A dream complex inhibitor for use in treating a medical condition induced by disruption of a circadian rhythm

Abstract

The invention relates to an inhibitor of the protein complex Dimerization partner, RB-like, E2F and multi-vulval class B (DREAM complex inhibitor) for use in the treatment of a medical condition induced by disruption of a circadian rhythm. The invention further relates to a DREAM complex inhibitor for use as a sleep mimetic. The invention further relates to a pharmaceutical composition comprising a DREAM complex inhibitor with one or more pharmaceutically acceptable excipients for use in treating a medical condition induced by disruption of a circadian rhythm.

Description

[0001] A DREAM COMPLEX INHIBITOR FOR USE IN TREATING A MEDICAL CONDITION INDUCED BY DISRUPTION OF A CIRCADIAN RHYTHM

[0002] DESCRIPTION

[0003] The invention is in the field of biochemistry and medicine, more specifically in the field of chronobiology and the treatment of medical conditions induced by disruption of a circadian rhythm.

[0004] The invention relates to an inhibitor of the protein complex Dimerization partner, RB-like, E2F and multi-vulval class B (DREAM complex inhibitor) for use in the treatment of a medical condition induced by disruption of a circadian rhythm. The invention further relates to a DREAM complex inhibitor for use as a sleep mimetic.

[0005] The invention further relates to a pharmaceutical composition comprising the DREAM complex inhibitor with one or more pharmaceutically acceptable excipients for use according to the present invention.

[0006] BACKGROUND OF THE INVENTION

[0007] As a molecular time-keeping mechanism, the internal circadian clock generates a biological rhythm and regulates various physiological processes such as blood pressure, hormone secretion, sleepwake cycles, and body temperature in mammals, and in particular, in humans. Circadian clocks are highly conserved throughout evolution.

[0008] The general mechanism of circadian disruption involves a disturbance in the finely tuned orchestration of endogenous circadian rhythms that regulate various physiological and behavioral processes in a roughly 24-hour cycle. At the heart of this regulatory system is the suprachiasmatic nucleus (SCN), often referred to as the "master clock," located in the anterior hypothalamus. This central pacemaker coordinates and aligns circadian clocks in different brain regions and peripheral tissues with external synchronizing agents, ensuring a harmonious temporal organization of bodily functions.

[0009] Circadian rhythms are intricately modulated by a combination of endogenous factors, such as genetic and physiological influences, and external and behavioral factors, including exposure to light, activity, and feeding schedules. Disruption of these components can lead to circadian dysregulation, broadly termed "circadian disruption."

[0010] One key aspect of circadian disruption is its bidirectional relationship with various health outcomes. Not only does circadian disruption increase the severity of diseases, but many diseases, in turn, can disrupt circadian rhythms, creating an interplay between circadian regulation and health. This reciprocal relationship spans multiple organizational levels, from molecular and cellular changes to misalignments among physiological systems and behavioral cycles.

[0011] Measures of circadian disruption include parameters like circadian phase (timing), alignment between internal-internal or internal-external rhythms, and the period and amplitude of circadian rhythms. Disturbance in circadian phase alignment and amplitude are particularly associated with adverse health consequences. These disruptions can occur at various levels, ranging from intrinsic changes at the molecular and cellular levels to misalignments among different organizational levels and with behavioral and environmental cycles.

[0012] The molecular circadian clock, governed by genes involved in autoregulatory feedback loops (e.g., circadian locomotor output cycles kaput [CLOCK], brain and muscle ARNT-like [BMAL], period [PER], rev-erb / nuclear receptor subfamily 1 , group D [NR1D], and cryptochrome [CRY]), represents a fundamental mechanism driving circadian rhythms. While the SCN rhythm itself cannot be directly measured in humans, biomarkers like the timing of melatonin onset and amplitude serve as crucial indicators of circadian rhythms. Moreover, disturbances in sleep-wake rhythms, often accompanying circadian disruption, contribute significantly to overall health consequences.

[0013] Lifestyle factors and environmental changes, such as shift work, constant light exposure, or irregular sleep patterns, contribute to circadian disruption. This disruption is not merely a consequence but a significant contributing factor to a range of health issues, including neurologic, psychiatric, cardiometabolic, and immune disorders. Circadian clock impairment and sleep deprivation disrupt multiple aspects of organismal fitness, from cognition to the immune response. Such multifold influence can partially be explained by the requirement of sleep for the fluid flow and molecular damage clearance in the brain, with the central nervous system (CNS) mediating a plethora of organismal responses.

[0014] Without being bound by existing theory, sleep deprivation and disruption of the circadian rhythms may impinge on master regulator pathways, which control fundamental aspects of cellular physiology and thereby modulate the function of multiple cell types. The discovery of such core regulatory mechanisms represents a crucial and novel step towards developing interventions that restore homeostasis under circadian misalignment and lack of sleep.

[0015] Treatment methods are available for circadian disruptions, including lifestyle modifications, light therapy, and pharmaceutical interventions. Lifestyle adjustments entail establishing consistent daily routines and optimizing sleep environments, potentially managing symptoms associated with circadian rhythm disorders. However, it is essential to consider the potential drawbacks associated with these treatments. Implementing lifestyle changes may demand a significant commitment, and their effectiveness can vary among individuals. Light therapy, while beneficial, may result in side effects such as eye strain and headaches.

[0016] Medications offer pharmacological solutions to align sleep patterns with the desired schedule. Melatonin receptor agonists, a type of melatonin medicine, are prescribed to regulate melatonin production and treat non-24-hour sleep-wake rhythm disorder. However, they may result in side effects like dizziness and fatigue. Melatonin supplements, lab-made versions of the sleep hormone, are recommended for disorders such as delayed sleep-wake phase disorder. Not regulated by the FDA, these supplements may vary in dose and purity between brands, with potential side effects including excess sleepiness and headaches.

[0017] Caffeine is suggested to prevent daytime sleepiness, but it is advised to avoid it within eight hours of the desired bedtime. Sleep-promoting medicines like benzodiazepines and zolpidem may be prescribed for faster sleep onset, but they can cause side effects such as muscle weakness and confusion, especially in older adults. Wake-promoting medicines like modafinil and armodafinil help individuals stay alert during shift work, but their effects may be short-term, and some sleepiness may persist.

[0018] While these medications offer symptomatic relief, they often do not address the underlying causes of the cellular and organ malfunctions triggered by circadian disruptions. Moreover, potential side effects and complications necessitate careful consideration and consultation with healthcare professionals before initiating any pharmacological intervention.

[0019] In the prior art, only a few studies investigated the underlying mechanisms of clock disruption and proposed methods to restore the circadian rhythm.

[0020] For example, in JP2011195560A, harmine or harmane alkaloids are described to improve or restore the circadian rhythm as such. The methods disclosed therein primarily focus on improving or restoring the circadian rhythm itself and make no mention of treating downstream effects or medical conditions arising from circadian rhythm disruption.

[0021] EP4091611A discloses an inhibitor of DREAM complex assembly and / or function for use in repairing DNA damage in a cell of a subject. The inhibitor can be selected from the group comprising an inhibitory nucleic acid, an antibody and a small molecule inhibitor.

[0022] Abbassi et al. (Pharmacology & Therapeutics, 2015) disclose the role of DYRK1A and its inhibition in neurodegenerative diseases and cancer. It establishes a link between DYRK1A inhibition and the expression of the DREAM complex, highlighting its impact on cell proliferation.

[0023] Naert et al. (European Neuropsychopharmacology, 2015) disclose the use of Leucettine 41 as a DYRK1 A inhibitor for preventing memory impairment and neurotoxicity induced by oligomeric Ap25- 35 peptides in mice. It further suggests that DYRK1 A inhibition, including through other inhibitors such as harmine, has a beneficial effect on reducing oxidative stress and memory impairment.

[0024] None of the prior art discloses methods specifically targeting the adverse effects resulting from circadian rhythm disruption, but is primarily focused either on reversal of distinct cell and tissue pathologies unrelated to sleep or on restoring the circadian rhythm itself. Furthermore, no prior art provides methods or approaches for treating conditions or disorders arising directly as a consequence of circadian rhythm disruption. Moreover none of the prior art proposes to use DREAM inhibitors as mimetics of restorative sleep.

[0025] Thus, one objective of the present invention addresses an unmet need for treating medical conditions induced by disruption of the circadian rhythm, independent of whether circadian rhythm itself is restored.

[0026] SUMMARY OF THE INVENTION

[0027] In light of the prior art, the technical problem underlying the invention was providing alternative or improved means for treating medical conditions induced by disruption of a circadian rhythm.

[0028] Another objective was to provide alternative or improved means for treating medical conditions induced by disruption of the circadian rhythm that are independent of restoring a circadian rhythm as such, and / or means that are effective in addressing medical detriments arising from disruption of a circadian rhythm but are downstream of the circadian rhythm itself.

[0029] One focus of the invention was on developing interventions with the capacity to improve health even in the cases when clock and sleep functions cannot be restored either by choice (social jetlag, shift work) or due to environmental and / or pathological reasons.

[0030] These problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims. The present invention therefore relates to an inhibitor of the protein complex Dimerization partner, RB-like, E2F and multi-vulval class B (DREAM complex inhibitor) for use in the treatment of a medical condition induced by disruption of a circadian rhythm.

[0031] For the first time, the DREAM complex has been characterized as the core regulator of clock-altered pathway activities, where pharmacological and genetic inhibition of the DREAM complex alleviates molecular and organismal dysfunction instigated by distorted clock rhythms.

[0032] The discovery of such core regulatory mechanisms represents an important step toward developing interventions that restore homeostasis and / or patient health under circadian rhythm misalignment and lack of sleep. In embodiments, the restoration of homeostasis and / or patient health occurs independent of circadian rhythm realignment or an alleviation of a lack of sleep. In embodiments, the medical effect of the DREAM complex inhibitor occurs independently from (and / or downstream of) correcting or realigning the circadian rhythm itself.

[0033] Circadian rhythms, intrinsic biological cycles with an approximate 24-hour period, intricately regulate fundamental physiological processes, including feeding behaviors, sleep-wake cycles, hormonal release, and metabolic functions. Governed by the suprachiasmatic nucleus (SCN) in the anterior hypothalamus, the circadian system is entrained to external stimuli or“zeitgebers”, such as light exposure. Modern lifestyles, marked by artificial lights, shift work, and constant 24 / 7 work environments, challenge the endogenous circadian rhythm, leading to various health disorders. Disruption of circadian rhythms is associated with adverse health outcomes, including cancer, neurodegenerative diseases, cardiovascular issues, and diabetes.

[0034] The SCN, acting as the master timekeeper, orchestrates circadian processes by relaying signals from external stimuli to peripheral clocks. The light-dark detection pathway, mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs), influences melatonin secretion, regulating the sleepwake cycle. The molecular genetic mechanism involves a transcription-translation negative-feedback loop featuring key factors like BMAL1, CLOCK, PER, and CRY genes. Mutations in these genes can disrupt circadian functioning.

[0035] Circadian rhythm sleep disorders (CRSD) result from misalignments between endogenous biological rhythms and external cues. Intrinsic disorders include delayed and advanced sleep phase disorders, irregular sleep-wake rhythm disorder, and non-24-hour sleep-wake disorder. Extrinsic disorders, like shift work sleep disorder and jet lag, arise from the mismatch between the biological clock and imposed schedules, leading to sleep disturbances and daytime sleepiness.

[0036] A number of pharmacological and behavioral interventions that restore circadian function and regular sleep patterns are known in the art, such as melatonin supplementation, sleeping pills, and bright light therapy.

[0037] The natural hormone melatonin is known to facilitate proper sleep timing in the context of sleep disorders and jetlag. Sleeping pills vary in molecular targets with key examples being benzodiazepine receptor agonists (BZRAs) (eszopiclone, zaleplon, and zolpidem), benzodiazepine hypnotics (estazolam, flurazepam, temazepam, triazolam, and quazepam), dual orexin receptor antagonists (DORAs) (daridorexant, lemborexant, and suvorexant), histamine receptor antagonists (doxepin), and melatonin receptor agonists (Ramelteon). While effective in inducing sleep, most of these compounds elicit side effects, which may impede quality of life (drowsiness, dizziness, headache, diarrhea, and addiction). Bright light therapy consisting of repeated and defined (same light intensity, same duration) bright light exposures at a fixed time of day was recently put forward as an effective non-pharmacological intervention in circadian sleep disorders, insomnia, and sleep disruption in Alzheimer's disease and dementia patients. This approach, however, remains controversial.

[0038] Collectively, all existing solutions are focused on inducing and / or maintaining sleep by restoring the circadian rhythm. However, the restoration of circadian rhythms is not always feasible due to inherent challenges in adjusting lifestyle or behavior that may be ongoing and in contrast to circadian rhythm realignment. This limitation arises, by way of example, from either a complete absence of a clock component or ongoing disruption, attributed for example to genetic mutations, or the presence of an irreversible clock-disturbing stimulus, such as extended sleep deprivation.

[0039] In such cases, where restoring defective clock components and / or removing the disturbing stimulus interventions prove ineffective, the present invention aims to restore and / or improve the health of target peripheral cells even in the absence of restored, restorable or permanently disrupted circadian rhythm.

[0040] Therefore, one objective of the present invention was the decoupling of sleep and / or sleep disturbance from its physiological (i.e., health or medical) consequences in the body. Another objective of the invention was to provide means that address medical conditions or impairments associated with and / or induced by circadian rhythm disruption, without being dependent on having to restore the circadian rhythm itself.

[0041] All current interventions that target sleep deprivation typically rely on the restoration of sleep, which is not entirely feasible in all cases. For example, this approach often fails in patients affected by severe brain dysfunction like dementia. Also, night shift workers still suffer from the health consequences of circadian misalignment even if they sleep during the day with the help of conventional sleep medication. Moreover, all existing pharmacological interventions, including melatonin, induce side effects that impair general well-being, cognitive function and even involve addiction.

[0042] In the present invention, the inventors discovered that circadian rhythm disruption is connected to many negative health effects by the activity of a chromatin remodeling complex called DREAM (Dimerization partner, RB-like, E2F and multi-vulval class B).

[0043] Subsequently, as demonstrated in the examples below, genetic inactivation of the DREAM complex restored the health of animals affected by persistent circadian impairment. The same rescue was observed following exposure to DREAM inhibitors.

[0044] No suggestion exists in the art that inhibition of the DREAM complex represents an effective intervention that rapidly rescues organismal health amid continued circadian dysfunction, acting downstream of the circadian clock. The findings of the present invention are therefore unexpected, and the DREAM inhibitors of the present invention exhibit unexpected and beneficial effects with respect to health restoration in clock impaired organisms independent of or downstream of the circadian clock reinstatement.

[0045] The present invention thus also presents a novel clinical situation over earlier uses of similar molecules, such as harmine, or other DREAM complex inhibitors. The discovery or observation that DREAM complex inhibition leads to addressing medical conditions of clock-disrupted organisms and their underlying pathologies independently of restoring circadian clock, indicates a novel technical effect underlying the present invention, not previously identified in the art. From this novel technical effect, a novel clinical situation arises, wherein such DREAM complex inhibitors may be administered to different patient populations, at different doses or in different dosage regimes as previously known from earlier studies on harmine, or other DREAM complex inhibitors. The identification of the novel biological / technical effect underlying the invention also enables the treatment of novel patient groups and achieving novel therapeutic effects in said groups, thus defining novel medical treatments.

[0046] In embodiments, the inhibition of the DREAM complex rapidly rescues organismal health amid continued circadian dysfunction.

[0047] In embodiments, the inhibition of the DREAM complex acts downstream of the circadian clock.

[0048] In embodiments, the inhibition of the DREAM complex acts downstream of the circadian clock and uncouples circadian disruption from its negative health effects.

[0049] In some embodiments, the inhibitor is a DYRK1A inhibitor, also known as an inhibitor of a “dualspecificity tyrosine phosphorylation-regulated kinase 1A” (DYRK1A inhibitor).

[0050] As DYRK1 A emerges as a therapeutic target, a variety of inhibitors have been identified. In embodiments, DYRK1A inhibitors may be categorized into three types: ATP-competitive (Type I), non-ATP competitive with partial binding (Type II), and non-ATP competitive with specific pocket binding (Type III). Natural products like harmine and synthetic compounds with indazole, azaindole, or benzothiazole moieties show activity as DYRK1 A inhibitors. Clinical trials, such as those involving epigallocatechin-3-gallate (EGCG), a polyphenolic component from green tea, underscore the potential therapeutic applications of DYRK1 A inhibitors in addressing diseases associated with DYRK1A dysregulation.

[0051] Notable DYRK1 A inhibitors include but are not limited to harmine, leucettine-L41 , 5-IT, GNF4877, and Epigallocatechin gallate (EGCG). Other promising compounds like PST-001 , Silmitasertib, Lorecivivint, and FRTX-02 are in various stages of clinical development.

[0052] In embodiments, the DYRK1A inhibitor is a non-selective ATP competitive DYRK1 A inhibitor.

[0053] In embodiments, the DYRK1 A inhibitor is harmine, leucettine-L41 , 5-IT, GNF4877, and / or Epigallocatechin gallate (EGCG).

[0054] In embodiments, the DYRK1A inhibitor is PST-001 , Silmitasertib, Lorecivivint, and / or FRTX-02.

[0055] In some embodiments, the inhibitor is a p-carboline alkaloid, preferably a harmala alkaloid, or a pharmacologically acceptable salt or ester thereof.

[0056] In embodiments, the inhibitor is harmine or a pharmacologically acceptable salt or ester thereof.

[0057] The alkaloid harmine, a DREAM complex inhibitor used in the present invention to inactivate DREAM in the context of circadian disruption, is among the traditional remedies known to humans for a long time. The spectrum of its recent therapeutic benefits includes depression, Parkinson's, and Alzheimer's diseases, and side effects are only detectable at very high doses. Unlike conventional sleep medications, DREAM inhibition by harmine acts directly upon downstream molecular targets that mediate loss of health in the case of circadian disruption. In this way, the health impairments are rescued rapidly, and the rescue is achieved even if circadian behavior itself remains disrupted (like in shift workers and dementia patients).

[0058] It was entirely surprising that DREAM inhibition by harmine does not require restoration of coordinated circadian behavior in order to alleviate the associated loss of health. This is a unique feature absent in any of the existing treatments. Furthermore, all current sleep medications indirectly modulate health with sleep restoration as the obligatory intermediate step. Conversely, DREAM inhibition, e.g., by harmine, acts directly upon downstream molecular targets responsible for loss of health, eliciting a faster positive effect, without the need of resetting the circadian rhythm itself. To the best of the inventors’ knowledge, harmine has not been reported to elicit side effects comparable to conventional sleep medications.

[0059] In embodiments, the inhibitor is an inhibitor of Dyrk (INDY), or a pharmacologically acceptable salt or ester thereof.

[0060] In embodiments, a subunit of the DREAM complex is inhibited, wherein the inhibited subunit of the DREAM complex is LIN9, LIN54, and / or RBBP4 / RBBP7.

[0061] In one embodiment, the inhibited subunit of the DREAM complex is LIN9.

[0062] In one embodiment, the inhibited subunit of the DREAM complex is LIN54.

[0063] In one embodiment, the inhibited subunit of the DREAM complex is lin-53 / RBBP4 / RBBP7.

[0064] While the small molecule DYRK1 A inhibitor INDY is known in the art for its ability to inhibit the DREAM complex, no mention has been made of employing such a substance in the treatment of a medical condition associated with and / or induced by a disrupted circadian rhythm. Notably, EP4091611A1 teaches inhibition of DREAM complex assembly and / or function to repair DNA damage in a subject's cell. EP4091611A1 discloses inhibitors like harmine and INDY. However, it explicitly emphasizes the use of such DREAM inhibitors to target lin-52, lin-35, efl-1 , and dpl-1 subunits of the DREAM complex, demonstrating substantial differences in the specific subunits within the DREAM complex that are affected, in comparison to the present invention.

