Dhodh inhibitors for use in the chronopharmacoligical treatment and prevention of obesity and obesity-related metabolic disorders

Abstract

Targeting mitochondrial function is a strategy for preventing metabolic disorders, but restoring mitochondria diurnal regulation remains to be explored. Using targeted metabolomics, we identified 24-hour mitochondrial rhythms promoted by nocturnal time-restricted feeding (TRF) in mice fed a high-fat diet (HFD). Preceding its metabolic benefits, TRF promoted mitochondrial daily rhythms by converging on the enzyme dihydroorotate dehydrogenase (DHODH), which coordinates pyrimidine biosynthesis and mitochondrial oxidative metabolism. The inhibition of DHODH activity by the short half-life inhibitor BAY-2402234 modulated mitochondrial oxidative metabolism, thereby preventing diet-induced obesity, but only when injected at the transition between night and day. This timed inhibition of DHODH promoted oscillations in mitochondrial metabolic pathways and oscillations in the CoQ / CoQH2 ratio, improving metabolic adaptation to the HFD challenge. Our results highlight the role of mitochondrial daily rhythms in the pathophysiology of obesity and open avenues for chronopharmacological treatments targeting these rhythms. Thus, the present invention pertains to a method for treating or preventing obesity in a subject in need thereof. This method includes administering to the subject, at a specific time of the circadian rhythm, a therapeutically effective amount of a dihydroorotate dehydrogenase inhibitor that exhibits an inhibitory duration of less than 24 hours, and more specifically, a short half-life of less than 24 hours.

Description

[0001] DHODH INHIBITORS FOR USE IN THE CHRONOPHARMACOLIGICAL TREATMENT AND PREVENTION OF OBESITY AND OBESITY-RELATED METABOLIC DISORDERS

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to the timed use of a dihydroorotate dehydrogenase (DHODH) enzyme inhibitor for the treatment of obesity and obesity-related metabolic disorders.

[0004] BACKGROUND OF THE INVENTION:

[0005] In 2016, 39% of adults were overweight worldwide. Although obesity is considered preventable, the failure to understand its multifaceted genetic and environmental origins has led to an overall inability to address its increasing prevalence. Among the causes of the obesity pandemic, circadian disruptive behaviours (jet lag, shift work, etc.) of modern lifestyles play a key role. In mammals, daylight-synchronized neurons in the hypothalamic suprachiasmatic nuclei (SCN) (1-2) pace sleep-wake and feeding behaviors, blood pressure, and body temperature. But peripheral organs also harbour an intrinsic molecular clock that regulates their function (3-4). In hepatocytes, feeding time synchronizes the clock to control metabolism (5- 7). Currently, how diurnal rhythmic metabolism can mitigate the effects of excessive calorie consumption is generating interest in chrononutrition or chronopharmacology approaches, i.e., exploiting the cyclical nature of biological phenomena to pace drug delivery to optimize patient benefit (8-9).

[0006] Overfeeding mice with a high-fat diet (HFD) diet disturbs biological rhythms. HFD dampens feeding rhythms within a few days (10). HFD at daytime (physiologic fasting phase in mice) worsens weight gain (11). HFD impairs the rhythmic hepatic core clock genes expression (10), impairs the rhythmic metabolic transcriptional regulation (12-13) and rhythmic hepatic metabolome, and impairs rhythmic mitochondrial beta-oxidation (14). Conversely, HFD restricted to the night-time (physiologic feeding phase), known as time-restricted feeding (TRF), prevents metabolic dysfunction and liver pathologies. TRF reduces steatosis, normalizes glucose intolerance, insulin resistance, and fat mass (15). TRF improves the rhythmicity of circadian genes and metabolic effectors expression such as gluconeogenic genes (15-16). TRF also favours rhythmic expression of genes involved in lipid metabolism and lowers de novo lipogenesis (15-16). Furthermore, the benefits of TRF persist in the absence of a functional molecular circadian clock (17), suggesting the role of other key metabolic players.

[0007] It has been reported evidence of rhythmic changes in the mitochondria composition relying on a functional clock. Proteomics on whole liver extracts and isolated mitochondria revealed daily changes in mitochondrial proteome regulated by rhythmic mitochondrial protein import (14; 18). Functional clustering of the rhythmic mitochondrial proteome showed daily oscillations of catabolic and oxidative functions (14; 18). In fact, circadian clock driven NAD+ cycle impacts mitochondrial oxidative metabolism in mice notably via SIRT3 -dependent mitochondrial protein deacetylation processes (19-21). Hepatic mitochondrial daily rhythms also regulate hepatic metabolism and, by impairing mitochondrial dynamics, circadian clock disruption triggers metabolic diseases (22;23). The mitochondrial lipidome in mouse liver has daily oscillations that are lost in the absence of the core clock proteins PERI and PER2 (24). Together, these studies indicate a rhythmic regulation of mitochondrial composition, with major implications for the rhythmic function of mitochondria in daily homeostasis.

[0008] Interestingly, published works on mice fed a HFD indicate that TRF ameliorates mitochondrial oxidative functions. TRF increases the relative mitochondrial mass in hepatocytes, potentially through the correction of defective Ppar-alpha oscillations prompted by ad libitum HF diets (15). TRF ameliorates expression of pyruvate carboxylase leading to increase levels of TCA intermediates, and reduces glutathione (15). Fatty acid oxidation is also improved by TRF which restores CPT1 activity and levels of 3 -hydroxybutyrate, an endproduct of P-oxidation. Simultaneous quantification of temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmall)-deficient animals, demonstrated that mitochondrial genes for mitochondrial proteins are translated rhythmically under both the influence of the circadian clock and feeding rhythms (25). Mitochondria integrate circadian rhythms and daily metabolic signals. However, it is unclear whether this function participates in the metabolic beneficial effects of TRF or can be targeted to prevent obesity.

[0009] Targeting mitochondrial function is a strategy for preventing metabolic disorders, but restoring mitochondria diurnal regulation remains to be explored.

[0010] SUMMARY OF THE INVENTION:

[0011] The inventors demonstrate that a chronopharmacological inhibition of Dihydroorotate dehydrogenase (DHODH), the only mitochondrial enzyme in the pyrimidine biosynthetic pathway and connecting pyrimidine biosynthesis to the functional respiratory, restores rhythms in mitochondria and de novo pyrimidine synthesis while preventing diet-induced obesity.

[0012] Therefore, the present invention relates to a method for treating obesity in a subject requiring such treatment. This method includes administering a therapeutically effective quantity of a dihydroorotate dehydrogenase inhibitor to the subject. This inhibitor has a brief half-life and is administered at a specific time of the day related to the subject’s circadian rhythm.

[0013] In particular, the invention is defined by the claims.

[0014] DETAILED DESCRIPTION OF THE INVENTION:

[0015] In this study, the inventor show that mitochondrial diurnal rhythms protect from diet induced obesity. They show that TRF promotes rhythmicity in de novo pyrimidine synthesis while improving rhythms in mitochondrial oxidative functions. Using targeted metabolomics, the inventors identified 24-hour mitochondrial rhythms promoted by nocturnal time-restricted feeding (TRF) in mice fed a high-fat diet (HFD). Preceding its metabolic benefits, TRF promoted mitochondrial daily rhythms by converging on the enzyme dihydroorotate dehydrogenase (DHODH), which coordinates pyrimidine biosynthesis and mitochondrial oxidative metabolism. A chronopharmacological inhibition of dihydroorotate dehydrogenase (DHODH) by the short half-life inhibitor BAY-2402234 restores rhythms in mitochondria and de novo pyrimidine synthesis while preventing diet-induced obesity. This results implicate mitochondria daily rhythms in the pathophysiology of obesity and implicate the enzyme DHODH as a druggable metabolic time switch.

[0016] Accordingly, a first object of the present invention refers to a method of treating or preventing obesity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase, wherein said inhibitor of dihydroorotate dehydrogenase exhibits a short inhibition duration and wherein said inhibitor of dihydroorotate dehydrogenase is administered at a specific time of the day related to the subject’s circadian rhythm.

[0017] As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By “therapeutic regimen” is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a “loading regimen”, which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase “maintenance regimen” or “maintenance period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0018] As used herein, a “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a patient. For example, a “therapeutically effective amount of the active agent” to a patient is an amount of the active agent that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression, or physical conditions associated with the disease affecting the patient. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 100 mg / kg of body weight per day.

[0019] As used herein the terms “administering” or “administration” refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., the DHODH inhibitor) into the subject, such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. In particular embodiment, the DHODH inhibitor is orally administered.

[0020] As used herein, the term "subject" refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like), that can be afflicted with obesity. In a particular embodiment of the present invention, the subject is a human being. The term "subject" does not denote a particular age, and thus encompasses children, teenagers, and adults.

[0021] In particular embodiment, the subject is an obese subject.

[0022] As used herein the term "obesity" refers to a condition characterized by an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), which is calculated as body weight per height in meter squared (kg / m2). Obesity refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg / m2, or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg / m2. An "obese subject" is an otherwise healthy subject with a BMI greater than or equal to 30 kg / m2or a subject with at least one co-morbidity with a BMI greater than or equal 27 kg / m2. A "subject at risk of obesity" is an otherwise healthy subject with a BMI of 25 kg / m2to less than 30 kg / m2or a subject with at least one co-morbidity with a BMI of 25 kg / m2to less than 27 kg / m2. The increased risks associated with obesity may occur at a lower BMI in people of Asian descent. In Asian and Asian-Pacific countries, including Japan, "obesity" refers to a condition whereby a subject with at least one obesity -induced or obesity -related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg / m2. An "obese subject" in these countries refers to a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg / m2. In these countries, a "subject at risk of obesity" is a person with a BMI of greater than 23 kg / m2to less than 25 kg / m2.

[0023] As used herein, the term "dihydroorotate dehydrogenase" or “DHODH” has its general meaning in the art and refers to the enzyme of the pyrimidine ribonucleotide synthesis pathway located in the mitochondria and supporting the respiratory chain. DHODH catalyses the oxidation of dihydroorotate to orotate by reducing ubiquinone into ubiquinol, in de novo pyrimidine biosynthesis. Thus, the term “dihydroorotate dehydrogenase inhibitor” or “DHODH inhibitor” refers to any compound capable of inhibiting DHODH expression or activity. In particular, an DHODH inhibitor can inhibit the pyrimidine synthesis and particularly inhibit the reduction of ubiquinone to ubiquinol. Typically, the DHODH inhibitor is an antibody, a peptide, a polypeptide, an aptamer, a small organic molecule, a siRNA, a shRNA, an antisense oligonucleotide or a ribozyme.

