.

[0088] Sleep dysfunction is intricately linked to gastrointestinal disorders. Pro-inflammatory cytokines, such as tumor necrosis factor, interleukin-1, and interleukin-6, play crucial roles in sleep regulation and certain gastrointestinal diseases, including gastroesophageal reflux disease, inflammatory bowel disease, liver disease, and colorectal cancer. The gut-brain axis, encompassing the central nervous system, autonomic nervous system, and enteric nervous system, connects sleep regulation pathways to gastrointestinal physiology, modulating immune activation, intestinal permeability, and enteroendocrine signaling. This complex relationship extends to digestive processes during sleep, influenced by factors such as esophageal susceptibility to gastric acid damage, gastric acid secretion patterns, motility complexes, small intestinal motility, and colonic contractions. Furthermore, sleep disturbances are associated with altered cytokine release, exacerbating inflammatory responses in various gastrointestinal diseases.

[0089] Sleep deprivation may contribute to the development and progression of inflammatory joint diseases, including rheumatoid arthritis (RA). RA is characterized by persistent synovitis and inflammation, leading to joint destruction. Studies have shown a correlation between sleep deprivation and RA, impacting quality of life, fatigue, and overall health. Experimental studies have linked sleep deprivation to alterations in inflammatory mediators, potentially involving impaired regulatory T-cell function. Circadian rhythms, particularly time-based therapy, have been suggested to optimize the efficacy of RA treatment. Assessing and addressing sleep disturbances in RA is crucial for improving patient outcomes, including quality of life and disease progression.

[0090] Sleep has a profound impact on breathing, affecting central respiratory control, lung mechanics, and muscle contractility. While these effects are often benign in healthy individuals, they can lead to significant hypoxemia and hypercarbon dioxide in patients with chronic obstructive pulmonary disease (COPD), especially during rapid eye movement (REM) sleep. Factors such as airflow obstruction, hyperinflation, respiratory muscle dysfunction, and medication use all contribute to significant hypoventilation in COPD patients during sleep. Sleep disturbances are prevalent in COPD, with approximately 40% of patients experiencing difficulty initiating or maintaining sleep, resulting in chronic fatigue and decreased quality of life.

[0091] Sleep disorders are associated with a variety of diseases, including cancer, with the highest prevalence observed in breast cancer patients. Approximately 30-70% of cancer patients report sleep problems, roughly twice the rate in the general population. Studies have shown a link between sleep disorders, circadian rhythm disruption, and tumor development and growth. Research has also revealed a negative correlation between sleep duration and breast cancer risk, highlighting the importance of assessing the contribution of sleep to tumorigenesis. Sleep disorders are also associated with altered immune responses, potentially influencing cancer outcomes.

[0092] Other aspects of the invention relate to a DREAM complex inhibitor, used as a sleep mimic or for simulating and / or achieving the effects of sleep, particularly sleep recovery.

[0093] In one implementation, the DREAM complex inhibitor is a sleep mimic.

[0094] In one embodiment, the DREAM complex inhibitor is a sleep mimic, or is administered as a sleep mimic. In another embodiment, the DREAM complex inhibitor used as a sleep mimic is formulated to induce a sleep-like recovery state in the absence of natural sleep. In yet another embodiment, the DREAM complex inhibitor mimics one or more physiological processes typically associated with sleep. In yet another embodiment, the DREAM complex inhibitor used as a sleep mimic replicates the restorative benefits of sleep.

[0095] In one embodiment, the present invention relates to a DREAM complex inhibitor for treating insomnia. In another embodiment, the present invention relates to a DREAM complex inhibitor for treating the negative effects of insomnia. In yet another embodiment, the present invention relates to a DREAM complex inhibitor for treating medical conditions (preferably conditions induced by circadian rhythm disruption) in patients suffering from insomnia or other sleep disorders.

[0096] In one embodiment, the present invention relates to a method for treating insomnia or for treating medical conditions induced by insomnia, comprising administering a DREAM complex inhibitor to a subject in need. In another embodiment, the present invention relates to a DREAM complex inhibitor in a cosmetic method for treating the negative effects of insomnia.

[0097] In a further embodiment, the present invention relates to a DREAM complex inhibitor for mitigating the effects of circadian rhythm disruption, comprising administering the DREAM complex inhibitor as a sleep mimic to provide restorative benefits equivalent to or similar to natural sleep, such as reducing fatigue, cognitive impairment and mood disorders associated with sleep loss or irregular sleep patterns, as well as alterations in metabolic, quality control and detoxification mechanisms caused by altered sleep.

[0098] In one embodiment, the present invention relates to a pharmaceutical composition comprising a DREAM complex inhibitor, said DREAM complex inhibitor being formulated for administration, for example, once daily, twice daily, or three times daily, wherein the composition provides therapeutic and / or effective plasma concentrations of the DREAM complex inhibitor for a duration of 4, 6, 8, 10, 12, 14, 16, or 18 hours after administration. In embodiments, such administration ensures effective treatment of insomnia and / or the negative effects of insomnia without significant cumulative and / or toxic effects with long-term use, preferably as measured by pharmacokinetic parameters. In a preferred embodiment, administration of the DREAM complex inhibitor does not result in chronic and / or long-term effective inhibitory levels in the subject. For example, the composition provides effective plasma concentrations of the DREAM complex inhibitor for a duration not exceeding 16 hours, preferably not exceeding 14 hours, or not exceeding 12 hours. For example, a single administration of a β-carboline alkaloid (preferably Halman alkaloid or Halmin), or a pharmacologically acceptable salt or ester thereof, having PK parameters that result in the half-life of the compound, such that chronic activity levels are not achieved in the subject. In this implementation, this allows subjects to also experience increased expression of the DREAM complex during the 24-hour circadian rhythm, resulting in chromatin densification, which may be necessary during the waking phase.

[0099] This invention is not bound by theory and seeks to provide DREAM complex inhibition as a sleep mimic based on DREAM complex activity and / or expression in subjects. For example, in cases of insomnia or other forms of sleep disorders, the DREAM complex can be overexpressed when it should be low or suppressed. One example is that sleep-deprived patients have unwanted DREAM complex activity and / or expression for most of the 24-hour period, inducing chromatin compression, which may prevent DNA repair and other cellular repair and detoxification activities. For instance, during periods of reduced sleep, the DREAM complex can be highly expressed, which can counteract the restorative effects that sleep would normally provide. Low levels of the DREAM complex are associated with DNA decompression, DNA repair, and other restorative functions that typically occur during sleep. With persistently activated DREAM complexes, such as in cases of insomnia, the restorative effects of sleep will not occur, (in part) due to high levels of DREAM complex expression and / or activity. In its implementation, the present invention seeks to reduce the activity and / or expression of the DREAM complex, thereby providing a restorative sleep effect, even in patients experiencing sleep loss or insomnia and therefore having unwanted expression and / or activity of the DREAM complex.

[0100] As used herein, the term "sleep mimic" refers to agents, compounds, etc., that induce one or more physiological effects commonly associated with sleep, preferably DREAM complex inhibitors.

[0101] In one embodiment, the present invention relates to a composition comprising a DREAM complex inhibitor for treatment by oral, rectal, intranasal, intrapulmonary or transdermal delivery, intramuscular, intraocular, subcutaneous, intravenous, intra-articular, intra-articular, intraperitoneal, intracystic, intravenous or parenteral administration.

[0102] In this implementation, the dosing regimen does not cover methods that are inconsistent with patient adherence or have side effects that significantly impair daily use. Long-term administration of DREAM complex inhibitors preferably does not induce dependence or tolerance, thus enabling both short-term and sustained long-term benefits.

[0103] In one embodiment, the present invention relates to a method for providing personalized sleep and / or circadian rhythm therapy using a DREAM complex inhibitor, including adjusting the dosage and timing of administration of the inhibitor based on the needs of the subject.

[0104] In one embodiment, the present invention relates to a method for inducing restorative sleep effects in subjects with sleep pattern disorders, the method comprising administering a DREAM complex inhibitor at a dose optimized for the patient’s needs, for example optimized to address a specific phase of sleep disorder or insomnia.

[0105] In one embodiment, the present invention relates to a method for treating a subject experiencing a sleep disorder, comprising suppressing DREAM to allow for chromatin decompression, DNA repair, and other restorative effects of sleep.

[0106] In another embodiment, the present invention relates to a method for simulating sleep recovery effects, wherein the method includes administering a DREAM complex inhibitor. In embodiments, the present invention provides a method for modulating DREAM complex activity under genotoxic stress conditions to protect genome integrity and / or enhance DNA repair.

[0107] Uniquely, DREAM complex inhibitors can be used for individuals who must intentionally avoid sleep for occupational reasons (e.g., shift work, etc.) to provide the same restorative benefits as those experienced by this group with chronic sleep disorders.

[0108] In one aspect, the invention also relates to a pharmaceutical composition comprising a DREAM complex inhibitor according to the invention and one or more pharmaceutically acceptable excipients.

[0109] All aspects of the present invention are unified, benefit from, based on, and / or associated with the following common and surprising discovery: inhibitors of the DREAM complex for treating medical conditions induced by circadian rhythm disorders, preferably for treating medical conditions including acute or chronic medical lesions associated with sleep deprivation and circadian rhythm disorders. Detailed Implementation

[0110] This invention relates to an inhibitor of protein complex dimerized mate bodies, RB-like structures, E2F, and multiple vulvar B types (DREAM complex inhibitor) for the treatment of medical conditions induced by circadian rhythm disorders.

[0111] Unless the context indicates a different meaning, all words and terms used herein shall have the same meaning as commonly given to them by those skilled in the art. All terms used in the singular shall include the plural form of the term, and vice versa.

[0112] As used in this article, "circadian rhythms" occur approximately every day (i.e., 24 hours) and can also be referred to or understood as endogenous biological rhythms, endogenous oscillations, a separate circadian clock, or internal biological clock. For example, circadian rhythms refer to the cycles of human oscillatory gene expression, hormone secretion, neurotransmitter secretion, and core body temperature rhythms that regulate bodily activities, including rest periods. External factors, including light, food, and temperature, adjust these oscillations to the local environment.

[0113] Circadian rhythms ensure that human biological rhythms (including sleep) repeat in 22- to 25-hour increments. These rhythms temporally constitute physiology and behavior to optimally utilize the fourth dimension (i.e., time) for adaptation, for example, by anticipating predictable changes in environmental conditions and by temporally merging compatible processes or temporally separating incompatible processes.

[0114] For example, the dark hormone melatonin is secreted by the pineal gland and is usually secreted a few hours before the usual bedtime; its secretion is inhibited by light. Cortisol, secreted by the adrenal glands, causes preparation for wakefulness and usually reaches a brief peak before waking.

[0115] As used in this article, the “dimerized budding coupler (DP), retinoblastoma-like (RB), E2F, and MuvB complex” (also known as the “DREAM complex” or “DREAM / LINC complex”) is an evolutionarily conserved protein component crucial for coordinating precise gene expression in cell cycle regulation. It comprises distinct components, including retinoblastoma-like protein 1 (RBL1), retinoblastoma-like protein 2 (RBL2), dimerized budding coupler 1 (DP1), dimerized budding coupler 2 (DP2), dimerized budding coupler 3 (DP3), and polygenital type B (MuvB), each playing a key role in suppressing gene expression during the quiescent (G0) phase. This suppression prevents gene expression during the G1 / S and G2 / M phases, maintaining cell quiescence. Notably, a key function of the complex involves suppressing specific genes, such as B-MYB (BMYB), during G0, thereby promoting their expression during the S and G2 / M phases when the cell cycle is active.

[0116] During cell cycle entry, the dissociation of specific proteins from the DREAM complex leads to the recruitment of activated E2F proteins, enabling the expression of late G1 and S phase genes. BMYB, initially repressed by the DREAM complex, is expressed during S phase, binds to MuvB, and promotes the expression of key G2 / M phase genes. Further gene expression in G2 promotes recruitment via the Forkhead Box M1 (FOXM1). Subsequently, the DREAM complex reforms at the end of mitosis, again repressing G1 / S and G2 / M phase genes, thus completing the cell cycle.

[0117] The assembly and functional regulation of the DREAM complex involve phosphorylation events and interactions with key kinases, such as dual-specific tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Phosphorylation of specific residues (e.g., S28 of LIN-52) is crucial for proper DREAM complex assembly and function, while DYRK1A activity influences association between p130 and MuvB, thereby affecting cell cycle progression.

[0118] The DREAM complex enhances binding affinity and gene repression by facilitating docking of the DREAM complex at gene promoters through specific DNA sequences. While the RB-E2F complex typically recruits chromatin modifiers for gene repression, the DREAM complex influences gene expression by potentially affecting nucleosome localization rather than directly recruiting modifiers.

[0119] In certain cancers with elevated levels of mitotic gene expression, dysregulation or alteration of the regulation of the DREAM complex has been observed to promote a shift from quiescence to increased proliferation.

[0120] In mammalian systems, members of the DREAM complex include, but are not limited to: LIN9, Rb, p107, p130, F25965, LOC91750, RBBP4 / RbAp48, RbAp46, tesmin, DP1, DP2, DP3, E2F4, E2F5, MYBL1, MYBL2, RBL1 / p107, RBL2 / p130, LIN-37, LIN-54, and LIN-52. In *Caenorhabditis elegans*, members of the DREAM (also known as DRM) complex include, but are not limited to: LIN-9, LIN-35, LIN-37, LIN-52, LIN-53, LIN-54, DPL-1, and EFL-1. In *Drosophila melanogaster*, members of the DREAM (also dREAM or MMB) complex include, but are not limited to: Mip130, RBF1, RBF2, Mip40, dLin52, p55 / Caf1, Mip120, dDP, Myb, and dE2F2; tMAC may include Tomb, Aly, Mip40, and p55 / Caf1. Any implementation relating to the inhibition of one or more complex subunits in any system (e.g., *Caenorhabditis elegans* or humans) also relates to the inhibition of orthologs or homologs in other systems, as illustrated in the examples above.

[0121] Inhibition of the DREAM complex represents a novel approach not previously proposed in the art, enabling alternative methods for restoring the health of organisms amid persistent circadian rhythm disruption and sleep deprivation.

[0122] As used in this article, the “circadian locomotor output cycles kaput” (CLOCK) is a gene encoding a transcription factor crucial for regulating circadian rhythms. Located at locus 4q12 on chromosome 4, this gene produces a protein called CLOCK, which plays a central role in the circadian pacemaker. Operating as a transcription factor, CLOCK forms a heterodimer with BMAL1, binds to E-box regulatory elements in DNA, and promotes the expression of circadian rhythm genes such as PER and CRY. This initiates a feedback loop in which the translated PER and CRY proteins inhibit the CLOCK-BMAL1 complex, ultimately suppressing its transcription. A unique feature of CLOCK is its inherent acetyltransferase activity, essential for circadian chromatin remodeling. This enzyme and its histone acetyltransferase (HAT) activity affect circadian rhythm gene expression, influencing processes such as metabolism, sleep, and cardiovascular function.

[0123] Inhibitors

[0124] According to the present invention, an "inhibitor" is considered any reagent, substance, compound, molecule, or other means that causes a slowing down, inhibition, blockade, or otherwise interference with or negatively impacting the activity, function, expression, or signaling of said target induced, carried out, or manifested in the absence of an inhibitor. The terms "reagent," "substance," "compound," and "molecule" are used interchangeably. The terms "analyte" and "derivative" are used interchangeably.

[0125] For example, the inhibitors of the present invention may comprise an affinity agent, antibody, or antigen-binding fragment thereof that binds to the DREAM complex or its subunits, or may comprise an antisense or interfering nucleic acid molecule, such as short interfering RNA (siRNA), that targets the DREAM complex or its subunits, or a protein degradation (TPD) molecule, such as a proteolytic target chimera (PROTAC). Preferred inhibitors are those described herein.

[0126] The term "DREAM complex inhibitor" or similar terms refer to a class of inhibitors known and established by those skilled in the art. For example, DREAM complex inhibitors are described in Bujarrabal-Dueso et al. (Nat Struct Mol Biol 30, 475–488 (2023)) and Wang et al. (J Clin Invest. 2022;132(15): e157086). Furthermore, those skilled in the art are familiar with the purposes and conventional means for determining whether any given substance falls within the functional characterization of a DREAM complex inhibitor. As a non-limiting example, means for determining DREAM complex inhibitors are established in the art and are illustrated in the following examples, such as Example 5.