[0065] No suggestion exists in the art that inhibiting the subunits LIN9, LIN54, and / or lin-53IRBBP4IRBBP7, either alone or together, would improve health, despite the persistent disruption of the circadian rhythm. The therapy according to the invention is neither aimed at recovering the circadian rhythm, nor is it a direct therapy for sleep disorders. The primary aim of the present invention is to address the medical conditions associated with sleep deprivation and their health consequences.

[0066] Therefore, a skilled person could not have expected that inhibiting the DREAM complex with DYRK1A inhibitors would impact subunits within the complex other than those mentioned in EP4091611 A1 . Moreover, it was not anticipated that such inhibition would offer alternative or improved methods for addressing medical conditions induced by disruptions in the circadian rhythm.

[0067] In embodiments, the inhibitor comprises or consists of an affinity reagent, antibody or antigenbinding fragment thereof, binding the DREAM complex or subunit thereof, or comprises an antisense or interfering nucleic acid molecule, such as short interfering RNA (siRNA), targeting the DREAM complex or subunit thereof.

[0068] In one preferred embodiment, the DREAM complex inhibition is transient. In embodiments, the transient inhibition comprises pharmacological activity as a DREAM inhibitor after administration for a certain time, and preferably not continuously, for example at some times throughout a 24 hour cycle the DREAM inhibition is not evident or is at low or negligible levels. The DREAM complex can be inhibited by various methods known to the skilled person in the art, e.g., small molecules disrupt interactions, antibodies block specific binding sites, and nucleic acid molecules may prevent the synthesis of proteins within the complex.

[0069] In one embodiment, the DREAM complex inhibitor is a small molecule, an affinity reagent, antibody or antigen-binding-fragment thereof, that targets the DREAM complex and / or one or more subunits of the DREAM complex, and leads to a reduction or inhibition in DREAM complex function.

[0070] In one embodiment, the DREAM complex inhibitor is a nucleic acid molecule, capable of reducing DREAM complex expression or function. By way of example, antisense oligonucleotides, or interfering nucleic acid species may be employed to target (reduce) the expression of one or more subunits of the DREAM complex. A skilled person is aware of nucleic acid-based approaches to reduce expression of a subunit of the DREAM complex.

[0071] In other embodiments, the DREAM complex can be inhibited by targeted protein degradation (TPD), e.g., by proteolysis-targeting chimera (PROTAC) molecules. As known to a skilled person, a proteolysis targeting chimera (PROTAC) is a heterobifunctional molecule composed 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 engaging an E3 ubiquitin ligase, and another that binds to a target protein meant for degradation. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein via the proteasome. By using this approach, effective degradation of one or more DREAM complex subunits may be achieved and thus DREAM complex inhibition according to the present invention.

[0072] In some embodiments, the medical condition is induced by and / or comprises a loss of tissue function, oxidative stress, and / or dysfunction of cellular homeostasis.

[0073] As demonstrated in the examples below, RNAi treatment against the C. elegans PER orthologue Un- 42 induced persistent but moderate clock disruption as a mimetic of lifestyle-inflicted clock irregularities. This intervention was also applied in C. elegans models of proteostasis stress and accelerated aging, followed by proteomics and molecular and functional tests. In addition, transcriptomic analyses in mice with altered circadian behavior and in PER1 knock-down human cells were conducted. These tests validated the link between clock disruption and homeostatic impairments, especially in the context of additional cellular stress. A plethora of conserved and ubiquitously expressed molecular targets altered by the distortion of clock were discovered in line with the multifold loss of health under clock impairment.

[0074] In embodiments, the patient suffers from sleep deprivation.

[0075] In embodiments, the treatment is independent of restoration of the circadian rhythm, said treatment and the relevant biological effect preferably occurring downstream of the circadian rhythm.

[0076] Sleep deprivation and disruption of circadian rhythms are common events in the modern world. It is well documented that a persistent lack of coordinated circadian behavior / sleep leads to a multitude of health impairments, from immune deficiency to cognitive decline. While sleep disruption can often be treated by specialized medications that actively promote sleep, this does not apply in all cases. Factors like insensitivity, cognitive side effects, or addiction may prevent individuals from relying on conventional sleeping pills. In addition, individuals may avoid sleep for extended periods for professional reasons (shift work and comparable occupations).

[0077] In mammals, a central circadian pacemaker in the suprachiasmatic nuclei (SCN) orchestrates peripheral oscillators throughout the body. Clock genes, including BMAL1, CLOCK, NPAS2, CRY1 / 2, and PER1-3, form auto-regulatory feedback loops driving circadian oscillations. Post- transcriptional modifications, such as phosphorylation by casein kinase family members, play a role in clock precision. Ancillary loops involve Rev-erba, Rora, DEC1, DEC2, and D-site albumin promoter binding protein. Sleep regulation therefore involves a circadian pacemaker and homeostatic sleep drive. The SCN influences sleep timing through neuronal projections. For example, a so-called Process C determines sleep timing, while Process S builds sleep drive. Mutations in clock genes correlate with sleep disorders, and polymorphisms in PER genes relate to diurnal preferences. The CLOCK:BMAL1 complex, casein kinase I, and clock gene mutations impact circadian period length, linking circadian rhythms to sleep regulation.

[0078] Conversely, prolonged wakefulness affects the expression of clock genes in the cerebral cortex, upregulating the genes Perl and Per2. The homeostatic sleep drive builds up an increased need for sleep in response to extended wake periods, independent of the time of day. Sleep deprivation impacts the firing rates 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 where clock genes influence sleep patterns.

[0079] Conversely, disturbances in sleep, such as sleep deprivation, can impact the expression and function of clock genes.

[0080] Thus, the present intervention acts upon downstream molecular effects of circadian impairment, thereby enabling a bypass of sleep regulation and acting on its downstream molecular targets to improve health, even if sleep itself is not restored.

[0081] In some embodiments, the medical condition to be treated comprises an acute medical impairment associated with sleep deprivation and disruption of a circadian rhythm.

[0082] In some embodiments, the acute medical impairment is one or more of fatigue, lethargy, cognitive impairment, brain fog, impaired memory, gastro-intestinal discomfort, cardiovascular impairment, blood pressure alteration, decline of immune-competence, change in mood, depression, anxiety and / or paranoia.

[0083] Sleep disruption leads to numerous health consequences. Short-term effects in healthy individuals may include increased stress susceptibility, somatic problems, reduced quality of life, emotional distress, mood disorders, and cognitive deficits. In children and adolescents, sleep disruption negatively affects psychosocial health, school performance, and risk-taking behaviors. Adults experience psychosocial issues such as emotional distress, mood disorders, and cognitive deficits. Sleep disruption alters cognition and performance, impacting attention, executive function, memory, decision-making, and judgment. In individuals with underlying medical conditions, sleep disruption reduces quality of life (QoL), affecting patients with conditions like chronic kidney disease or liver transplants. The DREAM inhibitors of the present invention are therefore suitable for the treatment and / or prevention of such disorders that result from circadian rhythm disruption and / or a lack of sleep. In another embodiment, the medical condition comprises a chronic medical condition associated with sleep deprivation and disruption of a circadian rhythm.

[0084] In embodiments, the chronic medical condition is a neurologic, psychiatric, cardiovascular, metabolic, allergic, immunologic, gastrointestinal, rheumatic, proliferative disease (cancer), microbiome, multi-organ and / or a pulmonary disorder.

[0085] Circadian disruption significantly impacts neurological disorders, influencing their development, expression, and severity. Conditions such as cerebrovascular disease, epilepsy, migraine, multiple sclerosis, and various neurodevelopmental disorders are linked to circadian disruption. In neurodevelopmental disorders like autism spectrum disorders (ASDs), misaligned cortisol rhythm and lower amplitude melatonin rhythm are observed. Single-nucleotide polymorphisms (SNPs), particularly in melatonin receptors, may contribute to ASD. Smith-Magenis syndrome (SMS) involves a deletion affecting the gene RAI1, associated with melatonin regulation. For SMS, strategies include pi -adrenergic antagonist administration and melatonin supplementation. In neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD), circadian disruption predicts disease development. Melatonin and light therapy show potential for managing circadian-related symptoms in PD and AD. Strategies focusing on circadian function may decelerate age-related cognitive decline in AD.

[0086] Circadian disruption is prevalent in psychiatric diseases, notably schizophrenia and mood disorders like depression, bipolar disorder, and seasonal affective disorder. Seasonal affective disorder, marked by depression during reduced daylight, demonstrates the impact of circadian misalignment on depressive symptoms. "Circadian depression" is a term coined for mood disorders exhibiting rhythmicity, seasonality, and treatment responses influenced by circadian factors. Human studies link altered clock gene expression and genetic polymorphisms in clock genes to mood disorders. Earlier sleep midpoint correlates with lower depression risk.

[0087] Circadian misalignment, where environmental or behavioral rhythms diverge from central or peripheral clocks, elevates cardiovascular disease (CVD) risk. Shift work associates with myocardial infarction (Ml) and stroke, potentially induced by hypertension and inflammation. In mouse stroke models, prolonged "shift work" upregulates inflammatory mediators, worsening stroke severity. Night shift workers face increased CVD risk due to a circadian disruption-induced IL-6-mediated inflammatory mechanism, evidenced by thicker carotid intimal-medial walls. Misalignment alters blood pressure regulation, impacting cardioprotective nocturnal blood pressure dipping. Arrhythmias exhibit diurnal variation, influenced by circadian misalignment and non-24-hour light / dark cycles.

[0088] The intricate link between circadian rhythms and metabolism is vital for optimal energy utilization. Circadian disruption significantly affects metabolic health, leading to impaired glucose tolerance, insulin resistance, and increased diabetes and obesity risks. Core clock gene disruptions, social jet lag, and misalignment in chronotype contribute to metabolic impairments. Timing of food intake, light exposure, and personalized interventions, such as time-restricted feeding, play crucial roles in mitigating these risks. Obesity is associated with CLOCK gene variants, and shift workers face elevated risks of metabolic disorders.

[0089] The circadian clock intersects with immune function, impacting health and susceptibility to infections. In the context of COVID-19, circadian misalignment, as seen in shift work, is associated with an increased risk of SARS-CoV-2 infection. The circadian system's influence extends to asthma, where genetic mutations affecting clock genes contribute to defects in epithelial barriers, potentially heightening asthma susceptibility. This misalignment, exacerbated by factors like hypoxia, may increase the risk of allergic reactions.

[0090] Sleep dysfunction is intricately linked to gastrointestinal disorders. Proinflammatory cytokines, such as tumor necrosis factor, interleukin-1 , and interleukin-6, play a pivotal role in both sleep regulation and certain gastrointestinal diseases like gastroesophageal reflux disease, inflammatory bowel disease, liver disorders, and colorectal cancer. The gut-brain axis, encompassing the central nervous system, autonomic nervous system, and enteric nervous system, connects sleep regulation pathways with gastrointestinal physiology, modulating immune activation, intestinal permeability, and enteroendocrine signaling. The intricate relationship extends to the digestive process during sleep, influencing factors such as esophageal susceptibility to gastric acid injury, gastric acid secretion patterns, migrating motor complexes, small intestinal motility, and colonic contractions. Moreover, disruptions in sleep have been associated with alterations in cytokine release, exacerbating inflammatory responses in various gastrointestinal disorders.

[0091] Sleep deprivation may contribute to the development and progression of inflammatory joint diseases, including rheumatoid arthritis (RA). RA, characterized by persistent synovitis and inflammation, leads to joint destruction. Studies show a reciprocal relationship, with sleep deprivation impacting RA patients' quality of life, fatigue, and health. Experimental studies link sleep loss to altered inflammatory mediators, potentially involving impaired regulatory T cell function. Circadian rhythm, particularly chronotherapy, is suggested to optimize RA treatment efficacy. Evaluating and addressing sleep disorders in RA is crucial for improving patient outcomes, including quality of life and disease progression.

[0092] Sleep profoundly influences breathing, impacting central respiratory control, lung mechanics, and muscle contractility. While such effects are generally benign in healthy individuals, they can lead to significant hypoxemia and hypercapnia in those with chronic obstructive pulmonary disease (COPD), especially during rapid eye movement (REM) sleep. Factors like airflow obstruction, hyperinflation, respiratory muscle dysfunction, and medication use contribute to pronounced hypoventilation in COPD patients during sleep. Sleep disturbance is prevalent in COPD, with approximately 40% experiencing difficulties initiating or maintaining sleep, contributing to chronic fatigue and diminished quality of life.

[0093] Sleep disruptions are linked to various diseases, including cancer, with breast cancer patients experiencing the highest prevalence. Approximately 30-70% of cancer patients report sleep problems, about twice as many as the general population. Research suggests a connection between sleep disruption, circadian misalignment, and tumor initiation and growth. Studies indicate an inverse association between sleep duration and breast cancer risk, emphasizing the importance of assessing sleep's contribution to tumorigenesis. Sleep disruption is also implicated in altering immune responses, potentially affecting cancer outcomes.

[0094] Further aspects of the invention relate to a DREAM complex inhibitor for use as a sleep mimetic, or for mimicking and / or obtaining the effect of sleep, in particular the restorative effect of sleep.

[0095] In one embodiment, the DREAM complex inhibitor is a sleep mimetic. In one embodiment, the DREAM complex inhibitor is a sleep mimetic, or is administered as a sleep mimetic. In embodiments, the DREAM complex inhibitor for use as a sleep mimetic is formulated to induce a sleep-like restorative state in the absence of natural sleep. In embodiments, the DREAM complex inhibitor mimics one or more physiological processes typically associated with sleep. In embodiments, the DREAM complex inhibitor for use as a sleep mimetic replicates the restorative benefits of sleep.

[0096] In one embodiment, the present invention relates to a DREAM complex inhibitor for use in the treatment of insomnia. In embodiments, the present invention relates to a DREAM complex inhibitor for use in the treatment of negative implications of insomnia. In embodiments, the present invention relates to a DREAM complex inhibitor for use in the treatment of a medical condition, preferably conditions induced by circadian rhythm disruption, in patients with insomnia or other sleep disorders.

[0097] In one embodiment, the present invention relates to method for the treatment of insomnia, or for the treatment of a medical condition induced by insomnia, comprising administering a DREAM complex inhibitor to a subject in need thereof. In embodiments, the present invention relates to a DREAM complex inhibitor for use in a cosmetic method for the treatment of negative implications of insomnia.

[0098] In further embodiments, 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 mimetic to provide restorative benefits equivalent or similar to natural sleep, such as reducing fatigue, cognitive impairment, and mood disturbances associated with sleep loss or irregular sleep patterns, as well as alterations of metabolism, quality control and detoxification mechanisms caused by altered sleep.

[0099] In one embodiment, the present invention relates to a pharmaceutical composition comprising a DREAM complex inhibitor formulated for e.g., once-daily, twice-daily, or thrice-daily administration, 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 following administration. In embodiments, such administration ensures effective treatment of insomnia and / or the negative implications of insomnia without significant accumulation and / or toxic effects over prolonged use, preferably as measured by pharmacokinetic parameters. In preferred embodiments, administration of the DREAM complex inhibitor does not lead to chronic and / or prolonged effective inhibitory levels in a subject. For example, the composition provides effective plasma concentrations of the DREAM complex inhibitor for a duration of not more than 16 hours, preferably not more than 14 hours, or not more than 12 hours. For example, a single administration of a p-carboline alkaloid, preferably a harmala alkaloid, or harmine, or a pharmacologically acceptable salt or ester thereof, has PK parameters that lead to a half-life of the compound, such that chronic, active levels are not achieved in the subject. In embodiments, this allows the subject to experience also an increase of the DREAM complex expression during the 24h circadian period, and thus chromatin compaction, as may be required during an awake phase.

[0100] Without being bound by theory, the invention seeks to provide DREAM complex inhibition as a sleep mimetic, in accordance with DREAM complex activity and / or expression in the subject. For example, in cases of insomnia or other forms of disrupted sleep, the DREAM complex may be overexpressed at a time in which said complex should be low or repressed in expression. One example is where the patient, who lacks sleep, has unwanted DREAM complex activity and / or expression during most of the 24h daily period, to induce chromatin compaction, which can prevent DNA repair as well as other cellular repair and detoxification activities. By way of example, during phases of reduced sleep, the DREAM complex may be highly expressed, which can work against the restorative effects that sleep would otherwise provide. Low levels of DREAM complex are associated with DNA decompaction, DNA repair, and other restorative functions, which usually occur during sleep. By having a chronically activated DREAM complex, for example in cases of insomnia, the restorative effect of sleep will not occur, due (in part) to high levels of DREAM complex expression and / or activity. In embodiments, the invention seeks to reduce DREAM complex activity and / or expression, thus providing the restorative effects of sleep, even in patients who are undergoing sleep loss or insomnia, and thus have unwanted DREAM complex expression and / or activity.

[0101] As used herein, the term “sleep mimetic” refers to an agent, compound, or the like, preferably a DREAM complex inhibitor, that induces one or more physiological effects typically associated with sleep.

[0102] In one embodiment, the present invention relates to a composition comprising a DREAM complex inhibitor for use in therapy by oral, rectal, , intranasal, intrapulmonary, or transdermal delivery, intramuscular, intraocular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral administration.

[0103] In embodiments, the dosage regimen does not cover approaches where the dosage regimen is not consistent with patient compliance, and where side effects significantly impair daily usage. A chronic administration of the DREAM complex inhibitor preferably does not induce dependency or tolerance, thus enabling short-term as well as sustained long-term benefits.

[0104] In embodiments, the present invention relates to a method of providing personalized sleep and / or circadian therapy using a DREAM complex inhibitor, comprising adjusting the dose and time of administration of said inhibitor based on the subject’s needs.

[0105] In embodiments, the present invention relates to a method for inducing restorative sleep effects in subjects with disrupted sleep patterns, the method comprising administering a DREAM complex inhibitor at a dose optimized for the patient’s needs, for example optimized to address sleep disruption, or particular phases of sleep disruption or insomnia.

[0106] In embodiments, the present invention relates to a method for the treatment of subjects experiencing sleep disturbance and comprises DREAM to be inhibited, to allow decompaction of chromatin, DNA repair, and other restorative effects of sleep.

[0107] In a further embodiment, the present invention relates to a method for mimicking the restorative effects of sleep, wherein the method comprises the administration of a DREAM complex inhibitor. In embodiments, the invention provides methods for modulating DREAM complex activity to protect genomic integrity and / or enhance DNA repair under conditions of genotoxic stress.

[0108] Uniquely, DREAM complex inhibitors can be utilized in individuals who must intentionally avoid sleep for professional reasons (e.g., shift work and the like), to deliver restorative benefits identical to the effects of sleep for this population, whose sleep is permanently disrupted. In one aspect, the present invention further relates to a pharmaceutical composition comprising the DREAM complex inhibitor with one or more pharmaceutically acceptable excipients for use according to the invention.

[0109] The various aspects of the invention are unified by, benefit from, are based on and / or are linked by the common and surprising finding of inhibitors of the DREAM complex for use in the treatment of a medical condition induced by disruption of a circadian rhythm, preferably for the treatment of medical conditions comprising acute or chronic medical impairment associated with sleep deprivation and disruption of a circadian rhythm.

[0110] DETAILED DESCRIPTION OF THE INVENTION

[0111] The present invention relates to an inhibitor of the protein complex Dimerization partner, RB-like, E2F and multi-vulval class B (DREAM complex inhibitor) for use in the treatment of a medical condition induced by disruption of a circadian rhythm.

[0112] All words and terms used herein shall have the same meaning commonly given to them by the person skilled in the art unless the context indicates a different meaning. All terms used in the singular shall include the plural of that term and vice versa.

[0113] As used herein, a "circadian rhythm" occurs over approximately one day, i.e., 24 hours, and may also be referred to or understood as an endogenous biological rhythm, endogenous oscillation, individual circadian clock, or internal clock. For example, the circadian rhythm refers to the cycle of human oscillatory gene expression, human hormone secretion, neurotransmitter secretion cycle, and core body temperature rhythm to regulate physical activity, including rest times. External cues, including light, food, and temperature, adjust such oscillation to the local environment.