[0024] Tests and assays for determining whether a compound is an inhibitor of DHODH are well known by the skilled person in the art such as described in Schblermann B, Bonowski J, Grigalunas M, Burhop A, Xie Y, Hoock JGF, Liu J, Dow M, Nelson A, Nowak C, Pahl A, Sievers S, Ziegler S. Identification of Dihydroorotate Dehydrogenase Inhibitors Using the Cell Painting Assay. Chembiochem. 2022 Nov 18;23(22):e202200475.

[0025] As used herein, the term “inhibition time” refers to the time that the DHODH inhibitor are able to inhibit DHODH activity (i.e inhibiting the pyrimidine synthesis and / or inhibiting the reduction of ubiquinone to ubiquinol). Tests for determining the inhibition time of a compound are well known to the person skilled in the art, for example kinetics of inhibition of DHODH can be realized as described in Knecht W, Henseling J, Loftier M. Kinetics of inhibition of human and rat dihydroorotate dehydrogenase by atovaquone, lawsone derivatives, brequinar sodium and polyporic acid. Chem Biol Interact. 2000 Jan or Christian S, Merz C, Evans L, Gradl S, Seidel H, Friberg A, Eheim A, Lejeune P, Brzezinka K, Zimmermann K, Ferrara S, Meyer H, Lesche R, Stoeckigt D, Bauser M, Haegebarth A, Sykes DB, Scadden DT, Losman JA, Janzer A. The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies. Leukemia. 2019 Oct.

[0026] As used herein, a DHODH inhibitor exhibiting a short inhibition time refers to an DHODH inhibitor exhibiting a transient inhibition on 24 hours (i.e the decrease of DHODH activity by said inhibitor is transient, the DHODH activity return to control / basal level within 24 hours after the administration of the inhibitor). In the context of the invention, a DHODH inhibitor exhibiting a short inhibition time refers to an DHODH inhibitor exhibiting an inhibition time equal or less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 hours.

[0027] In some embodiment, the terms “exhibiting a short inhibition time” also mean “exhibiting a short duration of action”.

[0028] In particular embodiment, regarding all the methods of the invention, the DHODH inhibitor is an DHODH inhibitor exhibiting an inhibition time equal or less than 12 hours.

[0029] In particular embodiment, regarding all the methods of the invention, the DHODH inhibitor is an DHODH inhibitor exhibiting an inhibition time comprising between 30 minutes and 12 hours, and particularly between 1 hours and 8 hours, particularly between 2 hours and 6 hours, and more particularly between 4 hours and 6 hours.

[0030] In particular embodiment, regarding all the methods of the invention, the DHODH inhibitor is an DHODH inhibitor exhibiting an inhibition time of 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 246 minutes, 250 minutes, 260 minutes, 270 minutes, 300 minutes, 330 minutes, 360 minutes, 390 minutes, 420 minutes, 450 minutes, 480 minutes, 510 minutes, 540 minutes, 570 minutes or 600 minutes.

[0031] It is well known by the skilled man in the art that the inhibition time can be influenced by the regimen / dose of the inhibitors as well as its half-life. As explained above it is well known within the skill of the art to determines doses of the compound to achieve the desired therapeutic effect (i.e the desired time inhibition).

[0032] Thus, in particular embodiment, the inhibitor of dihydroorotate dehydrogenase exhibiting a short inhibition time is an inhibitor of dihydroorotate dehydrogenase with a short half-life.

[0033] As used herein, the term “Half-life” has its general meaning in the art and refers to the elimination half-life. The definition of elimination half-life is the length of time required for the concentration of a particular substance (i.e DHODH inhibitor) to decrease to half of its starting dose in the body of the subject. Thus, after one half-life has passed, 50% of the initial drug (i.e DHODH inhibitor) amount is removed from the body. Tests and assays for determining half life of a compound are well known by the skilled person in the art such as described in Hallare J, Gerri ets V. Half Life. Treasure Island (FL), 2023.

[0034] As used herein, a DHODH inhibitor exhibiting a short half-life refers to an DHODH inhibitor exhibiting a half-life equal or less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 hours.

[0035] In particular embodiment, regarding all the methods of the invention, the DHODH inhibitor is an DHODH inhibitor exhibiting a half-life equal or less than 12 hours.

[0036] In particular embodiment, regarding all the methods of the invention, the DHODH inhibitor is an DHODH inhibitor exhibiting a half-life comprising between 30 minutes and 12 hours, and particularly between 1 hours and 8 hours, particularly between 2 hours and 6 hours, and more particularly between 4 hours and 6 hours.

[0037] In particular embodiment, regarding all the methods of the invention, the DHODH inhibitor is an DHODH inhibitor exhibiting a half-life of 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 246 minutes, 250 minutes, 260 minutes, 270 minutes, 300 minutes, 330 minutes, 360 minutes, 390 minutes, 420 minutes, 450 minutes, 480 minutes, 510 minutes, 540 minutes, 570 minutes or 600 minutes.

[0038] In particular embodiment, the DHODH inhibitor is a small organic molecule

[0039] As used herein, the term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

[0040] DHODH inhibitor according to the invention are well known in the art and are typically described in the following patent application WO2018077923.

[0041] In particular embodiment, regarding all the methods of the invention, the DHODH inhibitor is BAY-2402234 having the following formula (I) :

[0042]

[0043] BAY-2402234 is a selective dihydroorotate dehydrogenase (DHODH) inhibitor exhibiting a half-life comprises between 4 to 6 hours. Its CAS Number is 2225819-06-5.

[0044] Other examples of DHODH inhibitors according to invention includes the following compounds:

[0045] ML390

[0046] As used herein, the term “circadian rhythm” has its general meaning in the art, and refers to the twenty four-hour central circadian clock, located in the suprachiasmatic nuclei (SCN) in the hypothalamus in the brain, controls the daily timing of multiple brain and body systems including sleep, alertness and performance, metabolism, reproductive rhythms, and immune function, among many others. In addition to this central or ‘master circadian clock' in the brain, other parts of the brain and peripheral organs and tissues, such as the lungs, liver, heart, pancreas and kidneys, also generate circadian rhythms and work synchronously with the SCN to control and fine-tune local circadian rhythms, such as twenty-four hour rhythms in cardiac, liver, or lung function. Methods for determining / estimating circadian rhythm are well known by the skilled person in the art such as described in Reddy S, Reddy V, Sharma S. Physiology, Circadian Rhythm. Treasure Island (FL): StatPearls Publishing; 2023 Jan; Reid KJ. Assessment of Circadian Rhythms. Neurol Clin. 2019 Aug;37(3):505-526.

[0047] Another important concept of circadian rhythm is zeitgeber time (ZT). As used herein, the term ’’zeitgeber” (literally, time giver or time cue) has its general meaning in the art and refers to environmental variables that are capable of acting as circadian time cues. The light / dark cycle is the most important zeitgeber, but other stimuli such as melatonin can also function as zeitgebers. ZT is the temporal relation of the circadian rhythm, marked by the dim light melatonin onset (DLMO), to entraining signals such as dawn (or the first introduction of light, i.e. wake time in Humans). For example, a person whose DLMO occurs 13 h after wake time is said to have a DLMO ZT of 13. The ZT of the DLMO can provide information about the phase of a person's circadian rhythm. Natural sunlight permits organisms to synchronize their physiology to the external world. Earth’s 24 h rotation induces cycles of day and night that synchronize the physiology of living organisms. Thus, sunlight is the most important natural signal to entrain our rhythms of sleep, feeding, body temperature, and metabolism. Moreover, light effects on physiology are modulated by seasons, when the length and intensity of natural light change. Therefore, sunlight impacts daily and seasonal physiology. The chronic exposure to light at nighttime has been correlated to mood alterations, metabolic dysfunctions, and poor cognition. The beginning of the light phase corresponds to Zeitgeber (ZT) 0, while ZT12 is the beginning of the dark phase. Animal models such as rodents, including mice, are principally nocturnal (i.e. sleep during the day but become active during the night). On the contrary, human beings are diurnal (i.e. sleep during the night and become active during the day). It is thus expected that a diurnal being will have a opposite circadian rhythm. Thus, an effect observed in ZT0 in nocturnal mammal will be expected at ZT12 in diurnal mammal. Indeed, the rhythmic expression of the circadian clock and the rhythmic expression of metabolic effectors have a 12-hour shift between diurnal and nocturnal mammals.

[0048] Thus, in particular embodiment regarding all the methods of the invention, when the subject is diurnal (i.e. in human beings), the DHODH inhibitor is administered at the beginning of the dark phase of subject’s circadian rhythm (i.e at the beginning of ZT12).

[0049] In particular embodiment regarding all the methods of the invention, when the subject is diurnal (i.e. human beings), the DHODH inhibitor is administered at a time selected from 4 hours, 3 hours, 2 hours, 60 min, 30 min, 15 min, 10 min, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute before the beginning of the dark phase of subject’s circadian rhythm. In particular embodiment regarding all the methods of the invention, when the subject is diurnal (i.e. human beings), the DHODH inhibitor is administered at a time selected from 4 hours, 3 hours, 2 hours, 60 min, 30 min, 15 min, 10 min, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute before the subject’s bedtime.

[0050] In particular embodiment regarding all the methods of the invention, when the subject is diurnal (i.e. human beings), the DHODH inhibitor is administered at a time selected from 4 hours, 3 hours, 2 hours, 60 min, 30 min, 15 min, 10 min, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute after the beginning of the dark phase of subject’s circadian rhythm.

[0051] In particular embodiment regarding all the methods of the invention, when the subject is diurnal (i.e. human beings), the DHODH inhibitor is administered at a time selected from 4 hours, 3 hours, 2 hours, 60 min, 30 min, 15 min, 10 min, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute after the subject’s bedtime.

[0052] In particular embodiment regarding all the methods of the invention, when the subject is nocturnal (i.e. rodents), the DHODH inhibitor is administered at the beginning of the light phase of subject’s circadian rhythm (i.e at the beginning of ZT0).

[0053] In particular embodiment regarding all the methods of the invention, when the subject is nocturnal (i.e. rodent), the DHODH inhibitor is administered at a time selected from 4 hours, 3 hours, 2 hours, 60 min, 30 min, 15 min, 10 min, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute before the beginning of the light phase of subject’s circadian rhythm.

[0054] In particular embodiment regarding all the methods of the invention, when the subject is nocturnal (i.e. rodent), the DHODH inhibitor is administered at a time selected from 4 hours, 3 hours, 2 hours, 60 min, 30 min, 15 min, 10 min, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute after the beginning of the light phase of subject’s circadian rhythm.

