Reactivating Circadian Rhythms as a Therapeutic Strategy: Insights from Basic Research

Abstract

One of the most significant conceptual changes brought about by the discovery of clock genes and development of circadian-clock mutant mice is the recognition that impaired circadian rhythmicity extends its impact far beyond sleep, driving pathogenesis of a wide variety of disorders such as cancer, obesity, and hypertension. However, despite this growing clinical evidence, chronobiology still lacks a coherent answer to the converse question: can restoration of circadian rhythms ameliorate—or even reverse—such diseases? In this review, three complementary pharmacological strategies—each still in preclinical development—are explored. First, direct modulation of the transcription-translation feedback loop (TTFL)—the core gene-regulatory circuit that generates 24-h rhythms in almost all nucleated cells—is reviewed as an approach to manipulation of cellular circadian biology. Second, the suprachiasmatic nucleus (SCN)-enriched G-protein-coupled receptor Gpr176 is highlighted as a central-clock target, given its ligand-independent, G_z-mediated control of cAMP signaling and demonstrated ability to reset the master pacemaker. Third, the concept of rhythmic enhancement of output function is introduced and exemplified by describing re-activation of circadian oxidized form of nicotinamide adenine dinucleotide (NAD⁺)-dependent 3β-hydroxy-steroid dehydrogenase (3β-HSD) activity in the meibomian gland—using nicotinamide mononucleotide (NMN)—to restore peripheral clock-driven steroidogenesis in this tissue, which leads to amelioration of meibomian gland dysfunction, a leading cause of dry eye disease. This review aims to highlight the molecular logic of each strategy; both mechanistic insights and safety/efficacy considerations are discussed.

1. INTRODUCTION

Can various diseases be reversed or alleviated by correcting the internal circadian clock? (Fig. 1). Impaired circadian rhythms have been linked to pathogenesis of a wide variety of diseases, including hypertension,^1,2) obesity,³⁾ diabetes mellitus,⁴⁾ cancer,⁵⁾ polyploidization of liver cells,⁶⁾ rheumatoid arthritis,⁷⁾ hepatic metabolism disorders,⁸⁾ jet-lag syndrome,⁹⁾ nocturia,¹⁰⁾ chronic kidney disease,^11,12) pain hypersensitivity,¹³⁾ and evaporative dry-eye disease,¹⁴⁾ among others. This growing evidence supports the potential value of developing drugs targeting the circadian clock system to prevent and treat a broad spectrum of related disorders. However, the lack of clinically validated clock-modulators has thus far delayed proof-of-concept in chronomedicine (Fig. 1, a future direction of chronobiology).

Fig. 1. The Core Question of This Review Regarding the Potential of Circadian Clock Re-Activation

In this review, a series of studies aimed at developing drugs targeting the circadian clock system will be explored. More specifically, this review highlights the logic of three different pharmacological strategies for drugging clock: i) direct transcription-translation feedback loops (TTFL)-mediated cellular clock modulation (see chapter 2.1), ii) central-clock modulation via suprachiasmatic nucleus (SCN)-specific G-protein coupled receptors (GPCRs) (see chapter 2.2), and iii) tissue-specific amplification of peripheral clock outputs (see chapter 2.3). Integrating and comparing these potential approaches, this review argues that reactivating circadian rhythms is a therapeutic avenue that merits further investigation.

2. MOLECULAR LOGIC FOR DRUG DISCOVERY TARGETING CENTRAL AND PERIPHERAL CLOCKS

2.1. Is the TTFL a Viable Therapeutic Target?

Notwithstanding intriguing reports of transcription-independent circadian oscillations of per-oxiredoxin superoxidation in red blood cells,¹⁵⁾ the field-wide consensus holds that, in every nucleated cell, whether central or peripheral, individual circadian rhythms are generated by TTFLs.^16–18) Reinforcing this model, mice carrying a site-specific mutation only at the E′-box element in the promoter of the core clock gene Per2 demonstrate that transcription via this element is essential for establishing cell-autonomous circadian oscillations.¹⁹⁾ Similarly, deleting the Cry1 intronic enhancer region, which contains a ROR-responsive element, led to alteration of circadian period.²⁰⁾ These data indicate that the mechanism to generate and maintain 24-h rhythmicity lies in the architecture of the TTFLs in each cell. In this regard, it is worth noting that in the Per2 E′-box mutant cells, all mammalian core clock proteins, including Per1, Per2, Clock, Bmal1, Cry1, Cry2, CK1, DBP, E4BP4, ROR and REV-ERB remain intact, the sole change being the mutated E′-box in the Per2 promoter, indicating that transcription is a critical nodal element for generating rhythmicity (Fig. 2).

