Circadian Clock-Based Therapeutics in Chronic Pulmonary Diseases

Abstract

The circadian clock is the biochemical oscillator that orchestrates the observable circadian rhythms in physiology and behavior. Disruption of the circadian clock in the lung during chronic pulmonary diseases itself becomes one of the key etiological risk factors that further drive pathobiology. Pre-clinical studies support that pharmacological manipulation of the circadian clock is a conceivable approach for the development of novel clock-based therapeutics. Despite recent advances, no effort has been undertaken to integrate novel findings for the treatment and management of chronic lung diseases. We, therefore, recognized the need to discuss the candidate clock genes that can be potentially targeted for therapeutic intervention. Herein, we aim to create the first roadmap that will further advance the development of circadian clock-based therapeutics and provide better outcomes in treating chronic pulmonary diseases.

Why target the circadian clock in the lung?

The circadian clock is the core biochemical oscillator with a period near 24 h that is responsible for the observed daily rhythms in physiology and behavior. Mounting evidence highlights that the circadian clock is tightly linked to lung function at multiple levels [1]. Early evidence came from chronic asthma and COPD patients, the majority of whom reported worsening symptoms during the nighttime [2,3]_. Later, epidemiological studies also supported that circadian clock disruption (in shift workers, or people with irregular sleep patterns and sleeping-related breathing disorders) increases the risks of developing inflammatory diseases such as asthma, and other chronic lung diseases such as pulmonary arterial hypertension (PAH), pulmonary fibrosis (PF) and lung cancer [4–7]. Although these studies well-establish the direct connection between circadian clock disruption and many chronic pulmonary diseases, the relationship at the molecular level remains elusive. This is perhaps the main reason the latest 2021 NIH Sleep Research Plan, “Advancing the science of sleep and circadian biology research” (which highlighted several aspects of circadian research on health and disease) had no mention of circadian clock modulation to treat or manage chronic diseases in the critical opportunity section, CO5: “develop chronotherapeutic approaches to prevent and treat chronic diseases” (https://www.nhlbi.nih.gov/sleep-research-plan/critical-opportunities). However, we propose here that existing literature provides ample evidence to strongly support pharmacological manipulation of the circadian clock as a conceivable approach for the development of novel clock-based therapeutics against chronic pulmonary diseases.

The circadian clock in the lung: scope for novel clock-based therapeutics

The circadian clock in the lung at the cellular level is a 1:1 stoichiometric balance between circadian activators including circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), and the repressors including Period (PER1/2) and Cryptochrome (CRY1/2) that are necessary to sustain the rhythmicity in mammals. These clock molecules make up the core loop of the circadian machinery where the repressors interact with the activators to suppress their own transcription. Additionally, another negative feedback loop directly operates on the core loop, consisting of the REV-ERBα/β (Nuclear Receptor Subfamily 1 Group D Member 1/2) and RORs (Retinoic Acid Receptor-Related Orphan Receptor) which controls the expression of the activator BMAL1. These two loops referred to as the transcriptional-translational feedback loop [TTFL] operate in concurrence with one another to generate circadian rhythms [8,9]. A detailed description of the lung circadian clock machinery can be found in our recent focused review on circadian clock disruption in chronic pulmonary diseases [9].

Previously, we established that circadian clock disruption in the lung is one of the major contributing factors that drive the pathogenesis of chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), PF, PAH, cystic fibrosis (CF), and lung cancer [9]. Recent discoveries of small molecules that selectively target the circadian molecular clock have been shown to restore internal rhythms thereby ameliorating the physiological consequences of circadian clock disruption [10]. However, detailed descriptions of the known pathways or proteins that can be targeted at the cellular level are often overlooked. This is critical in the development of novel clock-based therapeutics. Here, we provide our current interpretation of the circadian clock disruption in chronic lung disease and summarize the circadian clock modulators (small molecules) that selectively target the circadian clock proteins such as REV-ERBs, RORs, PERs, CRYs, CLOCK, and Casein Kinases (CKs). Then, we discuss how the restoration of the circadian clock function in the lung can reverse or ameliorate pathophysiological traits observed during chronic pulmonary diseases in light of the affected pathways or the etiology (Table 1 and Figures 1A–C; see Box 1). Finally, we briefly discuss chronotherapy (also known as the timed administration of drugs for reduced toxicity and enhanced efficacy) and highlight the gaps in our understanding which prevent proper implementation of chronotherapy. We refrain from the discussion of optimizing the circadian clock for better health because it is unlikely that patients suffering from chronic pulmonary diseases cannot adhere to a strict lifestyle.

Table 1.

Summary of circadian clock modulators as a novel therapeutic agent with an implication for the treatment of chronic lung diseases.