[0055] In particular, the method of the present invention is particularly suitable for controlling weight gain in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase, wherein said inhibitor of dihydroorotate dehydrogenase exhibits a short inhibition time and wherein said inhibitor of dihydroorotate dehydrogenase is administered at a specific time of the day related to the subject’s circadian rhythm.

[0056] The method of the present invention is also particularly suitable for stimulating weight loss in a subject in need thereof comprising administering to the subject to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase, wherein said inhibitor of dihydroorotate dehydrogenase exhibits a short inhibition time and wherein said inhibitor of dihydroorotate dehydrogenase is administered at a specific time of the day related to the subject’s circadian rhythm.

[0057] The method of the present invention is also particularly suitable for the treatment or prevention of obesity related diseases.

[0058] Thus, in another object the invention refers to a method for treating or preventing obesity -related metabolic diseases in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase, wherein said inhibitor of dihydroorotate dehydrogenase exhibits a short inhibition time and wherein said inhibitor of dihydroorotate dehydrogenase is administered at a specific time of the day related to the subject’s circadian rhythm.

[0059] The term "obesity-related metabolic diseases" encompasses disorders that are associated with, caused by, or result from obesity. Examples of obesity-related metabolic disorders include overeating and bulimia, diabetes, hypertension, elevated plasma insulin concentrations and insulin resistance, dyslipidemia, hyperlipidemia, breast, prostate, endometrial and colon cancer, heart disease, cardiovascular disorders, abnormal heart rhythms and arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, angina pectoris, cerebral infarction, cerebral thrombosis and transient ischemic attack, and osteoarthritis. Other examples include pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass. Further examples of obesity-related disorders include metabolic syndrome, also known as syndrome X, insulin resistance syndrome, type II diabetes, impaired fasting glucose, impaired glucose tolerance, inflammation, such as systemic inflammation of the vasculature, atherosclerosis, hypercholesterolemia, hyperuricaemia, as well as secondary outcomes of obesity such as left ventricular hypertrophy. Obesity-related disorders also include the liver abnormalities associated with obesity such as metabolic dysfunction-associated steatotic liver disease (MASLD) a rising cause of cirrhosis associated to obesity and metabolic syndrome. Indeed, MASLD can present as simple steatosis or evolve towards inflammation and steatohepatitis (Metabolic dysfunction-associated steatohepatitis (MASH)), with a 20 % risk of cirrhosis after 20 years. “Dyslipidemia” is a major risk factor for coronary heart disease (CHD). Low plasma levels of high-density lipoprotein (HDL) cholesterol with either normal or elevated levels of low density (LDL) cholesterol is a significant risk factor for developing atherosclerosis and associated coronary artery disease in humans. Dyslipidemia is often associated with obesity.

[0060] In particular embodiment, the obesity-related metabolic disease is type 2 diabetes or metabolic syndrome.

[0061] In particular, the method of the present invention is particularly suitable for improving insulin sensitivity in a subject in need thereof (e.g. a subject living with obesity) comprising administering to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase, wherein said inhibitor of dihydroorotate dehydrogenase exhibits a short inhibition time and wherein said inhibitor of dihydroorotate dehydrogenase is administered at a specific time of the day related to the subject’s circadian rhythm.

[0062] In other words, the present invention relates to a method of treating or preventing insulin resistance in a subject in need thereof comprising administering to the subject to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase, wherein said inhibitor of dihydroorotate dehydrogenase exhibits a short inhibition time and wherein said inhibitor of dihydroorotate dehydrogenase is administered at a specific time of the day related to the subject’s circadian rhythm.

[0063] According to the invention, the DHODH inhibitor is administered to the subject in the form of a pharmaceutical composition. Typically, the DHODH inhibitor may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The IDO inhibitor can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0064] As used herein, the term “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0065] Thus, in another aspect, the invention refers to a pharmaceutical composition comprising a DHODH inhibitor exhibiting a short inhibition time for use for treating or preventing obesity in a subject in need thereof, wherein said inhibitor of dihydroorotate dehydrogenase exhibits a short inhibition time and wherein said inhibitor of dihydroorotate dehydrogenase is administered at a specific time of the day related to the subject’s circadian rhythm. In another aspect, the inventors have demonstrated that exogenous glutamine is both necessary and sufficient for DHODH inhibition to affect de novo pyrimidine biosynthesis. Specifically, they show that BAY 2402234 inhibits the de novo pyrimidine biosynthesis pathway in a glutamine-dependent manner.

[0066] In some embodiments, the level of glutamine is measured, in particular exogenous glutamine is measured.

[0067] In some embodiments, a therapeutically effective amount of a DHODH inhibitor is administered to the subject when a high level of glutamine is detected in a subject,. In some embodiments, the DHODH inhibitor is administered at a time selected from 4 hours, 3 hours, 2 hours, 60 minutes, 30 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute before or after the onset of the subject’s dark phase, as determined by the subject’s circadian rhythm when a high level of glutamine is detected in a subject.

[0068] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0069] FIGURES:

[0070] Figure 1: Inhibition of DHODH activity does not affect day night food intake distributions

[0071] (A) BAY concentration in serum, liver, epididymal white adipose tissue and brain lysates following an acute intraperitoneal injection (n=3 / timepoint). (B) Abundance of circulating dihydroorotate (DHO) in mouse serum after single injection of BAY or vehicle (VEH ; n=3-4 / timepoint / condition). (C) Serum GDF15 measured 4 h or 16 h in BAY- or VEH- DIO mice treated for 4 weeks every other day either at ZT0 or ZT12 (n=3-4 / ZT / condition). (D) Daily water intake (n=3-4 / ZT / condition). (E) Food intake and daily intake distribution in DIO mice after 1 week of daily injections of VEH or BAY 2402234 at respective ZT (n=5 / condition).

[0072] Figure 2: Time-dependent inhibition of DHODH prevents diet-induced obesity

[0073] (A) Experimental design. Mice were fed a HFD for 12 weeks. Starting from week 8, mice were injected with vehicle (VEH) or BAY-2402234 (BAY; 1,6 mg / kg) every other day for four weeks. The IP injection was at either ZT0 or ZT12 (n=10 / condition). (B) Mouse body weight at the end of the protocol (n=10 / condition). (C) Cumulative weight gain in VEH- and BAY-injected obese mice over the 4-week treatment (n=10 / condition). (D) Masses of liver, eWAT and sWAT reported as percentage of body weight (n=9-10 / condition). (E) Liver triglyceride content (n=8 / condition). (F-G). Glucose tolerance tests for mice treated at ZTO (F) or ZT12 (G). GTTs (left panels) were performed 6 hours after VEH / BAY 2402234 injection (n=6 / condition). Middle panels report AUC-GTT and right panels fasting glycemia (n=6 / condition). (H) Food intake over the four-weeks treatment (n=10 / condition). Cumulative food intake. (I) Daily food intake during light and dark phase (n=3 independent 24 h measurements evenly spread over the 4-week treatment). (Data presented as mean ± SEM, *p < 0.05, **p < 0.005, ***p < 0.001.

[0074] Figure 3. Time-dependent inhibition of DHODH alters mitochondrial oxidative metabolism and metabolome in DIO mice.

[0075] (A) Time-dependent effects of DHODH inhibition on metabolites involved in pyrimidine biosynthesis (n=3-4 / ZT / condition). (B) Ubiquinone redox status in mouse liver following injections at ZTO or ZT12 (n=3-4 / ZT). (C) Mitochondrial complex III enzymatic activity in mitochondria isolated at ZT4 and ZT16 in mice treated at ZTO (top) or ZT12 (bottom). n=3-4 / ZT / condition. (D) ATP to ADP ratio (n=3-4 / ZT / condition). Data presented as mean ± SEM, *p < 0.05, ***p < 0.001.

[0076] Figure 4: Exogenous glutamine is necessary and sufficient for DHODH inhibition to impact de novo pyrimidine biosynthesis.

[0077] (A) Concentrations of key intermediates in the pyrimidine biosynthesis pathway in the presence or absence of DHO and BAY 2402234 (BAY), n = 6 independent experiments. (B) Concentrations glutamine (GLN) and aspartate (ASP) in the presence or absence of each amino acids in the media. (C) Concentrations of key intermediates in the pyrimidine biosynthesis pathway in the presence or absence of GLN, aspartate ASP, and BAY 2402234. n = 3 independent experiments. * denoting statistical significance at p < 0.05.

[0078] Figure 5: BAY 2402234 inhibits the de novo pyrimidine biosynthesis pathway in a glutamine-dependent manner.

[0079] (A-C) The bar graphs show metabolite concentrations across four conditions: with and without glutamine (GLN) and with or without DHODH inhibition by BAY 2402234 (BAY). A. Metabolites of the de novo pyrimidine pathway. (B) Ratio of orotate DHO to orotate. (C) Uridine metabolites. (D) The bar graphs show de novo pyrimidine pathway metabolites concentrations across four conditions, all with GLN: with and without uridine (URD) and with or without DHODH inhibition by BAY. Error bars represent the mean ± SEM, with * denoting statistical significance at p < 0.05. n = 6 (A-C) or 4 (D) independent experiments. In cases where fewer than 6 individual data points are shown, this is due to undetected metabolites.

[0080] EXAMPLE

[0081] Material & Methods

[0082] Animal studies

[0083] The local ethics committee approved the care and use of experimental animals (Pays de la Loire, France, project APAFIS#6697, compliant with directive 2010 / 63 / EU). Male C57BL / 6J mice were housed (4-5 / cage) in ventilated cabinets under 12 h light: 12 h dark conditions. After four weeks of acclimation, mice (9-10 weeks old) were fed a High Fat Diet (HFD) (60% fat, reference D12492, Research Diet). The beginning of the light phase corresponds to Zeitgeber (ZT) 0, while ZT12 is the beginning of the dark phase. Mice were fed ad libitum (AL) or exclusively during the dark phase (TRF). TRF mice had access to food for 8-9h as described in previous studies (15-16). Mice were euthanatized by exsanguination under isoflurane anesthesia.

[0084] Indirect calorimetry

[0085] Whole body metabolism was measured using metabolic cages (Panlab, Harvard apparatus). Mice were housed in adaptation cages for 2 days and then measure of oximetry and activity were performed for 3 days.

[0086] Glucose and insulin tolerance tests

[0087] GTTs and ITTs were performed on fasted animals. Mice were fasted for 16 hr (ZT22- ZT38) or 3 hr (ZT13-16) for GTTs or ITTs, respectively. Mice were given 1.5 g / kg body weight glucose (GTT) or 1 U / kg insulin (ITT), respectively.