Fig. 2. The Mammalian Circadian Clock System

Circadian rhythms are generated in nearly all cells in the body. In each cell, common core clock genes generate circadian rhythmicity (left). At the organismal level, the central clock located in the SCN directs harmonious oscillations of peripheral tissues (middle). GPCRs in the SCN help to generate robust circadian rhythmicity, either positively or negatively regulating cAMP signaling in a time-of-day-dependent manner (right). TTFL, transcription/translation feedback loop; AC, adenylate cyclase.

Drug-discovery efforts targeting core clock proteins within the TTFL have advanced most notably for the transcriptional repressor Cry and the nuclear receptors REV-ERB and ROR, for which potent and selective small-molecule modulators have been identified.^21–23) Cry2-targeting molecules improve glucose clearance in obese mice and inhibit glioblastoma cell growth in culture.^24–26) A synthetic REV-ERB agonist decreases obesity, accompanied by changes in expression of metabolic genes in liver, skeletal muscle and adipose tissue, in mice.²⁷⁾ Pharmacologic acceleration of REV-ERB proteolysis enhances the amplitude of circadian gene expression critical for liver metabolism and improves energy homeostasis in mice.²⁸⁾ A ROR agonist, Nobiletin, also acts as a circadian clock amplitude enhancer and helps combat metabolic disorder²⁹⁾ and cancer growth³⁰⁾ in mice. Because REV-ERB and ROR are multi-functional nuclear receptors, their activity affects multiple pathways even beyond the TTFL: The observed therapeutic benefits are likely mediated through a mix of clock-dependent and clock-independent mechanisms—and may even reflect a synergistic interplay of both.³¹⁾

The TTFL-targeting drugs necessitate further clinical evaluation from a pragmatic standpoint. Because clock genes are present in virtually all cells in the body (Fig. 2), systemic application is likely to cause complex effects. In both primates and rodents, the phases of rhythmic clock gene expression in the SCN and peripheral tissues are disparate from each other.^32,33) Therefore, drugs able to act on core clock-components would likely reset all oscillators simultaneously, jeopardizing the adaptive phasic hierarchy between SCN and peripheral tissues.^32,33) To avoid this risk, selective tissue-limited administration of drug may be necessary.

2.2. Avenues for Central Clock Modulation—GPCRs as Promising Targets

The mammalian circadian clock is organized in a systemic hierarchy of multiple oscillators, in which the SCN functions as a master pacemaker at the top of hierarchy^34–36) (Fig. 2). The SCN harmonizes the phase and amplitude of circadian rhythms of peripheral tissues. Because of this role, the SCN is a unique and ideal target for modulating the entire body clock system.

From a drug-development perspective, GPCRs selectively expressed in the SCN are attractive targets: GPCRs are the largest family of cell-surface receptors and comprise a major source of drug targets with diverse therapeutic applications.^37,38) The tissue-expression specificity is also key to confining drug action. Unlike drugs targeting TTFLs, SCN specificity makes it possible to design drugs that affect the central clock (SCN) while having no “direct” effect on peripheral clocks, leaving them subject to SCN-dependent cues, and thereby retaining normal between-tissue phasic organization in the body. However, despite these conceivable (or theoretical) advantages, therapeutic exploitation of SCN-specific GPCRs is still at an early, proof-of-concept stage of basic research, exemplified by studies on Gpr176 (see below).

Gpr176 is an SCN-enriched orphan GPCR that can set the pace of the SCN clock^39,40)—thus, this GPCR might be a target for drug discovery for circadian clock treatment. Gpr176 undergoes asparagine (N)-linked glycosylation, a post-translational modification required for its proper cell-surface expression.⁴¹⁾ Although its endogenous ligand remains unknown, this orphan receptor has the ability to exhibit ligand-independent basal activity.^39,42) Gpr176 couples to the unique G-protein subclass Gz (or Gx) and participates in reducing cAMP production during the night.³⁹⁾

The regulator of G-protein signaling 16 (Rgs16) is equally important for the regulation of cAMP synthesis in the SCN,^43,44) suggesting an interplay between Gpr176 and Rgs16 in the regulation of circadian cAMP pathway in the SCN⁴⁵⁾ (Fig. 2). In the SCN of Gpr176^−/− mice, expression of neuromedin U (Nmu) and neuromedin S (Nms) is markedly up-regulated, and this compensatory increase preserves the mice’s normal phase-resetting responses to light.⁴⁰⁾