Small molecule(s)	Circadian target(s) and specific activity	Circadian Modulation	Functional output of small molecule in pre-clinical studies (in vitro and in vivo)	References
GSK4112	Rev-erbα (Agonist)	Competitive with heme (ligand of Rev-erbs), inhibit expression of Bmal1	Inhibits Il6, Cxcl11, Ccl2, Cxcl6, Il19 expression in primary human monocyte-derived macrophages; Suppresses il6 and ccl2 mRNA in macrophages; Reduces pro-inflammatory cytokines IL-6 and IL-8 in primary human small airway epithelial cells and suppressed IL-6, KC, and MIP-2 cytokine release in LPS-treated mouse lung fibroblasts. Inhibits gluconeogenesis in mouse hepatocytes; Suppresses TGF-β-induced αSMA (Acta) and collagen-1 (Col1a1) expression in primary human fibroblasts.	[6,20–23]
SR9009	Rev-erbα (Agonist)	Inhibits expression of Bmal1	Suppress Il17a expression and Th17 cell development and proliferation; Inhibits IgE and IL-33-mediated mast cell activation in the development of allergic disease; Enhances mononuclear cell phagocytic activity (in vitro) and increases pathogen elimination (in vivo); Decrease IL-1β-induced expression of pro-inflammatory cytokine genes Ccl2, Cxcl10, Cox-2, Inos, Il6 and Il8, and proteins MMP3 and MMP13 in a dose-dependent manner; Inhibits MAPK signaling targets (pJNK, p-p38, p-ERK) and NF-κB (p-P65, p-IκBα, p-IKKα/β); Improves mitochondrial function, increase energy expenditure, reduce fat mass, plasma triglycerides and cholesterol in mice; Selective anticancer activity, inhibits autophagy and promotes apoptotic activity in cancer cells.	[29,44,49,6 7,107,108]
GSK1362	Rev-erbα (Inverse Agonist)	Increases Bmal1 expression while stabilizing REV-ERBα protein by inhibiting SUMO and ubiquitin	Repressed Il6, Ccl2, and G-csf gene expression in alveolar macrophages; Stabilized Rev-erbα abundance in LA-4 and NHBE cells; Blocks IL-1β mediated SUMO-2 ligation of Rev-erbα.	[13]
		modification
GSK2667	Rev-erbα (Agonist)	Reduces Bmal1 promoter activity (decreased Bmal1 expression)	Inhibits replication of Hepatitis C and related flaviviruses (Dengue and Zika virus) by interfering with the lipid metabolism in Huh-7 cells; Reduces pre-miR-122 levels which regulates cholesterol and lipid metabolism; Suppress Il6 expression in THP-1 cells stimulated with LPS.	[109,110]
GSK2945	Rev-erbα (Agonist)	Suppresses Bmal1 expression	Reduces Il6 expression in THP-1 cells stimulated with LPS; Potent antagonist with better bioavailability when given via oral route.	[110]
T0901317	RORα/γ (Inverse Agonist)	Inhibits transactivation of  RORα and RORγ, but not RORβ; Repressed  RORα/γ-dependent transactivation of RORresponse reporter genes	Inhibits Th17 cell development and proliferation by suppressing Il17a expression in mouse splenocytes in vivo; Blocked IL-23 mediated expression of Il17a	[58,59]
SR1001	RORα/γ (Inverse Agonist)	Suppression of ROR transcriptional activity	Inhibits the development of Th17 cells by blocking Il17a gene expression; Inhibited the expression of cytokines (Il17a, Il21, Il17f and Il22) in Th17 cells; Suppressed clinical severity of autoimmune disease (autoimmune encephalomyelitis) in mice.	[47]
SR1555	RORγ (Inverse Agonist)	Suppress IL-17 promoter activity driven by RORγ but not RORα	Inhibits Th17 cell development and increases the frequency of Treg cells; Enhances energy expenditure, reduced body weight in obese diabetic mice and improved insulin sensitivity.	[36,48]
SR2211	RORγ (Inverse Agonist)	Efficacious RORγ modulator and activity repressor	Inhibits gene expression of Il17a and Il23r in EL4 cells; Inhibits pro-inflammatory cytokine expression (Il1β, Il6, and Tnfα) in LPS stimulated RAW 264.7 cells.	[39,56]
TMP778	RORγt (Inhibitor)	Mimics the effect of RORγt deletion	Blocks human Th17 and Tc17 cell differentiation; Acutely modulate IL-17A production and inflammatory gene expression of Il17a, Il17f, Il22, Il26, Il23, and Ccr6 in effector and memory Th17 cells; Blocks IL-23 induced IL-17A production.	[57]
VTP-938	RORγt (Inverse Agonist)		Attenuated allergen-specific Th17 cell development in lung-draining lymph nodes; Decreased allergen-induced production of IL-17 and neutrophilic inflammation; Markedly reduced airway hyperresponsiveness.	[37]
GSK805	RORγt (Inhibitor)	Inhibits RORγt transcriptional activity	More potent than TMP778 at inhibiting Th17 cell responses; Limits inflammation by reducing Th17 cells but preserves group 3 innate lymphoid cells.	[60,61]
SR3335	RORα (Inverse Agonist)	Selectively binds to RORα but no other RORs; suppress the expression of RORα target genes	Inhibit gluconeogenesis by suppressing transcription of RORα target genes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase.	[111]
CLK8	CLOCK (Inhibitor)	Selectively inhibits CLOCK and prevents the translocation of CLOCK:BMAL1 complex to the nucleus	Stabilizes the negative arm of the transcriptional-translational feedback loop with no effect on the lengthening of the period; Corrects dampened circadian rhythms in chronic diseases and may have possible implications to treat different lung carcinomas that are associated with increased expression of CLOCK and reduced PER and CRY.	[94,95]
KS15	Cry1/2 (Inhibitor)	Enhance E-box mediated transcription by inhibiting CRY1/2- mediated repression, and activating CLOCK:BMAL1 induces E-box-mediated transcription	Reduced Circadian amplitude; Delay circadian phase in fibroblasts; Decreases proliferation of MCF-7 breast cancer cells and increases chemosensitivity to anti-tumor drugs.	[97,98,112]
CK1-7	CK1 (Inhibitor)	Prevent PER1 degradation; Delays nuclear entry of PER1; Lengthens nuclear residence	Blocks PER1 degradation and delays the nuclear entry of PER1 in normal human fibroblasts; Induce cytotoxicity in primary acute leukemia tumor cells and SCID-Beige mouse systemic tumor model.	[80,81]
PF670462	CK1δ/ε (Inhibitor)	Dose- and timedependent phase delay	Prevents TGF-β-induced epithelial-mesenchymal transition; Prevents both acute and chronic bleomycin-induced pulmonary fibrosis in mice; Inhibits fibrogenic marker expression (Col1a1 and Ctgf) in 2D and 3D culture systems.	[86,101]
PF4900567	Selective CK1ε (Inhibitor)	Inhibits PER2 degradation and PER3 nuclear localization	Potent CK1ε inhibitor with 20-fold greater selectivity over CK1δ.	[82]
NCC007	CKα/δ (Inhibitor)	Derivative of Longdaysin (more potent); Further lengthens circadian period	Exhibit in vivo efficacy and lengthens mouse behavioral rhythms.	[85]
GO289	CK2 (Inhibitor)	Lengthens circadian period; Inhibits phosphorylation and degradation of PER2	Exhibits cell-type dependent inhibition of cancer cell growth	[90]