[0127] As used in this article, “Bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A)” is an enzyme encoded by the human DYRK1A gene. It belongs to the dual-specific tyrosine phosphorylation-regulated kinase (DYRK) family and is crucial in cellular regulation. The gene undergoes selective splicing, producing multiple transcript variants and at least five distinct isoforms. DYRK1A is characterized by its nuclear targeting signaling sequence, protein kinase domain, leucine zipper motif, and a highly conserved 13-hitidine repeat sequence. It catalyzes the autophosphorylation of serine / threonine and tyrosine residues, potentially influencing cell proliferation and brain development. DYRK1A is located in a critical region of Down syndrome on chromosome 21 and is involved in learning deficits associated with Down syndrome. Additionally, it has clinical significance in HIV-1 replication and autism spectrum disorders and is a potential therapeutic target for diseases such as diabetes and various cancers.

[0128] DYRK1A inhibitors are generally classified into three types: ATP-competitive (Type I), partially bound non-ATP-competitive (Type II), and specifically pocket-bound non-ATP-competitive (Type III). Natural products such as halamine and synthetic compounds containing indazole, azaindole, or benzothiazole moieties have shown activity as DYRK1A inhibitors. Clinical trials, such as those involving epigallocatechin-3-gallate (EGCG) (a polyphenolic component derived from green tea), provide examples of the potential therapeutic applications of DYRK1A inhibitors in addressing diseases associated with DYRK1A dysregulation.

[0129] Several non-selective ATP-competitive DYRK1A inhibitors, such as halamine, INDY, 5-IT, leucettine-L41, and GNF4877, are available from commercial suppliers. CX-4945 is currently in phase 1 and 2 clinical trials for cancer. Lorecivivint has been tested in a phase 3 trial for knee osteoarthritis, administered intra-articularly at a dose of 0.07 mg per year. FRTX-02 was well tolerated in a MAD study in healthy volunteers at an oral dose of 150 mg / day. Therefore, the inhibitors of the present invention are commercially available and in clinical development, and thus those skilled in the art will not find it difficult to identify or procure the inhibitors of the present invention.

[0130] As used herein, “β-carboline” (9H-pyrido[3,4-b]indole) is characterized by a tricyclic structure comprising a benzene ring fused to a pyridine ring, and may include various substitutions on the ring. Structurally, β-carboline belongs to the group of indole alkaloids and has a pyridine ring fused to an indole skeleton. β-carboline alkaloids are widely found in prokaryotes, plants, and animals. Some β-carboline, particularly tetrahydro-β-carboline, can be naturally formed in plants and humans using tryptophan, serotonin, and tryptamine as precursors. They are widely used in prokaryotes, plants, and animals, with different derivatives found in Banisteriopsis caapi, Peganum harmala, sea squirts, cyanobacteria, various foods (e.g., fish, meat, raisins), and scorpion skin. β-carboline exhibits a variety of biological activities, including DNA intercalation, enzyme inhibition, and receptor interaction, and displays sedative, anti-anxiety, hypnotic, antitumor, antiviral, antiparasitic, and antimicrobial properties. The pharmacological effects of specific β-carboline depend on their substituents. Various substituents and derivatives are known in the art and can be identified by those skilled in the art without excessive effort.

[0131] Examples of β-carboline include, but are not limited to, pinoline, harmane, harmine, harmaline, harmalol, tetrahydroharman, 9-methyl-β-carboline, 3-carboxy-tetrahydrononharman, and / or their pharmacologically acceptable salts or esters.

[0132] In this embodiment, β-carboline inhibits the DREAM complex. In a preferred embodiment, the inhibitor is harala alkaloid.

[0133] As used herein, "harala alkaloids" are derived from β-carboline molecules such as harmammin, haramarin, and tetrahydroharmammin, sharing a common structural framework characterized by an indole skeleton fused to a pyridine ring. These alkaloids act as reversible inhibitors of monoamine oxidase A (RIMA), selectively affecting the MAO-A isoform of the enzyme at appropriate doses. This inhibition enhances neurotransmitters such as serotonin and norepinephrine, prolonging their activity in the central nervous system. Harala alkaloids exhibit specificity for MAO-A, thus reducing their risk when combined with foods containing tyramine.

[0134] In a preferred embodiment, the inhibitor is halamine or a pharmacologically acceptable salt or ester thereof.

[0135] Harmine (7-methoxy-1-methyl-9H-pyrido[3,4-b]indole) is an alkaloid derived from the harmine alkaloid group, and therefore also a β-carboline. It is primarily found in cappuccino and Syrian rue. Harmine is also known by various other names, such as anisterin, banisterine, telopathin, telepathine, leucoharmine, and yageine. In humans, it acts as a reversible MAO inhibitor (monoamine oxidase inhibitor) with an excitatory effect on the central nervous system. The enzyme MAO-A is blocked, but not MAO-B, which has a similar effect. As an MAO inhibitor, harmine prevents the breakdown of monoamines by monoamine oxidase. Therefore, it delays the metabolism of the neurotransmitters serotonin and dopamine, the hormone melatonin, and various hallucinogenic tryptamines (such as DMT, psilocybin, and mescaline). Only its delayed function as a MAO inhibitor can delay the DMT also contained in the drug, so that it can be effective when taken orally.

[0136] The biosynthesis of halamine involves a complex biochemical pathway. While the exact precursor in the biosynthesis (free tryptophan or L-tryptophan) remains a subject of study, L-tryptophan is presumed to be the major precursor. The proposed biosynthetic scheme proceeds via the shikimic acid pathway, leading to the formation of halamine through a series of steps, including decarboxylation, rearrangement, and hydroxylation.

[0137] At the molecular level, halamine interacts with a variety of molecular targets. It exhibits a significant inhibitory affinity for bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) (serine / threonine kinase). The interaction between halamine and DYRK1A is significant in the neural environment; its inhibition is associated with altered tau phosphorylation, suggesting potential implications for neurodegenerative diseases. Furthermore, halamine modulates neurotransmitter pathways, acting as an inverse agonist at the benzodiazepine site of the GABA-A receptor.

[0138] Halamine is also used as a fluorescent pH indicator. Its fluorescence decreases as the pH value increases.

[0139] Halmin derivatives may exist, but are not limited to, the following chemical structures: C6H5CH2-, 4-Cl-C6H4CH2-, C6H 11 CH2-, 4-NO2-C6H4CH2-, C6H5-(CH2)3CH2-, 4-CN-C6H4CH2-, 4-Br-C6H4CH2-.

[0140] In another embodiment, the DREAM complex inhibitor used according to the present invention is a "Dyrk inhibitor", also known as "INDY", or a pharmacologically acceptable salt or ester thereof.

[0141] INDY ((1Z)-1-(3-ethyl-5-hydroxy-2(3H)-benzothiazole subunit)-2-propanone) is a potent ATP-competitive inhibitor of Dyrk1A and Dyrk1B kinases, with IC50 values ​​of 0.24 μM and 0.23 μM, respectively. At the molecular level, INDY achieves inhibition by competitively binding to the ATP-binding pocket of the Dyrk1A enzyme. The benzothiazole structure of INDY promotes extensive hydrophobic interactions with amino acid residues such as Val173, Ala186, Phe238, Leu241, Leu294, and Val306, resulting in a robust binding interface. In addition to its specific inhibition of Dyrk1A, INDY exhibits broad inhibition of related kinases such as DYRK2, DYRK3, CLK1, CLK4, casein kinase 1 (CSNK1D), and PIM1, and distinguishes itself from Halmin by the lack of inhibitory activity against monoamine oxidase A.

[0142] In some embodiments, the inhibitor comprises an affinity agent, antibody, or antigen-binding fragment thereof that binds to the DREAM complex or its subunits, or an antisense or interfering nucleic acid molecule, such as short interfering RNA (siRNA), that targets the DREAM complex or its subunits. Various inhibition techniques and reagents are familiar to those skilled in the art.

[0143] Medical conditions

[0144] The terms “symptom,” “disease,” or “medical condition” used in this article are used interchangeably.

[0145] In implementation, circadian rhythm-related disorders can be associated with or caused by variations in the intensity, amplitude, phase shift, and internal asynchrony of the circadian rhythm. These disorders can be caused by endogenous factors such as genetic variations, age, sex, menstruation, disrupted melatonin or cortisol secretion, and sleep phase delay or advance syndromes, or by external factors such as environmental factors, including cross-time zone travel, night shift work, shift work, stress, jet lag, and social jet lag. Circadian rhythm disturbances include non-24-hour disturbances, types of circadian rhythm sleep disturbances (including jet lag-related and shift work-related disturbances), and sleep phase alteration-related disturbances (including sleep phase delay syndrome and sleep phase advance syndrome). Circadian rhythm sleep rhythm disturbances can also occur in patients with other conditions, including but not limited to neurodegenerative diseases (such as Alzheimer's or Parkinson's disease), patients with head trauma or encephalitis, patients with mental illness, and patients in intensive care units.

[0146] Circadian rhythm sleep disorders typically refer to asynchrony between the internal sleep-wake rhythm and the light-dark cycle. Patients often experience insomnia, excessive daytime sleepiness, or both, which usually subsides as the body's biological clock realigns itself. If the rhythm adapts, symptoms may subside within days, or in some patients (e.g., older patients) within weeks or months.

[0147] As is known to those skilled in the art, light exposure is a powerful synchronizing factor for circadian rhythms. As a therapeutic measure, exposure to bright light (sunlight or artificial light of 5,000 to 10,000 lux) and activity after the desired wake-up time, and the use of sunglasses to reduce light exposure before the desired bedtime, are beneficial for rapid adaptation to circadian rhythms. An important factor here is the accurate determination of the circadian rhythm's phase and amplitude.

[0148] In the implementation, the medical condition is induced by and / or includes loss of tissue function, oxidative stress and / or dysfunction of intracellular homeostasis.

[0149] As used in this article, the term "tissue" refers to a group of cells with similar structures and functions, and is the basic organizational level between cells and complete organs in biology. There are four main tissue types: epithelial tissue, connective tissue, muscle tissue, and neural tissue, each designed for a specific function. Epithelial tissue covers the surface of organs and provides protection, secretion, and absorption. Connective tissue, with variations such as blood and bone, supports and connects organs, aiding in nutrient transport and tissue repair. Muscle tissue produces movement and includes skeletal muscle, smooth muscle, and cardiac muscle types. Neural tissue, comprising neurons and glial cells, forms the central and peripheral nervous systems, facilitating rapid communication. Each tissue type is essential for supporting and maintaining overall bodily health.

[0150] Oxidative stress is caused by an imbalance between reactive oxygen species (ROS) and the biological system's ability to counteract or repair the damage they produce. At the molecular level, disturbances in the cellular redox state lead to toxic effects, producing peroxides and free radicals that harmfully affect cellular components, including proteins, lipids, and DNA. Base damage and strand breaks in DNA induced by ROS such as superoxide radicals and hydrogen peroxide result in long-term effects, with severe stress leading to cell death. The production of highly reactive substances, such as free radicals and peroxides, exacerbates cellular damage, particularly in DNA. DNA damage, including complex tandem damage, frequently forms and is associated with oxidative stress, aging, and cancer. Repair mechanisms continuously address oxidative DNA damage, but cellular damage exceeds their repair capacity under severe stress, leading to ATP depletion and uncontrolled cell death. Polyunsaturated fatty acids, as major ROS targets, undergo oxidation, producing a variety of products that not only serve as markers of oxidative stress but also contribute to tissue and DNA damage.

[0151] Intracellular homeostasis is the maintenance of a stable state within the cell, which is essential for optimal function, metabolism, and survival. Key processes include, but are not limited to, autophagy, oxidative phosphorylation (OXPHOS), protein ubiquitination, and SUMOylation. In mitochondria, OXPHOS generates ATP through nutrient breakdown, which is the primary cellular energy source. Abnormal regulation can lead to conditions such as mitochondrial myopathy. Autophagy (a recycling process) removes damaged cellular components essential for cellular health. Protein ubiquitination marks proteins for degradation, ensuring proper cell cycle progression, DNA repair, and signal transduction. Abnormal regulation can lead to cancer and neurodegenerative diseases.

[0152] As used herein, “sleep deprivation” is a prevalent condition characterized by insufficient or inadequate duration and quality of sleep required for optimal alertness, performance, and health. Sleep is a critical physiological process supporting a wide range of functions, including memory consolidation, mood regulation, immune function, and overall health maintenance. Recommended sleep duration varies with age, ranging from 9–11 hours for school-aged children and 7–9 hours for adults aged 18–64. Various factors contribute to sleep deprivation, including but not limited to lifestyle choices, sleep environment, work-related problems, sleep disorders, and other medical conditions. Lifestyle behaviors such as inconsistent bedtimes, use of electronic devices before bedtime, and shift work can disrupt normal sleep patterns. Sleep disorders such as insomnia, sleep apnea, restless legs syndrome, and bruxism can affect both the duration and quality of sleep. Additionally, medical conditions such as chronic pain, mental health disorders, diabetes, and substance abuse can lead to sleep deprivation.

[0153] Symptoms of sleep deprivation include, but are not limited to, changes in sleep patterns, mood alterations, cognitive impairment, and physiological effects. Common signs include involuntary falling asleep, fatigue, experiencing stimuli, mood changes, difficulty concentrating, and increased appetite. While the direct effects of sleep deprivation are on cognitive and emotional function, chronic sleep deprivation has long-term consequences for brain function, mental health, and the risk of chronic diseases.

[0154] The effects of sleep deprivation extend to all aspects of health, including brain function, immune health, and the risk of chronic conditions such as obesity, diabetes, and cardiovascular disease. Sleep deprivation is closely associated with mental health conditions such as anxiety and depression, and this bidirectional relationship demonstrates the complex interaction between sleep and mental health.

[0155] In a medical context, the term "acute" describes a disease characterized by a recent onset or short duration. The term implies suddenness, indicating a rapid appearance, change, or worsening of symptoms. This is in contrast to "chronic," which refers to a condition that persists over a longer period. Additionally, "acute" implies urgency, typically indicating a rapidly progressing disease that requires immediate attention. The timescale of a disease is measured by whether it is acute, subacute, or chronic. "Subacute" indicates a longer duration or slower change, while "chronic" implies an indeterminate duration or minimal change over time. The specific timescale varies based on the nature of the disease, and those skilled in the art can determine the different stages of the disease.

[0156] The terms “chronic condition,” “chronic disease,” or “chronic medical injury” are used interchangeably and are characterized by their sustained or long-term effects, typically lasting longer than three months. Chronic conditions generally affect multiple areas of the body, exhibit incomplete responsiveness to treatment, and persist for an extended period. Remission and relapse periods, characterized by temporary improvement or recurrence of symptoms, are common in chronic diseases. These conditions are frequently associated with non-communicable diseases with non-infectious causes and encompass a range of health states, including syndromes, physical disabilities, disabilities, and diseases.

[0157] Chronic conditions play a significant role in causing illness, disability, and reduced physical or mental capacity. Risk factors for these conditions vary by age and sex, and many common chronic diseases stem from dietary, lifestyle, and metabolic factors. Social determinants, including socioeconomic status, education level, and race / ethnicity, also play a crucial role in chronic diseases, contributing to observed differences in care. Barriers to access to medicine and delays in receiving care further exacerbate the challenges for patients from minority and underserved populations.

[0158] In one implementation, the medical condition includes acute medical injury associated with sleep deprivation and circadian rhythm disruption, wherein the acute medical injury is one or more of fatigue, somnolence, cognitive impairment, brain fog, memory impairment, gastrointestinal discomfort, cardiovascular injury, blood pressure changes, decreased immunity, mood changes, depression, anxiety, and / or paranoia.

[0159] As used in this article, “fatigue” refers to a state of tiredness or exhaustion that typically follows prolonged physical or mental activity. In a medical context, fatigue is complex and can be associated with a variety of conditions, such as autoimmune diseases, organ failure, chronic pain, mood disorders, heart disease, infectious diseases, and post-infectious conditions. Physical fatigue is caused by muscle fatigue resulting from strenuous physical activity, while mental fatigue is caused by prolonged cognitive activity, thereby impairing cognitive abilities. Fatigue can manifest as drowsiness, lethargy, or disorientation, which differs from normal fatigue caused by daily activities.

[0160] "Stupor" is a medical condition characterized by deep and persistent drowsiness in which the affected person can only be awakened with difficulty and temporarily. It indicates an abnormal decline in consciousness, unlike ordinary drowsiness or lethargy. The term is associated with conditions such as sleep disorders (African trypanosomiasis) and encephalitis dormantiasis. Stupor signifies slowed movement, difficulty in arousal, and impaired cognitive function, including thinking, concentration, and memory. Unlike fatigue involving physical exhaustion without mental impairment, stupor indicates an impact on the brain and overall cognitive function.