Another interesting feature of Gpr176 is its rhythmic expression in the SCN, peaking during the night.³⁹⁾ The neuropeptide vasoactive intestinal polypeptide (VIP) and its cognate receptor Vipr2 signaling is necessary for cells in the SCN to synchronize their timekeeping,^46,47) and a genetic ablation of Vipr2 in the SCN leads to reduced expression of Gpr176,⁴⁸⁾ suggesting a counterbalancing between Vipr2-Gs signaling and Gpr176-Gz signaling in the SCN (see a Yin-Yang model depicted in Fig. 2). At the mRNA level, light causes a rapid downregulation of Gpr176 expression in cholecystokinin (CCK)-positive neurons in the SCN.⁴⁹⁾ These mRNA data suggest that circadian clock-dependent and light dependent Gpr176 activity in the SCN is likely controlled by both its unknown ligand and the amount of receptor expressed. In spite of this wealth of information of gene expression and knockout mouse data studying Gpr176, we have little information about its ligand.⁵⁰⁾ Substantial additional effort—including cell-based high-throughput screening as well as structure-guided virtual screening—will be necessary to drive drug discovery for Gpr176.^51–54)

2.3. Reactivation of Rhythmic Output Pathways in Specific Peripheral Tissues

Besides directly targeting the rhythm-generation mechanism (i.e., oscillator or TTFL) itself, an equally possible avenue is to manipulate the circadian clock’s output pathways (i.e., outputs) (Fig. 3, left). Although the TTFL machinery is common to all tissues, each tissue converts this oscillator’s signal into diverse, tissue-unique functional output rhythms. Pharmacologically amplifying these specific output rhythms offers two advantages: (i) precision—interventions can be confined to the tissue of interest, and (ii) functional efficacy—by acting directly on the effector pathway, interventions can be effective and specific (Fig. 3, left). I term this strategy rhythmic enhancement of output function. Its potential can be exploited, in principle, in any tissue; while I apply it to the meibomian gland, a peripheral tissue in the eyelid (see below).

Fig. 3. A Strategy for Rhythmic Enhancement of Output Function

(Left) A conceptual diagram of enhancement of circadian rhythm of specific output. (Right) A graphic summary depicting the effect of rhythmic enhancement of steroidogenic activity in the meibomian gland leading to improvement of meibomian gland function. MGD, meibomian-gland dysfunction.

The meibomian glands in the eyelids produce oil (meibum) to the ocular surface to prevent dehydration (dry eye). Androgens are generated inside this tissue through a mechanism of local intracrine activity.^14,55) Canonically, hormones are produced by endocrine organs and delivered to target tissues. However, for steroids, the concept of “intracrinology,” whereby hormones are synthesized in the tissue where they exert their effect without being released into blood circulation, has been proposed⁵⁵⁾ and identified for a number of tissues including the meibomian gland¹⁴⁾ (Fig. 3, right). The circadian clock regulates this local intracrine activity through nicotinamide adenine dinucleotide (NAD⁺)-dependent circadian 3β-hydroxy-steroid dehydrogenase (3β-HSD) activity. Age-associated reduction in 3β-HSD activity caused meibomian gland dysfunction (MGD) leading to evaporative dry eye disease.^14,56) Conversely, reactivation of circadian 3β-HSD activity by boosting its coenzyme NAD⁺ availability (through topical nicotinamide mononucleotide (NMN) application) improved glandular cell proliferation and alleviated MGD and accompanying dry eye phenotype.¹⁴⁾ These data exemplify a model-case in which (re)activation of a specific output rhythm can reverse local tissue disorder.

The time-dependent and tissue-specific enhancement of rhythmic intracrine activity appears important to normalize meibomian gland function. To restore this rhythm, NMN-containing eye drop was supplied to mice specifically during daytime, which is when the endogenous meibomian intracrine activity is in its daily peak phase.¹⁴⁾ On the other hand, night-time eye-drop administration of NMN was less effective than the daytime application, indicating that appropriately timed up-regulation resembling the endogenous circadian rhythm is essential to draw the maximum drug effect.

The potential merit of topical (re)activation of local intracrine activity also involves fewer potential side-effects on extra-meibomian tissues. For example, the same NAD⁺-dependent 3β-HSD enzyme is known to contribute to aldosterone production in the adrenal gland.^57–62) Abnormally activated 3β-HSD activity in this tissue leads to hyperaldosteronism, associated with salt-sensitive hypertension in mice²⁾ and is linked to idiopathic hyperaldosteronism in humans.⁶²⁾ Therefore, avoidance of off-site activation of 3β-HSD enzymatic activity (for example, in the adrenal gland) is critical for drug-development and safety evaluation.