[0114] The circadian rhythm ensures that human biological rhythms, including sleep, repeat in 22- to 25- hour units. These rhythms temporally structure physiology and behavior to make optimal use of the fourth dimension (i.e., time) for fitness, e.g., by anticipating predictable changes in environmental conditions and by temporally merging compatible processes or temporally separating incompatible processes.

[0115] For example, the dark hormone melatonin is secreted by the pineal gland, and is typically secreted a few hours before habitual bedtime and its secretion is suppressed by light. Cortisol, secreted by the adrenal gland, causes preparation for waking and typically peaks briefly before waking.

[0116] As used herein, the “Dimerization Partner (DP), Retinoblastoma (RB)-like, E2F, and MuvB complex,” also so known as “DREAM complex” or “DREAM / LINC complex,” is an evolutionarily conserved protein assembly that orchestrates precise gene expression crucial for cell cycle regulation. It comprises distinct components, including Retinoblastoma-Like 1 (RBL1), Retinoblastoma-Like 2 (RBL2), Dimerization Partner 1 (DP1), Dimerization Partner 2 (DP2), Dimerization Partner s (DP3), and Multi-vulval class B (MuvB), each playing a pivotal role in repressing gene expression during quiescence (GO). This repression prevents the expression of G1 / S and G2 / M phase genes, maintaining cellular dormancy. Notably, the complex's pivotal function involves inhibiting specific genes, like B-MYB (BMYB), during GO, facilitating their expression during S and G2 / M phases when the cell cycle is active.

[0117] During entry into the cell cycle, the dissociation of specific proteins from DREAM leads to the recruitment of activating E2F proteins, enabling the expression of late G1 and S phase genes. BMYB, initially repressed by the DREAM complex, becomes expressed during the S phase, binding to MuvB and promoting the expression of critical G2 / M phase genes. Further gene expression facilitation in G2 occurs via Forkhead Box M1 (FOXM1) recruitment. Subsequently, the DREAM complex reforms at the end of mitosis, repressing G1 / S and G2 / M genes again, thus completing the cycle.

[0118] DREAM complex assembly and function regulation involves phosphorylation events and interactions with crucial kinases like Dual-Specificity Tyrosine-Phosphorylation-Regulated Kinase 1A (DYRK1 A). Phosphorylation of specific residues, e.g., LIN-52's S28, is crucial for proper DREAM complex assembly and function, while DYRKIA's activity influences the association between p130 and MuvB, affecting cell cycle progression.

[0119] Docking of the DREAM complex onto gene promoters is facilitated by specific DNA sequences, enhancing binding affinity and gene repression. While the RB-E2F complex typically recruits chromatin modifiers for gene repression, the DREAM complex influences gene expression by potentially affecting nucleosome positioning rather than direct recruitment of modifiers.

[0120] Disruptions or alterations in DREAM complex regulation contribute to shifts from quiescence towards increased proliferation, observed in certain cancers with elevated mitotic gene expression levels.

[0121] 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 C. elegans, members of the DREAM (also DRM) complex include but are not limited to LIN-9, LIN-35, LIN-37, LIN-52, LIN- 53, LIN-54, DPL-1 , EFL-1. In D. 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 embodiment related to inhibition of one or more complex subunits in any one system (e.g. C. elegans or in humans), also relates to inhibition of the orthologues or homologues in other systems, as mentioned by way of example above.

[0122] The inhibition of the DREAM complex represents a novel method not previously suggested in the art that enables an alternative approach toward repairing organismal health amid persistent clock impairment, and sleep deprivation.

[0123] As used herein, “circadian locomotor output cycles kaput” (CLOCK) is a gene that encodes a transcription factor crucial for the regulation of circadian rhythms. This gene, located on chromosome 4 at gene locus 4q12 , produces a protein known as CLOCK, which plays a central role in the circadian pacemaker. Operating as a transcription factor, CLOCK forms a heterodimer with BMAL1 , binding to the E-box regulatory elements in DNA and promoting the expression of circadian genes like PER and CRY. This initiates a feedback loop where the translated PER and CRY proteins inhibit the CLOCK-BMAL1 complex, ultimately repressing their transcription. CLOCK'S unique feature is its intrinsic acetyltransferase activity, which is crucial for circadian chromatin remodeling. This enzyme, with its histone acetyltransferase (HAT) activity, influences circadian gene expression, affecting processes such as metabolism, sleep, and cardiovascular function.

[0124] Inhibitors

[0125] According to the present invention, an “inhibitor” is considered to be any agent, substance, compound, molecule, or other means that results in slowing, repressing, blocking, or otherwise interfering with or negatively affecting the activity, function, expression, or signaling that said target induces, performs, or exhibits in the absence of the inhibitor. The terms “agent,” “substance,” “compound,” and “molecule” can be used interchangeably. The terms “analog” and “derivatives” can be used interchangeably.

[0126] For example, the inhibitor of the present invention may comprise an affinity reagent, antibody, or antigen-binding fragment thereof binding the DREAM complex or subunit thereof or comprise an antisense or interfering nucleic acid molecule, such as short interfering RNA (siRNA), targeting the DREAM complex or subunit thereof, or targeted protein degradation (TPD), such as proteolysis- targeted chimera (PROTAC) molecules. Preferred inhibitors are those described herein.

[0127] The term “DREAM complex inhibitor” or analogous term relates to an established class of inhibitors known to a skilled person and established 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, a skilled person is aware of objective and routine means for determining whether any given substance falls in the functional characterization of a DREAM complex inhibitor. By way of non-limiting example, means for determining a DREAM complex inhibitor are established in the art and shown in the Examples below, as seen in Example 5.

[0128] As used herein, “Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase 1 A (DYRK1 A)” is an enzyme encoded by the DYRK1A gene in humans. It belongs to the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family and is crucial in cell regulation. The gene undergoes alternative splicing, producing several transcript variants and at least five different isoforms. DYRK1A is characterized by a nuclear targeting signal sequence, a protein kinase domain, a leucine zipper motif, and a highly conservative 13-consecutive-histidine repeat. It catalyzes autophosphorylation on serine / threonine and tyrosine residues, potentially influencing cell proliferation and brain development. Positioned in the Down syndrome critical region on chromosome 21 , DYRK1A is implicated in learning defects associated with Down syndrome. Additionally, it has clinical significance in HIV-1 replication and autism spectrum disorder, and is a potential therapeutic target for diseases like diabetes and various cancers.

[0129] DYRK1A inhibitors are typically categorized into three types: ATP-competitive (Type I), non-ATP competitive with partial binding (Type II), and non-ATP competitive with specific pocket binding (Type III). Natural products like harmine and synthetic compounds with indazole, azaindole, or benzothiazole moieties show activity as DYRK1A inhibitors. Clinical trials, such as those involving epigallocatechin-3-gallate (EGCG), a polyphenolic component from green tea, provide an example of the potential therapeutic applications of DYRK1A inhibitors in addressing diseases associated with DYRK1A dysregulation.

[0130] Various non-selective ATP competitive DYRK1 A inhibitors, such as harmine, 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 Phase 3 trials for knee osteoarthritis at an annual dose of 0.07 mg administered intraarticularly. FRTX-02 was well-tolerated in a MAD study in healthy volunteers at an oral dose of 150 mg / day. The inhibitors of the present invention are therefore commercially available and in clinical development, thus a skilled person has no difficulty in determining or sourcing the inhibitors of the present invention.

[0131] As used herein, „p-carbolines” (9H-pyrido[3,4-b]indole) are characterized by a tricyclic structure comprising a benzene ring fused to a pyridine ring and may include various substitutions on the rings. Structurally, p-carbolines belong to the group of indole alkaloids, with a pyridine ring fused to an indole skeleton. p-Carboline alkaloids are widespread in prokaryotes, plants and animals. Some P-carbolines, notably tetrahydro-p-carbolines, may be formed naturally in plants and the human body with tryptophan, serotonin and tryptamine as precursors. They are widespread in prokaryotes, plants, and animals, with diverse derivatives in Banisteriopsis caapi, Peganum harmala, ascidians, cyanobacteria, various foods (e.g., fish, meat, raisins), and scorpion cuticles. p-Carbolines exhibit various biological activities, including DNA intercalation, inhibition of enzymes, and interactions with receptors, showcasing sedative, anxiolytic, hypnotic, antitumor, antiviral, antiparasitic, and antimicrobial properties. The pharmacological effects of specific p-carbolines are dependent on their substituents. Various substituents and derivatives are known in the field and can be identified by a skilled person without undue effort.

[0132] Examples of p-carbolines include but are not limited to pinoline, harmane, harmine, harmaline, harmalol, tetrahydroharmine, 9-methyl-p-carboline, 3-carboxy-tetrahydrononharman, and / or their pharmacologically acceptable salt or ester thereof.

[0133] In embodiments, p-carbolines inhibit the DREAM complex. In preferred embodiments, the inhibitor is a harmala alkaloid.

[0134] As used herein, “harmala alkaloids,” derived from the beta-carboline molecule, for example harmine, harmaline, and tetrahydroharmine, share a common structural framework characterized by an indole skeleton fused to a pyridine ring. These alkaloids function as reversible inhibitors of monoamine oxidase A (RIMAs), selectively affecting the MAO-A isoform of the enzyme at reasonable doses. This inhibition potentiates neurotransmitters like serotonin and norepinephrine, extending their activity in the central nervous system. Harmala alkaloids exhibit specificity for MAO-A, making them less risky when combined with foods containing tyramine.

[0135] In preferred embodiments, the inhibitor is harmine or a pharmacologically acceptable salt or ester thereof.

[0136] “Harmine” (7-Methoxy-1-methyl-9H-pyrido[3,4-b]indole) is an alkaloid from the harmane alkaloid group and therefore also a p-carboline. It occurs prominently in Banisteriopsis caapi and Syrian rue. It also known by various other names, such as anisterin, banisterine, telopathin, telepathine, leucoharmine, and yageine. In the human body, it has a stimulating effect on the central nervous system as a reversible MAO inhibitor (monoamine oxidase inhibitor). 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 the enzyme monoamine oxidase. It thus delays the metabolization of the neurotransmitters serotonin and dopamine, the hormone melatonin and various tryptamine- based hallucinogens such as DMT, psilocybin and mescaline. Only its function as an MAO inhibitor delays the breakdown of DMT, which is also contained in the potion, to such an extent that it can also have an effect when taken orally.

[0137] The biosynthesis of harmine involves complex biochemical pathways. While the precise precursor in the biosynthesis, free tryptamine or L-tryptophan, remains a subject of investigation, L-tryptophan is postulated as the primary precursor. The proposed biosynthetic scheme unfolds through the Shikimate acid pathway, resulting in the formation of harmine through sequential steps, including decarboxylation, rearrangement, and hydroxylation.

[0138] At the molecular level, harmine interacts with various molecular targets. It exhibits substantial inhibitory affinity towards dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), a serine / threonine kinase. Harmine's interaction with DYRK1A holds significance in neurological contexts, and its inhibition is associated with altered tau phosphorylation, suggesting potential implications for neurodegenerative disorders. Moreover, harmine modulates neurotransmitter pathways, acting as an inverse agonist at the benzodiazepine site of gamma-aminobutyric acid type A (GABA-A) receptors.

[0139] Harmine is also used as a fluorescent pH indicator. Its fluorescence decreases as the pH value rises.

[0140] Derivatives of harmine may be presented but not limited to the following chemical structures: C6H5CH2-, 4-CI-C6H4CH2-, C6HHCH2-, 4-NO2-C6H4CH2-, C6H5-(CH2)3CH2-, 4-CN-C6H4CH2-, 4-Br- C6H4CH2-.

[0141] In another embodiments, the DREAM complex inhibitor for use according to the present invention is “inhibitor of Dyrk,” also known as “INDY,” or a pharmacologically acceptable salt or ester thereof.

[0142] INDY ((1Z)-1-(3-ethyl-5-hydroxy-2(3H)-benzothiazoylidene)-2-propanone) is a potent and ATP- competitive inhibitor of the DyrklA and Dyrkl B kinases, with IC50 values of 0.24 pM and 0.23 pM, respectively. At a molecular level, INDY achieves inhibition by competitively binding to the ATP pocket of the DyrklA enzyme. The benzothiazole structure of INDY facilitates extensive hydrophobic interactions with amino acid residues such as Val173, Ala186, Phe238, Leu241 , Leu294, and Val306, creating a robust binding interface. Beyond its specific inhibition of DyrklA, INDY exhibits a broad spectrum of inhibitory effects on related kinases, such as DYRK2, DYRK3, CLK1 , CLK4, casein kinase 1 (CSNK1 D), and PIM1 and distinguishes itself from harmine by lacking inhibitory activity against monoamine oxidase A.

[0143] In some embodiments, the inhibitor comprises an affinity reagent, antibody or antigen-binding fragment thereof binding the DREAM complex or subunit thereof, or comprises an antisense or interfering nucleic acid molecule, such as short interfering RNA (siRNA), targeting the DREAM complex or subunit thereof. A skilled person is familiar with various inhibition techniques and reagents.

[0144] Medical conditions

[0145] The terms “disorder,” “disease,” or “medical condition” as used herein, can be used interchangeably.

[0146] In embodiments, circadian rhythm-related disorders may be associated with or induced by changes in strength of circadian rhythm, amplitude, circadian rhythm phase shift, and internal desynchronization. Such conditions can be caused by endogenous factors, such as a genetic variation, age, gender, menorrhea, disturbed melatonin or cortisol secretion, delayed or advanced sleep phase syndrome, or external factors, such as environmental factors, e.g., time zone crossing travel, working hours at night, rotating shift work, stress, jetlag, social jetlag. Circadian rhythm disorders comprise Non-24-Hour disorders, circadian rhythm sleep disorder types comprising jet lag type (jet lag disorder), shift work type (shift work disorder), and altered sleep phase types, including delayed sleep phase syndrome and advanced sleep phase syndrome. Circadian sleep rhythm disturbances may also occur in patients with other diseases, including, but not limited to, neurodegenerative disorders such as Alzheimer’s or Parkinson’s disease, patients who have had head trauma or encephalitis, patients with psychiatric diseases, and patients in intensive care units.

[0147] Circadian rhythm sleep disorders typically refer to a desynchronization between internal sleep-wake rhythms and the light-darkness cycle. Patients typically suffer from insomnia, excessive daytime sleepiness, or both, which usually resolve as the body clock realigns itself. If the rhythms adapt, the symptoms may subside over several days or, in some patients (e.g., elderly patients), over several weeks or months. As known to the skilled person, light is a strong synchronizer of circadian rhythms. As a therapeutic measure, exposure to bright light (sunlight or artificial light with an intensity of 5,000 to 10,000 lux) and activity after the desired wake-up time and using sunglasses to reduce light exposure before the desired bedtime is beneficial for rapid adaptation to the circadian rhythm. An important factor here is the correct determination of the circadian phase and amplitude of the circadian rhythm.

[0148] In embodiments, the medical condition is induced by and / or comprises a loss of tissue function, oxidative stress, and / or dysfunction of cellular homeostasis.

[0149] As used herein, the term “tissue” refers to a group of cells with similar structures and functions and is a fundamental organizational level between cells and complete organs in biology. There are four main tissue types: epithelial, connective, muscle, and nervous, each designed for specific functions. Epithelial tissue covers organ surfaces and provides protection, secretion, and absorption. Connective tissue, with variations like blood and bone, supports and binds organs, aiding in nutrient transport and tissue repair. Muscle tissue generates movement, including skeletal, smooth, and cardiac types. Nervous tissue, comprising neurons and glial cells, forms the central and peripheral nervous systems, facilitating rapid communication. Each tissue type is crucial in supporting and maintaining overall bodily health.

[0150] “Oxidative stress” arises from an imbalance between reactive oxygen species (ROS) and the biological system's capacity to neutralize them or repair the resulting damage. At the molecular level, disturbances in cellular redox states lead to toxic effects, generating peroxides and free radicals that detrimentally affect cellular components, including proteins, lipids, and DNA. The base damage and strand breaks in DNA, induced by ROS like superoxide radicals and hydrogen peroxide, contribute to long-term effects, with severe stress causing cell death. The production of highly reactive species, such as free radicals and peroxides, aggravates cellular damage, particularly in DNA. DNA lesions, including complex tandem lesions, are frequently formed and associated with oxidative stress, aging, and cancer. Repair mechanisms continuously address oxidative DNA damage, but cellular damage surpasses repair capabilities under severe stress, leading to ATP depletion and uncontrolled cell death. Polyunsaturated fatty acids, which are primary ROS targets, undergo oxidation, yielding various products that not only serve as markers of oxidative stress but also contribute to tissue and DNA damage.

[0151] “Cellular homeostasis” is the maintenance of stable conditions within cells, which is crucial 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, the primary cellular energy source, through nutrient breakdown. Dysregulation can lead to conditions like mitochondrial myopathies. Autophagy, a recycling process, removes damaged cellular components critical for cellular health. Protein ubiquitination tags proteins for degradation, ensuring proper cell cycle progression, DNA repair, and signaling. Dysregulation may contribute to cancer and neurodegenerative diseases.

[0152] As used herein, “sleep deprivation” is a pervasive condition characterized by an insufficient or inadequate duration and quality of sleep required for optimal alertness, performance, and health. Sleep is a crucial physiological process that supports various functions, including memory consolidation, emotional regulation, immune function, and overall health maintenance. The recommended sleep duration varies with age, ranging from 9-11 hours for school-age children to 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 issues, sleep disorders, and other medical conditions. Lifestyle behaviors such as inconsistent bedtimes, the use of electronic devices before bedtime, and shift work can disrupt normal sleep patterns. Sleep disorders like insomnia, sleep apnea, restless leg syndrome, and bruxism can impair both the duration and quality of sleep. Additionally, medical conditions such as chronic pain, mental health disorders, diabetes, and substance abuse can contribute to sleep deprivation.

[0153] Symptoms of sleep deprivation include but are not limited to changes in sleep patterns, mood alterations, cognitive impairments, and physical effects. Falling asleep unintentionally, feeling fatigued, experiencing irritability, changes in mood, difficulty concentrating, and increased appetite are among the common signs. While the immediate effects of sleep deprivation impact cognitive and emotional functions, chronic sleep deficiency has long-term consequences on brain function, mental health, and the risk of chronic conditions.

[0154] Sleep deprivation's impact extends to various aspects of health, including brain function, immune health, and the risk of chronic conditions like obesity, diabetes, and cardiovascular diseases. Sleep deprivation is closely linked to mental health disorders such as anxiety and depression, and its bidirectional relationship suggests a complex interplay between sleep and mental well-being.

[0155] In the medical context, the term "acute" describes a disease characterized by a recent onset or short duration. The term denotes suddenness, indicating that symptoms manifest, change, or worsen rapidly. This is antonymous to "chronic," signifying conditions that persist over an extended period. Additionally, "acute" implies a sense of urgency, often suggesting that the illness is rapidly progressive and requires immediate attention. The temporal scale of a disease is measured by whether it is acute, subacute, or chronic. "Subacute" indicates a longer duration or less rapid change, while "chronic" implies an indefinite duration or minimal change over time. The specific time scale varies based on the nature of the disease, and a skilled person in art is able to determine distinct stages of diseases.

[0156] The terms “chronic condition,” “chronic disease,” or “chronical medical impairment” are used interchangeably and are characterized by its persistent or long-lasting effects, typically lasting over three months. Chronic conditions often impact multiple areas of the body, exhibit incomplete responsiveness to treatment, and endure over an extended duration. Periods of remission and relapse, where the condition temporarily improves or reoccurs, are common in chronic diseases. These conditions are frequently linked to non-communicable diseases, which have non-infectious causes and encompass a range of health states, including syndromes, physical impairments, disabilities, and diseases.