[0161] Cognitive impairment refers to a medical condition characterized by difficulties in memory, learning, concentration, decision-making, and other mental processes that affect daily life. It ranges from mild to severe; mild impairment allows an individual to perform daily activities, while severe impairment can lead to loss of comprehension, communication, and independence. Common symptoms include memory loss, repetitive behaviors, difficulty recognizing people or places, mood changes, visual problems, and challenges in planning and performing tasks. Cognitive impairment encompasses a variety of conditions affecting cognition, including memory, attention, language, and executive function. Assessment, including neuropsychological tests such as the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA), helps in diagnosing cognitive impairment. The condition can be transient (delirium) or range from mild to severe, affecting an individual's memory, problem-solving abilities, and ability to interact with their environment.

[0162] As used in this article, "confusion," often referred to as "brain fog" or "mental fog," refers to a medical condition in which an individual experiences reduced arousal and consciousness, difficulty in judging time or their surroundings, and challenges in maintaining attention. Brain fog is associated with perceived cognitive impairment, characterized by fluctuating states of cognitive dysfunction affecting daily activities. This phenomenon is prevalent in various diseases, particularly those involving chronic pain, affecting 15% to 40% of individuals with chronic pain and serving as a major component of their condition. In such cases, chronic pain can deplete cognitive resources, thus hindering effective thinking. Brain fog can also be caused by factors such as lack of sleep, malnutrition, medications, or drugs.

[0163] The term "memory impairment" or "memory loss" refers to an impaired or inability to recall previously stored information, experiences, or events in the brain, thus affecting an individual's ability to remember and retrieve past memories. Sleep deprivation has a significant impact on episodic memory, affecting both object-level and associative forgetting. Comparative studies involving nighttime sleep, daytime wakefulness, and nighttime sleep deprivation have revealed enhanced forgetting during wakefulness and sleep deprivation, with additional deficits in associative memory observed particularly after prolonged periods of sleep deprivation. The effects of sleep deprivation on memory recall are severe, disrupting normal merging processes and leading to impaired accuracy in memory recall. Sleep deprivation may cause irregular fragmentation between episodic representations, thereby altering the qualitative nature of forgetting.

[0164] Blood pressure (BP) reflects the force exerted by circulating blood against the walls of blood vessels. This pressure is primarily caused by the pumping action of the heart within the circulatory system. Usually measured in millimeters of mercury (mmHg), blood pressure involves two values: systolic and diastolic pressure. Systolic pressure (the maximum force during a heartbeat) and diastolic pressure (the minimum force between heartbeats) are expressed as two numbers. The normal range for adult blood pressure is generally considered to be between 90 / 60 mmHg and 120 / 80 mmHg, but this can vary based on age, health, and individual factors.

[0165] As used in this article, “immune capacity” refers to the body’s ability to produce a normal immune response when exposed to antigens. It contrasts with immunodeficiency, which indicates a compromised immune system. Regarding the immune capacity of lymphocytes such as B cells and T cells, it means the ability to mature and recognize antigens, thereby achieving an effective immune response. Positive selection (involving the recognition of major histocompatibility complex (MHC) molecules) is crucial for lymphocytes to become immunely active.

[0166] As used herein, “mood fluctuations,” “emotional changes,” or “mood swings” encompass extreme or sudden changes in mood that can be constructive or destructive in problem-solving. Severe mood swings can indicate mental illnesses such as bipolar disorder, characterized by unstable and destructive mood swings. Terms such as mood swings, mood instability, affective variability, or mood variability describe fluctuating or oscillating emotions. These terms are used interchangeably, although they may have unique characteristics describing a particular pattern. Emotional dynamics are influenced by a variety of factors, resulting in unstable, volatile, or turbulent patterns. The range of mood swings can be from normal struggles around self-esteem to dramatic oscillations in bipolar disorder, lasting from extremely rapid to prolonged, lasting days or weeks. External triggers, stressors, and psychosis can contribute to mood oscillations. Sleep plays a crucial role in mood regulation, and sleep deprivation can affect mood, leading to negative emotions and increasing the risk of mood disorders such as depression and anxiety. Conversely, mood disorders can also disrupt sleep patterns, thus highlighting the bidirectional relationship between sleep and mood.

[0167] "Depressive disorder," commonly known as "depression," is a prevalent mental illness characterized by prolonged depressed mood or loss of interest in activities. It differs from normal mood changes and can affect all aspects of life, including relationships, work, and daily functioning. Depression can affect anyone, but it is more common in people who have experienced abuse, severe loss, or stress. Women are more susceptible than men, with global estimates suggesting that approximately 3.8% of the population (including 5% of adults and 5.7% of adults over 60 years of age) experiences depression. Symptoms of depression include persistent sadness, sleep disturbances, changes in appetite, feelings of guilt or hopelessness, and thoughts of death or suicide. It can manifest in different forms, such as major depressive disorder, persistent depressive disorder, and bipolar disorder, each with its own unique characteristics and severity.

[0168] Anxiety is an emotion characterized by inner turmoil, worrying thoughts, and physical changes such as elevated blood pressure. Anxiety disorders, including generalized anxiety disorder (persistent anxiety that interferes with daily life), panic disorder (frequent, unexpected panic attacks), social anxiety disorder (intense fear of being judged in social situations), and various phobia-related disorders, all involve excessive and persistent worry or fear. Symptoms range from restlessness and irritability to physical manifestations such as sweating and trembling. Phobias involve irrational fears that lead to avoidance behaviors. Anxiety disorders can be persistent and affect daily activities and relationships. Separation anxiety disorder is commonly associated with children but can also affect adults, causing fear of separation from loved ones. Anxiety, when persistent and severe, can develop into an anxiety disorder, last for a longer period, and often coexists with other mental disorders.

[0169] Paranoia is characterized by organized delusions and may lead to a diagnosis of personality disorder; antipsychotic medications are usually beneficial. It involves irrational thinking influenced by anxiety, doubt, and fear, featuring persecutory delusions and conspiracy theories distinct from phobias. Paranoia is accompanied by false insecurity and pervasive distrust, and individuals may exhibit attribution biases, perceiving unexpected behavior as intentional threats. Common symptoms include feelings of apathy, depression, isolation, and resignation. Paranoia is associated with psychosis and exists in various subtypes, such as delusions of passion, persecutory delusions, delusions of litigation, and exalted delusions. Singleness is common in the paranoia population, affecting interpersonal relationships. Types of paranoia can be categorized by commonalities, with social anxiety being the most frequent manifestation. Severe paranoia can lead to isolation behaviors, resulting in a diagnosis of personality disorder.

[0170] Circadian rhythm disturbances play a significant role in various “neurological disorders,” influencing their development, presentation, and severity. “Neurodevelopmental disorders” can include autism spectrum disorders (ASD) and rare genetic conditions such as Angleman syndrome, Williams syndrome, Prad-Willi syndrome, Fragile X syndrome, and Smith-Magini syndrome, and often manifest as circadian rhythm disturbances and poor sleep quality. Specifically, ASD is associated with biomarkers of circadian rhythm disturbances, such as cortisol dysregulation and low-amplitude melatonin rhythms. Treatment strategies for circadian rhythm disturbances in conditions such as Smith-Magini syndrome involve the administration of β1-adrenergic antagonists and melatonin supplementation for repositioning. A bidirectional relationship with circadian rhythm disturbances has been observed in “neurodegenerative diseases” such as Parkinson's disease (PD) and Alzheimer's disease (AD). Circadian rhythm dysregulation and reduced rhythm amplitude predict the development of neurodegenerative diseases. PD, characterized by dopamine regulation disorder, exhibits circadian rhythm disturbances at various disease stages, suggesting a potential role in pathogenesis. Bright light therapy and melatonin have been developed as circadian rhythm-based PD therapies. AD, associated with SCN neuronal loss and impaired light input pathways, displays circadian rhythm disturbances at multiple levels, correlated with the degree of neuronal damage.

[0171] Circadian rhythm disruption is a common factor in mental illnesses, including conditions such as schizophrenia and mood disorders, including depression, bipolar disorder, and seasonal affective disorder (SAD). SAD, characterized by depressive symptoms during the fall and winter months when daylight is reduced, demonstrates the impact of seasonally induced circadian rhythm dysregulation on mental health. Another condition is sleep-wake phase delay disorder, where deviations between melatonin onset and bedtime increase the likelihood of depressive symptoms. The term "circadian rhythm depression" has been coined to describe this clinical phenotype, emphasizing the need for circadian rhythm-targeted therapies in mood disorders with specific rhythms, seasonality, and treatment responses. Preclinical evidence in mice establishes a bidirectional relationship between mood disorders and the circadian rhythm system, where serotonin receptor regulation induces circadian rhythm disruption. External factors such as inappropriate timing of light exposure induce depressive symptoms mediated by projections from retinal ganglion cells to key brain regions. Human functional MRI studies have shown that light exposure affects mood by inhibiting amygdala activity and enhancing prefrontal cortex connectivity. Altered expression of circadian rhythm genes and genetic polymorphisms within these genes are mechanistic factors in mood disorders. Synchronizing the brain's biological clock with the external environment has been associated with a reduced risk of depression.

[0172] The influence of light conditions on the efficacy of psychotropic medications supports the potential of circadian rhythm-based treatments for mental illness. Chronotype, or individual preference for the timing of activity, also affects treatment outcomes. Lithium, a treatment for bipolar disorder, shows varying effects based on chronotype, with responders tending towards the "early-onset" type. Bright light therapy has emerged as a relevant intervention across a variety of mood disorders, including major depressive disorder, bipolar disorder, and seasonal affective disorder.

[0173] As used herein, the terms “cardiovascular disease,” “heart disease,” and “cardiac disease” are interchangeable with or at least related to the terms “cardiomyopathy” or “cardiovascular-related conditions.” Vascular disease is a large category of diseases affecting the heart and / or blood vessels (arteries and veins). Cardiovascular diseases include arrhythmias, vascular diseases, myocardial infarction, heart failure, myocarditis, atherosclerosis, restenosis, coronary artery disease, coronary artery disease, atherosclerotic cardiovascular disease, hypertension, cardiac fibrosis, stroke, sudden cardiac death syndrome, heart failure, ischemic heart disease, ischemic cardiomyopathy, myocardial infarction, and coronary artery calcification. These diseases share similar causes, mechanisms, and treatments. Most cardiovascular diseases have common risk factors, including inflammation, fibrosis, diabetes, cholesterol, and vascular deposits.

[0174] Circadian rhythms have profound implications for cardiovascular health, influencing the timing of myocardial infarction, stroke, and ventricular arrhythmias. The peak incidence of cardiovascular events (morning) is partly attributed to the circadian regulation of thrombotic factors. Circadian rhythm dysregulation between environmental rhythms and the internal biological clock increases the risk of cardiovascular disease (CVD), contributing to hypertension, inflammation, and elevated CVD risk in night shift workers. Disruptions in blood pressure regulation and arrhythmias have been observed, and the complex interaction between circadian rhythm disturbances and CVD has been illustrated in individuals with type 2 diabetes. Implementing circadian rhythm-based therapeutic strategies, such as bright light therapy to induce expression of core circadian clock genes, holds promise for playing a role during the morning period when CV events are more likely to occur. Timing-based therapies for CVD are gaining attention, with studies supporting bedtime-based treatment in hypertensive patients. In children, circadian rhythms vary widely with age, and factors such as obesity and pediatric diseases disrupt cardioprotective physiology, highlighting the need for interventions to mitigate long-term CVD risk in this population. Modifiable behavioral factors (including screen time and school start time) provide opportunities for targeted interventions for adolescents.

[0175] Metabolic disorders encompass a range of conditions affecting genetic, cellular, and systemic metabolism. These include impaired glucose tolerance, insulin resistance, and an increased risk of conditions such as diabetes and obesity. Furthermore, metabolic disorders adversely alter the body's processing and distribution of substrates, supplements, and nutrients (including proteins, carbohydrates, fatty acids, and lipids), leading to abnormal chemical reactions in the body that are associated with pathophysiological changes. The complex relationship between the circadian rhythm system and metabolism significantly impacts diabetes and obesity. Circadian rhythm disruption leads to impaired glucose tolerance, insulin resistance, and an increased risk of metabolic disorders. Mutations in core circadian clock genes (such as ClockΔ19) and disturbances in Bmal1 result in metabolic damage. Social jet lag, intra- and social timing differences, and late-night sleep patterns are associated with diabetes risk. The timing of food consumption and light exposure characteristics affect metabolic function.

[0176] Obesity is associated with ClockΔ19 mutations and specific CLOCK gene variants. Polymorphisms in the CLOCK and melatonin receptor genes are associated with increased BMI. Shift workers face an increased risk of metabolic disorders and obesity, and social jet lag is associated with overweight. Timing of meals and light exposure are recommended strategies for calibrating energy intake with metabolic rhythms, potentially improving health and weight regulation.

[0177] Immunological disorders, also known as immune disorders or immunological diseases, are conditions in which the immune system malfunctions due to either overactivity (autoimmune disorders) or underactivity (immunodeficiency disorders). Examples of immunological disorders include autoimmune diseases such as rheumatoid arthritis, lupus (systemic lupus erythematosus), type 1 diabetes, and multiple sclerosis. Immunodeficiency disorders can include primary immunodeficiency diseases (such as severe combined immunodeficiency disease - SCID), acquired immunodeficiency syndromes (AIDS), and common variant immunodeficiency (CVID and DiGeorge syndrome). Allergies and hypersensitivity reactions can include allergic rhinitis, asthma, and anaphylactic reactions.

[0178] Allergic symptoms, or allergies, are caused by a hypersensitive response of the immune system to normally harmless environmental substances, triggering a variety of symptoms in different organs. The immune basis of allergy involves two phases: sensitization and the development of memory T-cell and B-cell responses, followed by IgE production and effector function. Eosinophils, intrinsic lymphoid cells, dendritic cell subsets, epithelial cells, tissue inflammation, the epithelial barrier, tissue remodeling, and chronicity play key roles in allergic diseases such as asthma, atopic dermatitis (AD), and allergic rhinitis (AR). Different molecular mechanisms, biomarkers, and responses to biotherapies characterize different disease phenotypes and endotypes.

[0179] Allergies are conditions in which the immune system overreacts to normally harmless substances. These include hay fever, food allergies, atopic dermatitis, allergic asthma, and anaphylactic reactions. Symptoms manifest in various organs, such as red eyes, itchy rashes, sneezing, coughing, runny nose, shortness of breath, and swelling. Allergens such as pollen, certain foods, metals, insect bites, and medications can trigger allergies. Genetic and environmental factors contribute to the development of allergies. Immunoglobulin E antibodies (IgE) bind to allergens, triggering the release of inflammatory chemicals that cause symptoms. Diagnosis involves a medical history and, in some cases, skin or blood tests. Treatment includes avoiding allergens, medications such as steroids and antihistamines, and, in severe cases, injections of adrenaline. Allergen immunotherapy is effective for specific allergies.

[0180] “Gastrointestinal disorders” (GI disorders) encompass conditions affecting the entire gastrointestinal tract, including the esophagus, stomach, small and large intestines, rectum, and accessory organs such as the liver, gallbladder, and pancreas. While gastrointestinal symptoms are common, functional gastrointestinal disorders (FGIDs) such as irritable bowel syndrome (IBS) and functional dyspepsia are prevalent and lack a structural explanation. The pathophysiology involves a bidirectional dysregulation of the gut-brain axis, microbial dysbiosis, altered immune function, visceral hypersensitivity, and abnormal gastrointestinal motility. These disorders are often accompanied by psychosocial comorbidities. Biopsychosocial models emphasize the complex interplay between physical symptoms and psychological factors. FGIDs are characterized by dysregulation of the gut-brain axis, rather than structural abnormalities. Low-grade intestinal inflammation, altered microbiota, and immune activation are the pathogenic mechanisms leading to FGIDs. In contrast, structural gastrointestinal disorders involve abnormalities visible on examination and affect motility, sometimes requiring surgical intervention. Examples can include strictures, hemorrhoids, colonic polyps, and inflammatory bowel disease. Sleep dysfunction is intricately associated with gastrointestinal conditions such as gastroesophageal reflux disease (GERD), inflammatory bowel disease (IBD), liver disorders, and colorectal cancer. In GERD, a clear link between sleep disturbances and nocturnal symptoms is observed, influencing the risk of esophageal complications. In peptic ulcer disease, shift workers have a higher incidence of ulcers, likely due to unpredictable meal times, sleep dysfunction, work stress, and the use of nonsteroidal anti-inflammatory drugs (NSAIDs). Sleep disturbances are prevalent in irritable bowel syndrome (IBS) and functional dyspepsia, affecting sleep quality and exacerbating gastrointestinal symptoms. Studies have also highlighted a bidirectional relationship between IBD and sleep dysfunction, emphasizing the potential for sleep disturbances to lead to disease relapse. Patients with cirrhosis experience sleep disturbances caused by circadian rhythm disruption, even without hepatic encephalopathy. Furthermore, chronic hepatitis C infection is associated with sleep disturbances, but not with psychiatric conditions. Obstructive sleep apnea affects 3% to 7% of adults and plays a role in various gastrointestinal conditions, including nonalcoholic fatty liver disease and cirrhosis.