Clinically, the loss of sex-steroid hormones in men (andropause) and women (menopause) is a critical hallmark of aging. Both male and female express 3β-HSD in the meibomian gland.^14,63) Androgen metabolites, namely, 5α-androstane-3,17-dione, 3α-hydroxy-5α-androstan-17-one, and 3β-hydroxy-5α-androstan-17-one, have been identified to be secreted in meibum samples regardless of genders.⁶³⁾ These metabolic biomarkers may serve to assess meibomian gland activity in clinical settings. Future clinical study will therefore require evaluating the impact of “circadian” NMN application on these markers in patient cohorts. This might become a representative trial for rhythmic enhancement of output function for new drug-discovery.

3. CONCLUSION AND FUTURE DIRECTION

This review explored the potential of leveraging three complementary circadian strategies—TTFL modulation, SCN-specific GPCR targeting, and tissue-specific rhythmic enhancement of output function—to create next-generation chronotherapeutics. It is my view that these strategies will enable us to propose new drugs and therapies for disease groups that are still in need of novel medications, such as MGD and sleep disorders. Importantly, such strategies may be effective in addressing the age-related natural decrease in circadian rhythmicity and restoring clock function in older populations to alleviate age-associated diseases.^64,65)

Although this review focused on drug-based chronomedicine, the circadian system is also remarkably responsive to drug-free behavioral cues. Preclinical studies show that regularly timed exercise, structured feeding schedules, and the resulting predictable fluctuations in core body temperature can robustly amplify peripheral clock rhythms and restore rhythmic gene expression across multiple tissues.^66–69) Harnessing such lifestyle-driven entrainment signals not only offers a low-cost, low-risk means of reinforcing endogenous clocks but may also act synergistically with TTFL-, SCN-, or output-focused therapeutics to maximize clinical benefit. On the basis of pharmacological interventions designed to strengthen (or re-activate) endogenous circadian rhythms, these approaches may help to restore temporal coordination of behavior and physiologies and thereby modulate disease outcomes. I envisage that insights gleaned from circadian-clock research over the coming decade will not only satisfy fundamental scientific curiosity but also shape the development of novel therapeutic approaches to improve human health and wellness.

Acknowledgments

I thank Dr. Hitoshi Okamura, Professor Emeritus, Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan for his continued guidance to our laboratory research. I also thank all previous and current staffs in my laboratory: Dr. Yoshiaki Yamaguchi, Dr. Takahito Miyake, Dr. Emi Hasegawa, and Dr. Yoichi Tanaka for their support. This work and studies described in this review were supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (22H04987 and 24H02306), the Cyclic Innovation for Clinical Empowerment of the Japan Agency for Medical Research and Development (JP22pc0101069), SRF, the Basis for Supporting Innovative Drug Discovery and Life Science Research program of the Japan Agency for Medical Research and Development (JP21am0101092), and Astellas Foundation for Research on Metabolic Disorders.

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2024 Pharmaceutical Society of Japan Award for Divisional Scientific Promotion.