Figure 1. Circadian clock-based modulators and their potential therapeutic implications in chronic lung diseases.

(A) Rev-erb agonists are direct regulators of core clock gene Bmal1. Both REV-ERBs and RORs are transcribed from the E-box promoter upon CLOCK:BMAL1 binding and fine-tune expression of Bmal1 by competing with each other at the ROR response element (RORE). Rev-erb agonists modulate inflammation, Th17 proliferation and differentiation, autophagy, and production of αSMA, Col1A1, triglycerides, and cholesterol levels, which have major implications in COPD, asthma, fibrosis, and atherosclerosis. (B) RORγt inverse agonists inhibit the association of RORγt transcription factor to the RORE that drives the expression of Il17, and Th17 cell differentiation and proliferation. Notably, VTP938 has been shown to attenuate allergen-specific Th17 cell development in draining lymph nodes, allergen-induced production of IL-17, neutrophilic inflammation, and markedly reduced airway hyperresponsiveness that has implications for asthma and COPD. (C) PER and CRY are transcribed from the E-box promoter and interact with the CLOCK:BMAL1 complex to suppress their transcription. CK1/2 inhibitors prevent the degradation of the PER and CRY in the cytoplasm by inhibiting CK1δ/ε, thereby increasing their time in the cytosol. CRY1/2 inhibitors function by inhibiting CRY1/2-mediated repression, and activating CLOCK:BMAL1 induced E-box-mediated transcription. Both CK1/2 and CRY1/2 inhibitors increase the abundance of PER and CRY proteins, which has been shown to reduce TGFβ-induced epithelial-to-mesenchymal transition, Col1a1 and Ctgf expression, collagen deposition, and cancer cell growth that have implications for fibrosis and lung cancer treatment. The blue arrow pointing down represents the reduction of the above-mentioned key pathophysiological phenotypes.

Box 1: Casein Kinase (CK) 1/2, Period, Cryptochrome and Clock in lung cancer and PF.