[0181] "Rheumatic diseases," also known as "rheumatic conditions," encompass more than 200 conditions that cause chronic, intermittent pain in the joints or connective tissues. It includes arthritis and "non-articular rheumatic diseases," which overlap with the term soft tissue conditions. Major categories of rheumatic diseases can include diffuse connective tissue diseases (such as rheumatoid arthritis, lupus), spondylitis-related arthritis (such as ankylosing spondylitis, psoriatic arthritis), osteoarthritis, infectious rheumatic syndromes, metabolic and endocrine disorders (such as gout), tumors, neurovascular diseases, bone and cartilage diseases, and extra-articular diseases (such as bursitis, tendinitis). Rheumatic diseases, such as osteoarthritis and rheumatoid arthritis, can cause severe joint pain due to cartilage damage.

[0182] Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by long-term respiratory symptoms and airflow limitation. Common types include emphysema and chronic bronchitis. While emphysema involves enlarged airspace and permanent lung tissue damage, chronic bronchitis is defined as a cough lasting at least three months per year for two consecutive years. COPD is usually caused by smoking, but other risk factors include air pollution, occupational irritants, and genetic factors such as alpha-1 antitrypsin deficiency. Diagnosis is based on poor airflow measured by spirometry. COPD causes symptoms such as shortness of breath, chronic cough, and wheezing. It is associated with low-grade systemic inflammation and often coexists with conditions such as asthma, cardiovascular disease, and lung cancer. Treatment includes smoking cessation, vaccination, medications, and interventions such as oxygen therapy or lung transplantation.

[0183] Cancer is characterized by the abnormal growth of cells with the potential to invade other parts of the body. There are over 100 types, contrasting with non-metastatic benign tumors. Causes include smoking, obesity, poor diet, infection, ionizing radiation, and genetic defects. Treatments include surgery, radiation therapy, chemotherapy, and targeted therapy. Symptoms are diverse, and cancer diagnosis can be challenging, often mimicking other conditions. Local symptoms are caused by a tumor mass or ulceration, while systemic symptoms are caused by bodily reactions. Metastasis, the spread of cancer, usually occurs in later stages and affects organs such as the lungs, liver, brain, and bones. Cancers are classified by cell type (carcinoma, sarcoma, lymphoma, leukemia, germ cell tumors, blastoma), and are usually named based on organs or tissues.

[0184] Alterations in genes such as proto-oncogenes, tumor suppressor genes, and DNA repair genes can become cancer drivers. Triggers include errors during cell division, DNA damage caused by environmental factors, and genetic mutations. Cancer cells differ from normal cells in their uncontrolled growth, invasion, immune system evasion, and altered nutrient dependence.

[0185] Various causes exist for the aforementioned symptoms. In one embodiment, the cause of these symptoms is a disruption of the circadian rhythm. In a further embodiment, the aforementioned condition can be treated using the DREAM complex inhibitors and methods disclosed herein.

[0186] treat

[0187] As used herein, the terms “individual” and “subject” are generally used interchangeably and refer to any animal exhibiting symptoms of a disease, condition, or condition treatable with the DREAM complex inhibitors and methods disclosed herein. In a preferred embodiment, a subject includes any animal exhibiting symptoms of a disease, condition, or condition associated with sleep deprivation and circadian rhythm disruption, such as acute medical injuries including fatigue, somnolence, cognitive impairment, brain fog, memory impairment, gastrointestinal upset, cardiovascular injury, blood pressure changes, decreased immunity, mood changes, depression, anxiety, and / or paranoia, or chronic medical conditions including neurological, psychiatric, cardiovascular, metabolic, allergic, immune, gastrointestinal, rheumatic, proliferative (cancer), and / or pulmonary conditions treatable with the methods disclosed herein. Suitable subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and domesticated animals or pets (e.g., cats or dogs). Non-human primates are included, with human patients being preferred.

[0188] As used in this article, the terms “(medical) disease,” “(medical) symptom,” and “(medical) condition” are generally used interchangeably.

[0189] As used herein, “treatment” or “treating” includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally include a reduction or improvement in the symptoms of a disease or condition, or a delay in the progression of the disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of a disease, condition, or associated symptoms. The term “treatment effective” is intended to include, within reasonable medical judgment, excessive toxicity, irritation, and / or other problems or complications, but in proportion to a reasonable benefit / risk ratio.

[0190] As used herein, “prevention” and similar terms, such as “prevented,” “being prevented,” or “preventative,” refer to methods used to prevent, suppress, or reduce the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the appearance or recurrence of its symptoms. As used herein, “prevention” and similar terms also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition before its onset or recurrence.

[0191] This invention relates to the use of inhibitors of protein complex dimerizing mate bodies, RB-like structures, E2F, and multiple vulvar B types (DREAM complex inhibitors) for the treatment of medical conditions induced by circadian rhythm disorders.

[0192] In implementation, the use of DREAM complex inhibitors in subjects in need of treating medical conditions induced by circadian rhythm disturbances includes administering an effective dose (e.g., a therapeutically effective dose) of the inhibitors considered herein. The dosage and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but clinical trials may determine an appropriate dosage.

[0193] In embodiments, a pharmaceutical composition containing a DREAM complex inhibitor for administration to a subject may include at least one additional pharmaceutically acceptable additive, such as a carrier, thickener, diluent, buffer, preservative, surfactant, etc., and selected molecules. The pharmaceutical composition may also include one or more other active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc. Pharmaceutically acceptable carriers that can be used in these formulations are conventional. Those skilled in the art are familiar with compositions and formulations suitable for the drug delivery of the bispecific agents disclosed herein. Typically, the nature of the carrier will depend on the specific method of administration employed. For example, parenteral formulations typically contain injectable fluids, including pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc., as a medium. For solid compositions (e.g., powders, pills, tablets, or capsules), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biological neutral carriers, the drug composition to be administered may contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitol monolaurate.

[0194] In implementation, the DREAM complex inhibitor can be combined with pharmaceutically acceptable carrier substances as needed to approximate physiological conditions, such as pH adjusters and buffers, tonicants, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitol monolaurate, and triethanolamine oleate. For solid compositions containing bispecific reagents, conventional, non-toxic, pharmaceutically acceptable carriers can be used, including, for example, pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. Liquid pharmaceutical compositions, whether solutions, suspensions, or other similar forms, may include one or more of the following: a sterile diluent, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, or isotonic sodium chloride; a fixing oil, such as synthetic mono- or diglycerides of glycerol, polyethylene glycol, glycerol, propylene glycol, or other solvents that can be used as a solvent or suspension medium; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate; and a reagent for adjusting osmotic pressure, such as sodium chloride or glucose. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0195] The DREAM complex inhibitor contained in the (pharmaceutical) composition can be administered to subjects via a variety of mucosal administration routes, including oral, rectal, intranasal, intrapulmonary or transdermal delivery, intramuscular, intraocular, subcutaneous, intravenous, intra-articular, intra-articular, intraperitoneal, intrathecal, intravenous or parenteral routes.

[0196] According to the disclosure herein, in an implementation, a preventative or therapeutically effective amount of a DREAM complex inhibitor may be administered to a subject requiring such treatment at a time and under conditions sufficient to prevent, suppress, and / or improve a selected condition or one or more of its symptoms, wherein the condition is caused by a circadian rhythm disorder.

[0197] The attending clinician can adjust the dosage to maintain the desired concentration at the target site (e.g., the lungs or systemic circulation). Higher or lower concentrations can be selected based on the delivery mode, such as transdermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. The dosage can also be adjusted based on the release rate of the administered formulation, such as the release rate of an intrapulmonary inhaler versus a powder, a sustained-release oral formulation versus an injectable granule, or a transdermal delivery formulation.

[0198] This disclosure also includes kits, packages, and multi-container units containing the DREAM complex inhibitor described herein or pharmaceutical compositions comprising thereof and / or methods of administration thereof, for use in the prevention and treatment of the conditions described herein and other conditions in human subjects.

[0199] It should be understood that the specific embodiments described herein are shown by way of illustration and not as limitations on the invention. The main features of the invention may be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to determine many equivalents of the specific procedures described herein using most conventional research. These equivalents are considered to be within the scope of the invention and covered by the claims. All publications and patent applications referenced in the specification indicate the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated as incorporated by reference.

[0200] When used in the claims and / or specification in conjunction with the words "a" or "an," the term "comprising" may mean "an," but it also has the same meaning as "one or more," "at least one," and "one or more." Unless explicitly stated otherwise, the use of the term "or" in the claims is intended to mean "and / or." However, this disclosure only supports the definitions of alternatives and "and / or." Throughout this application, where applicable, the term "about" indicates an inherent error variation between the value and the means, the method used to determine the value, or the object of study. Attached Figure Description

[0201] The invention is illustrated by way of example in the following figures. The figures will provide further description of potential preferred embodiments to enhance support for one or more non-limiting embodiments of the invention.

[0202] Brief description of the attached diagram:

[0203] Figure 1 RNAi-mediated knockdown of lin-42 / PER impairs circadian rhythm-controlled developmental timing and exacerbates aggregation-induced tissue exhaustion.

[0204] Figure 2 Genetic damage to the biological clock triggers changes in histone expression, chromatin organization, and the abundance of the DREAM complex.

[0205] Figure 3 Inactivation of different components of the DREAM complex alleviated increased protein aggregation and tissue dysfunction caused by circadian rhythm disruption.

[0206] Figure 4 The increase in histone expression triggered by circadian rhythm disruption depends on DREAM activity.

[0207] Figure 5 Disruption of the circadian rhythm disrupts multiple core pathways for cellular maintenance in a dream-dependent manner.

[0208] Figure 6 The DREAM-circadian clock interaction is evolutionarily conserved, and pharmacological DREAM dysregulation restores homeostasis in animals with impaired circadian clocks.

[0209] Figure 7 RNAi-mediated lin-42 / PER inactivation disrupts circadian rhythm-dependent developmental timing.

[0210] Figure 8 RNAi-mediated circadian rhythm disruption exacerbates muscle paralysis in Caenorhabditis elegans models of Alzheimer's and Huntington's diseases.

[0211] Figure 9 Protein-protein interaction analysis places the DREAM complex at the heart of proteomic changes induced by circadian rhythm disruption.

[0212] Figure 10 The changes in the whole proteome were comparable between animals that underwent lin-42 / PER inactivation during development and adulthood.

[0213] Figure 11 The interaction between DREAM and circadian rhythm damage shows subunit specificity.

[0214] Figure 12 :lin-53 / DREAM inactivation restoration in protein homeostasis of circadian rhythm disordered animals expressing polyglutamine-linked YFP.

[0215] Figure 13 : Disruption of the biological clock enhances histone expression in a dream-dependent manner.

[0216] Figure 14 : Disruption of the biological clock increases histone expression in a dream-dependent manner.

[0217] Figure 15 Disruption of the circadian rhythm disrupts core pathways for cell maintenance in a dream-dependent manner.

[0218] Figure 16 The same basic cellular processes in Caenorhabditis elegans and mice are regulated by the biological clock.

[0219] Figure 17The same basic cellular processes in Caenorhabditis elegans and humans are regulated by the biological clock.

[0220] Figure 18 DREAM expression is reduced during sleep, and sleep deprivation prevents this reduction. The circadian rhythm and sleep disturbances interfere with basic cellular processes in a DRM / DREAM-dependent manner.

[0221] Figure 19 High expression of DREAM protects DNA from wakefulness-related damage, while low expression of DREAM promotes repair during sleep.

[0222] Figure 20 Circadian rhythm disruption increases histone expression in a DRM / DREAM-dependent manner.

[0223] Figure 21 Circadian rhythm disruption interferes with basic cellular processes in a DRM / DREAM-dependent manner.

[0224] Figure 22 The DREAM-dependent pathway is a functional driver of the negative effects of circadian rhythm disruption.

[0225] Figure 23 DREAM expression changes during the natural sleep-wake cycle.

[0226] Figure 24 The DREAM subunit follows circadian rhythm dynamics and its expression is increased by melatonin treatment.

[0227] Figure 25 The same basic cellular processes are altered by circadian rhythm disruption in mice and nematodes.

[0228] Figure 26 : The same basic cellular activity is regulated through the biological clock / DREAM axis in nematode and human cells.

[0229] Figure 27 Experimental design for evaluating developmental timing following UV-B and lin-42 RNAi treatment.

[0230] Figure 28 The role of DREAM in normal sleep and sleep deprivation.

[0231] Detailed description of the attached diagram:

[0232] Figure 1RNAi-mediated knockdown of lin-42 / PER impairs circadian rhythm-controlled developmental timing and exacerbates aggregation-induced tissue exhaustion. (A) Luminescent experimental design. (B) Developmental timing is shown for wild-type, lin-42 (n1089), and lin-42 (ok2385) populations. Nematodes were fed ev or lin-42 RNAi in addition to D-luciferin. Quantitative analysis of larval developmental stages at 20°C based on luminescence is presented. Mean duration of each stage is indicated by black horizontal lines. n = 13–20 nematodes per condition, n = 4 independent experiments. One-way ANOVA was used for statistical evaluation. p < 0.000001. (C) Wild-type N2 nematodes were age-synchronized and fed ev or lin-42 RNAi from the L1 stage. Survival was scored daily. Animals were transferred to new plates every other day until AD10, then every four days thereafter, with n = 140 nematodes per condition. This figure represents three independent experiments. Significance was measured by the log-rank Mantel-Cox test. (D) Age-synchronized Q40-YFP (unc-54p::Q40::YFP) animal populations were fed ev or lin-42 RNAi. Paralysis was scored daily. Nematodes were transferred to new plates every other day. n = 140 nematodes / condition. This figure represents three independent experiments. Significance was measured by the log-rank Mantel-Cox test. p<0.000001.

[0233] Figure 2 Genetic damage to the biological clock triggers changes in histone expression, chromatin organization, and DREAM complex abundance. (a) Experimental design. (b) Principal component analysis comparing the whole proteome of wild-type *C. elegans* treated with ev or lin-42 RNAi from the L1 stage. Proteins were extracted at AD2, with small dots representing a single replicate, n=800 nematodes per replicate. (c) Bar chart showing significantly enriched GO biological process items (FDR≤0.05) in animals treated with lin-42 and ev RNAi. (d) Volcano plot depicting different expressed proteins in lin-42 and ev RNAi-treated nematodes, with histones highlighted in red. Horizontal dashed lines indicate a p-value threshold of 0.05, and vertical lines indicate logarithmic values. 10 The fold change threshold was ±0.05. (e) shows a heatmap of the selected DREAM subunits and interacting factors. Color codes represent the log2 (fold change) values ​​between lin-42 and ev RNAi-treated nematodes; including L1 and L4 stages of RNAi treatment. (f) A mechanistic model is proposed.

[0234] Figure 3Inactivation of different DREAM complex components alleviated increased protein aggregation and tissue dysfunction caused by circadian rhythm disruption. (a) Experimental design. (b) From L1 stage, Q40-YFP animals were fed ev or lin-42 RNAi in combination with RNAis targeting specific subunits / interacting factors of the DREAM complex; paralysis scores were assessed daily. Co-targeting with lin-9(b), lin-54(c), lin-53(d), and the DREAM interactor his-41(e) is shown, with n=140 nematodes per condition. Figures represent at least three independent experiments. Significance was measured by the log-rank Mantel-Cox test. p < 0.0001. (f) Age synchronization of Q40-YFP nematodes was performed, feeding the specified RNAi construct from the L1 stage and imaging was conducted at three distinct time points: L4, AD1, and AD2. The number of aggregates divided by the corresponding YFP intensity is shown, with n = 26–32 nematodes per condition; this plot represents three independent experiments. Error bars represent standard errors (sem). Significance was measured using one-way ANOVA within each age group and Sidak multiple comparison test. p<0.0001; p<0.01; p < 0.0001; ns, not significant.