[0088] In vivo inhibition of DHODH activity

[0089] In vivo pharmacological inhibition of DHODH in obese mice was performed by IP injection of BAY-2402234 (1.6 mg / kg, reference HY-112645, Cliniscience) every other day either at ZT0 or ZT12. Mice were sacrificed for organ collection at ZT4 and ZT16.

[0090] Isolation of liver mitochondria

[0091] Liver mitochondria were isolated by differential centrifugation as previously described, with minor modifications (23). All manipulations were then performed on ice or at 4°C. Briefly, liver (-150 mg) was washed twice, minced and homogenized in mitochondrial isolation buffer (MIB: 70 mM sucrose, 210 mM mannitol, 5 mM HEPES, 1 mM EGTA, and 0.5% BSA fatty acid free). Samples were centrifuged at 800g for 8 minutes and mitochondria-containing supernatants filtered through a 70 pm mesh filter. Samples were centrifuged for 8 minutes at 8000g and mitochondrial pellets were washed in MIB and centrifuged at 8000g for 5 minutes. Mitochondrial pellets were suspended in 100 pL of MIB. If the mitochondrial suspension was not used directly, mitochondria were pelleted and stored at -80°C.

[0092] Bioenergetics analyses

[0093] The oxygen consumption rate was determined on a Seahorse XF HS Mini Analyzer. For mitochondrial coupling assays, isolated mitochondria were incubated in an initial buffer (mitochondrial assay buffer: 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgC12, 2 mM HEPES, 1 mM EGTA, and 0.2% fatty acid free BSA, pH 7.2) containing 10 mM succinate and 2 pM rotenone. Sequential addition of 4 mM ADP, 2 pg / mL oligomycin, 4 pM FCCP, and 4 pM antimycin A were used to determine state 3 and state 4 respiration.

[0094] Mitochondrial respiratory chain complex III activity

[0095] Enzymatic activity of complex III was determined on freshly isolated mitochondria using the mitochondrial complex III activity assay kit (Clinisciences, Ref. K520).

[0096] Immunoblot of mitochondrial respiratory complexes.

[0097] Mitochondrial pellets were dissolved in lysis buffer containing 8 M urea, a protease, phosphatase and sirtuins inhibitors ((PhosphoSTOP, Roche; Complete Roche EDTA Free, Roche; AGK7, Bertin ; Salermide, Bertin). Mitochondrial extracts were prepared and protein separated following the manufacturer’s recommendations (Novex immunoblot systems, Thermo). Proteins were transferred on PVDF membranes using the TransBlot transfer system (Biorad), and mitochondrial respiratory complexes were monitored with total OXPHOS primary antibody cocktail (458099) from Invitrogen. Loading was controlled with naphtol blue black (Sigma) staining. Western blot signal was detected using a Bio-Rad imaging system and quantified by Imaged.

[0098] Triglyceride measurement

[0099] Liver TG concentration was determined with the Infinity™ Triglycerides Liquid Stable kit (Thermofisher Scientific) based on an enzymatic and colorimetric reaction.

[0100] Untargeted metabolomics on liver and hepatic mitochondria

[0101] We performed analyses on liver and isolated hepatic mitochondria. A piece of liver was pulverized and weighed before the extraction of metabolites. For mitochondria, we used samples preserved as a pellet and taken up in Potassium -Phosphate buffered saline (KPBS) solution. After determination of the protein concentration of the mitochondrial samples, an aliquot was taken and again diluted in KPBS to obtain a final amount of 250 pg in 35 pL.

[0102] Metabolites were extracted in a solution of 50% methanol, 30% acetonitrile (ACN) and 20% water. The volume was adjusted to the mass of tissue collected (1 mL for 50 mg) or to the protein content of the purified mitochondria (1 mL for 1 mg of total protein). After addition of the extraction solution, the samples were vortexed for 5 min at 4°C and centrifuged at 16,000g for 15 min at 4°C. Supernatants were collected and stored at -80°C until analysis.

[0103] LC / MS analyses were performed on a QExactive Plus Orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe coupled to a Dionex UltiMate 3000 UPLC system (Thermo). Five microliters of samples were injected onto a ZIC-pHILIC column (150 mm x 2.1 mm; i.d. 5 pm) with a guard column (20 mm * 2.1 mm; i.d. 5 pm) (Millipore) for liquid chromatographic separation. Buffer A consisted of 20 mM ammonium carbonate, 0.1% ammonium hydroxide (pH 9.2), while buffer B was 100% acetonitrile. The chromatographic gradient was performed at a flow rate of 0.200 pl / min as follows: 0-20 min, linear gradient from 80% to 20% buffer B; 20-20.5 min, linear gradient from 20% to 80% buffer B; 20.5-28 min, 80% buffer B. The mass spectrometer was operated in full scan, with a spray voltage set at 2.5 kV and a capillary heated and maintained at 320 °C. Sheath gas flow was set to 20 units, auxiliary gas flow to 5 units, and scan gas flow to 0 units. Metabolites were detected in a mass range of 75 to 1000 m / z at a resolution of 35,000 (at 200 m / z) with the automatic gain control target at 106 and the maximum injection time at 250 ms. Data were acquired with Thermo Xcalibur software (Thermo). Metabolite peak areas were determined using Thermo TraceFinder software (Thermo), identified by the exact mass of each charged ion and the known retention time on the HPLC column.

[0104] Serum Quantitative metabolomics

[0105] Targeted metabolomics analyses were performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS). Serum samples did not require any preparation prior to extraction. On the other hand, tissue samples (liver, adipose tissue, and brain) were pulverized, weighed and diluted l :5 in PBS (l g = 5 mL, adding a volume in pL equal to five times the mass in mg). Homogenization of the samples was then performed mechanically using small metal balls and a tissue lyser (TissueLyser II, Qiagen). A portion of the recovered lysate was diluted 1 :5 in UPLC ultrapure water before proceeding with a protein assay (Thermofisher, Pierce BCA Protein Assay Kit), in order to normalize the concentrations obtained by mass spectrometry by the total protein concentration.

[0106] Detection of metabolites of the pyrimidine biosynthetic pathway: DHO. Orotate and Uridine

[0107] To determine the concentration of uridine, dihydro-orotate (DHO) and orotic acid, calibration ranges were prepared. DHO (Sigma, ref. D7003), orotate (Sigma, ref. 02750) and uridine (Sigma, ref. U3750) standards were prepared at 1 mM in UPLC water, mixed and serially diluted to obtain the following calibration concentrations: 100, 50, 25, 5, 1, 0. An aliquot of each range point was then taken according to the biological matrix to be analyzed: 10 pL for sera and lOOpL for tissues. An exogenous internal standard, 5-fluoroorotic acid (Sigma, ref. F5013), was used at a concentration of 50 mM in order to normalize the signals obtained and to compensate for potential matrix effects or other variabilities inherent to mass spectrometry. Since the extraction protocols for the three metabolites of interest differ in several aspects, the methods applied according to the matrix are detailed below:

[0108] Extraction into serum: Ten microliters (10 pL) of internal standard (50 mM) was added to a 10 pL aliquot of serum. After addition of 80 pL of acetonitrile (ACN), the samples were vortexed for 10 seconds and then centrifuged at 10,000 rpm (equivalent to 10,000 x g) for 10 minutes (10°C). The supernatant (80 pL) was collected in a glass bottle compatible with LC- MS / MS.

[0109] Tissue extraction (liver and adipose tissue): Twenty-five microliters (25 pL) of internal standard (50 mM) were added to a 100 pL aliquot of tissue homogenate. After addition of 400 pL of ACN, the samples were vortexed for 10 seconds and then centrifuged at 10,000 rpm for 10 minutes (10°C). The supernatant (450 pL) was collected in a glass bottle compatible with LC-MS / MS and evaporated under nitrogen flow before being re-suspended in 80 pL of ACN.

[0110] For both extractions, the calibration samples were processed in the same way.

[0111] Ubiquinone redox status

[0112] The determination of the redox status of ubiquinone involves the detection of its oxidized form, CoQ, and its reduced form CoQH2. The concentration of each form of ubiquinone was also determined using a standard range. CoQ 10 (Sigma, ref. C9538), CoQ9 (Bertin, ref. 16866) and COQ10H2 (Sigma, ref. 1705334) standards were prepared at 1 mM in methanol (MeOH), mixed and serially diluted to give the following concentrations: 50, 25, 10, 5, 2.5, 1, 0.5, 0.1, and 0 pM. A 100 pL aliquot of each calibration point was taken for extraction. The internal standard CoQ4 (Sigma, ref. C2470), an exogenous form in rodents, was prepared in MeOH at 10 pM. To extract ubiquinones from liver, 20 pL of internal standard was added to 100 pL of tissue homogenate or calibration solution, and then 900 pL of a methanol / chloroform mixture (2:1, v:v) was added. After 10 seconds of vortexing, the samples were centrifuged at 10000 rpm for 10 minutes (10°C). The supernatant (800 pL) was collected and transferred to a glass vial compatible with LC-MS / MS and dried under nitrogen flow before being taken up in a mixture of 50% ACN, 50% UPLC water and 0.1% formic acid.

[0113] Pharmacokinetics of BAY 2402234 Blood and tissues of interest were collected at indicated times after intraperitoneal injection of BAY (1.6 mg / kg) in mice. For calibration, a standard curve of BAY 2402234 (0- 5 pM) was realized by dilution in appropriate serum or tissue lysates. For extraction, serum and tissue homogenates were mixed with CAN (1 :5, v:v), centrifuged at 10,000 rpm for 10 minutes (10°C), and supernatant was recovered before drying under nitrogen flow. Sample recovery was performed with 25% CAN buffer contining 0.1% aqueous formic acid. The samples were then analyzed by LC-MS / MS (AQUITY H-Class, Xevo TQD (Waters) system ) with a UPLC BEH Cl 8 column (Waters).

[0114] 3' UTR sequencing

[0115] RNA extraction was performed on frozen liver using Nucleozol (Macherey Nagel). The 3’seq RNA profiling protocol was performed as previously described (Soumillon et al. 2013). Libraries were prepared from 10 ng of total RNA in 4 pl. Poly(A) tails of mRNAs are labeled with universal adapters, well-specific barcodes, and unique molecular identifiers (UMIs) during template-switching reverse transcriptase. Barcoded cDNAs from multiple samples are then pooled, amplified, and labeled using a transposon fragmentation approach that enriches the 3Zends of the cDNA: 200ng of full-length cDNA are used as input to the Nextera™ DNA Flex Library Prep kit (ref #20018704, Illumina) and Nextera™ DNA CD indexes (24 indexes, 24 samples) (ref #20018707, Illumina) according to the manufacturer's protocol (Nextera DNA Flex Library Document, ref #1000000025416 v04, Illumina). The library size is controlled on the 2200 Tape Station Sytem (Agilent Technologies). A 350-800 bp long library is run on a NovaSeq 6000 using the NovaSeq 6000 SP Reagent Kit 100 cycles (ref #20027464, Illumina) with reads of 17*-8-105* cycles.