CK inhibitors (CK1-7, PF670462, PF4900567, NCC007, GO289) function to lengthen the circadian period by stabilizing core clock proteins including period and cryptochromes [80–86]. Per1/2/3 and Cry1/2 are significantly altered in several cancer types including breast, pancreatic, and colorectal cancer [87]. Per1/2 has a tumor-suppressive role in mice through its involvement in ATM-Chk1/Chk2 DNA damage response [88]. Low Per1/2 expression plays a pivotal role in the progression and development of cancer [89]. CK2 inhibitors (G0289) not only inhibit phosphorylation and degradation of PER2 but exhibit cell type-dependent inhibition of cancer cell growth [90]. Downregulation of Per2 in non-small cell lung cancer (NSCLC) is associated with tumor progression and metastasis [91]. This acquired neoplastic growth of cancer cells can be ameliorated by stimulating the overexpression of Per1/2 [92]. Patients with higher Per1/2 expression show a greater survival rate in NSCLC suggesting loss of PER(s) function in tumor progression [93]. Interestingly, with the downregulation of PER and CRY, CLOCK is significantly upregulated in human lung adenocarcinomas [94]. Thus, it would be interesting to test the therapeutic potential of CLK8 (CLOCK inhibitor) in lung cancer progression in different human adenocarcinoma models in vitro and in vivo without affecting PER and CRY levels [95]. Overexpression of CK2 is associated with the promotion of tumorigenesis in the lung [90]. Ck2 knockdown show enhanced radiosensitivity to various NSCLC cells. Additionally, exposure to both CK2 inhibition and ionizing radiation together, delays radiation-induced DNA damage and reduce tumor growth [96]. Cryptochromes can also be targeted for cancer therapy. For instance, Cry1/2 inhibitor KS15 works by repressing the effect of Cry1/2 on the CLOCK:BMAL1 complex, thereby enhancing the transcription of E-box genes including Per1/2/3 and Rev-erbα/β [97,98]. Downregulation of Rev-erbα/β and Per1/2 genes have been associated with the progression of several types of lung adenocarcinomas and NSCLC, and thus warrants investigation if upregulation of the Per1/2 using KS15 might have therapeutic implications [91,99,100] (Table 1 and Figure 1C).

Studies have reported the anti-fibrotic effect of PF670462 in acute and chronic bleomycin-induced lung injury models as well as in vitro on spheroids made from primary human lung fibroblast from patients with PF and healthy controls. Increased expression of CK1δ and CK1ε in patients with PF compared to healthy control is observed, which corresponded to higher phospho-Per2. PF670462 can prevent TGFβ-induced EMT in vitro and reduce stiffness of the PF-derived spheroids. Furthermore, TGFβ-induced fibrogenic gene expression is inhibited by PF670462 and prevents bleomycin-induced fibrosis in mice [101]. Thus, inhibiting the overexpression of CK1 δ/ε in PF combined with the anti-fibrogenic effect of PF670462 may provide better therapy for fibrotic lung disease (Table 1 and Figure 1C).

Circadian clock and lung inflammation

The circadian clock is a known modulator of inflammation with humans displaying a time-of-day variation in symptoms and severity of inflammatory phenotypes in chronic lung diseases such as asthma and COPD [9]. Prior studies have repeatedly highlighted how the disruption of the circadian clock leads to pulmonary inflammation. Particularly the absence of the core clock protein BMAL1 which promotes pulmonary inflammation has been of great interest to researchers [11,12]. Although these studies have helped elucidate how the circadian clock regulates lung inflammation to a very large extent, it however does not explain how the altered status of oxidative stress and lung inflammation during chronic pulmonary diseases such as asthma or COPD can result in circadian clock disruption.

Chronic inflammation in the lung is one of the signature phenotypes that underlie the pathobiology of several chronic lung diseases including asthma and COPD. Understanding how chronic inflammation leads to circadian disruption might be essential for the development of clock-based therapeutics. Although this thrust area is still in its infancy, it is reasonable to surmise that there is some sort of inflammation-mediated reprogramming occurring at the cellular level in the lung that leads to circadian clock disruption (altered transcriptional and translational rhythms of core clock genes in the lungs). It was recently highlighted that inflammatory stimuli in the lung can not only promote the degradation of the important circadian transcription factor REV-ERBα through post-translational modifications such as SUMOylation and ubiquitination but also markedly change the stability of the protein [13]. The recent finding supports how significant downregulation of core clock genes such as Clock, Cry1, Cry2, Rorα, and Per2 in the lung tissues of patients with COPD may further contribute to the altered stability of circadian clock proteins [14].

The nuclear factor-kappa-B (NF-κB) transcription factor is another important modulator of the circadian clock. NF-κB regulates a variety of cellular processes including the innate immune response to inflammation. Hong et al., recently demonstrated that the circadian clock function is particularly vulnerable to unusual triggers of NF-κB activation, which is a common occurrence in chronic inflammatory diseases. NF-κB activity strongly inhibits the expression of core clock genes Per1/2, and Cry2 (repressors) leading to disruption of the core circadian loop of the clock machinery. Furthermore, inflammatory stimuli can modulate circadian gene expression epigenetically, which is associated with a marked reduction in H3K27ac (decreased histone acetylation leading to suppression in transcriptional activity) and RNA Polymerase II binding to the promoter of the core clock repressor gene Rev-erbα [15]. Together, this recent evidence helps explain for the first time how chronic inflammation leads to circadian clock disruption in the lung.