[0235] Figure 4 Elevated histone expression triggered by circadian rhythm disruption depends on DREAM activity. (ad) WT nematodes were age synchronized and treated with specified RNAi combinations from L1 to AD2. Western blot analysis of H1 and H2 histones was performed using tubulin as a loading control, and quantifications were given. n = 800 nematodes per condition; each figure includes results from 5 independent experiments. Error bars are standard errors (sem), representative WB images are shown in figure (e), and complete gels are visible. Figure 13 (f) Nematodes were treated as in (ad), and the mRNA expression levels of his-24(H1), hil-2(H1), and his-29(H2) were assessed by qPCR. n=250 for each condition, and the figure represents three independent experiments. Error bars are for SEM; by [data missing]. Methods: fold change in mRNA was calculated. Significance was assessed using one-way ANOVA within each group, followed by Sidak multiple comparison test. p<0.05; p<0.01; p<0.001; p < 0.0001; ns not significant. (g) Summary of the mechanisms presenting the data. (hm) Nematodes were treated with the specified RNAi combinations and control (ev) RNAi from L1 to AD2. Protein expression was resolved by proteomics. Box plots are provided showing the relative expression of proteins belonging to xenobiotic metabolism (h), glutathione metabolism (i), glycolysis (j), collagen (k), transcription initiation (l), and ribosome biosynthesis (m) (ev+lin-42 vs ev, and lin-42+lin-53 vs ev). Individual proteins are shown as dots, and the fold change of the median for each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. n = 800 nematodes per condition, and 4 independent populations were measured for each condition. In each boxplot, significance was assessed using the Mann-Whitney-Wilcoxon rank-sum test, and the Wilcoxon test was used to compare fold changes between the two groups. Two-tailed p-values ​​were calculated in all cases. -p<0.005; -p<0.01; -p<0.001; -p<0.0001. (no) From L1 stage, age-synchronized unc-54p::Q40::YFP animals were fed EV or lin-42 RNAi, combined with RNAi targeting specific genes associated with transcription initiation and ribosomal biogenesis, and paralysis scores were assessed daily. Co-targeting with rpb-11(N) and fib-1(o) is shown. n=140 nematodes per group, and the figure represents three independent experiments. Significance was measured by log-rank Mantel-Cox test, and two-tailed p-values ​​were calculated. -p<0.0001. (p) describes the mechanism model.

[0236] Figure 5 Circadian rhythm disruption interferes with multiple core pathways of cell maintenance in a dream-dependent manner. (a) Experimental design. Nematodes were treated with specified RNAi combinations from L1 to AD2. Protein expression was addressed using proteomics. (bg) Box plots are provided showing the relative expression of proteins belonging to the antioxidant response (b), glycolysis (c), collagen (d), lysosomes (e), transcription initiation (f), and ribosomes (g). Individual proteins are shown as dots, and the fold change of the median for each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. n = 800 nematodes per condition, and four independent populations were measured for each condition. Significance was assessed by Wilcoxon rank-sum test and two-tailed test. p < 0.05; p < 0.01; p < 0.001; p < 0.0001. (h) describes the mechanism model.

[0237] Figure 6 DREAM-circadian clock interactions are evolutionarily conserved, and pharmacological DREAM dysregulation restores homeostasis in clock-impaired animals. (ab) Transcriptomic data from the hippocampus of WT mice or mice expressing human AD-like APP variants (TG mice), which were either allowed free access (ALF) or timed feeding (TLF, Whittaker et al., 2023), were analyzed. Data from ZT0 and ZT12 were analyzed. Box plots show the relative expression (Log2FC) of mitochondrial (a) and OXPHOS (oxidative phosphorylation) (b) genes under specified conditions. Individual genes are shown as dots, and the fold change of the median for each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. n = 6–7 mice per condition. Significance was measured by the Wilcoxon test. p < 0.05; p < 0.01; p < 0.001; p < 0.0001. (c) Human retinal pigment epithelial cells (RPE) were treated with siRNA (10 nM) targeting PER1 for 48 h, and co-treated with halamine (10 μM) or DMSO (vector control) for 20 h. This showed that... The expression fold change was calculated using the method. Significance was measured within each group using one-way ANOVA and Sidak multiple comparison test. p<0.05; p<0.01; p<0.001; p < 0.0001; ns not significant. (de) Human RPE cells were treated as in (c) and whole transcriptome analysis was performed by mRNA sequencing. Box plots show the relative expression (Log2FC) of mitochondrial (d) and OXPHOS (e) genes. Individual genes are shown as dots, and the fold change of the median in each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. Significance was measured by the Wilcoxon test. p<0.05; p<0.01; p<0.001; p < 0.0001. n = 5 independent cultures per condition. (f) Age-synchronized Q40-YFP (unc-54p::Q40::YFP) nematode populations from L1 stage were treated with ev or lin-42 RNAi in plates containing 0.1% DMSO (vector) or halamine (60 μM). Paralysis scores were assessed daily, with n = 140 nematodes per condition. Figures represent at least three independent experiments. Significance was measured by the log-rank Mantel-Cox test. p<0.0001. (g) shows a model depicting the role of DREAM in cellular degradation following circadian rhythm disruption.

[0238] Figure 7 RNAi-mediated lin-42 / PER inactivation disrupts circadian rhythm-dependent developmental timing. (a) Developmental timing was analyzed in wild-type, lin-42 (n1089), and lin-42 (ok2385) populations. Nematodes were fed ev or lin-42 RNAi in addition to D-luciferin. A luminescence-based quantitative analysis of the duration of the molting phase at 20°C is shown. Mean duration is represented by black horizontal bars in each case, with n = 13–20 nematodes per condition. The figure represents the sum of four independent experiments. One-way ANOVA, p < 0.05; p < 0.01; p < 0.001; p < 0.0001.

[0239] Figure 8 RNAi-mediated circadian rhythm disruption exacerbated muscle paralysis in *C. elegans* models of Alzheimer's and Huntington's diseases. (a) Age-synchronized GMC101 (unc-54p::human Aβ1-42) nematodes were treated with ev or lin-42 RNAi from stage L1. The nematodes were cultured at 20°C until stage L4, then transferred to 25°C to induce amyloid-β toxicity. (b) From stage L4, age-synchronized Q40-YFP (unc-54p::Q40::YFP) animal populations were fed ev or lin-42 RNAi. (c) Age-synchronized GMC101 (unc-54p::human Aβ1-42) nematodes were treated with ev or lin-42 RNAi from stage L4. Transfer to 25°C was performed as described in (a). (d) Age-synchronized Q40-YFP (unc-54p::Q40::YFP) animal populations were fed ev or lin-14 RNAi. Paralysis was scored daily in all cases (ad), with n=140 nematodes per condition; the figures show representative results from at least three independent experiments. Significance was measured by the log-rank Mantel-Cox test. p < 0.05; p < 0.0001, ns, not significant.

[0240] Figure 9 Protein-protein interaction analysis places the DREAM complex at the heart of proteomic changes induced by circadian rhythm disruption. Figure 2 In protein b, the PC1 group that contributed the most to the network was subjected to STRING analysis. The threshold for the absolute rotation weights of the included proteins was >0.023, and 121 proteins showed significant network enrichment (p<1.0e). -16 Use different colors to depict the components or interacting factors of the chromatin repression complex DREAM.

[0241] Figure 10 The total proteomic changes were comparable between animals that underwent lin-42 / PER inactivation during development and adulthood. (a) Experimental design. (b) Principal component analysis of the total proteomic composition of wild-type *C. elegans* treated with ev or lin-42 RNAi from the L4 stage is shown. Dots represent single replicates, n=800 nematodes per sample. (c) Using... Figure 10 STRING analysis was performed on the protein group that contributed most to PC1 separation in b. The threshold for absolute rotation weights of proteins was >0.01, and 46 proteins showed significant network enrichment (p<7.72). -10 Use different colors to depict the components or interacting factors of the chromatin repression complex DREAM.

[0242] Figure 11 The interaction between DREAM and circadian rhythm disruption showed subunit specificity. (a) Age synchronization of 250 nematodes per condition was performed, starting from L1 stage with a specified RNAi combination, and expression of lin-42 and lin-53 genes was detected by qPCR at AD2. The figure represents three independent experiments. Mean values ​​and sem are shown. mRNA fold changes were detected by... Methods were calculated. Significance was assessed using an unpaired t-test. p<0.01; p < 0.001; ns, not significant. (be) Q40-YFP (unc-54p::Q40::YFP) were age synchronized and fed ev or lin-42 RNAi from L1 stage, combined with RNAi targeting specific DREAM subunits or interactors: lin-37(b), lin-52(c), lin-35(d), htz-1(e). Paralysis was scored daily, n = 140 nematodes per condition, and the figures represent at least three independent experiments. Significance was measured by log-rank Mantel-Cox assay. p<0.0001; p<0.05, ns, not significant.

[0243] Figure 12 :lin-53 / DREAM inactivation restores protein homeostasis in circadian rhythm-disordered animals expressing polyglutamine-linked YFP. Q40-YFP nematodes were age-synchronized and fed a specified RNAi combination from the L1 stage, and imaged at three distinct time points: L4, AD1, and AD2. (a) shows the YFP intensity per body surface area in Q40-YFP nematodes. Each condition had n = 26–32 nematodes, and this figure represents three independent experiments. Error bars are sem. Significance was measured separately within each age group using one-way ANOVA and Sidak multiple comparison test. p<0.05; p < 0.0001; ns not significant. (b) Representative images of AD2 stage Q40-YFP animals treated with the specified RNAi combination. The nematodes were imaged in an AxioZoom v.16 microscope at 90x magnification, with an exposure time of 150 ms for YFP and a bright field of 4.8 ms. Scale bar: 200 μm.

[0244] Figure 13 : Disruption of the biological clock enhances histone expression in a dream-dependent manner. Figure 4 a- Figure 4 Representative whole-gel scans of d. (a) H1, (b) H2B1L, (c) H2AZ1, (d) H2AZ2. Bands used for quantification are marked with arrows. H = histone, T = tubulin.

[0245] Figure 14 : Disruption of the biological clock increases histone expression in a dream-dependent manner. (ab) Figure 4 a- Figure 4The nematodes were treated and analyzed using antibodies specific to histones H3 and H4. Representative whole-gel scans (left) and corresponding quantifications (right) are shown. The figures are based on five independent experiments. n=800 for each condition, and error bars are sem. Significance was measured using one-way ANOVA. ns, not significant.

[0246] Figure 15 Disruption of the circadian rhythm disrupts core cellular maintenance pathways in a dream-dependent manner. (af) Figure 5 The animals were treated and analyzed. Box plots showed the relative expression of proteins involved in xenobiotic metabolism (a), fatty acid β-oxidation (b), oxidative phosphorylation (c), splicing (d), ribosome biosynthesis (e), and translation factors (f). Individual proteins were shown as dots, and the fold change of the median for each group was shown as a horizontal line; the upper and lower limits of the box plots indicated the first and third quartiles, and the whisker lines extended from each box boundary to 1.5 times the interquartile range. Each condition used n=800 nematodes, and four independent populations were measured for each experimental cohort. Significance was measured using the Wilcoxon rank- and two-tailed test. p < 0.05; p < 0.01; p < 0.001; p < 0.0001.

[0247] Figure 16 The same basic cellular processes in *C. elegans* and mice are regulated by the circadian rhythm. (ae) Transcriptome data are from Whittaker et al. (2023), categorized by... Figure 6 The analysis described in sections ab. (a) illustrates the in vivo experimental design. Box plots are provided showing the relative expression of ribosome (b), spliceosome (c), lysosome (d), and ECM (e) genes. Individual genes are shown as dots, and the fold change of the median for each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. n = 6–7 mice per condition. Significance was measured using the Wilcoxon test. p < 0.05; p < 0.01; p < 0.001; p < 0.0001.

[0248] Figure 17 The same basic cellular processes in *C. elegans* and human cells are regulated by the biological clock. (a) Processing and analysis of human RPE cells according to... Figure 6As described above. qPCR analysis of the P21 gene is shown. Significance was measured using one-way ANOVA within each group, followed by Sidak multiple comparison test. p<0.0001. (be) Processing and analysis of human RPE cells as follows: Figure 6 d- Figure 6 e. Box plots are provided showing the relative expression (Log2FC) of spliceosome (b), ribosome (c), ECM (d), and ribosome biosynthesis (e) genes. Individual genes are shown as dots, and the fold change of the median for each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. Significance was measured using the Wilcoxon assay. p<0.05; p<0.01; p<0.001; p < 0.0001. n = 5 independent cultures per condition. (f) Age-synchronized Q40-YFP (unc-54p::Q40::YFP) nematode populations were treated with ev or lin-42 RNAi in plates containing 0.1% DMSO (vector) or halamine (40 μM). Paralysis scores were assessed daily, with n = 140 nematodes per condition. Figures represent at least three independent experiments. Significance was measured using the log-rank Mantel-Cox test. p<0.0001.

[0249] Figure 18 DREAM expression is reduced during sleep, and sleep deprivation prevents this reduction. The circadian rhythm and sleep disturbances disrupt fundamental cellular processes in a DRM / DREAM-dependent manner. (ab) Transcriptome data from mouse cortex were analyzed, with samples collected during both sleep and wakefulness phases (experimental protocol). Figure 23 The complete Log2CPM data were filtered to visualize DREAM subunit expression, depicting Lin37 and E2f4 under normal sleep and wakefulness (left panel) and under sleep deprivation (SD) (right panel). Expression values ​​at ZT3 and ZT6 (NSD and SD) were normalized to ZT0, and the expression dynamics of both Lin37 and E2f4 were observed by comparing the NSD and SD cohorts. Significance was measured by unpaired t-tests for each condition (n=3–5), and mean and SEM values ​​are given. -p<0.005; -p<0.01; -p<0.001; -p<0.0001; ns, not significant. (c) The same Log2CPM values ​​were analyzed for another DREAM syncytial subunit, Lin9, as shown in the figure above. Wild-type mice were treated with melatonin or the vector for 2 weeks (experimental protocol). Figure 24 Hippocampus samples were collected during both light and dark periods. Lin9 gene expression was analyzed by RT-qPCR (see figure below). Data showed that... The method calculates the fold change in mRNA expression. Significance was measured by two-way ANOVA with Bonferroni correction for multiple comparisons. For each condition, n = 6–9, and mean and SEM values ​​are given. -p<0.005; -p<0.01; -p<0.001; -p<0.0001; ns, not significant. (d) Box plots show the relative expression of genes belonging to mitochondrial, OXPHOS, and ribosome biosynthesis (d) (SD(ZT3)-NSD(ZT3) and SD(ZT6)-NSD(ZT6) comparisons). Individual genes are shown as dots, and the median fold change for each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. In each case, n=3–5 mice were measured for each condition. Significance in each box plot was assessed by the Mann-Whitney Wilcoxon rank-sum test, and the fold change between the two groups was compared using the Wilcoxon test. Two-tailed p-values ​​were calculated in all cases. -p<0.005; -p<0.01; -p<0.001; -p<0.0001. (e) Human retinal pigment epithelium (RPE) cells were treated for 48 h with either control siRNA (siC1, 10 nM) or siRNA targeting PER1 (siPER1, 10 nM), followed by co-treatment with halamine (Har, 10 μM) or DMSO (vector control) for 20 h. Expression analysis of the PER1 and CLOCK genes was performed by mRNA sequencing. The figure shows the transcript count per million (TPM). Significance was measured using one-way ANOVA within each group, and the two-tailed p-value was calculated using the Sidak multiple comparison test. -p<0.005; -p<0.01; -p<0.001; -p<0.0001; ns, not significant. Box plots (f) are provided, showing the relative expression of genes involved in mitochondrial, OXPHOS, and ribosome biosynthesis (PER1 DMSO vs. ctrl DMSO, PER1 halamine vs. ctrl DMSO) (f). Individual genes are shown as dots, and the median fold change for each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. For each case, n = 3–5 mice were measured per condition. Significance in each box plot was assessed using the Mann-Whitney Wilcoxon rank-sum test, and the Wilcoxon test was used to compare fold changes between groups. Two-tailed p-values ​​were calculated in all cases. -p<0.005; -p<0.01; -p<0.001; -p<0.0001.