[0157] Chronic conditions play a significant role in contributing to disease, disability, and reduced physical or mental capacity. Risk factors for these conditions vary based on age and gender, with many prevalent chronic diseases stemming 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 disparities in care. Barriers to medical access and delays in receiving care further compound challenges for patients from minority and underserved populations.

[0158] In one embodiment, the medical condition comprises an acute medical impairment associated with sleep deprivation and disruption of a circadian rhythm, wherein the acute medical impairment is one or more of fatigue, lethargy, cognitive impairment, brain fog, impaired memory, gastrointestinal discomfort, cardiovascular impairment, blood pressure alteration, decline of immune-competence, change in mood, depression, anxiety and / or paranoia. As used herein, “fatigue” refers to a state of tiredness or exhaustion, often following prolonged physical or mental activity. In a medical context, fatigue is complex and can be associated with various conditions such as autoimmune diseases, organ failure, chronic pain, mood disorders, heart disease, infectious diseases, and post-infectious states. Physical fatigue results from muscle fatigue due to intense physical activity, while mental fatigue arises from prolonged cognitive activity, impairing cognitive abilities. Fatigue can manifest as sleepiness, lethargy, or directed attention fatigue, distinct from normal tiredness caused by daily activities.

[0159] “Lethargy” is a medical condition characterized by profound and persistent drowsiness, where the affected person can be aroused only with difficulty and temporarily. It signifies an unusual decrease in consciousness, distinct from usual drowsiness or sleepiness. The term is associated with conditions such as sleeping sickness (African trypanosomiasis) and encephalitis lethargica. Lethargy implies slow movement, difficulty in arousal, and impaired cognitive functions, including thinking, concentrating, and remembering. Unlike fatigue, which relates to physical exhaustion without mental impairment, lethargy indicates an impact on the brain and overall cognitive functions.

[0160] “Cognitive impairment” refers to a medical condition comprising difficulties in memory, learning, concentration, decision-making, and other mental processes that impact daily life. Ranging from mild to severe, mild impairment allows individuals to perform everyday activities, while severe impairment can lead to a loss of understanding, communication abilities, and independence. Common signs include memory loss, repetitive behavior, difficulty recognizing people or places, mood changes, vision problems, and challenges in planning and executing tasks. Cognitive impairment encompasses various conditions affecting cognition, including memory, attention, language, and executive functions. Assessments, including neuropsychological tests like the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA), help diagnose cognitive impairment. The condition can be temporary (delirium) or range from mild to severe, impacting an individual's ability to remember, solve problems, and engage with their surroundings.

[0161] As used herein, “clouding of consciousness,” commonly known as “brain fog” or “mental fog,” refers to a medical condition where an individual experiences reduced wakefulness and awareness, struggles to grasp time or surroundings, and faces challenges in maintaining attention. Brain fog is associated with perceived cognitive impairment. It is characterized by fluctuating states of cognitive dysfunction that impact daily activities. This phenomenon is prevalent in various illnesses, particularly those involving chronic pain, affecting 15% to 40% of individuals with chronic pain as a major component of their condition. In such cases, chronic pain may deplete cognitive resources, hindering effective thinking. Brain fog can also result from factors like lack of sleep, poor nutrition, medications, or drugs.

[0162] The term “memory impairment” or “memory loss” refers to the impairment or inability to recall information, experiences, or events previously stored in the brain, impacting an individual's ability to remember and retrieve past memories. Sleep deprivation has significant implications for episodic memory, influencing item-level and associative forgetting. Comparative studies involving overnight sleep, daytime wakefulness, and overnight sleep deprivation reveal heightened forgetting during wakefulness and sleep deprivation, with an additional deficit in associative memory observed specifically after extended periods of sleep deprivation. The impact of sleep deprivation on memory recall is severe, disrupting the normal consolidation process and leading to impairments in memory recall accuracy. Sleep deprivation may induce irregular fragmentation among episodic representations, altering the qualitative nature of forgetting. “Blood pressure” (BP) reflects the force of circulating blood against blood vessel walls. This pressure results mainly from the heart's pumping action within the circulatory system. Typically measured in millimeters of mercury (mmHg), blood pressure involves two values: systolic pressure 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 blood pressure in adults is typically considered to be between 90 / 60 mmHg and 120 / 80 mmHg, though this can vary based on age, health, and individual factors.

[0163] As used herein, “immunocompetence” refers to the body's capacity to generate a normal immune response when exposed to an antigen. It stands in contrast to immunodeficiency, which indicates a compromised immune system. Immunocompetence, concerning lymphocytes like B and T cells, implies maturity and the ability to recognize antigens, enabling an effective immune response. Positive selection, a process involving the recognition of major histocompatibility complex (MHC) molecules, is crucial for lymphocytes to become immunocompetent.

[0164] As used herein, “mood changes,” “change in mood,” or “mood swings” encompass extreme or sudden shifts in mood and can serve either a constructive role in problem-solving or be disruptive. Severe mood swings may be indicative of mental illnesses like bipolar disorder, characterized by erratic and disruptive mood fluctuations. Terminology such as mood swings, mood instability, affective lability, or emotional lability describes fluctuating or oscillating emotions. These terms are used interchangeably, although they may have unique characteristics describing specific patterns. Mood dynamics are influenced by various factors, resulting in erratic, labile, or unstable patterns. Mood swings can range from normal struggles around self-esteem to the wild oscillations of bipolar disorder, with durations varying from ultrarapid to extending over days or weeks. External triggers, stressors, and psychiatric illnesses can contribute to mood swings. Sleep plays a crucial role in mood regulation, and sleep deficiency can impact mood, contributing to negative emotions and increasing the risk of mood disorders such as depression and anxiety. Conversely, mood disorders can disrupt sleep patterns, emphasizing the bidirectional relationship between sleep and mood.

[0165] “Depressive disorder,” commonly known as “depression,” is a prevalent mental illness characterized by a prolonged depressed mood or loss of interest in activities. It differs from normal mood changes and can impact various aspects of life, including relationships, work, and daily functioning. Depression can affect anyone but is more prevalent in individuals who have experienced abuse, severe losses, or stress. Women are more susceptible than men, with global estimates indicating that around 3.8% of the population, including 5% of adults, and 5.7% of adults older than 60, experience depression. Symptoms of depression include persistent sadness, disrupted sleep, 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 unique characteristics and severity levels.

[0166] “Anxiety” is an emotion characterized by inner turmoil, worried thoughts, and physical changes like increased blood pressure. Anxiety disorders, including generalized anxiety disorder (persistent anxiety interfering with daily life), panic disorder (frequent, unexpected panic attacks), social anxiety disorder (intense fear of judgment in social situations), and various phobia-related disorders, involve excessive and persistent worry or fear. Symptoms range from restlessness and irritability to physical manifestations like sweating and trembling. Phobias involve irrational fears leading to avoidance behaviors. Anxiety disorders can persist, affecting daily activities and relationships. Separation anxiety disorder, often associated with children, can also affect adults, causing fear of separation from loved ones. Anxiety, when persistent and severe, may develop into anxiety disorders, lasting for extended periods and often coexisting with other mental disorders. “Paranoia” is characterized by delusions, often organized, and can lead to a personality disorder diagnosis; antipsychotic medication is often beneficial. It involves irrational thoughts influenced by anxiety, suspicion, and fear, with persecutory beliefs and conspiracy theories distinct from phobias. False accusations and general distrust accompany paranoia, and individuals may exhibit attribution bias, perceiving accidental behaviors as intentional threats. Common symptoms include feelings of powerlessness, depression, isolation, and relinquishing activities. Paranoia is linked to psychosis, and various subtypes like erotic, persecutory, litigious, and exalted exist. Single status is common among paranoid individuals, impacting interpersonal relationships. Types of paranoia are categorized by commonality, with social anxiety being the most frequently exhibited. Severe paranoia may result in isolating behaviors, leading to a diagnosis of personality disorder.

[0167] Circadian disruption plays a significant role in various “neurologic disorders,” influencing their development, expression, and severity. “Neurodevelopmental disorders” may include autism spectrum disorders (ASDs) and rare genetic conditions like Angelman, Williams, Prader-Willi, fragile X, and Smith-Magenis syndromes and often exhibit circadian disturbances and poor sleep quality. ASDs, in particular, are associated with biomarkers of circadian disruption, such as misaligned cortisol and lower-amplitude melatonin rhythms. Strategies to address circadian disruption in conditions like Smith-Magenis syndrome involve the administration of pi -adrenergic antagonists and melatonin supplementation for realignment. In “neurodegenerative disorders” like Parkinson's disease (PD) and Alzheimer's disease (AD), bidirectional relationships with circadian disruption are observed. Circadian misalignment and reduced rhythm amplitudes are predictive of neurodegenerative disease development. PD, characterized by disrupted dopamine regulation, shows circadian disruption at various disease stages, suggesting a potential role in pathogenesis. Bright light therapy and melatonin are explored as circadian-based therapeutics for PD. AD, associated with SCN neuron loss and impaired light input pathways, exhibits circadian disruption across multiple levels, correlating with the degree of neurologic impairment.

[0168] Circadian disruption is a common factor in “psychiatric diseases,” encompassing conditions like schizophrenia and mood disorders, including depression, bipolar disorder, and seasonal affective disorder. Seasonal affective disorder, marked by depressive symptoms during fall and winter with reduced daylight, demonstrates the impact of seasonally induced circadian misalignment on mental health. Another instance is delayed sleep-wake phase disorder, where misalignment between melatonin onset and bedtime increases the likelihood of depressive symptoms. The term "circadian depression" has been coined to describe this clinical phenotype, emphasizing the need for circadian- targeted treatment in mood disorders with specific rhythmicity, seasonality, and treatment responses. Preclinical evidence in mice establishes a bidirectional relationship between mood disorders and the circadian system, with serotonin receptor manipulation inducing circadian disruption. External factors like light at inappropriate times induce signs of depression, mediated by retinal ganglion cells projecting to key brain regions. Functional MRI studies in humans suggest light influences mood by suppressing amygdala activity and enhancing prefrontal cortex connectivity. Clock gene expression alterations and genetic polymorphisms in clock genes are mechanistic contributors to mood disorders. Synchronizing the brain clock with the external environment has been associated with decreased depression risk.

[0169] The influence of light conditions on the efficacy of psychiatric drugs supports the potential for circadian-based treatment approaches in psychiatric diseases. Chronotype, or individual preferences for activity timing, also impacts therapeutic effectiveness. Lithium, a treatment for bipolar disorder, shows varied effects based on chronotype, with responders tending to be "morning larks." Bright light therapy emerges as a relevant intervention across various mood disorders, including major depression, bipolar disorder, and seasonal affective disorder.

[0170] As used herein, the terms “cardiovascular disorder,” “heart disease,” and “cardiac disease” are interchangeable with, or at least related to, the terms “cardiomyopathy” or “cardiovascular-related disorders.” Cardiovascular disorders are a large disease class that affects the heart and / or blood vessels (arteries and veins). Cardiovascular disorders include arrhythmias, vascular disease, myocardial infarction, heart failure, myocarditis, atherosclerosis, restenosis, coronary heart disease, coronary artery disease, atherosclerotic cardiovascular disease, arterial hypertension, cardiac fibrosis, stroke, sudden cardiac death syndrome, heart failure, ischemic heart disease, ischemic cardiomyopathy, myocardial infarction, coronary artery calcification. These diseases have similar causes, mechanisms, and treatments. Most cardiovascular disorders have common risk factors, including inflammation, fibrosis, diabetes, cholesterol, and vascular deposits.

[0171] Circadian rhythms profoundly influence cardiovascular health, impacting the timing of myocardial infarctions, strokes, and ventricular arrhythmias. The vulnerable morning window for cardiovascular events is partly attributed to the circadian regulation of prothrombotic factors. Circadian misalignment between environmental rhythms and internal clocks increases the risk of cardiovascular diseases (CVD), inducing hypertension, inflammation, and heightened CVD risk in night shift workers. Disruptions in blood pressure regulation and arrhythmias are observed, and the intricate interplay between circadian disruption and CVD is exemplified in individuals with type 2 diabetes. Implementing circadian-based therapeutic strategies, such as bright light therapy to induce core clock gene expression, shows promise in targeting the vulnerable morning period for CV events. Chronotherapy for CVD is gaining attention, with studies supporting bedtime treatment for hypertensive patients. In children, circadian rhythms vary widely by age, and factors like obesity and pediatric diseases disrupt cardioprotective physiology, emphasizing the need for interventions to mitigate long-term CVD risk in this population. Modifiable behavioral factors, including screen time and school start times, present opportunities for targeted interventions in adolescents.

[0172] “Metabolic disorders” encompass a range of conditions affecting genetic, cellular, and systemic metabolism. This includes impaired glucose tolerance, insulin resistance, and an increased risk of disorders such as diabetes and obesity. Moreover, metabolic disorders negatively alter the body’s processing and distribution of substrates, supplements, and nutrients, including proteins, carbohydrates, fatty acids, and lipids, resulting in abnormal chemical reactions in the body which is associated with pathophysiological changes in the body. The intricate relationship between the circadian system and metabolism significantly impacts diabetes and obesity. Circadian dysregulation leads to impaired glucose tolerance, insulin resistance, and an increased risk of metabolic disorders. Mutations in core clock genes, like C / ocAA19, and disruptions in BmaH contribute to metabolic impairment. Social jet lag, internal and social timing discrepancies, and evening chronotypes are associated with diabetes risk. The timing of food consumption and light exposure characteristics influence metabolic function.

[0173] Obesity is linked to C / ocA 19 mutation and specific CLOCK gene variants. Polymorphisms in CLOCK and melatonin receptor genes are associated with increased BMI. Shift workers face an elevated risk of metabolic disorders and obesity, with social jet lag linked to overweight. Time- restricted feeding and light exposure timing are proposed strategies for aligning energy intake with metabolic rhythms, potentially improving health and weight regulation.

[0174] “Immunologic disorders,” also known as “immune disorders” or “immunological diseases,” refer to conditions in which the immune system malfunctions, either by overactivity (autoimmune disorders) or underactivity (immunodeficiency disorders). Examples of immunologic disorders include Autoimmune Disorders, such as Rheumatoid Arthritis, Lupus (Systemic Lupus Erythematosus), Type 1 Diabetes, and Multiple Sclerosis. Immunodeficiency Disorders may include Primary Immunodeficiency Diseases (e.g., Severe Combined Immunodeficiency - SCID), Acquired Immunodeficiency Syndrome (AIDS), Common Variable Immunodeficiency (CVID, and DiGeorge Syndrome). Allergic and Hypersensitivity Reactions may include Allergic Rhinitis, Asthma, and Anaphylaxis.

[0175] “Allergic disorders” or “allergies” result from an immune system hypersensitivity to typically harmless environmental substances, triggering various symptoms in different organs. The immunological foundation of allergies involves two phases: sensitization and the development of memory T and B cell responses, followed by IgE production and effector functions. Eosinophils, innate lymphoid cells, dendritic cell subsets, epithelial cells, tissue inflammation, epithelial barrier, tissue remodeling, and chronicity play crucial roles in allergic diseases like asthma, atopic dermatitis (AD), and allergic rhinitis (AR). Distinct molecular mechanisms, biomarkers, and responses to biological therapy characterize different disease phenotypes and endotypes.

[0176] Allergies encompass conditions from the immune system's hypersensitivity to typically harmless substances. These include hay fever, food allergies, atopic dermatitis, allergic asthma, and anaphylaxis. Symptoms manifest in various organs, such as red eyes, itchy rash, sneezing, coughing, runny nose, shortness of breath, and swelling. Allergens like pollen, certain foods, metals, insect stings, and medications can induce allergies. Genetic and environmental factors contribute to allergy development. Immunoglobulin E antibodies (IgE) binding to allergens trigger the release of inflammatory chemicals, causing symptoms. Diagnosis involves medical history and, in some cases, skin or blood testing. Treatments include allergen avoidance, medications like steroids and antihistamines, and, in severe cases, injectable adrenaline. Allergen immunotherapy is useful for specific allergies.

[0177] “Gastrointestinal diseases” (Gl diseases) encompass disorders affecting the entire gastrointestinal tract, spanning the esophagus, stomach, small and large intestines, rectum, and accessory organs like the liver, gallbladder, and pancreas. While gastrointestinal symptoms are prevalent, functional gastrointestinal disorders (FGIDs) like irritable bowel syndrome and functional dyspepsia are common, lacking structural explanations. The pathophysiology involves bidirectional dysregulation of gut-brain interaction, microbial dysbiosis, altered immune function, visceral hypersensitivity, and abnormal gastrointestinal motility. Psychological comorbidity often accompanies these disorders. The biopsychosocial model emphasizes the complex interplay between physical symptoms and psychological factors. FGIDs are characterized by brain-gut axis dysregulation rather than structural abnormalities. Low-grade intestinal inflammation, altered microbiome, and immune activation contribute to FGID pathogenesis. Structural gastrointestinal diseases, in contrast, involve abnormalities visible upon examination and affect motility, requiring surgical intervention in some cases. Examples may include strictures, hemorrhoids, colon polyps, and inflammatory bowel disease. Sleep dysfunction is intricately linked to gastrointestinal disorders, e.g., gastroesophageal reflux disease, inflammatory bowel disease, liver disorders, and colorectal cancer.

[0178] Gastroesophageal reflux disease demonstrates a clear connection between sleep disturbances and nocturnal symptoms, impacting the risk of esophageal complications. In peptic ulcer disease, the prevalence of ulcers is higher in shift workers, possibly due to unpredictable meal timings, sleep dysfunction, work stress, and nonsteroidal anti-inflammatory drug use. Sleep disturbances are prevalent in irritable bowel syndrome and functional dyspepsia, affecting sleep quality and exacerbating gastrointestinal symptoms. Studies also highlight the bidirectional relationship between inflammatory bowel disease and sleep dysfunction, emphasizing the potential for disturbed sleep to contribute to disease relapse. Patients with cirrhosis, even without hepatic encephalopathy, experience sleep disturbances attributed to circadian rhythm dysfunction. Furthermore, chronic hepatitis C infection is associated with sleep disturbances, independent of psychiatric conditions. Obstructive sleep apnea, affecting 3% to 7% of adults, plays a role in various gastrointestinal conditions, including nonalcoholic fatty liver disease and cirrhosis.

[0179] “Rheumatism,” also known as “rheumatic disorders,” encompasses over 200 conditions causing chronic, intermittent pain in joints or connective tissue. It includes arthritis and "non-articular rheumatism," which overlap with the term soft tissue disorder. Major categories of rheumatic disorders may include diffuse connective tissue diseases (e.g., rheumatoid arthritis, lupus), arthritis associated with spondylitis (e.g., ankylosing spondylitis, psoriatic arthritis), osteoarthritis, rheumatic syndromes with infectious agents, metabolic and endocrine diseases (e.g., gout), neoplasms, neurovascular disorders, bone and cartilage disorders, and extraarticular disorders (e.g., bursitis, tendinitis). Rheumatic diseases, like osteoarthritis and rheumatoid arthritis, can cause severe joint pain due to cartilage breakdown.

[0180] “Chronic obstructive pulmonary disease” (COPD) is a progressive lung disease characterized by long-term respiratory symptoms and airflow limitation, with common types being emphysema and chronic bronchitis. While emphysema involves enlarged airspaces and permanent lung tissue damage, chronic bronchitis is defined by a productive cough lasting at least three months each year for two years. COPD often results from tobacco smoking, but other risk factors include air pollution, occupational irritants, and genetic factors like 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 like asthma, cardiovascular disease, and lung cancer. Treatment includes smoking cessation, vaccinations, medications, and interventions like oxygen therapy or lung transplantation.

[0181] “Cancer,” characterized by abnormal cell growth with the potential to invade other body parts, encompasses over 100 types, contrasting with benign tumors that do not spread. Causes include tobacco use, obesity, poor diet, infections, ionizing radiation, and genetic defects. Treatment methods include surgery, radiation, chemotherapy, and targeted therapy. Symptoms vary, and cancer may be challenging to diagnose, often imitating other conditions. Local symptoms result from the tumor's mass or ulceration, while systemic symptoms arise from the body's response.