[0116] Primary Analysis

[0117] The raw fastq pairs correspond to the following criteria: the 16 bases of the first read correspond to 6 bases for a barcode specific to the designed sample and 10 bases for a unique molecular identifier (UMI). The second read (104 bases) corresponds to the sequence of the captured poly(A) RNAs. Demultiplexing was performed with an in-house python script. Raw paired-end fastq files were transformed into a single-end fastq file for each sample. Alignment on Ensembl Mus Musculus annotation ((assembly GRCm38 / mml0) reference transcriptome (Flicek P, et al. (2013) Ensembl 2013. Nucleic Acids Res 41(Database issue, D1):D48-D55) was performed using bwa (Li and Durbin 2009). Aligned reads were parsed and UMIs counted for each gene to create an expression matrix containing the absolute abundance of mRNAs in all samples. Reads aligned on multiple genes or containing more than 3 mismatches with the reference were discarded. The expression matrix is normalized and differentially expressed genes (DEG) are searched using the R package DESeq2 (Love, Huber, and Anders 2014).

[0118] Rhythmicity analysis

[0119] Analysis of both 3’UTR sequencing and metabolomics data were performed with Circacompare (Parsons et al. 2020). Resulting p values were adjusted using the Benjamini- Hochberg method to control for the false-discovery rate (FDR). Genes expression or metabolites abundances with q-value<0.05 were considered as rhythmic.

[0120] Results

[0121] Example 1 :

[0122] Mitochondrial diurnal rhythms are promoted early and persistently by nocturnal TRF,

[0123] To uncover the temporal relationship linking mitochondrial diurnal rhythms to the metabolic benefits of TRF in DIO in mice, we compared hepatic and mitochondrial metabolic oscillations in both short (4 days) and long-term (12 weeks) TRF (data not shown). There was no effect of short-term TRF on markers of DIO compared to ad libitum HFD counterparts. However, consistent with previous reports the long-term adverse effect of DIO on body weight (data not shown), food intake (data not shown), glucose tolerance, insulin sensitivity (data not shown) and serum insulin concentration (data not shown) were prevented by long-term TRF (data not shown). Remarkably, despite the absence of phenotypic differences, short-term TRF strengthened amplitudes of core-clock gene transcripts level (i.e. Per2, Bmall, Nrldl, Cryl) (data not shown) in a similar manner to long-term TRF (data not shown), suggesting that enhancement of molecular rhythms in mouse liver precedes metabolic effects. Hence, we used short-term TRF to monitor early molecular events preceding the metabolic benefits of longterm TRF. We investigated how TRF differentially promoted metabolic oscillations in whole liver and mitochondrial extracts from both short and long-term experiments. Metabolites were extracted from mouse livers and hepatic mitochondria isolated every 4 hours and analyzed by targeted metabolomics (LC-MS). We first considered mean (independent of the sampling hours) metabolites levels and observed metabolome remodeling by TRF was not apparent under short-term TRF (data not shown), as opposed to long-term TRF where phenotypic differences were accompanied by altered metabolite levels (data not shown).

[0124] Conversely, when examining rhythmicity using Circacompare, short-term TRF increased the number of oscillating metabolites similarly to long-term TRF in both liver and mitochondria. Remarkably, the promotion of rhythmicity was the highest in mitochondria during short-term TRF over a wide range of FDR (data not shown). Using a more stringent threshold (FDR = 0.05), we confirmed TRF impacted mitochondria more than livers with ~4- fold (for short-term) and ~2-fold (for long-term) increases in rhythmic metabolites compared to AL controls (data not shown). To further investigate how TRF differentially promoted metabolic oscillations in mitochondria and whole liver extracts, we compared rhythmicity patterns across conditions (data not shown). In liver, most rhythmic metabolites were identified both in AL and TRF conditions, (51 out of 99 in short-term TRF and 50 out of 79 in long-term TRF). In mitochondria, metabolic oscillations were almost exclusive to the TRF condition (51 out of 62 in short-term TRF and 27 out of 35 in long-term TRF). Overall, liver and mitochondrial metabolomes had opposite phase specificity with most metabolites peaking during the dark or the light phase, respectively (data not shown). Mitochondrial metabolites conserved a similar sharp light phase specificity (ZT6-ZT8) both in short- and long-term TRF, while liver rhythmic metabolites showed different preferential phase accumulation. Daily distribution of metabolite classes showed that differences between TRF and Ad lib conditions were more obvious in mitochondria compared to liver. Mitochondria metabolites peaked 3-6 hours earlier under short- or long-term TRF, denoting specific and persistent promotion in mitochondrial rhythms (data not shown). Overall, TRF impacts on metabolic rhythmicity was greater on isolated mitochondria compared with liver extracts. The distinct phase distributions of the metabolites indicate that liver mitochondria are specifically and early targeted by TRF.

[0125] TRF promotes rhythms in mitochondrial oxidative and pyrimidine biosynthesis pathways

[0126] In addition to the metabolome, HFD misaligns the hepatic daily transcriptome. TRF has been associated with modifications in hepatic transcriptome and metabolome temporal signature. In this framework, we hypothesized TRF promotes and coordinates rhythmic mitochondrial metabolic pathways at both transcriptomic and metabolomic level. We sequenced 3’ mRNAs in mouse liver from short-term and long-term experiments. TRF expanded rhythmic mRNA transcripts accumulation for both short and long-term experiments (data not shown). Gene ontology enrichment analysis for cellular component showed mitochondria as highly enriched within rhythmic transcripts promoted by TRF (data not shown) including for instance genes involved in mitochondrial respiration (data not shown). Taking advantage of our transcriptome and metabolome datasets, we performed a joint-analyses using MetaboAnalyst revealing that mitochondrial oxidative pathways linked to energy metabolism displayed enhanced rhythmicity in short and long-term TRF experiments (data not shown). Overall, promoted amplitudes were detected in both short- and long-term TRF with opposite phases in liver (nighttime) and mitochondria (daytime) for most metabolic pathways, once again indicating a specific mitochondrial signature in TRF. Glycolysis and pyruvate-related metabolites displayed enhanced rhythmicity, suggestive of a better metabolic switch from lipid to carbohydrate as energy substrate over the light / dark cycle, consistent with improved phenotypes and daily feeding patterns (data not shown). Glycolysis and pyruvate metabolism contribute to mitochondrial redox homeostasis through NAD+ / NADH shuttling and import of energetic substrate in TCA cycle (data not shown). Accordingly, nicotinamide metabolism, which participates in circadian clock mediated and feeding / fasting cycles mediated mitochondrial control, as well as TCA cycles intermediates also showed improved rhythmicity under TRF in both liver and mitochondria with opposite phase distribution (data not shown). By controlling mitochondrial respiration, these pathways modulate mitochondrial redox status and glutathione metabolism (GSH). GSH is a potent mitochondrial antioxidant and was also differentially affected by TRF in the liver compared to the mitochondria (data not shown). GSH synthesis notably relies on cytoplasmic amino acid metabolism, including arginine biosynthesis. GSH is then imported into mitochondria where its oxidation into glutathione disulfide (GSSG) neutralizes reactive oxygen species and protects mitochondria from oxidative damages. Conversely, TRF prompted mitochondrial rhythms in GSH to GSSG ratio while reducing lactate to pyruvate ratio, a marker of mitochondrial defect (data not shown), suggesting enhanced mitochondria integrity.

[0127] Looking for metabolites reflecting TRF control on mitochondrial oscillations in short and long-term experiments, we observed that three of the five most amplified or promoted metabolic rhythms included metabolites involved in de novo pyrimidine biosynthesis, namely carbamoyl aspartate, dihydroorotate (DHO) and orotate (data not shown). De novo pyrimidine biosynthesis relies on cytosolic enzymatic reactions, with the exception of the conversion of DHO to orotate by the dihydroorotate Dehydrogenase (DHODH). DHODH, an enzyme located on the inner mitochondrial membrane, uses ubiquinone as an electron acceptor and links pyrimidine biosynthesis to the functional respiratory chain (data not shown). Downstream of DHODH, orotate is converted into uridine mono phosphate (UMP) which serves as nucleoside precursor for DNA / RNA synthesis. Carbamoyl aspartate, DHO and orotate displayed a similar pattern of temporal accumulation peaking during the dark phase. These patterns were amplified by short-term TRF (data not shown) and persisted in long-term TRF (data not shown). Of note, the rhythmicity of UMP was promoted only in the long-term TRF, suggesting TRF early preferentially acts on upstream mitochondria-linked events. To test this hypothesis, we monitored ubiquinone Q cycle and succinate oxidation into fumarate, two proxies of DHODH activity. Similarly to metabolites involved in pyrimidine biosynthesis, both ubiquinone-to- ubiquinol and succinate-to-fumarate ratios showed improved temporal variation peaking during the dark phase (data not shown). Together, the data suggest TRF specifically amplifies daily mitochondrial oxidative functions by favoring DHODH-linked mitochondrial electron transport.