COPD results from the exaggerated inflammatory response in the lung caused by cigarette smoke (CS) exposure, a major etiological risk factor for the pathogenesis of COPD. The innate immune response facilitates the secretion of pro-inflammatory cytokines which play an important role in orchestrating lung inflammation that leads to increased infiltration of inflammatory cells of the immune system including neutrophils, macrophages, and lymphocytes (e.g., CD4⁺, and CD8⁺ T lymphocytes). These activated immune cells further release cytokines/chemokines which promote the progression of chronic inflammation [16]. Rev-erbα is one of the core clock genes that regulates the circadian expression of Bmal1 and has been identified as a key modulator of inflammation. Although the mechanistic pathways are yet to be elucidated, in human macrophages, NLRP3 inflammasomes (a critical component of the innate immune response that facilitates the secretion of pro-inflammatory cytokines) are expressed in a diurnal fashion. In Rev-erbα knockout (KO) mice, this diurnal expression pattern of NLRP3 inflammasomes is completely abolished and thus alters the expression patterns of pro-inflammatory cytokines (IL-1β and IL-18) compared to macrophages that expressed Rev-erbα [17]. Genetic ablation of Rev-erbα greatly aggravates inflammatory phenotypes in pre-clinical mouse models, thus providing strong evidence that REV-ERBs can modulate the innate immune response to inflammation [17–19]. Rev-erbα is significantly reduced both at the mRNA and protein level during chronic inflammation leading to circadian clock disruption in asthma and COPD, and thus allows us to selectively target REV-ERBs.

Rev-erb agonist as a novel clock-based therapeutic target for lung inflammation

Since the downregulation of Rev-erbα at the mRNA and protein level is implicated in exaggerated pulmonary inflammation, upregulation of REV-ERB using synthetic ligands/agonists looks promising to ameliorate pro-inflammatory cytokines. Indeed, activation of Rev-erbα using a selective Rev-erbα agonist (GSK4112) inhibits the LPS-induced production of pro-inflammatory cytokines IL-6, CXCL11, CXCL6, and CCL2 in primary human monocyte-derived macrophages, and represses the expression of Il6 and Ccl2 mRNA in LPS-treated peritoneal macrophages and RAW 264 cells, suggesting that Rev-erbα-mediated repression occurs at the DNA level [20–22]. Similarly, GSK4112 treatment attenuates CS extract and LPS-induced proinflammatory cytokine/chemokine (e.g., IL-6, IL-8, KC, MIP-2) release in human small airway epithelial cells and mouse lung fibroblasts respectively [23].

The two different subtypes of Rev-erbs (Rev-erbα and its paralog Rev-erbβ) are both responsible for circadian clock maintenance and immune regulation [24]. Double mutant Rev-erbα/β KO mice show augmented inflammatory response and chemokine activation that initiates an observable basal inflammatory state. However, Rev-erbα is deemed more potent in modulating inflammation. Rev-erbβ KO alone does not initiate the same basal inflammatory response (although more compared to the control group having functional Rev-erbα/β) [13]. This implies that selective targeting of Rev-erbα using a synthetic agonist might be more potent than targeting Rev-erbβ. Inflammatory challenges promote the degradation of Rev-erbα. Recently, pro-inflammatory cytokine IL-1β-mediated SUMO-2 ligation of REV-ERBα, which targets it for degradation, was shown to be successfully blocked by Rev-erbα inverse agonist GSK1362. This suggests that inflammation-mediated circadian clock disruption can be blocked at the molecular level. GSK1362 not only increased REV-ERBα abundance but markedly reduced inflammation [13] (Table 1 and Figure 1A).

Pro-inflammatory cytokines including TNFα, IL-1β, IL-6, and IL-8 are found to be increased in the sputum and bronchoalveolar lavage (BAL) fluid of patients with asthma and COPD. These cytokines are responsible for the amplified inflammation in part through the activation of the NF-κB pathway [25], which in turn promotes the expression of multiple other inflammatory genes. Unusual triggers from NF-κB activation lead to the disruption of the circadian clock by altering the transcription of the core clock genes. As reported earlier, REV-ERBα is significantly downregulated in asthma, COPD, and PF, which explains the exaggeration in the inflammatory response during the disease [9]. Thus, it would be interesting to observe how the overproduction of pro-inflammatory cytokines can be attenuated in asthma or COPD by the use of Rev-erb agonists. Further elucidating the affected pathways and how changes in Rev-erbα expression correspond to increased expression of pro-inflammatory cytokines in asthmatics or COPD patients at night hours might help devise better therapeutics targeting the circadian clock to control lung inflammation.

REV-ERB(s) and ROR(s) modulators as a novel therapeutic target for allergic asthma and COPD

The diurnal nature of asthma and COPD symptoms strongly suggests the involvement of the circadian clock machinery in the pathogenesis of chronic airway disease. Of the many core circadian clock proteins, REV-ERBs and RORs are the key modulators of inflammation in allergic asthma and COPD. Both REV-ERB and ROR (positive and negative respectively) compete with one another to bind to the RORE response element to fine-tune the expression of BMAL1, one of the key proteins of the core circadian loop. As highlighted earlier, repressed/reduced REV-ERBs in asthma and COPD and activation of ROR in COPD correspond to the aggravation of the diseases. Only recently, our understanding of how the circadian clock components interact with key transcription factors that control inflammatory pathways enables us to postulate how the circadian clock in the lung can be targeted to treat inflammation-associated chronic airway diseases.