[0182] Metastasis, the spread of cancer, commonly occurs in the late stages, impacting organs like the lungs, liver, brain, and bones. Cancers are classified by cell types (carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, blastoma), often named based on organ or tissue.

[0183] When altered, genes like proto-oncogenes, tumor suppressors, and DNA repair genes become cancer drivers. Causes include errors during cell division, DNA damage from environmental factors, and inherited mutations. Cancerous cells differ from normal cells in their uncontrolled growth, invasion, immune system evasion, and altered nutrient reliance.

[0184] Various causes for above-mentioned disorders exist. In embodiments, the cause of these disorders are due to circadian rhythm disruptions. In further embodiments, the above-mentioned conditions can be treated with the inhibitors of the DREAM complex and methods disclosed herein.

[0185] Treatment

[0186] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits a symptom of a disease, disorder, or condition that can be treated with the inhibitors of the DREAM complex and methods disclosed herein. In preferred embodiments, a subject includes any animal that exhibits symptoms of a disease, disorder, or condition associated with sleep deprivation and disruption of a circadian rhythm, e.g., an acute medical impairment, including fatigue, lethargy, cognitive impairment, brain fog, impaired memory, gastrointestinal discomfort, cardiovascular impairment, blood pressure alteration, decline of immune-competence, change in mood, depression, anxiety and / or paranoia, or a chronic medical condition, including neurologic, psychiatric, cardiovascular, metabolic, allergic, immunologic, gastrointestinal, rheumatic, proliferative disease (cancer) and / or a pulmonary disorder, that can be treated with methods disclosed herein. Suitable subjects include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs). Non-human primates and, preferably, human patients are included.

[0187] As used herein, the terms “(medical) disease,” “(medical) disorder,” and “(medical) condition” are often used interchangeably.

[0188] As used herein, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can optionally involve either the reduction or amelioration of symptoms of the disease or condition or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease, condition, or associated symptoms. The phrase “therapeutically effective” is intended to include, within the scope of sound medical judgment, excessive toxicity, irritation, and / or other problems or complications but commensurate with a reasonable benefit / risk ratio.

[0189] As used herein, "prevent" and similar words such as "prevented," "preventing," or "prophylactic," etc., indicate an approach for preventing, inhibiting, or reducing 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 occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0190] The present invention relates to an inhibitor of the protein complex Dimerization partner, RB-like, E2F and multi-vulval class B (DREAM complex inhibitor) for use in the treatment of a medical condition induced by disruption of a circadian rhythm.

[0191] In embodiments, an inhibitor of the DREAM complex for use in the treatment of a medical condition induced by disruption of a circadian rhythm in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount of the inhibitors contemplated herein. The quantity and frequency of administration will be determined by such factors as the condition of the patient and the type and severity of the patient's disease, although clinical trials may determine appropriate dosages.

[0192] Pharmaceutical compositions comprising a DREAM complex inhibitor for administration to a subject can include, in embodiments, at least one further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents, and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional. The person skilled in the art is aware of compositions and formulations suitable for pharmaceutical delivery of the bi-specific agent disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

[0193] The DREAM complex inhibitor can, in embodiments, be combined with pharmaceutically acceptable carrier substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions comprising the bi-specific agent, conventional non- toxic pharmaceutically acceptable vehicles can be used, which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutical compositions, whether they are solutions, suspensions, or other like forms, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0194] A DREAM complex inhibitor comprised in a (pharmaceutical) composition can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, , intranasal, intrapulmonary, or transdermal delivery, intramuscular, intraocular, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes.

[0195] In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of DREAM complex inhibitor(s) may, in embodiments, be administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate a selected condition or one or more symptom(s) thereof, wherein the condition is caused by disruption of a circadian rhythm.

[0196] The attending clinician can vary dosage to maintain a desired concentration at a target site (for example, the lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth.

[0197] The instant disclosure also includes kits, packages, and multi-container units containing the herein- described DREAM complex inhibitor(s) or pharmaceutical compositions comprising the same and / or means for administering the same for use in the prevention and treatment of conditions described herein and other conditions in human subjects. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain, using most routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification indicate the skill level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0198] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. However, the disclosure supports a definition of only alternatives and "and / or." Throughout this application, where relevant, the term "about" indicates that a value includes the inherent variation of error for the device, the method employed to determine the value or the variation among the study subjects.

[0199] FIGURES

[0200] The invention is demonstrated by way of example in the following figures. The figures are to provide a further description of potentially preferred embodiments that enhance the support of one or more non-limiting embodiments of the invention.

[0201] Brief description of the figures:

[0202] Figure 1 : RNAi-mediated knock down of lin-42 / PER impairs clock-controlled developmental timing and exacerbates aggregation-induced tissue failure.

[0203] Figure 2: Genetic impairment of clock triggers changes in histone expression, chromatin organization, and DREAM complex abundance.

[0204] Figure 3: Inactivation of distinct DREAM complex components alleviates increased protein aggregation and tissue dysfunction caused by disruption of clock.

[0205] Figure 4: Elevation of histone expression triggered by clock impairment is dependent on DREAM activity.

[0206] Figure 5: Circadian clock disruption interferes with multiple core pathways of cellular maintenance in a DREAM-dependent fashion.

[0207] Figure 6: DREAM-clock interaction is evolutionary conserved, and pharmacological DREAM disruption restores homeostasis of clock-impaired animals.

[0208] Figure 7: RNAi-mediated inactivation of lin-42 / PER interferes with clock-dependent developmental timing.

[0209] Figure 8: RNAi-mediated disruption of clock exacerbates muscle paralysis in C. elegans models of Alzheimer’s and Huntington’s diseases.

[0210] Figure 9: Protein-protein interaction analysis places DREAM complex at the core of proteome changes induced by clock impairment. Figure 10: Whole proteome changes are comparable between animals subjected to lin-42IPER inactivation during development and adulthood.

[0211] Figure 11 : The interplay between DREAM and clock impairment demonstrates subunit specificity.

[0212] Figure 12: / n-53 / DREAM inactivation restores protein homeostasis in clock-disrupted animals expressing poly-glutamine linked YFP.

[0213] Figure 13: Clock disruption enhances the expression of histones in a DREAM-dependent manner.

[0214] Figure 14: Clock disruption elevates expression of histones in a DREAM-dependent manner.

[0215] Figure 15: Circadian clock disruption interferes with core pathways of cellular maintenance in a DREAM-dependent manner.

[0216] Figure 16: Same basic cellular processes are modulated by clock in C. elegans and mice.

[0217] Figure 17: Same basic cellular processes are modulated by clock in C. elegans and human cells.

[0218] Figure 18: DREAM expression is lowered during sleep and sleep deprivation prevents the lowering, clock and sleep disruption interfere with fundamental cellular processes in a DRM / DREAM- dependent fashion.

[0219] Figure 19: High DREAM expression protects DNA from the damage associated with wakefulness while low DREAM expression facilitates repair during sleep.

[0220] Figure 20: Clock disruption elevates the expression of histones in a DRM / DREAM-dependent manner.

[0221] Figure 21 : Circadian clock disruption interferes with fundamental cellular processes in a DRM / DREAM-dependent manner.

[0222] Figure 22: DREAM-dependent pathways are functional drivers of the negative effects of circadian disruption.

[0223] Figure 23: DREAM expression changes during the natural sleep-wake cycle.

[0224] Figure 24: DREAM subunits follow a clock oscillation dynamics and expression is increased by Melatonin treatment.

[0225] Figure 25: Same fundamental cellular processes are altered by clock disruption in mice and nematodes.

[0226] Figure 26: Same fundamental cellular activities are regulated by clock / DREAM axis in nematodes and human cells.

[0227] Figure 27: Experimental design for assessing developmental timing upon UV-B and lin-42 RNAi treatment.

[0228] Figure 28: Role of DREAM in normal sleep and sleep deprivation.

[0229] Detailed description of the figures:

[0230] Figure 1 : RNAi-mediated knock down of lin-42 / PER impairs clock-controlled developmental timing and exacerbates aggregation-induced tissue failure. (A) Luminometry experimental design. (B) Developmental timing of wild-type, Un-42 (n1089), Un-42 (pk2385) populations is shown. Worms were fed with ev or iin-42 RNAi in addition to D-Luciferin. Quantitative analysis of the developmental larvae stages at 20°C based on luminescence is presented. The average duration of respective stages is shown as black horizontal bar. n= 13-20 worms per condition, n=4 independent experiments. One-way ANOVA was used for the statistical assessment, *p<0.000001. (C) Wild-type N2 worms were age-synchronized and fed with ev or Un-42 RNAi from the L1 stage. Survival was scored daily. Animals were transferred to new plates every second day until AD10, and every fourth day thereafter. n=140 worms per condition, graph representative of three independent experiments. Significance was measured by the Log-rank Mantel-Cox test, ns, not significant. (D) Age synchronized population of Q40-YFP (t / nc-54p::Q40::YFP) animals were fed with ev or Un-42 RNAi. Paralysis was scored daily. Worms were transferred to new plates every second day. n=140 worms per condition, graphs representative of three independent experiments. Significance was measured by the Log-rank Mantel-Cox test, ****p<0.000001 .

[0231] Figure 2: Genetic impairment of clock triggers changes in histone expression, chromatin organization, and DREAM complex abundance, (a) Experimental design, (b) Principal component analysis comparing whole proteomes of wild-type C. elegans treated with ev or Un-42 RNA from the L1 stage is shown. Proteins were extracted on AD2, and small dots are representative of individual replicas, n=800 worms per replica, (c) Bar plot of significantly enriched GO biological process terms in Un-42 versus ev RNAi-treated animals is shown (FDR < 0.05). (d) Volcano plot depicting differently expressed proteins in Un-42 versus ev RNAi-treated worms is shown, with histones highlighted in red. The horizontal dashed line indicates a p-value cut-off of 0.05, and the vertical lines have a log - fold change cut-off of ±0.05. (e) Heatmap of selected DREAM subunits and interactors is shown. The color code refers to Iog2 (fold change) values between Un-42 and ev RNAi-treated worms; RNAi treatments from both L1 and L4 stages are included, (f) Mechanistic model is presented.

[0232] Figure 3: Inactivation of distinct DREAM complex components alleviates increased protein aggregation and tissue dysfunction caused by disruption of clock, (a) Experimental design, (b-e) Q40-YFP animals were fed with ev or Un-42 RNAi in combination with RNAis targeting specific subunits / interactors of the DREAM complex from the L1 stage; paralysis was scored daily. Cotargeting with lin-9 (b), lin-54 (c), lin-53 (d), and DREAM interactor his-41 (e) is shown. n=140 worms per condition, graphs representative of at least three independent experiments. Significance was measured by the Log-rank Mantel-Cox test. ****p<0.0001 . (f) Q40-YFP worms were age- synchronized, fed with indicated RNAi constructs from the L1 stage, and imaged at three different time points: L4, AD1 , and AD2. Number of aggregates divided by respective YFP intensity is shown, n=26-32 worms per condition; the graph is representative of three individual experiments. Error bars are s.e.m. Significance was measured by one-way ANOVA within each age group separately with Sidak’s multiple comparison test. ****p<0.0001 ; **p<0.01 ; ****p<0.0001 ; ns, not significant.

[0233] Figure 4: Elevation of histone expression triggered by clock impairment is dependent on DREAM activity, (a-d) WT worms were age-synchronized and treated with indicated RNAi combinations from the L1 stage until AD2. Western blot analysis of H1 and H2 histones was conducted by using tubulin as a loading control, and quantification is presented. n=800 worms per condition; each graph includes the results of 5 independent experiments. Error bars are s.e.m, representative WB images are shown in panel (e), and full gels can be seen in Fig. 13. (f) Worms were treated as in (a-d), and mRNA expression levels of his-24 (H1), hil-2 (H1), and his-29 (H2) were assessed by qPCR. n=250 per condition, graphs are representative of three independent experiments. Error bars are s.e.m.; mRNA fold change was calculated by the AA Ct method. Significance was measured by one-way ANOVA within each group separately with Sidak’s multiple comparisons test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 ; ns, not significant, (g) Mechanistic summary of the data is presented, (h- m) Worms were treated with indicated RNAi combinations and control (ev) RNAi from the L1 stage until AD2. Protein expression was resolved by proteomics. Box plots showing relative expression (ev+lin-42 over ev, and / / n-42+ / / n-53 over ev comparisons) of proteins belonging to xenobiotic metabolism (h), glutathione metabolism (i), glycolysis (j), collagens (k), transcription initiation (I) and ribosome biogenesis (m) are presented. Individual proteins are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the box plot indicate the first and third quartile and the whiskers extend 1 .5 times the interquartile range from the limits of each box. n=800 worms per condition and 4 independent populations were measured in each case. Within each box plot, the significance was assessed by the Mann-Whitney Wilcoxon rank-sum test, and Wilcoxon test was used for the comparison between the two sets of fold changes. Two-tailed p values were computed in all cases. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****- p<0.0001. (n-o) Age-synchronized t / nc-54p::Q40::YFP animals were fed with EV or lin-42 RNAi in combination with RNAis targeting specific genes related to transcription initiation and ribosome biogenesis from the L1 stage, paralysis was scored daily. Co-targeting with rpb-11 (N) and fib-1 (o) is shown. n=140 worms per condition, graphs are representative of three independent experiments. Significance was measured by the Log-rank Mantel-Cox test, two-tailed p values were computed. ****-p<0.0001. (p) Mechanistic model is depicted.

[0234] Figure 5: Circadian clock disruption interferes with multiple core pathways of cellular maintenance in a DREAM-dependent fashion, (a) Experimental design. Worms were treated with indicated RNAi combinations from the L1 stage until AD2. Protein expression was resolved by proteomics, (b-g) Boxplots showing relative expression of proteins belonging to antioxidant response (b), glycolysis (c), collagens (d), lysosome (e), transcription initiation (f), and ribosome (g) are presented. Individual proteins are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the boxplot indicate the first and third quartile, and the whiskers extend 1 .5 times the interquartile range from the limits of each box. n=800 worms per condition, and 4 independent populations were measured in each case. Significance was assessed by Wilcoxon rank-sum and two-tailed test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 . (h) Mechanistic model is depicted.

[0235] Figure 6: DREAM-clock interaction is evolutionary conserved, and pharmacological DREAM disruption restores homeostasis of clock-impaired animals, (a-b) Analysis of transcriptomic data from hippocampi of mice either WT or expressing human AD-liked APP variant (TG mice) and either fed ad libitum (ALF) or subjected to time-restricted feeding (TLF, Whittaker et al., 2023). Data for ZTO and ZT12 were analyzed. Box plots show relative expression (Log2FC) of mitochondrial (a) and OXPHOS (oxidative phosphorylation) (b) genes between indicated conditions. Individual genes are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the boxplot indicate the first and third quartile, and the whiskers extend 1 .5 times the interquartile range from the limits of each box. n=6-7 mice per condition. Significance was measured by Wilcoxon test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 . (c) Human retinal pigment epithelial cells (RPE) cells were treated with siRNA against PER1 (10 nM) for 48h and co-treated with Harmine (10pM) or DMSO (vehicle control) for 20h. qPCR analysis of indicated clock genes was performed in harvested cells. Expression fold changes calculated by the AACt method are shown. Significance was measured by one-way ANOVA within each group separately with Sidak’s multiple comparisons test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 ; ns, not significant, (d-e) Human RPE cells were treated as in (c), and full transcriptomic analysis by mRNA sequencing was conducted. Box plots show relative expression (Log2FC) of mitochondrial (d) and OXPHOS (e) genes. Individual genes are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the boxplot indicate the first and third quartile, and the whiskers extend 1 .5 times the interquartile range from the limits of each box. Significance was measured by Wilcoxon test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 . n=5 independent cultures per condition, (f) Age- synchronized population of Q40-YFP (t / nc-54p::Q40::YFP) nematodes was treated with ev or Un-42 RNAi from the L1 stage in plates containing either 0.1 % DMSO (vehicle) or Harmine (60 pM). Paralysis was scored daily. n=140 worms per condition, graphs representative of at least three independent experiments. Significance was measured by the Log-rank Mantel-Cox test.

[0236] ****p<0.0001 . (g) The model depicting the role of DREAM in cell deterioration following clock disruption is shown.

[0237] Figure 7: RNAi-mediated inactivation of lin-42 / PER interferes with clock-dependent developmental timing, (a) Developmental timing of wild-type, Un-42 (n1089), and Un-42 (ok2385) populations are analyzed. Worms were fed with ev or Un-42 RNAi in addition to D-Luciferin. Luminescence-based quantitative analysis of the duration of the molting stages at 20°C is shown. The average duration is represented by the black horizontal bar in each case. n=13-20 worms per condition, graphs represent the sum of four independent experiments. One-way ANOVA, *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 .

[0238] Figure 8: RNAi-mediated disruption of clock exacerbates muscle paralysis in C. elegans models of Alzheimer’s and Huntington’s diseases, (a) GMC101 (unc-54p . human Api -42) worms were age- synchronized and treated with ev or Un-42 RNAi from the L1 stage. Until L4 stage, the worms were cultivated at 20°C and then shifted to 25°C to trigger amyloid-beta toxicity, (b) Age-synchronized population of Q40-YFP (t / nc-54p::Q40::YFP) animals was fed with ev or Un-42 RNAi from the L4 stage, (c) GMC101 (t / nc-54p::human Api-42) worms were age-synchronized and treated with ev or Un-42 RNAi from the L4 stage. The shift to 25°C was performed as described in (a), (d) Age- synchronized population of Q40-YFP (t / nc-54p::Q40::YFP) animals were fed with ev or lin-14 RNAi. Paralysis was scored daily in all cases (a-d). n=140 worms per condition; graphs show a representative result of at least three independent experiments. Significance was measured by the Log-rank Mantel-Cox test. *p<0.05; ****p<0.0001 , ns, not significant.

[0239] Figure 9: Protein-protein interaction analysis places DREAM complex at the core of proteome changes induced by clock impairment. STRING analysis was performed with the protein groups that contributed strongest to the PC1 separation in Fig. 2b. The absolute rotational weight of the included proteins had a cutoff of >0.023, and 121 proteins had a significant network enrichment of p<1 .Oe-16. Depicted with different colors are the components or interactors of the chromatin repressor complex DREAM.

[0240] Figure 10: Whole proteome changes are comparable between animals subjected to lin-42 / PER inactivation during development and adulthood, (a) Experimental design, (b) Whole-proteome principal component analysis of wild-type C. elegans treated with ev or Un-42 RNAi from the L4 stage is shown. Small dots are representative of individual replicas, n=800 worms per sample, (c) STRING analysis was performed with the protein groups that contributed the strongest to the PC1 separation in Fig. 10b. The absolute rotational weight of the proteins had a cutoff of >0.01 , and 46 proteins had a significant network enrichment of p<7.72e'10. Depicted with different colors are the components or interactors of the chromatin repressor complex DREAM.

[0241] Figure 11 : The interplay between DREAM and clock impairment demonstrates subunit specificity, (a) 250 worms per condition were age-synchronized and treated with indicated RNAi combinations from the L1 stage, and Un-42 and lin-53 gene expression was measured by qPCR on AD2. Graphs are representative of three independent experiments. Mean and s.e.m. are presented. mRNA fold change was calculated by the AA Ct method. Significance was measured by unpaired t-test. **p<0.01 ; ***p<0.001 ; ns, not significant, (b-e) Q40-YFP (t / nc-54p::Q40::YFP) were age synchronized and fed from L1 stage with ev or Un-42 RNAi and in combination with RNAi against specific DREAM subunits or interactors: lin-37 (b), lin-52 (c), lin-35 (d), htz-1 (e). Paralysis was scored daily. n=140 worms per condition, graphs are representative of at least three independent experiments. Significance was measured by the Log-rank Mantel-Cox test. ****p<0.0001 ; *p<0.05, ns, not significant.