REFERENCES

• 1) Doi M. Circadian clock-deficient mice as a tool for exploring disease etiology. Biol. Pharm. Bull., 35, 1385–1391 (2012).
• 2) Doi M, Takahashi Y, Komatsu R, Yamazaki F, Yamada H, Haraguchi S, Emoto N, Okuno Y, Tsujimoto G, Kanematsu A, Ogawa O, Todo T, Tsutsui K, van der Horst GTJ, Okamura H. Salt-sensitive hypertension in circadian clock-deficient Cry-null mice involves dysregulated adrenal Hsd3b6. Nat. Med., 16, 67–74 (2010).
• 3) Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E, Laposky A, Losee-Olson S, Easton A, Jensen DR, Eckel RH, Takahashi JS, Bass J. Obesity and metabolic syndrome in circadian Clock mutant mice. Science, 308, 1043–1045 (2005).
• 4) Perelis M, Marcheva B, Ramsey KM, Schipma MJ, Hutchison AL, Taguchi A, Peek CB, Hong H, Huang W, Omura C, Allred AL, Bradfield CA, Dinner AR, Barish GD, Bass J. Pancreatic beta cell enhancers regulate rhythmic transcription of genes controlling insulin secretion. Science, 350, aac4250 (2015).
• 5) Kinouchi K, Sassone-Corsi P. Metabolic rivalry: circadian homeostasis and tumorigenesis. Nat. Rev. Cancer, 20, 645–661 (2020).
• 6) Chao HW, Doi M, Fustin JM, Chen H, Murase K, Maeda Y, Hayashi H, Tanaka R, Sugawa M, Mizukuchi N, Yamaguchi Y, Yasunaga JI, Matsuoka M, Sakai M, Matsumoto M, Hamada S, Okamura H. Circadian clock regulates hepatic polyploidy by modulating Mkp1-Erk1/2 signaling pathway. Nat. Commun., 8, 2238 (2017).
• 7) Hashiramoto A, Yamane T, Tsumiyama K, Yoshida K, Komai K, Yamada H, Yamazaki F, Doi M, Okamura H, Shiozawa S. Mammalian clock gene Cryptochrome regulates arthritis via proinflammatory cytokine TNF-alpha. J. Immunol., 184, 1560–1565 (2010).
• 8) Fustin JM, Doi M, Yamada H, Komatsu R, Shimba S, Okamura H. Rhythmic nucleotide synthesis in the liver: temporal segregation of metabolites. Cell Rep., 1, 341–349 (2012).
• 9) Yamaguchi Y, Suzuki T, Mizoro Y, Kori H, Okada K, Chen Y, Fustin JM, Yamazaki F, Mizuguchi N, Zhang J, Dong X, Tsujimoto G, Okuno Y, Doi M, Okamura H. Mice genetically deficient in vasopressin V1a and V1b receptors are resistant to jet lag. Science, 342, 85–90 (2013).
• 10) Negoro H, Kanematsu A, Doi M, Suadicani SO, Matsuo M, Imamura M, Okinami T, Nishikawa N, Oura T, Matsui S, Seo K, Tainaka M, Urabe S, Kiyokage E, Todo T, Okamura H, Tabata Y, Ogawa O. Involvement of urinary bladder Connexin43 and the circadian clock in coordination of diurnal micturition rhythm. Nat. Commun., 3, 809 (2012).
• 11) Yoshida Y, Matsunaga N, Nakao T, Hamamura K, Kondo H, Ide T, Tsutsui H, Tsuruta A, Kurogi M, Nakaya M, Kurose H, Koyanagi S, Ohdo S. Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis. Nat. Commun., 12, 2783 (2021).
• 12) Ohdo S. Chrono-drug discovery and development based on circadian rhythm of molecular, cellular and organ level. Biol. Pharm. Bull., 44, 747–761 (2021).
• 13) Koyanagi S. Chrono-pharmaceutical approaches to optimize dosing regimens based on the circadian clock machinery. Biol. Pharm. Bull., 44, 1577–1584 (2021).
• 14) Sasaki L, Hamada Y, Yarimizu D, et al. Intracrine activity involving NAD-dependent circadian steroidogenic activity governs age-associated meibomian gland dysfunction. Nat. Aging, 2, 105–114 (2022).
• 15) O’Neill JS, Reddy AB. Circadian clocks in human red blood cells. Nature, 469, 498–503 (2011).
• 16) Takahashi JS. Transcriptional architecture of the mammalian circadian clock. Nat. Rev. Genet., 18, 164–179 (2017).
• 17) Patke A, Young MW, Axelrod S. Molecular mechanisms and physiological importance of circadian rhythms. Nat. Rev. Mol. Cell Biol., 21, 67–84 (2020).
• 18) Nakagawa S, Nguyen Pham KT, Shao X, Doi M. Time-restricted G-protein signaling pathways via GPR176, G_z, and RGS16 set the pace of the master circadian clock in the suprachiasmatic nucleus. Int. J. Mol. Sci., 21, 5055 (2020).