Initiation of allergic immune response in asthma begins with the interaction of the lung epithelium with the allergen followed by the subsequent release of alarmins (cytokines: IL-25, IL-33, and TSLP), which facilitate B cell-mediated IgE production. IgE binds to a high-affinity IgE receptor, FcεRI on mast cells, basophils, and dendritic cells to facilitate allergen presentation to memory Th2 cells. Activation in turn leads to enhanced B cell maturation, impairment of the T regulatory (Treg) function, macrophage activation, and eosinophil recruitment, which further contributes to allergic inflammation in asthma [26]. The circadian clock regulates the day-night variation in IgE and IL-33-mediated activation of mast cells in mouse models. Studies report that the dose of allergens needed to trigger bronchospasms in asthma varies with the time of allergen administration being most potent during the nighttime [27,28]. This is partly achieved by CLOCK:BMAL1 complex binding to the E-box promoter of the high-affinity IgE receptor FcεRIβ or IL-33 receptors in a circadian manner [29]. Surprisingly, the use of Rev-erbα agonist SR9009 inhibits IgE and IL-33-mediated activation of mast cells independent of functional CLOCK activity [29]. Although CLOCK analog protein NPAS2 activity can compensate for CLOCK in peripheral tissues, it is hard to predict if the observed effects are due to the upregulation of NPAS2 [30]. Gab/PI3K pathway is essential in FcεRIβ signaling leading to degranulation of mast cells, and the NF-κB pathway for the activation of IgE and IL-33-mediated cytokine expression. Since REV-ERB activation strongly inhibits the IgE and IL-33 production and activation of mast cells, it is reasonable to conclude that REV-ERB potentially interacts with those pathways leading to the observed effects but would require additional research to confirm. Per2 is certainly another key clock gene that is involved in the allergic response. A time-of-day response in IgE-mediated passive cutaneous anaphylactic reaction is observed in wild-type (WT) mice but not in Per2 mutant mice [31]. Furthermore, Per2 deletion abolishes the rhythmic secretion of endocrine factors such as corticosterone, a key suppressor of allergic inflammation. Interestingly, there is a clear nadir in the anaphylactic reaction at the onset of the night, the same time when Per2 expression is the highest and a time when corticosterone peaks. However, the circadian variation of corticosterone is absent in Per2 KO mice [32]. These circadian findings are in line with cutaneous hypersensitivity reactions in nocturnal asthma showing a diurnal rhythm in IgE-mediated response [33].

During COPD exacerbation, there is an insufficient amount of Treg cells to suppress the inflammation [34]. Particularly, levels of anti-inflammatory cytokines like IL-10 and IL-35 produced by Treg cells are greatly reduced in COPD patients [35]. RORγ inverse agonist SR1555 possesses a dual role of not only inhibiting the development and proliferation of Th17 cells but also increasing the frequency of Treg cells, and thus might be helpful to investigate how these findings may be beneficial in COPD patients [36]. Recently, RORγt inverse agonist (VTP-938) was shown to attenuate allergen-specific Th17 cell development in lung-draining lymph nodes, which significantly reduced allergen-induced production of IL-17 with markedly diminished airway hyperresponsiveness [37].

In patients with COPD, an exaggerated number of macrophages and macrophage-derived chemoattractants have been identified in the lung parenchyma and bronchial airway. This attributes to the heightened macrophage-derived products in the lung including IL-8, TNFα, and reactive oxygen species [38]. Treatment with RORγ inverse agonist SR2211 therapeutically represses inflammatory T cell function and the activation of macrophages in humans [39]. This may be beneficial for patients with CF as well since TNFα, IL-1β, and IL-8 are elevated in patients with CF, and the lack of IL-10-mediated suppression of inflammatory markers is one of the key contributors to the development of CF in the lung [40]. Furthermore, M1 and M2 macrophages have also been implicated in the pathogenesis of asthma. M1 macrophage-derived higher levels of IL-23 and IL-1β have been shown to prime substantial Th1 and Th17 responses in the lung. This in turn promotes airway neutrophilia and acute airway hyperresponsiveness [40]. Thus, attenuating macrophage activation and inhibiting Th17 cell development and proliferation by suppressing IL-17A expression using specific RORα/γ inverse agonists is promising for the development of new clock-based therapeutics for chronic inflammatory lung diseases. (Table 1 and Figure 1A–B).