[0242] Figure 12: f / n-53 / DREAM inactivation restores protein homeostasis in clock-disrupted animals expressing poly-glutamine linked YFP. Q40-YFP worms were age-synchronized and fed with indicated RNAi combinations from the L1 stage and imaged at three different time points: L4, AD1 , and AD2. (a) YFP intensity per total body area in Q40-YFP worms is shown. n=26-32 worms per condition, the graph is representative of three individual experiments. Error bars are s.e.m. Significance was measured by one-way ANOVA within each age group separately with Sidak’s multiple comparison test. *p<0.05; ****p<0.0001 ; ns, not significant, (b) Representative images of AD2 Q40-YFP animals treated with indicated RNAi combinations. Worms were imaged in an AxioZoom v.16 microscope with 90x magnification with an exposure time of YFP 150 ms and Brightfield at 4.8 ms. The scale bar is 200 pm.

[0243] Figure 13: Clock disruption enhances the expression of histones in a DREAM-dependent manner. Representative full gel scans for Fig.4a-d. (a) H1 , (b) H2B1 L, (c) H2AZ1 , (d) H2AZ2. Bands used for quantification are marked by arrowheads. H= histone and T= tubulin.

[0244] Figure 14: Clock disruption elevates expression of histones in a DREAM-dependent manner, (a-b) Worms were treated and analyzed as described in Figure 4a-d, using antibodies specific to histones H3 and H4. Representative full gel scans (left) and respective quantifications (right) are shown. Graphs are based on five independent experiments. n=800 per condition and error bars are s.e.m. Significance was measured by one-way ANOVA. ns, not significant.

[0245] Figure 15: Circadian clock disruption interferes with core pathways of cellular maintenance in a DREAM-dependent manner, (a-f) Animals were treated and analyzed as described in Figure 5. Boxplots show relative expression of proteins involved in xenobiotic metabolism (a), fatty acid betaoxidation (b), oxidative phosphorylation (c), splicing (d), ribosome biogenesis (e), and translation factors (f). Individual proteins are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the boxplot indicate the first and third quartile, and the whiskers extend 1 .5 times the interquartile range from the limits of each box. n=800 per condition, and 4 independent populations were measured for each experimental cohort. Significance was measured by Wilcoxon rank-sum and two-tailed test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 .

[0246] Figure 16: Same basic cellular processes are modulated by clock in C. elegans and mice, (a-e) The transcriptomic data from Whittaker et al., 2023, was analyzed as described in Figures 6 a-b. (a) Schematic in vivo experimental design is depicted. Boxplots showing relative expression of ribosome (b), spliceosome (c), lysosome (d), and ECM (e) genes are presented. Individual genes are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the boxplot indicate the first and third quartile, and the whiskers extend 1.5 times the interquartile range from the limits of each box. n=6-7 mice per condition. Significance was measured by Wilcoxon test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 .

[0247] Figure 17: Same basic cellular processes are modulated by clock in C. elegans and human cells, (a) Human RPE cells were treated and analyzed as described in Figure 6c. qPCR analyses of the P21 gene is shown. Significance was measured by one-way ANOVA within each group separately with Sidak’s multiple comparisons test. ****p<0.0001 . (b-e) Human RPE cells were treated and analyzed as in Figure 6d-e. Box plots showing relative expression (Log2FC) of spliceosome (b), ribosome (c), ECM (d) and ribosome biogenesis (e) genes are presented. Individual genes are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the boxplot indicate the first and third quartile and the whiskers extend 1 .5 times the interquartile range from the limits of each box. Significance was measured by Wilcoxon test. *p<0.05; **p<0.01 ;

[0248] ***p<0.001 ; ****p<0.0001 . n=5 independent cultures per condition, (f) Age-synchronized population of Q40-YFP (t / nc-54p::Q40::YFP) nematodes was treated with ev or Un-42 RNAi in plates containing either 0.1% DMSO (vehicle) or Harmine (40 Paralysis was scored daily. n=140 worms per condition, graphs representative of at least three independent experiments. Significance was measured by the Log-rank Mantel-Cox test. ****p<0.0001 .

[0249] Figure 18: DREAM expression is lowered during sleep and sleep deprivation prevents the lowering, clock and sleep disruption interfere with fundamental cellular processes in a DRM / DREAM- dependent fashion, (a-b) Analysis of transcriptomic data from mice cortex, with samples collected both at sleep and wake phases (experimental scheme Fig. 23). The complete Log2 CPM data was filtered to visualize DREAM subunits expression, with Lin37 and E2f4 depicted, both under normal sleep and wakefulness (left panels) and under sleep deprivation (SD) (right panels). The expression values at ZT 3 and 6 (NSD and SD) were normalized to ZT 0 and the expression dynamics of both Lin37 and E2f4 can be observed comparing NSD and SD cohorts. Significance was measured by unpaired t-test. n= 3-5 in each condition, mean and S.E.M values are presented. *-p<0.005; **- p<0.01 ; ***-p<0.001 ; ****-p<0.0001 ; ns, not significant, (c) The same Log2 CPM value was analyzed for Lin9, another DREAM complex subunit, upper panel. Wild-type mice were treated with melatonin or vehicle for 2 weeks (experimental scheme Fig. 24), hippocampi were collected at both light and dark phases. Expression analysis of Lin9 gene was performed by RT-qPCR (lower panel). Data is shown as mRNA expression fold changes calculated by the AACt method. Significance was measured by multiple comparisons two-way ANOVA with a Bonferroni correction. n= 6-9 in each condition, mean and S.E.M values are presented. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****-p<0.0001 ; ns, not significant, (d) Box plots showing relative expression (SD (ZT3) - NSD (ZT3) and SD(ZT6) - NSD(ZT6) comparisons) of genes belonging to Mitochondria, OXPHOS, and Ribosome Biogenesis (d). Individual genes are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the box plot indicate the first and third quartile and the whiskers extend 1 .5 times the interquartile range from the limits of each box. n=3-5 mice per condition were measured in each case. Within each box plot, the significance was assessed by the Mann-Whitney Wilcoxon rank-sum test, and the Wilcoxon test was used for the comparison between the two sets of fold changes. Two-tailed p-values were computed in all cases *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****-p<0.0001 . (e) Human retinal pigment epithelial cells (RPE) cells were treated with control siRNA (siC1 , 10 nM) or siRNA against PER1 (siPERI , 10 nM) for 48h and co-treated with Harmine (Har, 10JJ.M) or DMSO (vehicle control) for 20h. Expression analysis of PER1 and CLOCK genes by mRNA sequencing with graphs showing transcript per million values (TPM). Significance was measured by one-way ANOVA within each group separately, and applying Sidak’s multiple comparisons test, two-tailed p values were computed. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****- p<0.0001 ; ns, not significant, (f) Box plots showing relative expression (PER1 DMSO versus Ctrl DMSO and PER1 Harmine versus Ctrl DMSO comparisons) of genes belonging to Mitochondria, OXPHOS, and Ribosome Biogenesis are presented (f). Individual genes are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the box plot indicate the first and third quartile and the whiskers extend 1 .5 times the interquartile range from the limits of each box. n=3-5 mice per condition were measured in each case. Within each box plot, the significance was assessed by the Mann-Whitney Wilcoxon rank-sum test, and the Wilcoxon test was used for the comparison between the two sets of fold changes. Two-tailed p values were computed in all cases. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****-p<0.0001 . Figure 19: High DREAM expression protects DNA from the damage associated with wakefulness while low DREAM expression facilitates repair during sleep, (a) Human retinal pigment epithelial cells (RPE) cells were treated with control siRNA (siC1 , 10 nM) or siRNA against PER1 (siPERI , 10 nM) for 48h and co-treated with Harmine (Har, 10 .M) or DMSO (vehicle control) for 20h. Expression analysis of CDKN1A (a) gene was performed by RT-qPCR (left panel) and mRNA sequencing (right panel) in the same cell extracts. Data is shown as mRNA expression fold changes calculated by the AACt method and transcript per million values (TPM) respectively. Data in the left panel is representative of 5 independent trials, and 4-5 independent samples were combined in the right panel. Significance was measured by one-way ANOVA within each group separately, and applying Sidak’s multiple comparisons test, two-tailed p values were computed. *-p<0.005; **-p<0.01 ; ***- p<0.001 ; ****-p<0.0001 ; ns, not significant, (b) Heatmap depicting expression of P53 target genes. The color code refers to Z score values, (c) Wild-type animals were treated with UV-B (400 and 500 mJ / cm2) 4h post seeding at larval stage 1 (L1) and the larval development was evaluated. The % of L1-L2, L3 and L4 stages were counted 44 hours post UV treatment. Significance was measured by an unpaired t-test. n=40-60 in each condition, mean and S.E.M values are presented. *-p<0.005; **- p<0.01 ; ***-p<0.001 ; ****-p<0.0001 ; ns, not significant, (d-f) Re-analysis of transcriptomic data from cortex of C57BL / 6J mice, with samples collected both at sleep and wake phases. Mediators of DNA damage and repair responses were analyzed under normal sleep and normal sleep compared to sleep deprivation (SD). Graphs show the expression dynamics of Cdknla (d), Gadd45b (e), and Ddb2 (f). Significance was measured by unpaired t-test. n= 3-5 in each condition, mean and S.E.M values are presented. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****-p<0.0001 ; ns, not significant, (g) The model depicting the role of DREAM in mediating cellular impairments downstream of disrupted clock is presented.

[0250] Figure 20: Clock disruption elevates the expression of histones in a DRM / DREAM-dependent manner, (a) Worms were treated and analyzed as described in Figure 4a-b, using antibody specific to histone H2AZ. Representative full gel scans (left) and respective quantification (right) are shown. The graph summarizes the results of five independent experiments. n=800 per condition and error bars are S.E.M. Significance was measured by one-way ANOVA, two-tailed p values were computed. ****-p<0.0001 ; ns, not significant, (b) Worms were treated as in (a) and mRNA expression levels of his-24 (H1), hil-2 (H1), and his-29 (H2) were assessed by RT-qPCR. n=250 worms per condition, graphs are representative of three independent experiments. Error bars are S.E.M.; mRNA fold change was calculated by the AA Ct method. Significance was measured by one-way ANOVA within each group separately and applying Sidak’s multiple comparisons test, two- tailed p values were computed. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****-p<0.0001 ; ns, not significant.

[0251] Figure 21 : Circadian clock disruption interferes with fundamental cellular processes in a DRM / DREAM-dependent manner, (a-l) Animals were treated and analyzed as described in Figure 4h-m. Box plots show the relative expression of proteins involved in aldehyde dehydrogenase (b), tryptophan metabolism (c), short-chain dehydrogenases (d), fatty acid p-oxidation (e), TCA cycle (f), OXPHOS (g), spliceosome (h), mitochondrial ribosome (i), cytosolic ribosome (j), and translation factors (I). Individual proteins are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the boxplot indicate the first and third quartile and the whiskers extend 1 .5 times the interquartile range from the limits of each box. n=800 per condition and 4 independent populations were measured. Within each box plot, the significance was assessed by the Mann-Whitney Wilcoxon rank-sum test, and Wilcoxon test was used for the comparison between the two sets of fold changes. Two-tailed p values were computed in all cases. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****-p<0.0001 . Figure 22: DREAM-dependent pathways are functional drivers of the negative effects of circadian disruption, (a-d) Age-synchronized t / nc-54p::Q40::YFP animals were fed with EV or Un-42 RNAi in combination with RNAis targeting specific genes related to identified DREAM-dependent pathways - translation initiation and ribosome biogenesis from the L1 stage, paralysis was scored daily. Cotargeting with ife-2 (a), ife-3 (b), xpd-1 (c), and rpoa-2 (d) is shown. n=140 worms per condition, graphs are representative of at least three independent experiments. Significance was measured by the Log-rank Mantel-Cox test, two-tailed p values were computed. ****-p<0.0001 .

[0252] Figure 23: DREAM expression changes during the natural sleep-wake cycle, (a) Experimental design of Jan et al., 2024 is depicted, (b-c) The transcriptomic data was analyzed as in Fig. 18a, 18c, and Fig. 19d-f. The Log2 CPM data was filtered to visualize DREAM subunits expression, Rbbp7, Lin54, and Lin37, under normal sleep and wakefulness (b). The expression dynamics of a non-cycling gene Bex2 is also depicted with each ZT compared to the expression at ZT 0. The same Log2 CPM data values at ZT 3 and 6 (NSD and SD) were normalized to ZT 0 and the expression dynamics of Rbbp44 is shown by comparing NSD and SD cohorts (left panel). As in b, Rbbp4 expression was also measured both during normal sleep and wake. Significance was measured by unpaired t-test. n= 3-5 in each condition, mean and S.E.M values are presented. *-p<0.005; **- p<0.01 ; ***-p<0.001 ; ****-p<0.0001 ; ns, not significant.

[0253] Figure 24: DREAM subunits follow a clock oscillation dynamics and expression is increased by Melatonin treatment, (a) Experimental design, (b) Wild-type mice were treated with melatonin (100 mg / kg) or vehicle for 2 weeks. Hippocampi were collected at both light (sleep) and dark (sleep) phases and samples processed for RT-qPCR analysis. The expression analysis of DREAM subunits Rbbp4, Rbbp7, Lin54, Lin37, Lin52 and the non-cycling gene Bex2 were performed by RT-qPCR. Data is shown as mRNA expression fold changes calculated by the AACt method. Significance was measured by multiple comparisons two-way ANOVA with a Bonferroni correction. n= 6-9 in each condition, mean and S.E.M values are presented. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****-p<0.0001 ; ns, not significant.

[0254] Figure 25: Same fundamental cellular processes are altered by clock disruption in mice and nematodes. Transcriptomics data analyzed as on Fig. 18d. (a-h) Box plots showing relative expression of glycolysis (a), TCA cycle (b), mitochondria p-oxidation (c), OXPHOS (d), ribosome (e), spliceosome (f), collagens (g), and proteostasis (h) genes are presented. Individual genes are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the boxplot indicate the first and third quartile and the whiskers extend 1 .5 times the interquartile range from the limits of each box. n=6-7 mice per condition. Within each box plot, the significance was assessed by the Mann-Whitney Wilcoxon rank-sum test, and the Wilcoxon test was used for the comparison between the two sets of fold changes. Two-tailed p values were computed in all cases. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****-p<0.0001.

[0255] Figure 26: Same fundamental cellular activities are regulated by clock / DREAM axis in nematodes and human cells, (a-f) Human RPE cells were treated and analyzed as described in Fig. 19a, n=4-5 independent cultures per condition. Expression analysis of PER1, CLOCK and CRY2 (a) genes are shown. RT-qPCR (lower panels) and transcriptomic measurements (upper panels) are presented as described in Fig. 19a. Significance was measured by one-way ANOVA within each group separately and applying Sidak’s multiple comparisons test, two-tailed p values were computed. ****-p<0.0001 . (b-c) Transcriptomic data was analyzed as in Fig. 18f. Box plots showing relative expression of the ribosome (b), and RNA polymerase (transcription) (c) genes are presented. Individual genes are shown as dots, the median fold change of each group is shown as a horizontal line; the upper and lower limits of the boxplot indicate the first and third quartile and the whiskers extend 1 .5 times the interquartile range from the limits of each box. Within each box plot, the significance was assessed by the Mann-Whitney Wilcoxon rank-sum test, and the Wilcoxon test was used for the comparison between the two sets of fold changes. Two-tailed p values were computed in all cases. *-p<0.005; **-p<0.01 ; ***-p<0.001 ; ****-p<0.0001 . (g) t / nc-54p::Q40::YFP nematodes were treated with EV or lin-42 RNAi in plates containing either 0.1% DMSO (vehicle) or Harmine (Har, 40JJ.M and 60 Paralysis was scored daily. n=140 worms per condition, graph is representative of three independent experiments. Significance was measured by the Log-rank Mantel-Cox test, two-tailed p values were computed. ****-p<0.0001 .

[0256] Figure 27: Experimental design for assessing developmental timing upon UV-B and I in-42 RNAi treatment, (a) Experimental design corresponding to Fig. 19c. Age-synchronized wild-type nematodes were treated with ev or I in-42 RNAi from L1 stage. Four hours post seeding, animals were treated with UV-B 400 or 500 mJ / cm2. After treatment, worms were transferred to fresh ev or lin-42 RNAi plates to ensure continued exposure to the treatment. Forty-four hours post UV treatment, the different larval stages were counted as L1-L2, L3, or L4 stages.

[0257] Figure 28: Role of DREAM in normal sleep and sleep deprivation. Model of how DREAM mediates the effects of sleep and wakefulness at the cellular level: during awake phase DREAM activity is high, causing chromatin compaction and shielding DNA from damage while simultaneously suppressing gene expression required for detoxification and repair (A). During sleep instead, DREAM activity goes down and repair activates become possible, including DNA repair. Sleep deprivation keeps DREAM activity high and prevents repair leading to gradual decline of health (B). This negative scenario can be prevented by using DREAM inhibitors that mimic or replicate the benefits of sleep as shown in C. We thus propose DREAM inhibitors to be mimetics of restorative sleep.

[0258] EXAMPLES

[0259] The invention is demonstrated by way of the examples disclosed below. The examples provide technical support for and a more detailed description of potentially preferred, non-limiting embodiments of the invention.

[0260] In order to demonstrate the functionality and beneficial properties of the inhibition of the DREAM complex described herein, the following examples are to be considered:

[0261] Moderate distortion of the circadian clock leads to tissue dysfunction in combination with physiological stress.

[0262] Clock disruption elicits expression changes of chromatin components and regulators of DNA metabolism.

[0263] DREAM complex subunits and specific histone variants mediate tissue dysfunction in response to circadian clock disruption.

[0264] Clock disruption inactivates adaptive stress responses and cellular maintenance mechanisms in a DREAM and histone dependent manner.

[0265] The DREAM-clock interaction is evolutionary conserved, and DREAM inhibition restores homeostasis under clock and sleep disruption.

[0266] Clock disruption interferes with fundamental cellular activities in a DRM / DREAM-dependent manner DREAM expression fluctuates between sleep and wakefulness in mammals, impacting fundamental homeostatic mechanisms.

[0267] High DREAM abundance confers DNA shielding during wakefulness at the cost of inferior repair.

[0268] Summary of the Examples

[0269] Circadian clock impairment and sleep deprivation have a disruptive impact on multiple aspects of organismal fitness, from cognition to the immune response. Such multifold influence can partially be explained by the requirement of sleep for the fluid flow and molecular damage clearance in the brain, with CNS mediating a plethora of organismal responses. A parallel hypothesis could be that sleep deprivation and disruption of the circadian rhythms impinge on master regulator pathways, which control basic aspects of cellular physiology and thereby modulate the function of multiple cell types. The discovery of such core regulatory mechanisms could represent an important step toward developing interventions that restore homeostasis under circadian misalignment and lack of sleep. Here, we used RNAi against the C. elegans PER orthologue Un-42 to induce persistent but moderate clock disruption as a mimetic of lifestyle-inflicted clock irregularities. We next applied this intervention in C. elegans models of proteostasis stress and accelerated aging, followed by proteomics and molecular and functional tests. In addition, transcriptomic analyses were conducted in mice with altered circadian behavior and in PER1 knockdown human cells. These tests validated the link between clock disruption and homeostatic impairments, especially in the context of additional cellular stress. We identified a plethora of conserved and ubiquitously expressed molecular targets altered by the distortion of clock in line with the multifold loss of health under clock impairment. Finally, we revealed the DREAM complex as the core regulator of clock-altered pathway activities and found that pharmacological and genetic inhibition of DREAM alleviates molecular, cellular, tissue and organismal dysfunction instigated by distorted clock.

[0270] Example 1: Moderate distortion of the circadian clock leads to tissue dysfunction in combination with physiological stress.