[0250] Figure 19 High expression of DREAM protects DNA from wakefulness-related damage, while low expression of DREAM promotes repair during sleep. (a) Human retinal pigment epithelial (RPE) cells were treated for 48 h with control siRNA (siC1, 10 nM) or siRNA targeting PER1 (siPER1, 10 nM), and co-treated for 20 h with halamine (Har, 10 μM) or DMSO (vector control). The expression of the CDKN1A(a) gene was analyzed in the same cell extracts by RT-qPCR (left panel) and mRNA sequencing (right panel). Data showed that the expression of CDKN1A(a) was obtained by RT-qPCR (left panel) and mRNA sequencing (right panel). Methods and fold changes in mRNA expression calculated using transcripts per million (TPM). Data in the left figure represent 5 independent trials, while the right figure represents combinations of 4–5 independent samples. Significance was measured individually within each group using one-way ANOVA, and two-tailed p-values ​​were calculated using the Sidak multiple comparison test. -p<0.005; -p<0.01; -p<0.001; -p<0.0001; ns, not significant. (b) Heatmap depicts the expression of P53 target genes. Color coding refers to Z-score values. (c) 4 h after inoculation in larval stage 1 (L1), UV-B (400 and 500 mJ / cm) was used. 2 Wild-type animals were treated, and larval development was assessed. The percentages of L1-L2, L3, and L4 stages were counted 44 hours after UV treatment. Significance was measured using an unpaired t-test. Mean and SEM values ​​are given for each condition (n = 40–60). -p<0.005; -p<0.01; -p<0.001; -p<0.0001; ns, not significant. (df) Reanalysis of transcriptomic data from the cortex of C57BL / 6J mice, samples were collected during both sleep and wakefulness phases. Mediators of DNA damage and repair responses were analyzed under normal sleep and normal sleep versus sleep deprivation (SD). The expression kinetics of Cdkn1a(d), Gadd45b(e), and Ddb2(f) are shown in the figure. Significance was measured using unpaired t-tests for each condition n=3–5, and mean and SEM values ​​are given. -p<0.005; -p<0.01; -p<0.001; -p<0.0001; ns, not significant. (g) A model was proposed to depict the role of DREAM in mediating cell damage downstream of circadian rhythm disruption.

[0251] Figure 20 : Circadian rhythm disruption increases histone expression in a DRM / DREAM-dependent manner. (a) Using histone H2AZ-specific antibodies, according to Figure 4 a- Figure 4 b describes the treatment and analysis of nematodes. A representative whole-gel scan (left) and corresponding quantification (right) are shown. This figure summarizes the results of five independent experiments, n=800 for each condition, with error bars representing SEM. Significance was measured using one-way ANOVA, and two-tailed p-values ​​were calculated. -p<0.0001; ns, not significant. (b) Nematodes were treated as in (a), and the mRNA expression levels of his-24(H1), hil-2(H1), and his-29(H2) were assessed by RT-qPCR. n=250 nematodes per condition. The figure represents three independent experiments. Error bars are from SEM; Methods: fold change in mRNA was calculated. Significance was assessed using one-way ANOVA within each group, and the Sidak multiple comparison test was applied to calculate the two-tailed p-value. -p<0.005; -p<0.01; -p<0.001; -p<0.0001; ns, not significant.

[0252] Figure 21 Circadian rhythm disruption interferes with basic cellular processes in a DRM / DREAM-dependent manner. (al) Animal handling and analysis Figure 4As described by hm. Box plots show the relative expression of proteins involved in aldehyde dehydrogenase (b), tryptophan metabolism (c), short-chain dehydrogenase (d), fatty acid β-oxidation (e), TCA cycle (f), OXPHOS (g), spliceosome (h), mitochondrial ribosomes (i), cytoplasmic ribosomes (j), and translation factors (l). Individual proteins are shown as dots, and the fold change of the median for each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. n=800 for each condition, and four independent populations were measured. Significance in each box plot was assessed by the Mann-Whitney Wilcoxon rank-sum test, and the Wilcoxon test was used for comparisons of fold changes between two groups. Two-tailed p-values ​​were calculated in all cases. -p<0.005; -p<0.01; -p<0.001; -p<0.0001.

[0253] Figure 22 The DREAM-dependent pathway is a functional driver of the negative effects of circadian rhythm disruption. (ad) From L1 stage, age-synchronized unc-54p::Q40::YFP animals were fed EV or lin-42 RNAi and combined with RNAi targeting specific genes associated with the identified DREAM-dependent pathway—translation initiation and ribosome biosynthesis—daily for paralysis scoring. Co-targeting with ife-2(a), ife-3(b), xpd-1(c), and rpoa-2(d) is shown. n=140 nematodes per condition, and the figure represents at least three independent experiments. Significance was measured by log-rank Mantel-Cox test, and two-tailed p-values ​​were calculated. -p<0.0001.

[0254] Figure 23 DREAM expression changes during the natural sleep-wake cycle. (a) describes the experimental design of Jan et al. (2024). (bc) Analysis of transcriptome data is presented below. Figure 18 a, 18c and Figure 19The expression of the DREAM subunits Rbbp7, Lin54, and Lin37 was visualized during normal sleep and wakefulness (b). The expression kinetics of the non-circulating gene Bex2 at each ZT were also depicted compared to expression at ZT0. The same Log2CPM data values ​​(NSD and SD) at ZT3 and ZT6 were normalized to ZT0, and the expression kinetics of Rbbp44 were shown by comparing the NSD and SD cohorts (left panel). Rbbp4 expression was also measured during normal sleep and wakefulness, as in b. Significance was measured by an unpaired t-test. Mean and SEM values ​​are given for n=3–5 for each condition. -p<0.005; -p<0.01; -p<0.001; -p<0.0001; ns, not significant.

[0255] Figure 24 The DREAM subunits follow circadian rhythm oscillations and their expression is increased by melatonin treatment. (a) Experimental design. (b) Wild-type mice were treated with melatonin (100 mg / kg) or a vector for 2 weeks. Hippocampal samples were collected during light (sleep) and dark (sleep) phases and processed for RT-qPCR analysis. The expression of DREAM subunits Rbbp4, Rbbp7, Lin54, Lin37, Lin52, and the non-circulating gene Bex2 was analyzed by RT-qPCR. Data showed that the expression of these subunits was increased by melatonin treatment. The method calculates the fold change in mRNA expression. Significance was measured by multiple comparisons using two-way ANOVA with Bonferroni correction. For each condition, n = 6–9, and mean and SEM values ​​are given. -p<0.005; -p<0.01; -p<0.001; -p<0.0001; ns, not significant.

[0256] Figure 25 The same basic cellular processes are altered by circadian rhythm disruption in mice and nematodes. Transcriptomic data are arranged according to... Figure 18Analysis was performed using d. (ah) provides box plots showing the relative expression of genes involved in glycolysis (a), the TCA cycle (b), mitochondrial β-oxidation (c), OXPHOS (d), ribosomes (e), spliceosomes (f), collagen (g), and protein homeostasis (h). Individual genes are shown as dots, and the median fold change for each group is shown as a horizontal line; the upper and lower limits of the box plots indicate the first and third quartiles, and the whisker lines extend from each box boundary to 1.5 times the interquartile range. n = 6–7 mice per condition. Significance in each box plot was assessed by the Mann-Whitney Wilcoxon rank-sum test, and the Wilcoxon test was used for comparisons of fold changes between two groups. Two-tailed p-values ​​were calculated in all cases. -p<0.005; -p<0.01; -p<0.001; -p<0.0001.

[0257] Figure 26 The same basic cellular activities are regulated through the biological clock / DREAM axis in nematodes and human cells. (af) Processing and analysis of human RPE cells according to Figure 19 As described in section a, n=4-5 independent cultures were used for each condition. Expression analysis of the PER1, CLOCK, and CRY2(a) genes is shown. RT-qPCR (bottom panel) and transcriptome measurements (top panel) are provided, as shown below. Figure 19 As described in section a. Significance was measured using one-way ANOVA within each group, and the two-tailed p-value was calculated using the Sidak multiple comparison test. -p<0.0001. (bc) Transcriptomics data as follows Figure 18 Analysis was performed. Box plots were provided showing the relative expression of ribosome (b) and RNA polymerase (transcription) (c) genes. Individual genes were shown as dots, and the fold change of the median for each group was shown as a horizontal line; the upper and lower limits of the box plots indicated the first and third quartiles, and the whisker lines extended from each box boundary to 1.5 times the interquartile range. In each box plot, significance was assessed by the Mann-Whitney Wilcoxon rank-sum test, and the Wilcoxon test was used for comparisons of fold changes between two groups. Two-tailed p-values ​​were calculated in all cases. -p<0.005; -p<0.01; -p<0.001; -p<0.0001. (g) unc-54p::Q40::YFP nematodes were treated with EV or lin-42 RNAi in plates containing 0.1% DMSO (vector) or halamine (Har, 40 μM and 60 μM). Paralysis was scored daily. n=140 nematodes per condition. The figure represents three independent experiments. Significance was measured by log-rank Mantel-Cox test, and two-tailed p-values ​​were calculated. -p<0.0001.

[0258] Figure 27 Experimental design for evaluating the developmental timing of UV-B and lin-42 RNAi treatments. (a) and Figure 19 c corresponds to the experimental design. From the L1 stage, age-synchronized wild-type nematodes were treated with ev or lin-42 RNAi. Four hours after inoculation, they were treated with UV-B 400 or 500 mJ / cm². 2 Animal treatment. After treatment, nematodes were transferred to fresh EV or lin-42 RNAi plates to ensure continued exposure to the treatment. Forty-four hours after UV treatment, different larval stages were counted as L1-L2, L3, or L4 stages.

[0259] Figure 28 The role of DREAM in normal sleep and sleep deprivation. A model of how DREAM mediates the effects of sleep and wakefulness at the cellular level: During the wakefulness phase, DREAM activity is high, leading to chromatin compression, shielding DNA from damage, and suppressing gene expression required for detoxification and repair (A). Conversely, during sleep, DREAM activity decreases, and repair becomes available, including DNA repair. Sleep deprivation keeps DREAM activity high and suppresses repair, leading to a gradual decline in health (B). As shown in C, this negative effect can be prevented by using DREAM inhibitors that mimic or replicate the benefits of sleep. Therefore, this paper proposes that DREAM inhibitors are mimics for restoring sleep.

[0260] Example

[0261] The present invention is illustrated by the following disclosed embodiments. These embodiments provide technical support and a more detailed description of potential preferred, non-limiting implementations of the invention.

[0262] To demonstrate the functional and beneficial properties of the DREAM complex inhibitor described herein, the following examples should be considered:

[0263] - Moderate disruption of the circadian rhythm leads to a combination of tissue dysfunction and physiological stress.

[0264] - Disruption of the biological clock causes changes in the expression of chromatin components and DNA metabolism regulators.

[0265] - DREAM complex subunits and specific histone variants mediate tissue dysfunction in response to circadian rhythm disruption.

[0266] - Disruption of the biological clock inactivates adaptive stress responses and cell maintenance mechanisms in a dream- and histone-dependent manner.

[0267] - The DREAM-circadian rhythm interaction is evolutionarily conserved, and DREAM inhibits the restoration of homeostasis under circadian rhythm disruption and sleep interruption.

[0268] - Circadian rhythm disruption interferes with basic cellular activities in a DRM / DREAM-dependent manner.

[0269] - DREAM expression fluctuates between sleep and wakefulness in mammals, affecting basic homeostasis mechanisms.

[0270] High DREAM abundance results in DNA shielding during awakening at the cost of poor repair.

[0271] Implementation Examples Overview

[0272] Circadian rhythm disruption and sleep deprivation have devastating effects on multiple aspects of organismal health, from cognition to immune responses. This multifaceted impact can be partly explained by the need for fluid flow and molecular damage clearance in the brain during sleep, with the CNS mediating excessive organismal responses. A similar hypothesis is that sleep deprivation and circadian rhythm disruption affect master regulatory pathways controlling fundamental aspects of cellular physiology, thereby modulating the function of multiple cell types. The discovery of this core regulatory mechanism could represent an important step in developing interventions to restore homeostasis under circadian rhythm dysregulation and sleep deprivation. Here, this invention uses RNAi targeting the PER homolog lin-42 of *C. elegans* to induce persistent but moderate circadian rhythm disruption as a mimicry of lifestyle-induced circadian rhythm irregularities. This invention then applies this intervention to a *C. elegans* model of protein homeostasis stress and accelerated aging, followed by proteomics and molecular and functional testing. Furthermore, transcriptomic analysis was performed in mice with altered circadian rhythm behavior and in human cells with PER1 knockdown. These tests confirmed the link between circadian rhythm disruption and impaired homeostasis, particularly under conditions of additional cellular stress. This invention identified a large number of conserved and universally expressed molecular targets altered by circadian rhythm disruption, consistent with various health losses associated with circadian rhythm impairment. Finally, this invention reveals the DREAM complex as a core regulator of circadian rhythm-altering pathway activity and finds that the pharmacological and gene-repressive effects of DREAM alleviate molecular, cellular, tissue, and organismal dysfunctions caused by circadian rhythm disruption.

[0273] Example 1: Moderate disruption of the circadian rhythm clock leads to a combination of tissue dysfunction and physiological stress.

[0274] In *C. elegans*, this invention employs RNAi-mediated gene inactivation to knock down the core component of the circadian clock PER / lin-42. Previous studies have found that complete loss of lin-42 function leads to accelerated aging and premature death in nematodes. This result is consistent with recent findings of shortened lifespan and increased cellular stress proliferation in sleep-deprived *D. melanogaster* and mice. Unlike complete clock inactivation, the primary objective of this invention is to test the biological effects of partial clock disruption, conceptually equivalent to the incidental loss of circadian rhythm function due to sleep deprivation in mammals. Therefore, we chose an RNAi-mediated gene knockdown approach instead of using loss-of-function mutants. Thus, this invention demonstrates that lin-42 RNAi treatment from the L1 larval stage indeed disrupts the clock-dependent molting process in *C. elegans*. Figure 1 B and Figure 7 However, it does not lead to a subsequent shortened lifespan, consistent with the hypothetical moderate circadian rhythm impairment. Figure 1 C). Because previous studies in mice and Drosophila melanogaster have shown that sleep deprivation induces cellular stress, this invention next investigates whether moderate circadian rhythm disruption, in conjunction with other stress factors, leads to tissue dysfunction. In these experiments, this invention uses a transgenic protein aggregation model to subject the body wall muscles of *C. elegans* to endogenous physiological stress. Specifically, this invention uses nematodes expressing human amyloid β-peptide and polyglutamine-conjugated YFP protein (Q40-YFP) in muscle tissue. Interestingly, inactivation of lin-42 from the L1 larval stage significantly enhanced muscle dysfunction and generalized paralysis in both transgenic models. Figure 1 D and Figure 8 A). A similar trend was also observed in transgenic animals treated with lin-42 RNAi from the L4 larval stage before adulthood. Figure 8 B and Figure 8 C). These results indicate that adult-onset circadian rhythm disruption is sufficient to cause a decline in tissue homeostasis, along with stress. Furthermore, developmental circadian rhythm distortion has a stronger impact on lifelong tissue function, consistent with the prevalence and highest importance of sleep and coordinated rest during species growth and development. Finally, this invention found that RNAi inactivation of the downstream circadian rhythm gene lin-14 leads to increased paralysis in Q40-YFP animals, similar to knockdown of the upstream master regulator lin-42. Figure 8(D) This result validates that circadian rhythm disruption is a mechanism linking lin-42 inactivation to tissue dysfunction under stress. In summary, this invention finds that moderate but persistent loss of circadian rhythm integrity can combine with intrinsic cellular physiological stress factors, triggering organ dysfunction.

[0275] Example 2: Disruption of the biological clock causes changes in the expression of chromatin components and DNA metabolism regulators.

[0276] To elucidate the molecular mechanisms linking circadian rhythm disruption with organ failure, this invention performed unbiased proteomics analysis on animals exposed to lin-42 RNAi from the L1 and L4 stages. Figure 2 A and Figure 10 A) A total of 5,358 proteomes were analyzed. Principal component analysis (PCA) of the whole proteome showed a clear separation between the lin-42 RNAi-treated group and the control cohort exposed to the empty vector (EV) in the L1 treatment setting. Figure 2 B). Subsequently, gene set enrichment analysis was performed on differentially expressed proteins between L1 stage animals treated with lin-42 and control RNAi-treated animals, revealing strong enrichment of gene ontology terms related to chromatin dynamics and DNA metabolism (B). Figure 2 C). Meanwhile, unbiased volcano plot analysis of the same group of proteins revealed that different histone variants were the most clearly regulated groups (C). Figure 2 D). In summary, these two independent and unbiased whole-proteome analyses suggest that chromatin dynamics and composition are involved in the effects of lin-42 knockdown on cells. To identify the hypothetical core regulators linking lin-42 inactivation to chromatin changes, this invention next computationally extracted along... Figure 2 In B, PC1 was the protein that contributed most to the separation between the lin-42 RNAi and EV control proteomes, and these proteins were analyzed using the STRING interaction assessment tool. Interestingly, a clear single hub with most interactions was detected, including components and interacting factors of the DREAM complex involved in chromatin remodeling and gene expression regulation during the cell cycle and quiescent phase. Figure 9 and Figure 2 E). Notably, although there was a lack of clear separation between the entire proteome of the control and L4-stage treated animals in PCA (E). Figure 10 B), but in animals exposed to lin-42 RNAi at the L1 and L4 stages, both the DREAM subunit and its interacting factors were upregulated (B). Figure 2 E). Furthermore, in both the L1 and L4 treatment cohorts, a significant number of DREM-related pivot genes were identified unbiasedly among the proteins that contributed most to PC1 separation. Figure 9 and Figure 10 C). These results are consistent with the stronger impact of L1-induced circadian rhythm disruption on organismal health. Figure 1 D, Figure 8 These findings reinforce the recognition of the DREAM complex as a key target of circadian rhythm disruption regardless of age, and demonstrate that the molecular effects of circadian rhythm damage are similar across development and adulthood, albeit with varying intensities.