REV-ERB(s) and ROR(s) role in autoimmune lung diseases

Autoimmune lung diseases are caused when a person’s own immune system targets the lung, leading to severe scarring and partial impairment of lung function. Autoimmunity in COPD develops when environmental factors, such as cigarette smoke, transform benign autoantibodies into pathogenic antibodies. This leads to immune complexes and complement deposition in the lung, and autoimmune-mediated damage to the airway [41]. Th17 cells play a pathogenic role in autoimmune COPD mainly through the release of cytokines including IL-17A and IL-21 [42]. Adaptive immunity to a large extent is under the circadian clock influence with T cells documented to exhibit strong circadian oscillation in the blood [43]. Quite expectedly, core clock proteins including REV-ERBs and RORs possess the ability to modulate Th17 cell development and proliferation [18,44,45]. Th17 cells are a subset of CD4⁺ T helper cells that produce IL-17 and other pro-inflammatory cytokines that are regulated by the circadian clock involved in the pathobiology of autoimmune and inflammatory diseases [18,44,46–49].

Prior studies show that the circadian clock gene Nfil3 can suppress the development of Th17 cells by inhibiting Rorc transcription [thus preventing the RORγt (a thymus-specific version of RORγ) expression while itself being repressed by Rev-erbα]. Anti-phasic oscillation of Nfil3 and Rorc is observed with peak expression at night and day respectively. Chronic circadian disruption greatly increases the amount of Th17 cell frequency, and this increased frequency of Th17 cells is suppressed in Rev-erbα KO mice, suggesting a role of REV-ERBα in Th17 cell proliferation [50]. Specifically, Rev-erbα is upregulated during Th17 cell development and possesses a dual role. At low levels, Rev-erbα facilitates the transcription of RORγt by suppressing the activity of Nfil3. Conversely, at high levels, Rev-erbα acts as a transcriptional repressor for RORγt since both compete for the shared DNA consensus sequence, and thus negatively regulate Th17 cell development and proliferation [44]

REV-ERB(s) and ROR(s) modulators as a novel therapeutic target for autoimmune lung diseases

Recent evidence strongly suggests that modulators of REV-ERB and ROR can be used as therapeutics for autoimmune diseases such as COPD and CF [44]. While RORs are strong promoters of Th17 differentiation, REV-ERBs inhibit differentiation and Th17-mediated pro-inflammatory cytokine expression. Th17 response plays a major role in autoimmunity, and very recently has been implicated in the pathogenesis of COPD [51]. Specifically, RORγt is one of the key promoters of Th17 cell differentiation in proinflammatory CD4+ T cells in the thymus [46]. As Th17-dependent exacerbations are common in autoimmune COPD, it is reasonable to speculate that the use of ROR inverse agonists, ROR antagonists, or REV-ERB agonists might be helpful in the management of COPD exacerbations (Figure 1A). Indeed, deletion of Rev-erbα enhances Th17-mediated pro-inflammatory cytokine expression such as IL-17A, while treatment with a Rev-erbα agonist (SR9009) significantly suppresses Th17 cell development as shown in pre-clinical models of colitis and encephalomyelitis [18,44].

Single nucleotide polymorphisms (SNPs) in the Rorα gene have also been reported as a risk factor for the development of COPD. Although these SNPs do not lead to functional changes in the protein, they may lead to transcriptional changes in gene expression but will require additional research to confirm [52]. Whether these SNPs enhance ROR function in vivo or suppress REV-ERB functions to enhance susceptibility for the development of COPD remains unclear. However, patients with COPD have a significantly higher level of IL-17A in the serum compared to healthy smokers and non-smokers. Higher IL-17A positively correlates with COPD severity and negatively correlates with airflow limitation. An increase in IL-17 is associated with the increased proportion of Th17 cells and IL-17-producing CD8⁺ T cells in patients with COPD often leads to autoimmunity [41,42,53]. Furthermore, the decreased expression of FOXP3 in COPD patients results in the upregulation of the RORγt in COPD patients which parallels the aggravation of the disease [54]. This strongly suggests that the circadian clock modulators including REV-ERB agonists or ROR inverse agonists and antagonists can be utilized to suppress the Th17 development and the overproduction of proinflammatory cytokines. However, it is also important to take into consideration how these small molecules can modulate T cell repertoire in the thymus. While RORγt antagonists have been reported to reshape the T cell repertoire in the thymus, REV-ERB agonists are deemed much safer with no effect on the T cell repertoire [44].

IL-17 signaling also plays a crucial role in CF pathogenesis, and a recent study found significantly higher levels of IL-17A and IL-17F in the sputum of CF patients [55]. A higher percentage of Th17 cells may be negatively correlated with lung function in CF patients. Several inverse agonists of the RORα/γ have also been identified including T0901317, SR1001, SR1555, SR2211, TMP778, and GSK805 that have been shown to markedly inhibit the production of IL-17 and the development of Th17 cells [36,39,47,48,56–61]. T0901317 and TP778 were also able to block IL-23-induced IL-17A production [57–59] (Table 1, Figure 1B). Overall, these studies strongly suggest that REV-ERBs and RORs are potential targets that possess the ability to ameliorate Th17-related autoimmune phenotypes in chronic lung diseases which need to be thoroughly explored using appropriate pre-clinical in vivo models.