[0271] In C. elegans, we employed RNAi-mediated gene inactivation to knock down a core component of the circadian clock PER / lln-42. The complete loss of Un-42 function was previously found to cause accelerated aging and early death in the nematodes. This result is consistent with recent findings observing reduced longevity and increased prevalence of cellular stress in sleep-deprived D. melanogaster and mice. Unlike complete clock inactivation, our main interest was to test the organismal effects of partial clock impairment, which is conceptually comparable to occasional loss of circadian function due to lack of sleep in mammals. Therefore, we opted for the RNAi-mediated gene knockdown approach instead of using loss-of-fu notion mutants. We found that Un-42 RNAi treatment from the L1 larval stage indeed caused the disruption of the clock-dependent molting process of C. elegans (Figure 1 B and 7) without causing subsequent reduction of lifespan (Figure 1C) in line with the envisaged moderate clock impairment. Because previous studies in mice and D. melanogaster suggested that sleep deprivation induces cellular stress, we next asked if moderate circadian clock disruption would cause loss of tissue function in combination with additional stressors. In these experiments, we used transgenic protein aggregation models to inflict endogenous physiological stress in the body wall muscle of C. elegans. Specifically, we used nematodes expressing human amyloid beta peptide and polyglutamine coupled YFP protein (Q40- YFP) in the muscle tissue. Interestingly, Un-42 inactivation from the L1 larval stage significantly enhanced muscle dysfunction and whole-body paralysis in both transgenic models (Figure 1 D and 8A). A similar trend was also observed in transgenic animals treated with Un-42 RNAi from the pre- adult L4 larval stage (Figure 8B and C). These results showed that clock disruption during adulthood is sufficient for causing the decline of tissue homeostasis in concert with stress. At the same time, developmental clock distortion had a stronger impact on the lifelong tissue functionality consistent with the highest prevalence and importance of sleep and coordinated quiescence during growth and development across species. Finally, we found that RNAi inactivation of the downstream clock gene lin-14 led to enhanced paralysis of the Q40-YFP animals, similar to the knockdown of the upstream master regulator Un-42 (Figure 8D). This result validates clock disruption as a mechanism linking Un-42 inactivation to tissue dysfunction under stress. Collectively, we found that moderate but persistent loss of clock integrity triggers organ dysfunction in combination with cell-intrinsic physiological stressors.

[0272] Example 2: Clock disruption elicits expression changes of chromatin components and regulators of DN A metabolism.

[0273] To elucidate the molecular mechanisms linking clock disruption to organ frailty, we performed unbiased proteomics analysis of the animals exposed to iin-42 RNAi from L1 and L4 stages (Figure 2A and 10A), detecting the total of 5.358 protein groups. The whole proteome principal component analysis (PCA) showed a clear separation between iin-42 RNAi treated and empty vector (EV) exposed control cohorts in the L1 treatment setting (Figure 2B). The subsequent gene set enrichment analysis of the proteins differentially expressed between animals treated with iin-42 and control RNAi from the L1 stage revealed strong enrichment of the gene ontology terms associated with chromatin dynamics and DNA metabolism (Figure 2C). Concurrently, the unbiased volcano plot analysis performed on the same set of proteins revealed diverse histone variants as the most clearly regulated group (Figure 2D). Collectively, these two independent and unbiased whole proteome analyses hinted towards the involvement of chromatin dynamics and composition in the cellular effects of the iin-42 knockdown. To identify the putative core regulators linking iin-42 inactivation to chromatin changes, we next computationally extracted the proteins that contributed strongest to the separation between iin-42 RNAi and EV control proteomes along the PC1 in Figure 2B and analyzed these by using the STRING interaction assessment tool. Interestingly, a clear single hub with most interactions was detected, which included components and interactors of the DREAM complex (Figure 9 and 2E) implicated in chromatin remodeling and gene expression regulation during cell cycle and quiescence. Notably, individual DREAM subunits and interactors were upregulated in animals exposed to iin-42 RNAi from both L1 and L4 stages (Figure 2E) despite the lack of strong separation between whole proteomes of control and L4 stage-treated animals in the PCA (Figure 10B). Moreover, comparable DREAM-linked hub genes could be identified in an unbiased manner among the proteins with the highest contribution to the PC1 -separation in both L1- and L4-treated cohorts (Figure 9 and 10C). These results are in line with the stronger effect of the L1 -initiated clock disruption on organismal health (Figure 1 D, Figure 8A-C). Concurrently, these findings reinforce the discovery of the DREAM complex as a key target of circadian clock disruption regardless of age and demonstrate that molecular effects of clock impairment are similar between development and adulthood, albeit different in strength.

[0274] Example 3: DREAM complex subunits and specific histone variants mediate tissue dysfunction in response to circadian clock disruption.

[0275] To validate the involvement of the DREAM complex in the tissue dysfunction caused by circadian clock disruption, we performed co-knock downs of iin-42 and individual DREAM subunits by double RNAi exposure of the Q40-YFP animals. In these tests, we focused on RNAi treatment initiated at the L1 larval stage because of the stronger functional response observed in this setting previously (Figure 3A). Strikingly, we found that RNAi-mediated depletion of DREAM subunits lin-9, lin-54, and lin-53 reversed muscle paralysis caused by the Un-42 gene knockdown in the Q40-YFP animals, with lin-53 inactivation showing the strongest reversal effect (Figure 3B-D). Notably, our double RNAi approach was effective in suppressing both genes simultaneously, as seen with the example of lin- 42 / lin-53 co-suppression (Figure 11 A). At the same time, the knockdown of other canonical DREAM subunits, such as lin-37, lin-52, and lin-35, did not affect tissue dysfunction elicited by the lin- 42 RNAi (Figure 11 B-D), demonstrating selective involvement of the distinct DREAM subunits in the effects of the circadian clock. Moreover, we showed that RNAi-mediated depletion of his-411 H2BC and htz-11 H2A.Z2 histone variants found to be prominent DREAM interactors in our STRING analysis (Figure 9) and in literature, also rescued enhanced paralyzes of the Q40-YFP nematodes treated with the iin-42 RNAi (Figure 3E and 11 E). Collectively, our results indicated that the enhanced activity of the specific DREAM complex variants might mediate the negative impact of clock inactivation on tissue homeostasis under stress. Our data also suggested that an increased abundance of specific DREAM interactor histones might play a role in this process by possibly enhancing chromatin compaction and broadly blunting gene expression, including genes implicated in the adaptive stress responses. In line with the hypothesis of broad gene expression blunting, YFP transgene expression was progressively reduced with age in Q40-YFP animals exposed to iin- 42 RNAi (Figure 12A and B) with reversal of this effect observed in worms co-treated with the anti- DREAM lin-53 RNAi. At the same time, relative YFP aggregation was progressively increased with age in the iin-42 RNAi exposed animals, and this distortion was also reversed by the co-knock down of the lin-53 DREAM subunit in line with respectively impaired and restored ability to counteract proteostasis stress (Figure 3F).

[0276] Example 4: Clock disruption inactivates adaptive stress responses in a DREAM and histonedependent manner.

[0277] We next used Western blot analysis to test if the increased abundance of histones in iin-42 KD animals was indeed regulated by DREAM. Consistent with this hypothesis and in line with the proteomics data shown in Figure 2D, iin-42 RNAi treatment resulted in elevated expression of all histone variants tested, and this elevation was reversed by the co-knock down of lin-53 in all cases (Figure 4A-E, Figures 13, and 14). Notably, the differential expression between control, iin-42 KD, and lin-42 / lin-53 co-knock down was achieved at the mRNA level (Figure 4F), suggesting that DREAM might act as a transcriptional inducer of these genes in line with previous reports of its dual repressor-activator role in gene expression.

[0278] Subsequently, we asked what molecular pathways become de-regulated in iin-42 KD nematodes and re-instated in lin-42 / his-41 and lin-42 / lin-53 co-knock down animals, representing molecular targets affected by the circadian clock disruption in a DREAM and histone-dependent manner. The comparative proteomics analysis of the three indicated cohorts and the EV RNAi-treated control sample (Figure 5A) strikingly revealed simultaneous alterations of multiple mechanisms implicated in both basal homeostasis of the cell and in adaptive stress responses. For instance, we found detoxification mechanisms such as antioxidant response (Figure 5B) and xenobiotic metabolism (Figure 15A) to be suppressed by iin-42 KD and re-activated by co-knock down of lin-42 / his- 41 and lin-42 / lin-53, and similar dynamics was seen for mechanisms of the adaptive metabolism such as glycolysis, fatty acid p-oxidation and OXPHOS (Figure 5C and 15B, C). Moreover, extracellular matrix components and mediators of lysosome acidification essential for the degradation of aggregates and autophagy flux were also comparably altered (Figure 5D and E). Of note, deregulation of the antioxidant response by iin-42 RNAi is in line with oxidative stress being the primary cause of the early demise in sleep-deprived Drosophila and mice, while lysosome dysfunction is in line with the impaired ability of iin-42 KD worms to clear Q40-YFP aggregates as seen in Figure 3F. Moreover, distorted expression of ECM components, including collagens, is linked to loss of tissue integrity and aging across species. Our findings are additionally supported by a recent report that detoxification and metabolic mechanisms are de-regulated in the livers of Per1 / Per2 double-knockout mice. Interestingly, basal cellular mechanisms such as Pol II transcription and splicing were differentially affected in our experimental system, showing upregulation following clock distortion and re-blunting by co-knock down of lin-42 / his-41 and lin- 42 / lin-53 (Figure 5F and 15D). Of note, changes in Pol II transcription and splicing were recently revealed to be hallmarks and functional drivers of normal aging, and the interference of the Un-42 KD with both processes provides yet another putative DREAM-dependent link between clock disruption and premature loss of tissue function. Finally, we observed ribosome, ribosome biogenesis, and translational proteins to be strongly downregulated by lin-42 / lin-53 co-knock down (Figure 5G, 15E, and F), suggesting that DREAM is implicated in the alteration of these activities during clock disruption. Of note, lowering of translation and ribosomal biogenesis are known adaptations to stress in C. elegans and yeast and contributors to the longevity benefits of rapamycin and metformin. In this context, our data suggests that adaptive changes of translation are restricted by Un-42 KD in a DREAM-dependent manner. Notably, by comparing the list of clock targets depicted in figures 4, 5 and 15 to the previously published DREAM ChiP-Seq C. elegans dataset, we determined that only a few of these genes are located in close proximity to genomic DREAM binding sites, reinforcing the model of their modulation via DREAM-dependent histone and chromatin changes. Overall, we found that clock disruption causes multiple alterations of basal and adaptive cellular functions that are restored by co-KD of DREAM and, in most cases, also histones.

[0279] Example 5: The DREAM-clock interaction is evolutionary conserved, and DREAM inhibition restores homeostasis under clock disruption.

[0280] To test if clock impairment interferes with the adaptive mechanisms also in mammalian tissues, we turned to publicly available transcriptomic data of mice affected by circadian disruption. Interestingly, a recent study explored circadian impairments and their alleviation by time-restricted feeding in the brains of transgenic mice expressing the AD-associated variant of the human amyloid precursor protein, which is conceptually similar to our experiments combining clock impairment with proteostasis stress in C. elegans. By reanalyzing the transcriptomic data of circadian-impaired and - restored animals provided in this study, we strikingly discovered that similar pathways were altered and restored in a clock-dependent manner in the mouse brain and C. elegans. For example, the expression of mitochondrial constituents and OXPHOS genes was reduced by circadian disruption and repaired by reinstatement of the clock in the mice (Figures 16A, 6A, and B). Similarly, ribosome and spliceosome genes, as well as genes encoding lysosome and ECM constituents, demonstrated opposite expression dynamics in circadian-disrupted and reinstated brains (Figures 16B, C, D and E). These analyses revealed that the same basic cellular processes are affected by circadian impairments in nematodes and mammals.

[0281] We next turned to a human cell culture system to test if PER inactivation is indeed sufficient to disrupt the circadian clock and if inhibition of the DREAM complex is able to reset the clock in the background of PER deficiency. Here, we used siRNA treatment to knock down PER1 in human RPE cells, and we used the pharmacological inhibitor Harmine to suppress the DREAM complex. The choice of PER1, in this case, was driven by a recent report showing it to be the key cycling PER paralogue across mammalian tissues. We found that PER1 knockdown (KD) is indeed able to disrupt the clock, as seen by altered expression of core clock genes BMAL1, CLOCK, and CRY2. Importantly, Harmine failed to restore the clock in PERf-deficient cells (Figure 6C) but caused expected changes in the cell cycle as seen by altered expression of the P21 / CDKN1A gene (Figure 17A), demonstrating Harmine’s validity as a DREAM inhibitor. Collectively, these results confirmed our hypothesis that the restorative effect of DREAM inhibition on the homeostasis of clock-disrupted cells is independent of clock reinstatement and likely occurs downstream of impaired clock.

[0282] Subsequently, we turned to deeper high-throughput transcriptomic analysis of PER1- and DREAM- inactivated RPE cells to identify the key homeostatic mechanisms regulated by clock in a DREAM- dependent manner in the human system. Strikingly, our findings were similar to observations obtained in the nematode and mouse models. For instance, we found that mitochondrial and OXPHOS genes are downregulated by clock disruption and reinstated by co-inhibition of DREAM also in human cells (Figure 6D and E), revealing mitochondrial function as the most consistently clock-regulated entity across species and cell types. Moreover, spliceosome genes, ribosome genes, as well as genes encoding ECM constituents and ribosome biogenesis factors showed opposite expression dynamics between clock-disrupted and DREAM co-inhibited cells (Figure 17B- E). This was again similar to findings in nematodes and mice and clearly suggested that the same core cellular processes are altered by clock impairment in a DREAM-dependent fashion across species.

[0283] Because DREAM inhibitor Harmine is bioavailable in vivo, we finally tested if Harmine treatment could alleviate the decline of fitness elicited by iin-42 KD in C. elegans comparably to the genetic DREAM inactivation. We found that co-treatment of the Q40-YFP animals with iin-42 RNAi and Harmine indeed reverses the paralysis induced by the clock disruption in this model (Figures 6F and 17F). These results demonstrate that the pharmacological inhibition of DREAM is a valid in vivo tool for rescuing the health of the organisms affected by persistent circadian dysfunction.

[0284] In summary, these examples reveal a conserved ability of clock disruption to interfere with multiple basic and adaptive cellular pathways and coined the DREAM complex as an active key mediator of these negative effects. These findings reveal DREAM as a novel intervention target for the repair of cellular functions under a lack of coordinated organismal quiescence.

[0285] Example 6: Clock disruption interferes with fundamental cellular activities in a DRM / DREAM- dependent manner

[0286] We next used Western blot analysis to test if the increased abundance of histones in iin-42 KD animals was indeed regulated by DRM / DREAM. Consistent with the proteomics data shown in Figure 2D, histone abundance was elevated following iin-42 RNAi exposure, and this elevation was reversed by the additional knockdown of lin-53 in all cases (Figure 4A-E, Figures 13, 14 and 20A). Notably, the strongest difference in protein abundance between control, iin-42 KD, and lin-42 / lin-53 co-knockdown cohorts was seen for H1 and H2B histone variants, known to regulate and enhance chromatin compaction (Figure 4A and B, Figure 13A-B), and their differential expression was observed also at the mRNA level (Figure 20B). These results indicate that DREAM / DRM facilitates higher histone levels (Figure 4P), likely leading to enhanced chromatin compaction.

[0287] Subsequently, we asked what molecular pathways become de-regulated in iin-42 knockdown nematodes and re-instated in lin-42 / lin-53 double knockdown animals, representing molecular targets affected by the circadian clock disruption in a DRM / DREAM-dependent manner, lin-53 inactivation was chosen due to its strongest ability of reversing the negative health effects of the Iin- 42 KD (Figure 3D), and because lin-53 / RBBP4 was reported to mediate the interaction between DRM / DREAM and histones. The comparative proteomics analysis of iin-42 KD, lin-42 / lin-53 coknockdown, and EV RNAi control-exposed cohorts (Figure 21A) strikingly revealed simultaneous alterations of multiple mechanisms implicated in both basal homeostasis of the cell and in adaptive stress responses. For instance, detoxification mechanisms such as xenobiotic metabolism (Figure 4H) and pathways implicated in the antioxidant response (Figures 41 and 21 B-D) were found to be suppressed by Un-42 KD and restored by the co-knockdown of lin-53. The antioxidant response was represented by simultaneous de-regulation of four distinct metabolic entities implicated in counteracting oxidative stress: aldehyde dehydrogenase protein family (Figure 21 B), glutathione metabolism (Figure 4I), tryptophan catabolism by kynurenine pathway (Figure 21 C) with intermediates known to exert ROS scavenging roles, and short chain dehydrogenase protein family implicated in xenobiotic and antioxidant defense responses (Figure 21 D). The outstanding enrichment of antioxidant mechanisms among the DRM-dependent clock targets fits well with oxidative stress being the primary cause of the early demise in sleep-deprived Drosophila and mice. At the same time, xenobiotic detoxification responses were previously found to be de-regulated in the livers of Per1 / Per2 double-knockout mice suffering from clock impairment, in line with our present findings. In addition, metabolic plasticity pathways such as glycolysis and fatty acid p- oxidation were diminished by Un-42 KD and reinstated by lin-53 co-inactivation (Figure 4J and 21 E), while key bioenergetics activities of mitochondria - TCA cycle and OXPHOS, were dampened by lin- 42 inactivation and partially rescued by lin-53 RNAi co-treatment (Figure 21 F and G). Finally, the expression of ECM components collagens was downregulated in iin-42 RNAi-exposed animals and restored by lin-42 / lin-53 co-knockdown (Figure 4K). Notably, both decline of metabolic plasticity and reduced expression of collagens are associated with loss of organ function during aging, establishing a link between clock disruption and accelerated tissue impairment. Moreover, an impairment of metabolic adaptive capacity was previously observed in Per1 / Per2 deficient mouse livers. The cell-protective interplay between metabolic and detoxification activities regulated by DRM / DREAM and clock can also be envisaged because tryptophan catabolism by kynurenine pathway is one of the key cellular sources of nicotinamide adenine dinucleotide (NAD+) - an energy carrier and enzymatic co-factor with key roles in mitochondrial maintenance and bioenergetics fitness, and with capacity to delay metabolic aging. The strong enrichment of metabolic pathways among DRM-dependent clock targets is in line with the large body of human clinical data designating clock and sleep dysfunction as confounding factors of metabolic diseases such as diabetes, obesity, and cardiovascular disorders.

[0288] Interestingly, basal cellular activities such as Pol II transcription and splicing were differentially affected in our experimental system showing upregulation following clock distortion and re-blunting by co-knockdown of lin-53 (Figure 4L and 21 H). Of note, increased speed of Pol II transcription and elevated splicing were recently revealed to be hallmarks and functional drivers of physiological aging, and the interference of the iin-42 KD with both processes provided yet another putative Hn- 53 / DRM-dependent link between clock disruption and premature organismal demise. Finally, we observed mitochondrial ribosome, cytosolic ribosome, ribosome biogenesis, and translational proteins to be strongly downregulated by lin-42 / lin-53 co-knockdown (Figure 4M and 21 l-L) suggesting that DRM / DREAM modulates these essential activities in the context of clock disruption. Because inhibition of translation is known to improve proteostasis by reducing protein folding stress, the strong blunting of this process at multiple levels likely contributes to superior capacity of lin- 42 / lin-53 double KD to reduce polyQ aggregates compared to Un-42 only inactivated worms (Figure 3F).