[0277] Example 3: DREAM complex subunits and specific histone variants mediate tissue dysfunction in response to circadian rhythm clock disruption.

[0278] To verify the involvement of the DREAM complex in tissue dysfunction caused by circadian rhythm disruption, this invention performed co-knockdown of lin-42 and a single DREAM subunit via dual RNAi exposure in Q40-YFP animals. In these tests, this invention focused on RNAi treatment starting from the L1 larval stage, as a stronger functional response was observed in previous studies in this setting. Figure 3 A). Notably, this invention found that RNAi-mediated depletion of the DREAM subunits lin-9, lin-54, and lin-53 reversed muscle paralysis induced by lin-42 gene knockdown in Q40-YFP animals, with lin-53 inactivation showing the strongest reversal effect. Figure 3 (BD). It is worth noting that the dual RNAi method of the present invention can effectively suppress two genes simultaneously, as shown in the example of co-suppression of lin-42 / lin-53 (BD). Figure 11 A). Meanwhile, knockdown of other typical DREAM subunits (e.g., lin-37, lin-52, and lin-35) did not affect the tissue dysfunction induced by lin-42 RNAi. Figure 11 (BD) indicates that different DREAM subunits selectively participate in the effects of the circadian rhythm clock. Furthermore, this invention demonstrates that RNAi-mediated depletion of his-41 / H2BC and htz-1 / H2A.Z2 histone variants is reflected in the STRING analysis of this invention ( Figure 9 ) and was found in the literature to be a prominent DREAM interactor, which also rescued the enhanced paralysis of Q40-YFP nematodes treated with lin-42 RNAi ( Figure 3 E and Figure 11E). In summary, the results of this invention suggest that enhanced activity of specific DREAM complex variants may mediate the negative impact of circadian rhythm inactivation on tissue homeostasis under stress. The data from this invention also suggest that increased abundance of specific DREAM interactor histones may play a role in this process by potentially enhancing chromatin compression and broadly inactivating gene expression, including genes involved in adaptive stress responses. Consistent with the hypothesis of broad gene expression inactivation, YFP transgene expression gradually decreased with age in Q40-YFP animals exposed to lin-42 RNAi. Figure 12 A and B) showed a reversal of this effect in nematodes co-treated with anti-DREAM lin-53 RNAi. Simultaneously, in animals exposed to lin-42 RNAi, relative YFP aggregation gradually increased with age, and this distortion was also reversed by co-knockdown of the lin-53 DREAM subunit, consistent with the ability to resist protein homeostasis stress, which was impaired and repaired, respectively. Figure 3 F).

[0279] Example 4: Circadian rhythm disruption inactivates adaptive stress response in a DREAM- and histone-dependent manner.

[0280] The present invention then uses Western blot analysis to test whether the increased abundance of histones in lin-42 KD animals is indeed regulated by DREAM. Consistent with this hypothesis, and with... Figure 2 Consistent with the proteomics data shown in D, lin-42 RNAi treatment resulted in increased expression of all tested histone variants, and in all cases, this increase was reversed by co-knockdown of lin-53. Figure 4 AE, Figure 13 and Figure 14 Notably, differential expression was achieved at the mRNA level among control, lin-42 KD, and lin-42 / lin-53 co-knockdown. Figure 4 F) indicates that DREAM can act as a transcription inducer for these genes, consistent with its previously reported dual repressor-activator role in gene expression.

[0281] Subsequently, this invention investigated which molecular pathways become dysregulated in lin-42 KD nematodes and recover in lin-42 / his-41 and lin-42 / lin-53 co-knockdown animals, representing molecular targets affected by circadian rhythm disruption in a DREAM-dependent and histone-dependent manner. Comparative proteomic analyses were performed on three indicated cohorts and control samples treated with EV RNAi. Figure 5A) This invention surprisingly reveals the simultaneous alteration of multiple mechanisms involved in cellular basal homeostasis and adaptive stress responses. For example, the invention discovers detoxification mechanisms, such as antioxidant responses ( Figure 5 B) and xenobiotic metabolism ( Figure 15 A) is inhibited by lin-42 KD and reactivated by co-knockdown of lin-42 / his-41 and lin-42 / lin-53, and is involved in adaptive metabolic mechanisms such as glycolysis, fatty acid β-oxidation, and OXPHOS. Figure 5 C and Figure 15 Similar kinetics were observed in B and C. Furthermore, considerable changes were also observed in the extracellular matrix components and lysosomal acidification mediators essential for aggregate degradation and autophagy flux. Figure 5 (D and E). Notably, the dysregulation of antioxidant responses by lin-42 RNAi is consistent with oxidative stress being a major cause of early death in sleep-deprived fruit flies and mice, while lysosomal dysfunction is consistent with the impaired ability of lin-42 KD nematodes to clear Q40-YFP aggregates, such as... Figure 3 As shown in F. Furthermore, distorted expression of ECM components (including collagen) is associated with loss of tissue integrity and transspecies aging. The findings of this invention are further supported by recent reports of downregulation of detoxification and metabolic mechanisms in the livers of Per1 / Per2 double knockout mice. Interestingly, in the experimental system of this invention, fundamental cellular mechanisms (e.g., Pol II transcription and splicing) were affected differently, showing upregulation after circadian rhythm distortion and re-passivation via co-knockdown of lin-42 / his-41 and lin-42 / lin-53. Figure 5 F and 15D). Notably, changes in PolII transcription and splicing have recently been revealed as markers and functional drivers of normal aging, and lin-42 KD disruption of both processes provides another hypothesized DREM-dependent link between circadian rhythm disruption and premature tissue dysfunction. Finally, this invention observed a significant downregulation of ribosomes, ribosomal biogenesis, and translational proteins by lin-42 / lin-53 co-knockdown (F and 15D). Figure 5 G, Figure 15 E and F) indicate that DREAM is associated with alterations in these activities during circadian rhythm disruption. Notably, reduced translation and ribosome biosynthesis are known adaptations to stress in *C. elegans* and yeast, as well as contributions to the longevity benefits of rapamycin and metformin. Against this background, the data of this invention suggest that adaptive changes in translation are limited by lin-42 KD in a DREAM-dependent manner. It is noteworthy that by... Figure 4 , Figure 5 and Figure 15The list of circadian clock targets described in this invention was compared with the previously published DREAM ChiP-Seq Caenorhabditis elegans dataset. This invention identified that only a few of these genes are located near genomic DREAM binding sites, thereby enhancing their regulatory model through DREAM-dependent histone and chromatin changes. In summary, this invention finds that circadian clock disruption leads to multiple alterations in basic and adaptive cellular functions, which are restored by common KD of DREAM, and in most cases, common KD of histones is also restored.

[0282] Example 5: The DREAM-circadian clock interaction is evolutionarily conserved, and DREAM inhibits the restoration of homeostasis under circadian clock disruption.

[0283] To test whether circadian rhythm disruption also interferes with adaptive mechanisms in mammalian tissues, this invention consulted publicly available transcriptomic data from mice affected by circadian rhythm disruption. Interestingly, recent studies have explored circadian rhythm disruption and its mitigation by time-restricted feeding in the brains of transgenic mice expressing AD-associated variants of human amyloid precursor protein, conceptually similar to the present invention's experiment combining circadian rhythm disruption with protein homeostasis stress in *C. elegans*. By reanalyzing the transcriptomic data from animals with disrupted and restored circadian rhythms presented in this study, this invention surprisingly found that similar pathways were altered and restored in a circadian rhythm-dependent manner in both the mouse brain and *C. elegans*. For example, the expression of mitochondrial components and the OXPHOS gene in mice was reduced by circadian rhythm disruption and restored upon circadian rhythm restoration. Figure 16 A, Figure 6 A and B). Similarly, ribosome and spliceosome genes, as well as genes encoding lysosomes and ECM components, exhibit opposite expression dynamics in the brain during circadian rhythm disruption and recovery. Figure 16 (B, C, D, and E). These analyses suggest that the same basic cellular processes in nematodes and mammals are affected by disruption of circadian rhythms.

[0284] Next, this invention turned to human cell culture systems to test whether PER inactivation was indeed sufficient to disrupt the circadian rhythm clock, and whether inhibition of the DREAM complex could reset the biological clock in the context of PER deficiency. Here, this invention used siRNA treatment to knock down PER1 in human RPE cells, and we used the pharmacological inhibitor harmine to inhibit the DREAM complex. In this case, PER1 selection was driven by recent reports indicating that it is a key circulating PER paraline across mammalian tissues. We found that PER1 knockdown (KD) did indeed disrupt the biological clock, as seen by altering the expression of core circadian clock genes BMAL1, CLOCK, and CRY2. Importantly, harmine failed to restore the biological clock in PER1-deficient cells. Figure 6 C), but caused the expected changes in the cell cycle, as seen by altering the expression of the P21 / CDKN1A gene ( Figure 17 A) demonstrates the effectiveness of halamine as a DREAM inhibitor. In summary, these results confirm the hypothesis of this invention that the restorative effect of DREAM inhibition on circadian rhythm disordered intracellular homeostasis is independent of circadian rhythm restoration and may occur downstream of the damaged circadian rhythm.

[0285] Subsequently, this invention turned to a more in-depth high-throughput transcriptomic analysis of PER1- and DREAM-inactivated RPE cells to identify key homeostatic mechanisms regulated by the circadian rhythm in a DREAM-dependent manner in human systems. Significantly, the findings of this invention are similar to observations obtained in nematode and mouse models. For example, this invention found that mitochondrial and OXPHOS genes in human cells are also downregulated by circadian rhythm disruption and restored by co-repression of DREAM (…). Figure 6 D and E) revealed mitochondrial function as the most consistent circadian rhythm regulator across species and cell types. Furthermore, spliceosome genes, ribosome genes, and genes encoding ECM components and ribosome biosynthesis factors exhibited contrasting expression kinetics between cells with circadian rhythm dysregulation and DREAM co-suppression. Figure 17 This is again similar to the findings in nematodes and mice, and clearly demonstrates that the same core cellular processes are altered across species in a dream-dependent manner by circadian rhythm disruption.

[0286] Because the DREAM inhibitor halamine is bioavailable in vivo, this invention ultimately tested whether halamine treatment could alleviate the health decline induced by lin-42 KD in *C. elegans*, comparable to genetic DREAM inactivation. This invention found that co-treatment of Q40-YFP animals with lin-42 RNAi and halamine did indeed reverse paralysis induced by circadian rhythm disruption in this model. Figure 6 F and Figure 17F). These results demonstrate that the pharmacological inhibition of DREAM is an effective in vivo tool for rescuing the health of organisms affected by persistent circadian rhythm dysfunction.

[0287] In summary, these examples reveal the conserved capacity of circadian rhythm disruption to multiple fundamental and adaptive cellular pathways and establish the DREAM complex as a key mediator of these negative effects. These findings reveal DREAM as a novel intervention target for repairing cellular function in uncoordinated organismal quiescence.

[0288] Example 6: Circadian rhythm disruption interferes with basic cellular activities in a DRM / DREAM-dependent manner.

[0289] Next, this invention uses Western blot analysis to test whether the increase in histone abundance in lin-42 KD animals is indeed regulated by DRM / DREAM. Figure 2 Consistent with the proteomics data shown in D, histone abundance increased after lin-42 RNAi exposure, and in all cases, this increase was reversed by additional lin-53 knockdown. Figure 4 AE, Figure 13 , Figure 14 and Figure 20 A). Notably, for the H1 and H2B histone variants, the strongest differences in protein abundance were observed among the control, lin-42 KD, and lin-42 / lin-53 co-knockdown cohorts, which are known to regulate and enhance chromatin compression (A). Figure 4 A and B, Figure 13 AB), and their differential expression was also observed at the mRNA level. Figure 20 B). These results indicate that DREAM / DRM promotes higher histone levels (B). Figure 4 P) can lead to enhanced chromatin compression.

[0290] Subsequently, this invention investigated which molecular pathways were downregulated in lin-42 knockdown nematodes and restored in lin-42 / lin-53 double knockdown animals, representing molecular targets affected by circadian rhythm disruption in a DRM / DREM-dependent manner. lin-53 showed the strongest ability to reverse the negative health effects of lin-42 KD. Figure 3 D), and because lin-53 / RBBP4 is reported to mediate interactions between DRM / DREAM and histones, lin-53 was selected for inactivation. Comparative proteomic analysis of lin-42 KD, lin-42 / lin-53 co-knockdown, and EV RNAi control exposure cohorts ( Figure 21A) Significantly revealed simultaneous alterations in multiple mechanisms involved in basal cellular homeostasis and adaptive stress responses. For example, detoxification mechanisms such as xenobiotic metabolism were discovered. Figure 4 H) and pathways involved in antioxidant reactions ( Figure 4 I and Figure 21 BD) is inhibited by lin-42KD and restored by co-knockdown of lin-53. Antioxidant responses are characterized by the simultaneous dysregulation of four distinct metabolic entities involved in counteracting oxidative stress: the aldehyde dehydrogenase protein family (…). Figure 21 B); glutathione metabolism ( Figure 4 I); Tryptophan catabolism via the kynurenine pathway ( Figure 21 C), in which intermediates are known to have ROS scavenging activity; and a family of short-chain dehydrogenase proteins associated with xenobiotics and antioxidant defense responses. Figure 21 D). The significant enrichment of antioxidant mechanisms in DRM-dependent circadian rhythm targets is highly consistent with oxidative stress being a major cause of early death in sleep-deprived fruit flies and mice. Furthermore, previous findings that xenobiotic detoxification responses are downregulated in the livers of Per1 / Per2 double knockout mice with circadian rhythm impairment are consistent with the current findings of this invention. In addition, metabolic plasticity pathways such as glycolysis and fatty acid β-oxidation are attenuated by lin-42 KD and restored by co-inactivation of lin-53. Figure 4 J and Figure 21 E), while the key bioenergetic activities of mitochondria—the TCA cycle and OXPHOS—are inhibited by lin-42 inactivation and partially restored by lin-53 RNAi co-treatment. Figure 21 F and G). Finally, the expression of ECM component collagen was downregulated in animals exposed to lin-42 RNAi and restored by lin-42 / lin-53 co-knockdown (F and G). Figure 4Notably, decreased metabolic plasticity and reduced collagen expression are both associated with organ dysfunction during aging, thus establishing a link between circadian rhythm disruption and accelerated tissue damage. Furthermore, impaired metabolic adaptation has previously been observed in the livers of Per1 / Per2 deficient mice. A cellular protective interaction between DRM / DREAM and circadian rhythm-regulated metabolic and detoxification activities can also be hypothesized, as tryptophan catabolism via the kynurenine pathway is one of the key cellular sources of nicotinamide adenine dinucleotide (NAD+), an energy carrier and enzymatic cofactor that plays a crucial role in mitochondrial maintenance and bioenergetic adaptation, and has the ability to delay metabolic aging. The strong enrichment of metabolic pathways in DRM-dependent circadian rhythm targets is consistent with a large body of human clinical data that designate circadian rhythm and sleep dysfunction as confounding factors in metabolic diseases such as diabetes, obesity, and cardiovascular disease.

[0291] Interestingly, in the experimental system of this invention, basal cell activities (e.g., Pol II transcription and splicing) were differentially affected, showing upregulation after circadian rhythm distortion and re-passivation through co-knockdown of lin-53. Figure 4 L and 21H). Notably, increased Pol II transcription rate and elevated splicing have recently been revealed as markers and functional drivers of physiological aging, and the interference of lin-42 KD on both processes provides yet another hypothesized lin-53 / DRM-dependent link between circadian rhythm disruption and premature death. Finally, this invention observed that mitochondrial ribosomes, cytoplasmic ribosomes, ribosome biosynthesis, and translation proteins were strongly downregulated via lin-42 / lin-53 co-knockdown (L and 21H). Figure 4 M and Figure 21 The results suggest that DRM / DREAM regulates these fundamental activities under conditions of circadian rhythm disruption. Since inhibition of translation is known to improve protein homeostasis by reducing protein folding stress, the strong inactivation of this process at multiple levels, compared to lin-42 inactivation alone, may contribute to the superior ability of lin-42 / lin-53 dual KD to reduce polyQ aggregates (IL). Figure 3 F).