• 19) Doi M, Shimatani H, Atobe Y, Murai I, Hayashi H, Takahashi Y, Fustin JM, Yamaguchi Y, Kiyonari H, Koike N, Yagita K, Lee C, Abe M, Sakimura K, Okamura H. Non-coding cis-element of Period2 is essential for maintaining organismal circadian behaviour and body temperature rhythmicity. Nat. Commun., 10, 2563 (2019).
• 20) Mermet J, Yeung J, Hurni C, Mauvoisin D, Gustafson K, Jouffe C, Nicolas D, Emmenegger Y, Gobet C, Franken P, Gachon F, Naef F. Clock-dependent chromatin topology modulates circadian transcription and behavior. Genes Dev., 32, 347–358 (2018).
• 21) Hirota T, Lee JW, St John PC, Sawa M, Iwaisako K, Noguchi T, Pongsawakul PY, Sonntag T, Welsh DK, Brenner DA, Doyle FJ 3rd, Schultz PG, Kay SA. Identification of small molecule activators of cryptochrome. Science, 337, 1094–1097 (2012).
• 22) Chen Z, Yoo SH, Takahashi JS. Development and therapeutic potential of small-molecule modulators of circadian systems. Annu. Rev. Pharmacol. Toxicol., 58, 231–252 (2018).
• 23) Miller S, Hirota T. Pharmacological interventions to circadian clocks and their molecular bases. J. Mol. Biol., 432, 3498–3514 (2020).
• 24) Dong Z, Zhang G, Qu M, Gimple RC, Wu Q, Qiu Z, Prager BC, Wang X, Kim LJY, Morton AR, Dixit D, Zhou W, Huang H, Li B, Zhu Z, Bao S, Mack SC, Chavez L, Kay SA, Rich JN. Targeting glioblastoma stem cells through disruption of the circadian clock. Cancer Discov., 9, 1556–1573 (2019).
• 25) Humphries PS, Bersot R, Kincaid J, Mabery E, McCluskie K, Park T, Renner T, Riegler E, Steinfeld T, Turtle ED, Wei ZL, Willis E. Carbazole-containing sulfonamides and sulfamides: discovery of cryptochrome modulators as antidiabetic agents. Bioorg. Med. Chem. Lett., 26, 757–760 (2016).
• 26) Miller S, Kesherwani M, Chan P, Nagai Y, Yagi M, Cope J, Tama F, Kay SA, Hirota T. CRY2 isoform selectivity of a circadian clock modulator with antiglioblastoma efficacy. Proc. Natl. Acad. Sci. U.S.A., 119, e2203936119 (2022).
• 27) Solt LA, Wang Y, Banerjee S, Hughes T, Kojetin DJ, Lundasen T, Shin Y, Liu J, Cameron MD, Noel R, Yoo SH, Takahashi JS, Butler AA, Kamenecka TM, Burris TP. Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists. Nature, 485, 62–68 (2012).
• 28) Zhao X, Hirota T, Han X, Cho H, Chong LW, Lamia K, Liu S, Atkins AR, Banayo E, Liddle C, Yu RT. Circadian amplitude regulation via FBXW7-targeted REV-ERBalpha degradation. Cell, 165, 1644–1657 (2016).
• 29) He B, Nohara K, Park N, Park YS, Guillory B, Zhao Z, Garcia JM, Koike N, Lee CC, Takahashi JS, Yoo SH, Chen Z. The small molecule nobiletin targets the molecular oscillator to enhance circadian rhythms and protect against metabolic syndrome. Cell Metab., 23, 610–621 (2016).
• 30) Kim E, Kim YJ, Ji Z, Kang JM, Wirianto M, Paudel KR, Smith JA, Ono K, Kim JA, Eckel-Mahan K, Zhou X, Lee HK, Yoo JY, Yoo SH, Chen Z. ROR activation by nobiletin enhances antitumor efficacy via suppression of IkappaB/NF-kappaB signaling in triple-negative breast cancer. Cell Death Dis., 13, 374 (2022).
• 31) Kim E, Yoo SH, Chen Z. Circadian stabilization loop: the regulatory hub and therapeutic target promoting circadian resilience and physiological health. F1000 Res., 11, 1236 (2022).
• 32) Mure LS, Le HD, Benegiamo G, Chang MW, Rios L, Jillani N, Ngotho M, Kariuki T, Dkhissi-Benyahya O, Cooper HM, Panda S. Diurnal transcriptome atlas of a primate across major neural and peripheral tissues. Science, 359, eaao0318 (2018).
• 33) Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki Y, Menaker M, Tei H. Resetting central and peripheral circadian oscillators in transgenic rats. Science, 288, 682–685 (2000).
• 34) Okamura H. Suprachiasmatic nucleus clock time in the mammalian circadian system. Cold Spring Harb. Symp. Quant. Biol., 72, 551–556 (2007).