[0289] To determine whether the identified pathways played a functional role in the negative health effects of clock impairment, we focused on the pathways upregulated by clock disruption in a DREAM- dependent manner (including transcriptional initiation, ribosome biogenesis, and translation). We suppressed these pathways using specific RNAi treatment in clock-disrupted unc-54p::Q40::YFP transgenic animals. Strikingly, all RNAi treatments mimicked the restorative effect of DREAM inhibition in this context (Figures 4N, O and 22A-D), showing that DREAM-dependent pathway alterations are functional drivers of the negative changes caused by clock disruption. Notably, by comparing the list of clock and DREAM co-targets depicted in Figures 4, 5 and 15 to the previously published DREAM ChiP-Seq C. elegans dataset, we determined that only few of these genes are located in the close proximity to the relevant genomic DREAM binding sites. This finding speaks in favor of indirect chromatin- and histone-dependent regulation of these targets by DRM / DREAM, which is also supported by all of the functional and molecular data we obtained thus far. Overall, we found that clock disruption causes multiple alterations of basal and adaptive cellular functions that are reduced by co-knockdown of DRM / DREAM likely by affecting histone levels and chromatin structure (Figure 4P). The amplitude of changes in the individual adaptive pathways was not drastic, but in combination, these alterations have the potential to impede cellular homeostasis and flexibility at multiple levels contributing to health decline associated with clock and sleep impairments.

[0290] Example 7: DREAM expression fluctuates between sleep and wakefulness in mammals, impacting fundamental homeostatic mechanisms.

[0291] We next asked if DREAM expression changes during the natural sleep-wake cycle by analyzing brain (cortex) transcriptomes of young mice. Interestingly, we found DREAM expression to be lowered during sleep and elevated during wakefulness (Figures 18A, C (upper panel) and 23B, D (right panel)), while sleep deprivation altered the circadian DREAM dynamics (Figures 18B and 23D (left panel)) and prevented distinct DREAM subunits from being timely downregulated (Figure 18B), similar to observations in clock-impaired C. elegans. To validate if DREAM expression is under active circadian control, we performed qPCR on cDNA isolated from brain tissue (hippocampi) of mice following exposure to a sleep hormone melatonin that increases the amplitude of circadian responses. We found expression of all tested DREAM subunits to be boosted by melatonin during the awake phase of the circadian cycle (Figures 18C (lower panel) and 24B), suggesting that daily fluctuations of DREAM expression are part of the circadian response.

[0292] We next asked if impaired DREAM lowering under sleep deprivation impacts fundamental cellular activities in mice as it does in worms. Here, we compared brain transcriptomes of sleep-deprived and sleep-intact mice during the circadian window designated to naturally deep sleep (Figure 23A). Of note, the same transcriptomic data and time window were used for the assessment of the relative DREAM dynamics between sleep and sleep deprivation in Figures 18 and 23. We found mitochondrial content, OXPHOS, TCA cycle, mitochondrial fatty acid p-oxidation, glycolysis as well as ECM components (collagens), proteostasis, ribosome biogenesis and content, and splicing to be altered by sleep deprivation (Figures 5D Fig 18D and 25 A-H), similar to findings in clock-impaired C. elegans. Notably, proteostasis seen to be dampened by sleep deprivation (Figure 25H), is known to be neuroprotective, while upregulation of collagens (Figure 25G) likely enhances the stiffness of the brain tissue impeding glymphatic clearance of toxic entities from the brain that occurs during sleep, and indeed de-regulation of brain collagens is linked to neurodegeneration and dementia in humans. Simultaneously, the downregulation of mitochondrial genes, including OXPHOS components (Figure 18D), along with an increase in glycolysis and TCA cycle gene expression (Figure 25A, B), resembles the "awake" metabolic state characterized by carbohydrate utilization, contrasting with the oxidative metabolism typical of restorative sleep. We thus show that DREAM expression is high during wakefulness and low during natural sleep. We also demonstrate that DREAM expression fails to be timely lowered in sleep-deprived mice similar to clock-impaired nematodes, and this failure correlates with de-regulation of similar repair and maintenance pathways as in clock-disrupted worms. We next turned to a human cell culture system to test if PER inactivation is indeed sufficient to disrupt circadian clock and if inhibition of the DREAM complex can alleviate clock-related molecular impairments in the background of PER deficiency. Here we used siRNA-mediated knockdown of PER1 in human retinal pigment epithelial (RPE) cells, and we used the DYRK1A inhibitor Harmine to pharmacologically suppress DREAM function, as seen in previous reports. PER1 was chosen as the key cycling PER paralogue across mammalian tissues as shown in a recent report. We found that PER1 knockdown was robust (Figures 18E and 26A) and indeed sufficient to cause clock impairments as seen by altered expression of CLOCK and CRY2 core clock genes in qPCR and transcriptomic tests (Figures 18E and 26A). Importantly, Harmine did not restore the clock in PER1- depleted cells (Figures 18E and 26A), confirming our hypothesis that the restorative effect of DREAM inhibition on the homeostasis of clock-disrupted cells is independent of clock reinstatement and likely occurs downstream of an impaired clock.

[0293] Subsequently, we turned to deeper high-throughput transcriptomic analysis of PER1- and Harmine- treated RPE cells to identify the key homeostatic mechanisms regulated by clock in a DREAM- dependent manner in human cells. Strikingly, our findings were similar to observations obtained in the nematode and mouse models. For instance, we found that mitochondrial and OXPHOS genes are downregulated by clock disruption and reinstated by co-inhibition of DREAM also in human cells (Figure 18F), revealing mitochondria and OXPHOS as the most consistently clock-regulated entities across species and cell types. Interestingly, this observation is consistent with the recently discovered key involvement of mitochondria and OXPHOS in the induction of sleep and damage clearance during sleep. Moreover, ribosome and ribosome biogenesis genes, and genes encoding the components of the transcriptional machinery showed opposite expression dynamics between clock-disrupted and DREAM co-inhibited cells (Figures 18F and 26B-C). These results matched the observations in nematodes and mice, and clearly suggested that similar fundamental cellular activities are altered by clock impairment in a DREAM-dependent fashion across species. Additionally, we found that co-administration of Harmine in t / nc-54p::Q40::YFP animals exposed to Un-42 RNAi indeed reversed the paralysis induced by clock disruption in this model (Figure 26D and E). These results demonstrate that pharmacological inhibition of DREAM is a promising in vivo tool for rescuing the health of organisms affected by sleep and clock disruption.

[0294] Example 8: High DREAM abundance confers DNA shielding during wakefulness at the cost of inferior repair.

[0295] We then explored the physiological significance of the circadian fluctuations of DREAM. Drawing from our findings that DREAM levels are elevated during wakefulness and contribute to increased histone abundance and likely histone loading, as well as literature evidence showing that histones and chromatin protect DNA from damage and that oxidative and DNA damage are heightened during wakefulness, we hypothesized that the transiently high levels of DREAM serve to shield genomic DNA from the excessive damage associated with wakefulness.

[0296] Indeed, inhibition of DREAM in cycling human cells led to elevated expression of P21 (Figure 19A) and P53 target genes (Figure 19B), in line with previous reports and consistent with genotoxic stress. At the same time, boosting of DREAM levels by iin-42 RNAi exposure protected C. elegans from the larval arrest triggered by ultraviolet B treatment known to induce helix-distorting DNA lesions (Figures 19C and 27), suggesting that DREAM elevation indeed protects DNA from the excessive damage.

[0297] On the other hand, previous reports found that DREAM inhibition upregulates a plethora of DNA repair pathways, and consistently, we found markers of genotoxic stress (P21 and Gadd45b) to be lowered during sleep in parallel with the reduction of DREAM expression, while representative DNA repair marker Ddb2 was upregulated (Figure 19D-F, left panel). Conversely, sleep deprivation hindered the lowering of P21 and Gadd45b expression levels (Figure 19D and E, right panel) like it impeded the lowering of DREAM expression, while Ddb2 failed to be upregulated in this context (Figure 19F, right panel).

[0298] Collectively, our findings suggest that DRM / DREAM has a dual role in protecting genome integrity during the circadian cycle: during wakefulness high levels of DRM / DREAM likely shield DNA from damage by increasing histone abundance, while during sleep DREAM levels are lowered allowing DNA repair to take place along with other repair and maintenance activities that remain suppressed during wakefulness as a trade-off for DNA protection. Under sleep deprivation, failure to downregulate DREAM likely interferes with repair, but as shown above, we mitigate this impairment with DREAM inhibitors such as Harmine.

[0299] Discussion of the Examples

[0300] As shown herein, the inventors have combined omics, genetics, functional and molecular analyses in three model organisms to elucidate the molecular basis of the body-wide health decline caused by persistent deregulation of the circadian clock and sleep. As a result, we discovered for the first time that clock disruption engages the DREAM complex and associated histone variants in distorting a multitude of basal and adaptive cellular activities ranging from translation and splicing to OXPHOS, oxidative stress response, and ECM remodeling. Our analyses suggest that this newly discovered conserved mechanism allows persistent circadian disruption to disable the capacity of the cells to effectively respond to stressors, which in turn causes organ dysfunctions that are particularly evident in the organisms predisposed to stress, such as nematode strains expressing aggregation-prone human A beta or Q40 YFP proteins. The disrupted processes are wide-ranging and influence many cell types, consistent with published observations that circadian / sleep disruptions impair the homeostasis of multiple organs. Our hypothesis is also consistent with the large body of reports linking sleep deprivation to human diseases and inferior organismal responses to extrinsic stressors ranging from infections and cognitive challenges to exercise and diet. In addition, our findings are in line with a recent report coining intestinal oxidative stress as the main cause of the premature demise of Drosophila and mice chronically deprived of sleep. The present invention therefore identifies key molecular aspects of the downstream consequences of circadian rhythm disruption and medical effects thereof, and present means to address such conditions. We show that pharmacological and genetic inhibition of the DREAM complex is able to repair the homeostasis of cells and organisms affected by circadian impairments. We demonstrate that this repair process does not entail the reinstatement of clock, which makes it unique among existing approaches to counteracting sleep and circadian disruption that all rely on the eventual repair of clock and sleep functions. The DREAM-based putative intervention is thus uniquely suited for cases when clock disruption must persist or cannot be reversed for any reason.

[0301] The present invention further highlights the ability of the DREAM complex to simultaneously regulate multiple cellular functions via controlling the abundance of specific histone variants and the accessibility of the respective chromatin regions. This model is supported by the previous report detecting a high abundance of the histone variant H2A.Z in the gene body of DREAM-repressed cell cycle targets, with HTZ-1 / H2A.Z being one of the principle histones we found to cooperate with DREAM in de-regulating the cellular resilience of clock-distorted animals. Consistently, only a minor fraction of the molecular targets altered by clock disruption in a DREAM-dependent manner harbors DREAM binding sites in their promoter regions, supporting the indirect chromatin-based regulation. Interestingly, this histone-dependent mode of DREAM action is distinct from the recently reported capacity of DREAM to repress multiple pathways implicated in DNA repair, which relies on the direct binding of DREAM to the regulatory elements of affected genes. Notably, also the predominantly implicated DREAM subunits differ between DNA repair and circadian functions of DREAM, with Un- 52, dpl-1, efl-1, and lin-35 having the strongest impact in DNA repair and lin-9, lin-54, and lin- 53 regulating the consequences of the circadian disruption. Our findings thus indicate the likely existence of two different modes of how specific variants of the DREAM complex regulate complex gene expression networks: (a) via direct binding and recruitment of co-factors and (b) via changes in chromatin accessibility (this study). The dependency of the circadian response on the presence of particular DREAM subunits enables targeted interventions that modulate the “sleep-specific” role of DREAM without interfering with its activities in other cellular processes. Finally, the inventors demonstrate that natural DREAM expression is regulated by circadian rhythms, with levels elevated during wakefulness and reduced during sleep. Increased DREAM expression during wakefulness leads to histone elevation, shielding genomic DNA from wakefulness-associated damage, but at the expense of dampened cellular repair. During sleep, the reduction of DREAM expression enables chromatin de-compaction and efficient repair processes, including DNA repair. However, sleep deprivation disrupts the downregulation of DREAM, chronically inhibiting repair mechanisms. Importantly, these detrimental effects of sleep deprivation can be reversed using pharmacological or other inhibitors of the DREAM complex, which thus act as mimetics of restorative sleep.

[0302] REFERENCES

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[0304] 2. Banks, S. & Dinges, D. F. Behavioral and physiological consequences of sleep restriction. J Clin Sleep Med 3, 519-528 (2007).

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[0306] 4. Knutson, K. L., Spiegel, K., Penev, P. & Van Cauter, E. The metabolic consequences of sleep deprivation. Sleep Med Rev 11 , 163-178, doi:10.1016 / j.smrv.2007.01 .002 (2007).

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[0310] 8. Tennessen, J. M., Gardner, H. F., Volk, M. L. & Rougvie, A. E. Novel heterochronic functions of the Caenorhabditis elegans period-related protein LIN-42. Dev Biol 289, 30-43, doi:10.1016 / j.ydbio.2005.09.044 (2006).

[0311] 9. Nelson, M. D. & Raizen, D. M. A sleep state during C. elegans development. Curr Opin Neurobiol 23, 824-830, doi:10.1016 / j.conb.2013.02.015 (2013).

[0312] 10. Monsalve, G. C., Van Buskirk, C. & Frand, A. R. LIN-42 / PERIOD controls cyclical and developmental progression of C. elegans molts. Curr Biol 21 , 2033-2045, doi:10.1016 / j.cub.2011.10.054 (2011).

[0313] 11 . Kalfalah, F. et al. Crosstalk of clock gene expression and autophagy in aging. Aging (Albany NY) 8, 1876-1895, doi:10.18632 / aging.101018 (2016).

[0314] 12. Vaccaro, A. et al. Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut. Cell 181 , 1307-1328 e1315, doi: 10.1016Zj.cell.2020.04.049 (2020).

[0315] 13. Morley, J. F., Brignull, H. R., Weyers, J. J. & Morimoto, R. I. The threshold for polyglutamine- expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc Natl Acad Sci U S A 99, 10417-10422, doi:10.1073 / pnas.152161099 (2002).

[0316] 14. McColl, G. et al. Utility of an improved model of amyloid-beta (Abeta(1)(-)(4)(2)) toxicity in Caenorhabditis elegans for drug screening for Alzheimer's disease. Mol Neurodegener 7, 57, doi: 10.1186 / 1750-1326-7-57 (2012).

[0317] 15. Gangwisch, J. E. et al. Short sleep duration as a risk factor for hypertension: analyses of the first National Health and Nutrition Examination Survey. Hypertension 47, 833-839, doi:10.1161 / 01. HYP.0000217362.34748.e0 (2006).

[0318] 16. Prinz, P. N. Age impairments in sleep, metabolic and immune functions. Exp Gerontol 39, 1739-1743, doi:10.1016 / j.exger.2004.06.023 (2004). Spiegel, K., Sheridan, J. F. & Van Cauter, E. Effect of sleep deprivation on response to immunization. JAMA 288, 1471-1472, doi:10.1001 / jama.288.12.1471-a (2002). Spiegel, K., Knutson, K., Leproult, R., Tasali, E. & Van Cauter, E. Sleep loss: a novel risk factor for insulin resistance and Type 2 diabetes. J Appl Physiol (1985) 99, 2008-2019, doi:10.1152 / japplphysiol.00660.2005 (2005). Anafi, R. C., Kayser, M. S. & Raizen, D. M. Exploring phylogeny to find the function of sleep. Nat Rev Neurosci 20, 109-116, doi:10.1038 / s41583-018-0098-9 (2019). Devlin, P. F. Signs of the time: environmental input to the circadian clock. J Exp Bot 53, 1535-1550, doi:10.1093 / jxb / erf024 (2002). Kanaya, H. J. et al. A sleep-like state in Hydra unravels conserved sleep mechanisms during the evolutionary development of the central nervous system. Sci Adv 6, doi:10.1126 / sciadv.abb9415 (2020). Nath, R. D. et al. The Jellyfish Cassiopea Exhibits a Sleep-like State. Curr Biol 27, 2984- 2990 e2983, doi:10.1016 / j.cub.2017.08.014 (2017). Siegel, J. M. Do all animals sleep? Trends Neurosci 31 , 208-213, doi: 10.1016 / j.tins.2008.02.001 (2008). Trojanowski, N. F. & Raizen, D. M. Call it Worm Sleep. Trends Neurosci 39, 54-62, doi:10.1016 / j.tins.2015.12.005 (2016). Ambros, V. & Horvitz, H. R. The lin-14 locus of Caenorhabditis elegans controls the time of expression of specific postembryonic developmental events. Genes Dev 1 , 398-414, doi:10.1101 / gad.1.4.398 (1987). Hristova, M., Birse, D., Hong, Y. & Ambros, V. The Caenorhabditis elegans heterochronic regulator LIN-14 is a novel transcription factor that controls the developmental timing of transcription from the insulin / insulin-like growth factor gene ins-33 by direct DNA binding. Mol Cell Biol 25, 11059-11072, doi:10.1128 / MCB.25.24.11059-11072.2005 (2005). Van Wynsberghe, P. M. & Pasquinelli, A. E. Period homolog LIN-42 regulates miRNA transcription to impact developmental timing. Worm 3, e974453, doi:10.4161 / 21624054.2014.974453 (2014). Szklarczyk, D. et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res 51 , D638-D646, doi:10.1093 / nar / gkac1000 (2023). Sadasivam, S. & DeCaprio, J. A. The DREAM complex: master coordinator of cell cycledependent gene expression. Nat Rev Cancer 13, 585-595, doi:10.1038 / nrc3556 (2013). Fischer, M. & Muller, G. A. Cell cycle transcription control: DREAM / MuvB and RB-E2F complexes. Crit Rev Biochem Mol Biol 52, 638-662, doi:10.1080 / 10409238.2017.1360836 (2017). Esterlechner, J. et al. LIN9, a subunit of the DREAM complex, regulates mitotic gene expression and proliferation of embryonic stem cells. PLoS One 8, e62882, doi:10.1371 / journal. pone.0062882 (2013).

Claims

CLAIMS1. An inhibitor of the protein complex Dimerization partner, RB-like, E2F and multi-vulval class B (DREAM complex inhibitor) for use in the treatment of a medical condition induced by disruption of a circadian rhythm.

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

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

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

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

6. The DREAM complex inhibitor for use according to claim 1 , wherein the inhibitor comprises an affinity reagent, antibody or antigen-binding fragment thereof binding the DREAM complex or subunit thereof, or comprises an antisense or interfering nucleic acid molecule, such as short interfering RNA (siRNA), targeting the DREAM complex or subunit thereof.

7. The DREAM complex inhibitor for use according to any one of the preceding claims, wherein a subunit of the DREAM complex is inhibited, wherein the inhibited subunit of the DREAM complex is lin-9, lin-54, and / or RBBP4 / RBBP7.

8. The DREAM complex inhibitor for use according to any one of the preceding claims, wherein the medical condition is induced by and / or comprises a loss of tissue function, oxidative stress, and / or dysfunction of cellular homeostasis.

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

10. The DREAM complex inhibitor for use according to any one of the preceding claims, wherein the treatment is independent of restoration of the circadian rhythm, preferably occurring downstream of the circadian rhythm.11 . The DREAM complex inhibitor for use according to any one of the preceding claims, for use as a sleep mimetic to provide one or more restorative effects of sleep.

12. The DREAM complex inhibitor for use according to any one of the preceding claims, wherein the medical condition comprises an acute medical impairment associated with sleep deprivation and disruption of a circadian rhythm.

13. The DREAM complex inhibitor for use according to the preceding claim, wherein the acute medical impairment is one or more of fatigue, lethargy, cognitive impairment, brain fog, impaired memory, gastro-intestinal discomfort, cardiovascular impairment, blood pressurealteration, decline of immune-competence, change in mood, depression, anxiety and / or paranoia.

14. The DREAM complex inhibitor for use according to any one of the preceding claims, wherein the medical condition comprises a chronic medical condition associated with sleep deprivation and disruption of a circadian rhythm.

15. The DREAM complex inhibitor for use according to the preceding claim, wherein the chronic medical condition is a neurologic, psychiatric, cardiovascular, metabolic, allergic, immunologic, gastrointestinal, rheumatic, proliferative disease (cancer), microbiome, multiorgan and / or a pulmonary disorder.

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

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