[0128] Time-dependent inhibition of DHODH improves whole body metabolism

[0129] Given its interconnection to the mitochondrial electron transport chain and the TCA cycle, we hypothesized that targeting DHODH activity to modulate mitochondrial metabolic oscillations would improve metabolic fitness in mice DIO. BAY 2402234 (BAY), is a short half-lived specific inhibitor of DHODH activity. By examining the evolution of its concentration over 24 hours in serum, brain, liver, and epididymal white adipose tissue, we estimated its biological half-life to be 4 to 6 hours (Fig. 1A), making it suitable to perform transient inhibition of DHODH enzyme activity. Accordingly, IP injection of BAY 2402234 at ZT0 resulted in temporary elevation in serum DHO peaking 8 hours post-injection (Fig. IB). Dosing time is fundamental for chronotherapies, exemplified by time-responsiveness of lipid- lowering Ppar agonists or Glucokinase activators. Considering the pharmacokinetics of BAY and that TRF promotes mitochondrial oscillations during the light phase, we speculated that the transient inhibition of DHODH would align better mitochondrial oxidative metabolism with energy demands when applied during the early light phase than during the dark phase. Hence, we decided to monitor the metabolic effects of timed DHODH inhibition in DIO mice treated either at ZT0 or ZT12. Mice were fed a HFD for 8 weeks prior to every other day injections of vehicle (VEH) or BAY for 4 weeks to avoid residual inhibitory effect across 2 consecutives diurnal cycles (Fig. 2A). Despite identical caloric intake, mice treated with BAY atZTO showed lower terminal body weight compared to other groups (Fig. 2B). Body weight gain on HFD was prevented by BAY injected at ZT0 whereas BAY injected at ZT12 had no effect on weight gain compared to the corresponding control (Fig. 2C). BAY injected at ZT0 also reduced fat mass while decreasing liver triglyceride content despite no reduction of liver weight, suggesting reduced liver steatosis (Fig. 2D and 2E). BAY injected at ZT0 also improved parameters of insulin sensitivity as fasting glycaemia was decreased and glucose tolerance improved (Fig. 2F). Of note, none of the aforementioned effects were observed when BAY was injected at ZT12 (Fig. 2G). Because BAY has been shown to alter concentrations of GDF15, an anorexigenic hormone, in db / db mice, we studied BAY effects on food intake in our model of DIO. Mice injected with BAY at ZTO or ZT12 had similar cumulative food intake (Fig. 2H) and day / night intake distribution (Fig. 21) over the protocol. Our results further indicate that metabolic benefits following timed DHODH inhibition in wild type mice is independent of appetite control. First, GDF15, remained unchanged in serum 4 hours and 16 hours following BAY injection at ZTO (Fig. 1C) but increased 4 hours after BAY injection at ZT12. Second, water consumption was unaffected in our experiments (Fig. ID) in contradiction to polydipsia correction in db / db mice. Third, daily injections of BAY in wild type mice the way they were performed in db / db mice, did not modify total nor daily distribution of food intake across light and dark phases (Fig. IE).

[0130] Altogether, our data show that the chrono-pharmacological inhibition of DHODH prevents metabolic complications in a time-specific manner without affecting caloric intake in HFD-fed obese mice.

[0131] DHODH inhibition alters mitochondrial oxidative metabolism in a time-dependent manner in DIO mice

[0132] The circadian clock controls mitochondrial dynamics, namely fusion, fission and mitophagy, to cope with varying nutrient availability and optimize mitochondrial bioenergetics in mouse live. Defective mitochondrial dynamics sensitizes mice to metabolic defects under DIO, and inhibition of DHODH activity elongates mitochondria in mouse cell lines. Hence, in combination with imaging, we analyzed daily expression levels of the different mitochondria- shaping genes following daily injections of BAY at ZTO in mice fed a HFD for 4 days. In a few days, BAY 2402234 injected at ZTO promoted rhythmic expression of genes involved in fusion, fission or mitophagy (data not shown) similarly to short-term TRF (data not shown). Despite apparent differences in morphologies (data not shown), transmission electron microscopy in liver slices collected at ZT4, i.e. 4 hours after VEH / BAY injections (data not shown) revealed AL. BAY and short-term VEH. TRF did not affect global mitochondrial area (data not shown) nor mitochondrial size distribution (data not shown), compared to the AL. VEH controls. However, when looking at mitochondrial roundness, we observed that, similarly to TRF, BAY injected at ZTO elongates mitochondria and corrected bulb-shaped morphologies observed in AL mouse livers (data not shown).

[0133] We next investigated whether time dependent metabolic benefits of DHODH inhibition in DIO mice was associated with changes in hepatic mitochondrial oxidative functions. Livers and mitochondria were collected 4 and 16 hours following the last BAY 2402234 injection at the end of the 4 week treatment period in DIO mice (Fig. 2A). Time-of-a-day specific inhibition of DHODH revealed that injection at ZTO led to a ~100-fold increase in Carbamoyl aspartate and DHO within 4 hours while metabolite levels was comparable to VEH controls 16 hours post-injection (Fig. 3A), indicating a strong and transient inhibition of DHODH over the protocol. In contrast, injection at ZT12 inhibited DHODH less effectively (~50-fold increase) but for a longer period as the increase in orotate precursors was similar 4 and 16 hours postinjection. Accordingly, time of inhibition differentially impacted mitochondrial electron transfer as shown with ubiquinone-to-ubiquinol ratio (Fig. 3B) which was increased at ZT16 only when injection was performed at ZTO. Principal-component analysis (PCA) across time of injection clustered mitochondrial metabolite profiles of BAY-injected mice away from VEH- injected mice and separated time of sampling when IP was performed at ZTO but not ZT12 (data not shown). BAY injected at ZTO reduced abundances of TCA cycle intermediates (citrate, cis-aconitate, succinate, alpha-ketoglutarate, and malate), energy metabolites (GDP, ADP, ATP) and mitochondrial cofactors (NAD+, NADP+) suggesting correction of mitochondrial energy metabolism associated with liver diseases. Circadian control of mitochondrial oxidative phosphorylation (OXPHOS) is ensured by accumulation of mitochondrial respiratory chain subunits during the light phase in mouse liver mitochondria. OXPHOS protein levels were similar at ZT4 and ZT16 in VEH control mice, indicating that DIO impairs rhythmic import of OXPHOS proteins (data not shown). By contrast, DHODH inhibition increased the accumulation of OXPHOS proteins during the light phase in mitochondrial fractions specifically when BAY was injected at ZTO and not at ZT12. To determine these whether OXPHOS protein patterns were associated with temporal changes in mitochondria energy metabolism, we measured the activity of complex III due to its proximity to DHODH. Complex III activity was increased at ZT4 in livers from mice injected with BAY at ZTO but not at ZT12 (Fig. 3C) leading to differences in ATP generation (Fig. 3D). These results led us to investigate whether the timed inhibition of DHODH also impacted mitochondrial oxidative metabolism. We observed increased GSH:GSSG ratio at ZT16, compared to ZT4 (data not shown) and different alterations of TCA cycle intermediates, including increased levels at ZT4 compared to ZT16 for fumarate and malate (data not shown), two metabolites connected to ubiquinone oxidation states, following BAY injection at ZTO only.

[0134] 4-day inhibition of DHODH activity promotes oscillations in mitochondrial oxidative metabolism in a time-dependent manner

[0135] The aforementioned data indicate that the timed DHODH inhibition may drive metabolic oscillations. We performed metabolomics in livers collected every 4 hours in mice subjected to HFD and injected daily with VEH or BAY for 4 days (data not shown). Timed- inhibition of DHODH activity inversed the temporal variation of orotate:DHO and succinate: fumarate ratios only when BAY was administered at ZTO (data not shown). Rhythmicity analysis (FDR<0.5) revealed that BAY reduced the number of oscillating metabolites, particularly when injected at ZT12 (data not shown). Although BAY injected at ZTO did not expand global rhythmicity (data not shown), phase distribution of oscillating metabolites was clearly modified, compared to VEH with an increased number of metabolites peaking from ZT6 to ZT12 (data not shown). In contrast, phase distributions were unaffected by BAY injected at ZT12 when compared to VEH counterparts (data not shown). Metabolites peaking during the late light phase in BAY ZTO livers were related to mitochondrial oxidative functions (data not shown). Indeed, NADP+ (data not shown), energy nucleosides (UTP, GTP, ATP; data not shown) or mitochondrial oxidative substrates (pyruvate, acetyl-CoA, acetylcarnitine) (data not shown) had improved oscillations when BAY was injected at ZTO compared to VEH. However, time-of-a-day injection of BAY poorly affected circadian clock gene expression (data not shown), indicating that DHODH inhibition primarily interacts with energy metabolism. Indeed, BAY injected at ZTO also induced temporal variations in ubiquinone to ubiquinol (data not shown) as well as ATP to ADP ratios (data not shown). In addition, mitochondria isolated from mouse liver injected with BAY at ZTO showed an increase in OCR at state 3u whereas BAY injected at ZT12 had no effect (data not shown). Respiratory control ratio (RCR) (state 3u / state 4o) further indicated improved mitochondrial efficiency following BAY injection at ZTO. Thus, our data indicate that timed DHODH inhibition can promote rhythmic mitochondrial oxidative metabolism, likely supporting diurnal metabolism in DIO.

[0136] To further test this hypothesis, we acutely inhibited DHODH with a single injection of BAY at ZTO or at ZT12 in mice after 3 days of HFD challenge. When measuring indirect calorimetry in metabolic cages during the fourth day, we observed the respiratory exchange ratio (RER) exhibited a nocturnal increase in mice injected with BAY at ZTO, but not at ZT12 (data not shown). This result reflects a higher rate of carbohydrate use at night illustrating the prompt protection from metabolic inflexibility provided by an injection of BAY at ZTO to mice under a HFD challenge.

[0137] Example 2 :

[0138] Exogenous glutamine is necessary and sufficient for DHODH inhibition by BAY

[0139] 2402234 to impact de novo pyrimidine biosynthesis We explored the role of glutamine in the modulation of the de novo pyrimidine biosynthesis pathway during DHODH inhibition by BAY 2402234. Glutamine and aspartate are key precursors in this pathway (data not shown). We assessed the pharmacodynamics of BAY 2402234 by using cellular DHO concentration as a proxy and selected 100 nM for the following experiment, as this concentration produced a near-plateau DHO level (data not shown). We also assessed the pharmacokinetics of BAY 2402234 by measuring its cellular concentrations and found that they did not decrease over time in the cells (data not shown). This assessment was necessary because the in vivo half-life of BAY 2402234 is approximately 4 hours (27). Therefore, we chose an 8-hour time frame for subsequent experiments, aligning with diurnal vs. nocturnal or fed vs. fasted paradigms. In the absence of the two precursors, glutamine and aspartate, DHO supplementation was necessary to achieve measurable DHO concentrations and to observe BAY 2402234's inhibitory effect on the de novo pyrimidine biosynthesis pathway (Fig. 4A). To identify nutritional conditions that modulate the action of BAY 2402234, we assessed its effects in the presence or absence of glutamine and aspartate, using DHO increase as a proxy for DHODH inhibition. The addition of either glutamine or aspartate raised the intracellular levels of both amino acids (Fig. 4B). As liver-derived cells, AML12 cells synthesize glutamine de novo using glutamate and ammonia through the action of glutamine synthetase, and they also produce aspartate internally via transamination reactions, allowing for the detection of aspartate even when glutamine and aspartate are not provided in the media (29). However, only glutamine addition effectively increased DHO concentration in the presence of BAY 2402234 (Fig. 4C). Glutamine therefore appears crucial for initiating pyrimidine synthesis by donating nitrogen to form carbamoyl phosphate. Adding glutamine boosts DHO production by overcoming this rate-limiting step, something aspartate cannot do directly. In situations where glutamine is not provided in the culture media, cells may not produce enough glutamine internally to meet all these competing demands simultaneously, because glutamine's multiple roles in metabolism create competition for its usage among essential processes, as highlighted by others (28). Our results indicate that exogenous glutamine is both necessary and sufficient in our model for DHODH inhibition by BAY 2402234 to impact de novo pyrimidine biosynthesis. This finding makes our model suitable for studying how nutrient-dependent DHODH activity affects cellular metabolism.