• 35) Herzog ED, Hermanstyne T, Smyllie NJ, Hastings MH. Regulating the suprachiasmatic nucleus (SCN) circadian clockwork: interplay between cell-autonomous and circuit-level mechanisms. Cold Spring Harb. Perspect. Biol., 9, a027706 (2017).
• 36) Miyake T, Doi M. Reconstitution of organismal liver clock function requires light. Trends Endocrinol. Metab., 30, 569–571 (2019).
• 37) Santos R, Ursu O, Gaulton A, Bento AP, Donadi RS, Bologa CG, Karlsson A, Al-Lazikani B, Hersey A, Oprea TI, Overington JP. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov., 16, 19–34 (2017).
• 38) Lorente JS, Sokolov AV, Ferguson G, Schioth HB, Hauser AS, Gloriam DE. GPCR drug discovery: new agents, targets and indications. Nat. Rev. Drug Discov., 24, 458–479 (2025).
• 39) Doi M, Murai I, Kunisue S, Setsu G, Uchio N, Tanaka R, Kobayashi S, Shimatani H, Hayashi H, Chao HW, Nakagawa Y, Takahashi Y, Hotta Y, Yasunaga J, Matsuoka M, Hastings MH, Kiyonari H, Okamura H. Gpr176 is a Gz-linked orphan G-protein-coupled receptor that sets the pace of circadian behaviour. Nat. Commun., 7, 10583 (2016).
• 40) Yamaguchi Y, Murai I, Takeda M, Doi S, Seta T, Hanada R, Kangawa K, Okamura H, Miyake T, Doi M. Nmu/Nms/Gpr176 triple-deficient mice show enhanced light-resetting of circadian locomotor activity. Biol. Pharm. Bull., 45, 1172–1179 (2022).
• 41) Wang T, Nakagawa S, Miyake T, Setsu G, Kunisue S, Goto K, Hirasawa A, Okamura H, Yamaguchi Y, Doi M. Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176. Sci. Rep., 10, 4429 (2020).
• 42) Martin AL, Steurer MA, Aronstam RS. Constitutive activity among orphan class-A G protein coupled receptors. PLOS ONE, 10, e0138463 (2015).
• 43) Doi M, Ishida A, Miyake A, et al. Circadian regulation of intracellular G-protein signalling mediates intercellular synchrony and rhythmicity in the suprachiasmatic nucleus. Nat. Commun., 2, 327 (2011).
• 44) Hayasaka N, Aoki K, Kinoshita S, Yamaguchi S, Wakefield JK, Tsuji-Kawahara S, Horikawa K, Ikegami H, Wakana S, Murakami T, Ramabhadran R, Miyazawa M, Shibata S. Attenuated food anticipatory activity and abnormal circadian locomotor rhythms in Rgs16 knockdown mice. PLoS One, 6, e17655 (2011).
• 45) Hastings MH, Maywood ES, Brancaccio M. Generation of circadian rhythms in the suprachiasmatic nucleus. Nat. Rev. Neurosci., 19, 453–469 (2018).
• 46) Harmar AJ, Marston HM, Shen S, Spratt C, West KM, Sheward WJ, Morrison CF, Dorin JR, Piggins HD, Reubi JC, Kelly JS, Maywood ES, Hastings MH. The VPAC₂ receptor is essential for circadian function in the mouse suprachiasmatic nuclei. Cell, 109, 497–508 (2002).
• 47) Aton SJ, Colwell CS, Harmar AJ, Waschek J, Herzog ED. Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons. Nat. Neurosci., 8, 476–483 (2005).
• 48) Hitrec T, Petit C, Cryer E, Muir C, Tal N, Fustin JM, Hughes ATL, Piggins HD. Timed exercise stabilizes behavioral rhythms but not molecular programs in the brain’s suprachiasmatic clock. iScience, 26, 106002 (2023).
• 49) Xu P, Berto S, Kulkarni A, Jeong B, Joseph C, Cox KH, Greenberg ME, Kim TK, Konopka G, Takahashi JS. NPAS4 regulates the transcriptional response of the suprachiasmatic nucleus to light and circadian behavior. Neuron, 109, 3268–3282.e6 (2021).
• 50) Foster SR, Hauser AS, Vedel L, Strachan RT, Huang XP, Gavin AC, Shah SD, Nayak AP, Haugaard-Kedstrom LM, Penn RB, Roth BL, Brauner-Osborne H, Gloriam DE. Discovery of human signaling systems: pairing peptides to g protein-coupled receptors. Cell, 179, 895–908.e21 (2019).
• 51) Kroeze WK, Sassano MF, Huang XP, Lansu K, McCorvy JD, Giguere PM, Sciaky N, Roth BL. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat. Struct. Mol. Biol., 22, 362–369 (2015).
• 52) Yang Z, Wang JY, Yang F, et al. Structure of GPR101-Gs enables identification of ligands with rejuvenating potential. Nat. Chem. Biol., 20, 484–492 (2024).