[0292] To determine whether the identified pathways play a functional role in the negative health effects of circadian rhythm disruption, this invention focuses on pathways (including transcription initiation, ribosome biosynthesis, and translation) that are upregulated in a DREAM-dependent manner by circadian rhythm disruption. This invention uses specific RNAi treatments to inhibit these pathways in circadian rhythm-disrupted unc-54p::Q40::YFP transgenic animals. Notably, all RNAi treatments in this paper mimicked the recovery effect of DREAM inhibition. Figure 4 N, O and Figure 22 The results (AD) indicate that alterations in the DREAM-dependent pathway are a functional driver of negative changes caused by circadian rhythm disruption. Notably, by... Figure 4 , Figure 5 and Figure 15 The list of co-targets of the circadian clock and DREAM described in this invention was compared with the previously published DREAMChip-Seq Caenorhabditis elegans dataset. This invention identified that only a few of these genes are located near DREAM binding sites in the relevant genomes. This finding facilitates the indirect, chromatin- and histone-dependent regulation of these targets via DRM / DREAM, and is supported by all the functional and molecular data we have obtained to date. In summary, this invention finds that circadian clock disruption leads to multiple alterations in basic and adaptive cellular function, which may be reduced by co-knockdown of DRM / DREAM affecting histone levels and chromatin structure. Figure 4 The changes in individual adaptive pathways are not drastic, but rather combined, and these alterations have the potential to impair intracellular homeostasis and flexibility at multiple levels, thereby contributing to a decline in health associated with circadian rhythm and sleep impairment.

[0293] Example 7: DREAM expression fluctuates between sleep and wakefulness in mammals, affecting basic homeostasis mechanisms.

[0294] Next, this invention investigated whether DREAM expression changes during the natural sleep-wake cycle by analyzing the transcriptome of the cerebral cortex of young mice. Interestingly, this invention found that DREAM expression decreases during sleep and increases during wakefulness. Figure 18 A, C (above) and Figure 23 B, D (right figure)), while sleep deprivation alters the DREAM dynamics of the circadian rhythm ( Figure 18 B and Figure 23 D (left figure)), and prevented different DREAM subunits from being downregulated in time ( Figure 18 B), similar to observations in *Caenorhabditis elegans* with disrupted circadian rhythms. To verify whether DREAM expression is under the control of an active circadian rhythm, this invention performed qPCR on cDNA isolated from mouse brain tissue (hippocampus) after exposure to the sleep hormone melatonin (which increases the amplitude of circadian rhythm responses). This invention found that during the wakefulness phase of the circadian rhythm cycle, the expression of all tested DREAM subunits was enhanced by melatonin ( Figure 18 C (see image below) and Figure 24 B) indicates that the daily fluctuations in DREAM expression are part of the circadian rhythm response.

[0295] Next, this invention investigates whether impaired DREAM reduction under sleep deprivation affects basal cellular activity in mice, as it does in nematodes. Here, this invention compares the brain transcriptomes of sleep-deprived and fully-slept mice during a circadian rhythm window designated as natural deep sleep. Figure 23 A). It is worth noting that, in Figure 18 and Figure 23 In this study, the same transcriptomic data and time windows were used to assess the relative dynamics of DREAM between sleep and sleep deprivation. This invention found that mitochondrial content, OXPHOS, TCA cycle, mitochondrial fatty acid β-oxidation, glycolysis, and ECM components (collagen), protein homeostasis, ribosome biosynthesis and content, and splicing are all altered by sleep deprivation. Figure 5 D, Figure 18 D and Figure 25 AH), similar to the discovery in *C. elegans*, a worm with disrupted biological clock. It is noteworthy that the observed inhibition of protein homeostasis by sleep deprivation is known to be neuroprotective. Figure 25 H), while the upregulation of collagen ( Figure 25 G) may enhance brain tissue stiffness, thereby hindering the clearance of toxic entities from the brain's lymphoid tissue during sleep. In fact, dysregulation of brain collagen is associated with neurodegenerative diseases and dementia in humans. Simultaneously, downregulation of mitochondrial genes (including OXPHOS components) Figure 18 D) and increased expression of genes involved in glycolysis and the TCA cycle ( Figure 25 A and B) resemble the “wakeful” metabolic state characterized by carbohydrate utilization, contrasting with the typical oxidative metabolism of restoring sleep. Therefore, this invention demonstrates that DREAM expression is high during wakefulness and low during natural sleep. This invention also demonstrates that DREAM expression fails to decrease in a timely manner in sleep-deprived mice similar to circadian rhythm-disrupted nematodes, and that this failure is associated with dysregulation of similar repair and maintenance pathways in circadian rhythm-disrupted nematodes.

[0296] Next, this invention turned to human cell culture systems to test whether PER inactivation was indeed sufficient to disrupt the circadian rhythm clock, and whether inhibition of the DREAM complex could mitigate clock-related molecular damage in the context of PER deficiency. Here, this invention used siRNA-mediated PER1 knockdown in human retinal pigment epithelial (RPE) cells and pharmacologically inhibited DREAM function using the DYRK1A inhibitor halamine, as previously reported. As recently reported, PER1 was selected as a key circulating PER paralog across mammalian tissues. This invention found that PER1 knockdown is robust ( Figure 18 E and Figure 26A), and indeed sufficient to cause circadian rhythm disruption, as seen in qPCR and transcriptome assays by altering the expression of the core circadian rhythm genes CLOCK and CRY2. Figure 18 E and Figure 26 A). Importantly, Halmin did not restore the biological clock in PER1-depleted cells ( Figure 18 E and Figure 26 A) confirms the hypothesis of this invention that the restorative effect of DREAM inhibition on the intracellular homeostasis of circadian rhythm disorder is unrelated to circadian rhythm restoration and may occur downstream of the damaged circadian rhythm.

[0297] Subsequently, this invention turned to more in-depth high-throughput transcriptomic analysis of PER1 and halamine-treated RPE cells to identify key homeostatic mechanisms regulated by the circadian rhythm in a DREAM-dependent manner in human cells. Notably, the findings of this invention are similar to observations obtained in nematode and mouse models. For example, this invention found that mitochondrial and OXPHOS genes are also downregulated by circadian rhythm disruption in human cells and restored by co-repression of DREAM. Figure 18 F) reveals mitochondria and OXPHOS as the most consistent circadian rhythm regulators across species and cell types. Notably, this observation is consistent with the recently discovered key involvement of mitochondria and OXPHOS in sleep induction and damage clearance during sleep. Furthermore, ribosome and ribosome biosynthesis genes, as well as genes encoding components of transcriptional mechanisms, exhibit opposite expression kinetics between cells with circadian rhythm dysregulation and DREAM co-suppression. Figure 18 F and Figure 26 These results are consistent with observations in nematodes and mice and clearly demonstrate that similar basic cellular activities are altered across species in a dream-dependent manner by circadian rhythm disruption. Furthermore, this invention found that co-administration of halamine in unc-54p::Q40::YFP animals exposed to lin-42 RNAi indeed reversed paralysis induced by circadian rhythm disruption in this model. Figure 26 (D and E). These results suggest that pharmacological inhibition of DREAM is a promising in vivo tool for rescuing the health of organisms affected by sleep and circadian rhythm disorders.

[0298] Example 8: High DREAM abundance provides DNA shielding during awakening at the cost of poor repair.

[0299] The present invention then investigated the physiological significance of the diurnal rhythm fluctuations of DREAM. From the findings of this invention, it is concluded that DREAM levels increase during wakefulness and promote an increase in histone abundance and possibly histone load. Furthermore, literature evidence suggests that histones and chromatin protect DNA from damage, and that oxidation and DNA damage increase during wakefulness. Therefore, this invention hypothesizes that transiently high levels of DREAM serve to protect genomic DNA from excessive wakefulness-related damage.

[0300] In fact, inhibition of DREAM in circulating human cells leads to P21 ( Figure 19 A) and P53 target gene ( Figure 19 Elevated expression of B was consistent with previous reports and was associated with genotoxic stress. Simultaneously, increased DREAM levels via lin-42 RNAi exposure protected *C. elegans* from larval arrest triggered by UV B treatment, which is known to induce helical twisting DNA damage. Figure 19 C and Figure 27 This indicates that elevated DREAM levels do indeed protect DNA from excessive damage.

[0301] On the other hand, previous reports have found that DREAM inhibition upregulates excessive DNA repair pathways, and this invention consistently finds that markers of genotoxic stress (P21 and Gadd45b) are reduced during sleep, while DREAM expression is decreased, and the representative DNA repair marker Ddb2 is upregulated. Figure 19 DF, left figure). Conversely, sleep deprivation inhibited the reduction of P21 and Gadd45b expression levels (DF, left figure). Figure 19 D and E (right figure), just as it prevents the reduction of DREAM expression, Ddb2 is not upregulated in this case. Figure 19 F, right figure).

[0302] In summary, the findings of this invention demonstrate that DRM / DREAM plays a dual role in protecting genome integrity during the circadian rhythm: during wakefulness, high levels of DRM / DREAM may shield DNA from damage by increasing histone abundance, while during sleep, DREAM levels decrease, allowing DNA repair to occur alongside other repair and maintenance activities that remain suppressed during wakefulness, as a trade-off for DNA protection. Under sleep deprivation, the inability to downregulate DREAM may interfere with repair, but as mentioned above, this invention mitigates this impairment using DREAM inhibitors such as halamine.

[0303] Discussion of Implementation Examples

[0304] As illustrated herein, the inventors combined omics, genetics, functional, and molecular analyses in three model organisms to elucidate the molecular basis of declining overall health caused by persistent dysregulation of the circadian rhythm and sleep. Thus, this invention reveals for the first time that circadian rhythm disruption causes the DREAM complex and associated histone variants to interfere with a wide range of fundamental and adaptive cellular activities, ranging from translation and splicing to OXPHOS, oxidative stress responses, and ECM remodeling. The analysis of this invention shows that this newly discovered conserved mechanism allows for persistent circadian rhythm disruption, preventing cells from responding effectively to stressors, which in turn leads to organ dysfunction, particularly evident in stress-prone organisms, such as nematode strains expressing the aggregation-prone human Aβ or Q40 YFP proteins. The disrupted processes are widespread and affect many cell types, consistent with published observations that circadian rhythm / sleep disruption impairs homeostasis in multiple organs. The assumptions of this invention are consistent with numerous reports linking sleep deprivation to human disease and poor biological responses to external stressors, ranging from infection and cognitive challenges to exercise and diet. Furthermore, the findings of this invention are consistent with recent reports indicating that gut oxidative stress is a leading cause of premature death in fruit flies and chronically sleep-deprived mice. Therefore, this invention identifies key molecular aspects of the downstream consequences and medical effects of circadian rhythm disruption and proposes means to address such conditions. This invention demonstrates that the pharmacological and genetic inhibition of the DREAM complex can restore homeostasis in cells and organisms affected by circadian rhythm impairment. This invention demonstrates that this repair process does not require restoration of the biological clock, making it unique among existing methods for counteracting sleep and circadian rhythm disruption, which all rely on the eventual restoration of biological clock and sleep function. Therefore, DREAM-based presumptive interventions are particularly suitable for situations where circadian rhythm disruption must persist or is irreversible for any reason.

[0305] This invention further emphasizes the ability of the DREAM complex to simultaneously regulate multiple cellular functions by controlling the abundance of specific histone variants and the accessibility of corresponding chromatin regions. This model is supported by previous reports that detected high abundance of the histone variant H2A.Z in the genomes of DREAM-repressed cell cycle targets, of which HTZ-1 / H2A.Z is one of the major histones identified in this invention that collaborate with DREAM to disrupt cellular resilience in circadian rhythm-distorted animals. Consistently, only a small fraction of the molecular targets altered by circadian rhythm disruption in a DREAM-dependent manner possess DREAM binding sites in their promoter regions, thus supporting indirect chromatin-based regulation. Interestingly, this histone-dependent mode of DREAM action differs from the recently reported ability of DREAM to inhibit multiple pathways involved in DNA repair, which relies on direct binding of DREAM to regulatory elements of affected genes. Notably, the key DREAM subunits involved also differ in their roles in DNA repair and the circadian rhythm function of DREAM. Lin-52, dpl-1, elf-1, and lin-35 have the strongest impact on DNA repair, while lin-9, lin-54, and lin-53 regulate the consequences of circadian rhythm disruption. Therefore, the findings of this invention suggest that specific variants of the DREAM complex may exist in two distinct modes of how the complex's gene expression network is regulated: (a) through direct binding and recruitment of cofactors, and (b) through altered chromatin accessibility (as in this study). The circadian rhythm response, dependent on the presence of specific DREAM subunits, enables targeted intervention that modulates the “sleep-specific” role of DREAM without interfering with its activity in other cellular processes. Finally, the inventors demonstrate that native DREAM expression is regulated by a circadian rhythm, with levels increasing during wakefulness and decreasing during sleep. Increased DREAM expression during wakefulness leads to elevated histone levels, shielding genomic DNA from wakefulness-related damage, but at the cost of inhibiting cellular repair. During sleep, reduced DREAM expression enables chromatin decompression and efficient repair processes, including DNA repair. However, sleep deprivation interferes with the downregulation of DREAM, thereby chronically suppressing repair mechanisms. Importantly, these detrimental effects of sleep deprivation can be reversed using pharmacological or other inhibitors of the DREAM complex, thus serving as a mimicry for restoring sleep.

[0306] References

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Claims

1. The use of an inhibitor of a protein complex dimerizing partner, RB-like, E2F and multiple vulvar B type (DREAM complex inhibitor) for the treatment of medical conditions caused by circadian rhythm disorders.

2. The use of the DREAM complex inhibitor according to claim 1, wherein, The inhibitor is a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor.

3. Use of the DREAM complex inhibitor according to any one of the preceding claims, wherein, The inhibitor is a β-carboline alkaloid, preferably a Halman alkaloid, or a pharmacologically acceptable salt or ester thereof.

4. Use of the DREAM complex inhibitor according to any one of the preceding claims, wherein, The inhibitor is halamine, or a pharmacologically acceptable salt or ester thereof.

5. The use of the DREAM complex inhibitor according to claims 1-3, wherein, The inhibitor is an inhibitor of Dyrk (INDY), or a pharmacologically acceptable salt or ester thereof.

6. The use of the DREAM complex inhibitor according to claim 1, wherein, The inhibitor comprises an affinity agent, antibody, or antigen-binding fragment thereof that binds to the DREAM complex or its subunits, or comprises an antisense or interfering nucleic acid molecule, such as a short interfering RNA (siRNA), that targets the DREAM complex or its subunits.

7. Use of the DREAM complex inhibitor according to any one of the preceding claims, wherein, The subunits of the DREAM complex are suppressed, wherein the suppressed subunits of the DREAM complex are lin-9, lin-54 and / or RBBP4 / RBBP7.

8. Use of the DREAM complex inhibitor according to any one of the preceding claims, wherein, The medical condition is caused by loss of tissue function, oxidative stress and / or dysfunction of cellular homeostasis, and / or includes loss of tissue function, oxidative stress and / or dysfunction of cellular homeostasis.

9. Use of the DREAM complex inhibitor according to any one of the preceding claims, wherein, The patient suffers from sleep deprivation and / or circadian rhythm disorder.

10. Use of the DREAM complex inhibitor according to any one of the preceding claims, wherein, The treatment is independent of the restoration of the circadian rhythm and preferably occurs downstream of the circadian rhythm.

11. Use of the DREAM complex inhibitor according to any one of the preceding claims, wherein the DREAM complex inhibitor is used as a sleep mimic to provide one or more sleep recovery effects.

12. Use of the DREAM complex inhibitor according to any one of the preceding claims, wherein, The medical conditions mentioned include acute medical injuries associated with sleep deprivation and circadian rhythm disorders.

13. Use of the DREAM complex inhibitor according to the preceding claim, wherein, The acute medical injury is one or more of the following: fatigue, drowsiness, cognitive impairment, brain fog, memory impairment, gastrointestinal discomfort, cardiovascular injury, changes in blood pressure, decreased immunity, mood changes, depression, anxiety and / or paranoia.

14. Use of the DREAM complex inhibitor according to any one of the preceding claims, wherein, The medical conditions mentioned include chronic medical conditions associated with sleep deprivation and circadian rhythm disorders.

15. Use of the DREAM complex inhibitor according to the preceding claim, wherein, The chronic medical conditions mentioned are neurological disorders, mental disorders, cardiovascular disorders, metabolic disorders, allergic disorders, immune disorders, gastrointestinal disorders, rheumatic disorders, proliferative diseases (cancer), microbiome disorders, multi-organ disorders, and / or pulmonary disorders.

16. A pharmaceutical composition comprising a DREAM complex inhibitor for use according to any one of the preceding claims and one or more pharmaceutically acceptable excipients.

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