[0140] BAY 2402234 modifies intermediary metabolism in a glutamine-dependent manner

[0141] We then examined how DHODH inhibition by BAY 2402234 affects pyrimidine biosynthesis under conditions both with and without glutamine. BAY 2402234 increased DHO levels as expected; however, this increase was over a hundred-fold higher in the presence of glutamine (Fig. 5A). Carbamoyl-aspartate levels showed a similar trend due to the reversible nature of the enzyme dihydroorotase (EC 3.5.2.3) (Fig. 5A) (30). Unexpectedly, we observed an increase in orotate levels with BAY 2402234 treatment. Previous studies on the pharmacodynamics of BAY 2402234 used dihydroorotate (DHO), not orotate, as a marker for DHODH inhibition (27). The carbamoyl-aspartate-to-orotate ratio has also been used by others as an indicator of DHODH inhibition (31). Consistent with DHODH inhibition, we noted an elevated DHO-to- orotate ratio with BAY 2402234, showing a roughly 100-fold increase in the presence of glutamine (Fig. 5B). Finally, DHODH inhibition was evidenced by a reduction in pyrimidine pathway metabolites (UMP, UDP, UTP, uridine) with BAY 2402234. Remarkably, this decrease occurred only in the presence of glutamine, indicating that BAY 2402234 inhibits pyrimidine biosynthesis in a glutamine-dependent manner (Fig. 5C). Finally, addition of uridine 200 pg / mL in the media was able to rescue uridine metabolites levels without affecting DHO accumulation (Fig. 5D).

[0142] We then studied the metabolic impact of DHODH inhibition with BAY 2402234 by analyzing Krebs cycle intermediates in the presence and absence of glutamine. The Krebs cycle also receives carbons from pyruvate, derived from glycolysis and converted into acetyl-CoA. As expected, glutamine supplementation raised glutamine levels; however, this increase was limited in the presence of BAY 2402234 (data not shown). In this experiment, endogenous glutamine was present even when no exogenous glutamine was provided. Glutamate, a direct product of glutamine through the glutaminase enzyme reaction displayed the same patterns as glutamine (data not shown). These results suggest that BAY 2402234 may alter the catabolism of both glutamine and glutamate. We also studied glutamine and BAY 2402234 effects on pyruvate levels as a source of carbon for the Kreb's cycle. Pyruvate increased in the presence of glutamine but not when BAY 2402234 was present (data not shown). By replenishing Krebs cycle intermediates, glutamine can indirectly support cellular energy production, potentially influencing glycolysis indirectly, but it is not known to directly increase pyruvate concentration. The alteration of carbon sources — glutamate and pyruvate — with BAY 2402234 prompted us to assess its effects on Krebs cycle intermediates. While glutamine raised Krebs cycle intermediate concentrations. BAY 2402234 reduced these concentrations solely when glutamine was present, with only malate showing significant reduction (data not shown). Unexpectedly, addition of uridine 200 pg / mL in the media rescued the effect of BAY 2402234 on glutamine and glutamate (data not shown) and on metabolite of the Krebs cycle (data not shown), although statistical significance was not reached because of the limited number of replicated independent experiments. These findings suggest that BAY 2402234 modifies intermediary metabolism in a glutamine-dependent manner, an effect that can be rescue by exogenous uridine.

[0143] Discussion:

[0144] Mammalian DHODH is the only enzyme of the pyrimidine ribonucleotide synthesis pathway located in the mitochondria and supporting the respiratory chain. DHODH catalyzes the oxidation of dihydroorotate to orotate by reducing ubiquinone into ubiquinol which can then interact with complex III of the respiratory chain. Mouse liver displays rhythmic pyrimidine synthesis. Downstream of DHODH, the oscillation of uridine decreases within three days of HFD and hepatic orotate is likely regulated by feeding rhythms, independently of a functional circadian clock. Uridine is a pyrimidine ribonucleoside, critical for RNA synthesis, glycogen deposition, and many other essential cellular processes. Plasma uridine governs energy homeostasis and thermoregulation in a mechanism involving adipocyte-dependent uridine biosynthesis and leptin signaling. However, the integration of uridine control of energy homeostasis with circadian metabolism is unknown. Interestingly, oral supplementation with uridine is able to alter the diurnal variations in liver nucleotide and lipid metabolism, and leads to weight loss in HFD-fed mice. However, our data do not support a primary role for uridine in the effect of BAY injected at ZT0 notably because uridine levels are antiphasic between nocturnal TRF and when BAY is injected at ZT0.

[0145] Our finding of TRF -promoted rhythmic metabolites in the de novo pyrimidine pathways, together with improved mitochondrial oxidative function, prompted us to study the effect of a timed DHODH inhibition. It should be noted that preclinical and clinical data do not suggest that the use of any known pharmacological inhibitor of DHODH is related to weight loss. Others have reported that DHODH inhibition with daily IP of BAY2402234, for 37 days resulted in decreased food intake in db / db mice with no effect on weight gain and improved glucose metabolism (Juan Zhang et al. 2021). This effect was attributed to translocation of GLUT4 to the plasma membrane and increase in the anorectic cytokine GDF15. These results differ from our observations and may be related to leptin signaling impairment and impaired regulation of food intake in db / db mice. Although the authors used BAY2402234 early in the morning, they did not investigate a potential time-of-day dependent effect. To date, the DHODH inhibitors approved for use in patients (leflunomide and its active metabolite, teriflunomide) have a half-life of two weeks, making them unsuitable for programmed DHODH inhibition. Modest weight loss is a side effect of Leflunomide in rheumatoid arthritis patients but not of Teriflunomide in with multiple sclerosis patients.

[0146] Our results confirmed reports that TRF over several weeks prevents DIO in mice and normalizes glucose tolerance and insulin resistance. TRF also improved the rhythmic expression of circadian genes and metabolic effectors such as gluconeogenic genes and lipid metabolism genes, as previously reported. An estimated 20% of transcripts are rhythmic in the liver of mice, but this is reduced by HFD feeding. We observed that TRF prevented rhythmicity of several hundreds, which is consistent with the notion that HFD also induces de novo rhythmicity in gene expression.

[0147] To isolate the metabolic effects of TRF, we used it for a shorter duration than required to improve the metabolic phenotype. This improved rhythmicity of liver mitochondrial metabolome and mitochondrial -related gene expression. This is consistent with the notion that mitochondria integrate circadian rhythms and daily metabolic signals. We have reported previously that the hepatic mitochondrial daily rhythmicity depends on a functional circadian clock and that mitochondrial proteins are translated rhythmically under the influence of the circadian clock and feeding rhythms. This TRF-enhanced rhythmicity is consistent with the idea of nocturnal TRF improving mitochondrial function. Increased mitochondrial density was reported in the seminal study showing that nocturnal TRF prevents DIO in mice. Other studies pointed out to feeding cues being able to alter mitochondrial rhythms. HFD, which disrupts feeding rhythms, blunts daily oscillations in mitochondrial respiration. TRF during the inactive phase abolishes the daily rhythm of mitochondrial respiration in rat skeletal muscle. Our findings extend the current knowledge by showing that TRF promotes mitochondrial rhythms in an early and specific manner at the level of metabolome and mRNA expression. We took advantage of this finding to identify altered pathways that may not have been apparent in longer TRF experiments.

[0148] Rhythmic respiration is facilitated by temporal expression of mitochondrial OXPHOS genes, suggesting the compartmentalization of mitochondrial oxidative metabolism in time is evolutionarily advantageous to optimize metabolic output. We have established previously that mitochondrial function is coupled to the daily fasting / feeding cycle in part through the circadian regulator Bmall-gene / protein regulation. TRF ameliorates rhythmic. Interestingly, a functional clock is not necessary for TRF benefits, indicating that other cellular components may integrate nutrient cues. TRF and BAY injected at ZTO promoted rhythms of reduced to oxidized CoQ (CoQ / CoQH2). The ratio has been suggested to act as a metabolic sensor that fine-tunes the mitochondrial electron transport chain configuration in order to match the prevailing substrate profile. The CoQ / CoQH2 ratio has been shown to be altered by HFD in rodents livers. The main function of CoQ is to act as an electron carrier in the ETC that drives all electrons to complex III. Most of the reduction reactions of CoQ come from NADH dependent complex I and FADH2-dependent complex II. Our results suggest the diurnal pattern of the CoQ / CoQH2 ratio could be improved both by TRF and BAY injected at ZTO to improve metabolic adaptation to the HFD challenge.

[0149] TRF or BAY modified the diurnal succinate / fumarate ratio. The succinate dehydrogenase complex (SDH), associated with the inner mitochondrial membrane, catalyzes the dehydrogenation of succinate to fumarate (a step of the citric acid cycle), reducing the FAD cofactor bound to the enzyme. As recently demonstrated, DHODH can, in some conditions such as hypoxia, maintains electron input into the ETC through fumarate reduction. The authors suggested that fumarate reduction could be important in diseases that cause tissue hypoxia such as obesity. Further investigations are necessary to establish whether timed-dependent beneficial effects of DHODH inhibition relies on rerouting electrons to fumarate to dissipate excess energy.

[0150] TRF in mice on HFD improves diurnal rhythms in the circadian clock. We and others have previously reported that mitochondrial dynamics and oxidative metabolism are controlled by the circadian clock. The clock enables synchronized architectural remodeling of mitochondria in the liver to accommodate the nutrient influx at the fasting-to-feeding transition and. Architectural / organizational changes of mitochondria serve specific metabolic purposes such that fusion increases metabolic efficiency, whereas fission promotes uncoupled respiration. However, TRF beneficial effects persist in the absence of a functional molecular circadian clock, suggesting the role of other key metabolic effectors. Our data indicate that mitochondrial rhythms could be such a metabolic actor. Indeed, our data suggest that timed inhibition of DHODH can engage an architectural remodeling that is similar to TRF, consistent with the notion that DHODH can directly regulate mitochondria morphology. With regards to mitochondrial function and morphology, an important connection with pyrimidine metabolism and has been recently elucidated. In human and mouse cell lines, DHODH inhibitors promoted abundance of mitochondrial fusion proteins (MNF1 / 2) and induced mitochondrial elongation.