• 53) Lees JA, Dias JM, Rajamohan F, et al. An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism. Nat. Commun., 14, 5938 (2023).
• 54) Lin X, Li M, Wang N, Wu Y, Luo Z, Guo S, Han GW, Li S, Yue Y, Wei X, Xie X, Chen Y, Zhao S, Wu J, Lei M, Xu F. Structural basis of ligand recognition and self-activation of orphan GPR52. Nature, 579, 152–157 (2020).
• 55) Labrie F. Intracrinology. Mol. Cell. Endocrinol., 78, C113–C118 (1991).
• 56) Hamada Y, Sasaki L, Uehara H, Suzuki T, Kinoshita S, Otsuka K, Kihara A, Yamaguchi Y, Miyake T, Doi M. Optimising the method for visualising mouse meibomian gland using eyelid whole-mount lipid staining. Ocul. Surf., 26, 268–270 (2022).
• 57) Otani T, Miyake T, Ota T, Yarimizu D, Nakagawa Y, Murai I, Okamura H, Hasegawa E, Doi M. Identification of angiotensin II-responsive circadian clock gene expression in adrenal zona glomerulosa cells and human adrenocortical H295R cells. Front. Endocrinol. (Lausanne), 16, 1525844 (2025).
• 58) Ota T, Fustin JM, Yamada H, Doi M, Okamura H. Circadian clock signals in the adrenal cortex. Mol. Cell. Endocrinol., 349, 30–37 (2012).
• 59) Ota T, Doi M, Yamazaki F, Yarimizu D, Okada K, Murai I, Hayashi H, Kunisue S, Nakagawa Y, Okamura H. Angiotensin II triggers expression of the adrenal gland zona glomerulosa-specific 3beta-hydroxysteroid dehydrogenase isoenzyme through de novo protein synthesis of the orphan nuclear receptors NGFIB and NURR1. Mol. Cell. Biol., 34, 3880–3894 (2014).
• 60) Yamamura K, Doi M, Hayashi H, Ota T, Murai I, Hotta Y, Komatsu R, Okamura H. Immunolocalization of murine type VI 3beta-hydroxysteroid dehydrogenase in the adrenal gland, testis, skin, and placenta. Mol. Cell. Endocrinol., 382, 131–138 (2014).
• 61) Yarimizu D, Doi M, Ota T, Okamura H. Stimulus-selective induction of the orphan nuclear receptor NGFIB underlies different influences of angiotensin II and potassium on the human adrenal gland zona glomerulosa-specific 3beta-HSD isoform gene expression in adrenocortical H295R cells. Endocr. J., 62, 765–776 (2015).
• 62) Doi M, Satoh F, Maekawa T, Nakamura Y, Fustin JM, Tainaka M, Hotta Y, Takahashi Y, Morimoto R, Takase K, Ito S, Sasano H, Okamura H. Isoform-specific monoclonal antibodies against 3beta-hydroxysteroid dehydrogenase/isomerase family provide markers for subclassification of human primary aldosteronism. J. Clin. Endocrinol. Metab., 99, E257–E262 (2014).
• 63) Nguyen Pham KT, Miyake T, Suzuki T, Kinoshita S, Hamada Y, Uehara H, Machida M, Nakajima T, Hasegawa E, Doi M. Identification of meibomian gland testosterone metabolites produced by tissue-intrinsic intracrine deactivation activity. iScience, 28, 111808 (2025).
• 64) Sato S, Solanas G, Sassone-Corsi P, Benitah SA. Tuning up an aged clock: circadian clock regulation in metabolism and aging. Transl. Med. Aging, 6, 1–13 (2022).
• 65) Olejniczak I, Pilorz V, Oster H. Circle(s) of life: the circadian clock from birth to death. Biology (Basel), 12, 383 (2023).
• 66) Miyake T, Inoue Y, Shao X, Seta T, Aoki Y, Nguyen Pham KT, Shichino Y, Sasaki J, Sasaki T, Ikawa M, Yamaguchi Y, Okamura H, Iwasaki S, Doi M. Minimal upstream open reading frame of Per2 mediates phase fitness of the circadian clock to day/night physiological body temperature rhythm. Cell Rep., 42, 112157 (2023).
• 67) Acosta-Rodríguez V, Rijo-Ferreira F, Izumo M, Xu P, Wight-Carter M, Green CB, Takahashi JS. Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice. Science, 376, 1192–1202 (2022).
• 68) Sato S, Dyar KA, Treebak JT, et al. Atlas of exercise metabolism reveals time-dependent signatures of metabolic homeostasis. Cell Metab., 34, 329–345.e8 (2022).
• 69) de Assis LVM, Kramer A. Circadian de(regulation) in physiology: implications for disease and treatment. Genes Dev., 38, 933–951 (2024).