[0151] To discover a new timed-based druggable pathway, we relied on rhythmicity analyses in the liver, an organ typified by its role in metabolic flexibility, rapidly shifting to meet the energetic needs of the organism during physiological fasting or feeding. But timed BAY injection in mice could directly, or indirectly, drive rhythmic metabolism in other organs. White adipose tissue is also characterized by metabolic flexibility. Creatine-driven substrate cycling enhances beige-fat mitochondrial respiration, creating a futile cycle required for HFD-induced energy expenditure in adipocyte, a process dependent on creatine import from the circulation. Creatine is synthesized in the liver and released in the circulation. Finally, adipocyte creatine metabolism was shown to be rhythmic an essential for the metabolic benefits during TRF. We observed improved creatine rhythm in liver of BAYZT0- injected mice, suggesting than the DHODH mediated benefits could operate through multiple organs and improve circadian alignment in peripheral organs. Also, Dhodh expression is regulated by feeding / fasting cycles in white adipose tissue. Further work is necessary to understand these multifaceted aspects of DHODH-driven rhythmicity.

[0152] Drugs targeting mitochondrial oxidative functions have been shown to alleviate metabolic defects in DIO rodents. However, despite the large body of evidence for the circadian nature of mitochondria, no time-based modulation of their function has yet been reported to modulate metabolic outcomes in animals. Circadian medicine aims to integrate knowledge of 24-hour biological rhythms to improve diagnosis and treatment. This can be achieved by targeting the molecular clock or exploiting its rhythmic outputs, as demonstrated in pre-clinical models of metabolic diseases. Pharmacological direct targeting the circadian clock has been investigated with the natural polymethoxylated flavone Nobiletin and a Clock amplitudeenhancing small molecule. In DIO mice, Nobiletin counteracted metabolic syndrome in a Clock gene-dependent manner. A recent example of targeting the clock output is the improvement of metabolism in obese Zucker rats only when glucokinase is activated pharmacologically to restore its physiological rhythmic activity. Interestingly, as for most fields of medicine, circadian precision medicine has not yet benefited the care of obesity. The current trend for sustained-release formulations holds the most promise for the pharmacological treatment of obesity, as illustrated by recent phase 3 clinical trials with incretin mimetics.

[0153] Here, this study highlights the essential role of glutamine in modulating the effects of dihydroorotate dehydrogenase (DHODH) inhibition on metabolic pathways, emphasizing its critical function in sustaining pyrimidine biosynthesis and supporting mitochondrial function under nutrient constraints. Our findings indicate that DHODH inhibition by BAY 2402234 impairs the de novo pyrimidine synthesis pathway in a glutamine-dependent manner, thereby affecting key intermediates and mitochondrial activity exclusively in the presence of glutamine. This dependency underscores glutamine's critical role in meeting biosynthetic and energy demands within the cell, positioning it as pivotal in maintaining metabolic adaptability. The glutamine dependency revealed in our study aligns with evidence that glutamine and aspartate, abundant in the human diet, are inversely associated with obesity prevalence and promote metabolic flexibility and health (33). Given that metabolic disorders such as obesity and type 2 diabetes are characterized by reduced flexibility, these amino acids may help protect against metabolic rigidity and related complications. The potential benefits of glutamine in mitigating high-fat diet-induced hepatic lipid accumulation and metabolic-associated steatotic liver disease (MASLD) further support its role as a protective factor against metabolic dysfunctions (34,35). Therefore, incorporating dietary strategies to optimize amino acid intake could complement pharmacological approaches targeting metabolic inflexibility.

[0154] It is notable that the results of our study demonstrate that uridine supplementation effectively rescues the metabolic disruptions caused by DHODH inhibition, emphasizing the interplay between nucleotide biosynthesis and cellular metabolic flexibility.

[0155] Interestingly, our findings diverge from previous studies that demonstrated inhibition of de novo pyrimidine biosynthesis impairs pyruvate oxidation (32), a key step in mitochondrial respiration. This discrepancy suggests that the interplay between pyrimidine metabolism and mitochondrial function is more complex than previously understood. By showing that DHODH inhibition does not uniformly disrupt pyruvate oxidation, our results open new avenues for exploring alternative mechanisms through which de novo pyrimidine synthesis interfaces with respiration and intermediary metabolism. These mechanisms may involve indirect metabolic signaling, compensatory pathways such as the salvage pathway, or uncharacterized interactions between nucleotide synthesis and the regulation of mitochondrial enzymes. Understanding these pathways could provide critical insights into how cells adapt to metabolic stress and may reveal novel therapeutic targets for diseases involving mitochondrial dysfunction or disrupted nucleotide metabolism.

[0156] Our study builds on these findings by emphasizing the critical role of glutamine in modulating these effects, identifying it as a limiting factor for metabolic adaptability under DHODH inhibition. The circadian rhythmicity of hepatic glutamine levels, with daily fluctuations impacting metabolic functions, adds another layer to this dependency (36).

[0157] BAY 2402234, as a short-acting DHODH inhibitor, could maximize its efficacy if administered in alignment with these natural oscillations in glutamine availability. This highlights a potential chronopharmacological approach, where the timing of DHODH inhibitor administration could improve therapeutic outcomes by aligning drug action with the body's metabolic rhythms, a concept gaining traction in metabolic disease treatment. Furthermore, our study aligns with research demonstrating glutamine’s role in preventing high-fat diet-induced hepatic lipid accumulation and metabolic-associated fatty liver disease (MAFLD). Specifically, DHODH inhibition led to altered mitochondrial and oxidative metabolism, which glutamine supplementation can counteract under high-fat conditions. Studies have shown that glutamine positively influences hepatic homeostasis by regulating enzymes involved in lipogenesis, like acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), promoting sirtuin 1 (SIRT1) expression, and modulating glucose metabolism via AKT / FOXO1 signaling pathways, as observed in high-fat diet-induced obese mice and oleic acid-treated HEPG2 cells (35). Furthermore, glutamine supplementation has been found to reduce serum lipid levels, hepatic lipid injury, and oxidative stress in MASLD models, although it may be more effective in preventing rather than reversing MASLD progression (34).

[0158] Our findings that dihydroorotate dehydrogenase (DHODH) inhibition alters carbon flux and respiration via glutamine pathways dependent on uridine nucleotides open promising avenues for translating these insights into clinical applications. DHODH inhibitors, already recognized for their potential in treating autoimmune diseases, could be further optimized by leveraging their effects on glutamine metabolism and mitochondrial function. In cancer, where altered glutamine metabolism is a hallmark of certain tumor types, targeting DHODH could disrupt metabolic flexibility, impair nucleotide biosynthesis, and compromise mitochondrial respiration essential for tumor growth (37). In autoimmune diseases, such as rheumatoid arthritis or multiple sclerosis, the ability of DHODH inhibitors to modulate uridine-dependent pathways could reduce the proliferation of activated lymphocytes, which rely on de novo pyrimidine synthesis (27,38). Furthermore, in viral infections, where rapid nucleotide turnover is essential for viral replication, disrupting uridine nucleotide availability via DHODH inhibition could enhance antiviral effects (39). Understanding the dependency of these applications on glutamine and uridine pathways provides a mechanistic basis for tailoring DHODH inhibitors to maximize therapeutic efficacy while minimizing off-target effects.

[0159] In conclusion, we report a timed pharmacological intervention targeting mitochondria that prevents obesity in mice fed a high-fat diet. Timed inhibition of DHODH promotes rhythms of mitochondrial oxidative functions to improve metabolic adaptation to the HFD challenge. As opposed to the chrononutrition intervention of TRF, where only the nighttime version prevents obesity in DIO mice, the observed effect were not related to altered food intake. Our findings open avenues for chrono-pharmacological treatments targeting mitochondrial rhythms. REFERENCES:

[0160] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of preventing or treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase (DHODH), wherein said inhibitor of dihydroorotate dehydrogenase exhibits a short inhibition time equals or less than 24 hours and wherein said inhibitor of dihydroorotate dehydrogenase is administered at a specific time of the subject’s circadian rhythm.

2. A method for controlling weight gain in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase, wherein said inhibitor of dihydroorotate dehydrogenase exhibits a short inhibition time equals or less than 24 hours and wherein said inhibitor of dihydroorotate dehydrogenase (DHODH) is administered at a specific time of the subject’s circadian rhythm.

3. A method for stimulating weight loss in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase (DHODH), wherein said inhibitor of DHODH exhibits a short half-life and wherein said inhibitor of DHODH is administered at a specific time of the subject’s circadian rhythm.

4. A method for treating or preventing obesity-related diseases in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase (DHODH), wherein said inhibitor of DHODH exhibits a short inhibition time equals or less than 24 hours and wherein said inhibitor of DHODH is administered at a specific time of the subject’s circadian rhythm.

5. A method for improving insulin sensitivity in a subject in need thereof (e.g. a subject living with obesity) comprising administering to the subject a therapeutically effective amount of an inhibitor of dihydroorotate dehydrogenase (DHODH), wherein said inhibitor of DHODH exhibits a short inhibition time equals or less than 24 hours and wherein said inhibitor of DHODH is administered at specific time of the subject’s circadian rhythm.

6. The method according to any one of claim 1 to 5, wherein the DHODH inhibitor exhibits a half-life equals or less than 24 hours.

7. The method according to claim 6, wherein the DHODH inhibitor exhibits a half-life ranging between 1 hour and 8 hours.

8. The method according to any one of claim 1 to 7, wherein the DHODH inhibitor is a small organic molecule.

9. The method according to claim 8, wherein the DHODH inhibitor is BAY-2402234 having the following formula (I) :

10. The method according to any one of claim 1 to 9, wherein the subject is diurnal.

11. The method according to claim 9, wherein the DHODH inhibitor is administered at a time selected from 4 hours, 3 hours, 2 hours, 60 min, 30 min, 15 min, 10 min, 9, 8, 7, 6, 5, 4, 3, 2 and 1 minute before or after the beginning of the dark phase of subject’s circadian rhythm.

12. The method according to any one of claims 1 to 11, where the DHODH inhibitor is administered to the subject when a high level of glutamine is detected in a subject.

13. A pharmaceutical composition comprising a dihydroorotate dehydrogenase (DHODH) inhibitor exhibiting a short inhibition time for use for treating obesity in a subject in need thereof, wherein said inhibitor of DHODH exhibits a short inhibition time equalsor less than 24 hours and wherein said inhibitor of DHODH is administered at specific time of the subject’s circadian rhythm.

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