REV-ERB agonists in pulmonary arterial hypertension and pulmonary fibrosis

Accumulating evidence undoubtedly suggests a major role of the circadian clock, particularly the involvement of the REV-ERBs, in the pathogenesis of chronic pulmonary diseases such as PAH and PF. A significant reduction in REV-ERBs in PAH and PF was recently reported in our focused review [9]. To date, there are no drugs that directly target the etiology of the diseases at the cellular and molecular level, and only involve symptom management upon onset of the disease. Here, we aim to demonstrate that the etiology of the diseases can be prevented at the molecular level using selective Rev-erb agonists.

Environmental factors including allergens, smoking, bacterial, and viral infections (from the past and current) can cause alveolar injury and contribute to the pathogenesis of PF. Injury to the lung epithelium results in the activation of the fibroblast-to-myofibroblast transition (FMT) leading to increased matrix deposition in the lung and scarring. Epithelium-to-mesenchymal transition (EMT) associated with fibrosis induces inflammation in the lung which further activates fibroblast differentiation [62,63]. Rev-erb agonist (GSK4112) can significantly repress TGFβ-induced mRNA expression of Acta2 and Col1a1 in human lung fibroblasts and precision-cut human lungs, and thus have major implications in fibrotic lung disease [6]. We recently demonstrated that Rev-erb agonists modulate both lung inflammation and EMT suggesting a role in fibrogenesis [19]. Additional evidence from single cell RNA-sequencing revealed that Rev-erbα expression is significantly reduced (undetectable) in alveolar type II (AT2) and transitional AT2 cells in patients with PF compared to healthy control [63]. Indeed, Rev-erbα is essential in attenuating fibrotic injury through its effect on FMT during bleomycin-induced pulmonary fibrosis in mice [6]. Interestingly, Rev-erbα/β was also shown to be strongly downregulated in aged mice compared to their younger counterparts. Whether this is true in humans is not yet known. However, if true, the findings may help explains why pulmonary fibrosis is an aging disease and most common among older adults [64].

One of the main causes of PAH involves the remodeling and increased proliferation of both the endothelium as well as smooth muscle cells of the pulmonary arteriole. Studies reports that REV-ERBα abundance is significantly reduced in pulmonary arterial smooth muscle cells (PASMC) obtained from PAH patients. Endothelial dysfunction in PAH is associated with abnormal smooth muscle proliferation and reduced apoptosis. Treatment with Rev-erb agonist (SR9009) reduces PASMC proliferation and reverses the resistance to apoptosis [65]. Patients with COPD also have a high prevalence of carotid plaquing, and an increased risk of atherosclerosis, which also contributes to the etiology of PAH and other cardiovascular events [66]. Rev-erb agonists have been shown to decrease obesity in mice, and markedly improve dyslipidemia and hyperglycemia implying that they may be protective against the development of pulmonary artery atherosclerosis as well [67] (Table 1 and Figure 1C). Future studies should investigate whether Rev-erb agonists will be beneficial to treat chronic lung diseases in obese asthma and COPD associated with comorbid conditions such as diabetes using pre-clinical mouse models. Furthermore, genetic screening of individuals and their close family members diagnosed with familial PAH or PF can help predict the individual’s susceptibility to the disease, and thus early intervention may have better outcomes and should be further investigated (See Box 2: Challenges and limitations in translational circadian medicine and chronotherapy).

Box 2: Challenges and limitations in translational circadian medicine and chronotherapy.

Although the pre-clinical studies demonstrate promising results, it is essential to highlight the barriers in circadian medicine. First, it is important to consider the diametrically different sleep-wake cycles in rodents vs. humans. Most circadian studies in vivo are performed in rodents (like mice and rats) who have evolved as nocturnal animals, unlike humans who are diurnal. Furthermore, rodents exhibit polyphasic sleep patterns which need careful consideration [102]. Second, there is an urgent need for the identification of novel circulating biomarkers that will help determine circadian rhythms in humans. Today, exposure to artificial light combined with several other factors including shiftwork, stress, sleep deprivation, and social jet lag significantly contributes to altered circadian rhythms. Additionally, there are different types of human chronotypes (morning vs evening). Laboratory animals are raised in controlled environments with strict 12:12 h light-dark cycles and thus differ significantly from humans with unique lifestyles. For reliable determination of human circadian rhythms, sampling of human biospecimens every 2 to 4 hours is necessary but difficult. Circadian rhythms of internal organs in humans have mostly been determined from samples obtained during surgical procedures (e.g., biopsies, organ transplantation, and deceased donors) [103]. Third, many circadian studies in vivo are often performed in global circadian gene knockout mouse models. Given the wide role of the circadian clock in controlling various aspects of physiology, more organ-specific (lung) or cell-type-specific circadian gene knockout studies should be conducted. The lung comprises several cell types (>40) and research has already established that specialized cells like the Clara cells are mostly responsible for determining the circadian rhythms of the lung [11,104].

Synthetic ligands must be tested extensively before their implementation in humans, or they could potentially be harmful. For instance, while REV-ERBs have been shown to ameliorate several inflammatory markers, inhibit Th17 cell proliferation and differentiation, or reduce triglycerides and cholesterol, Rev-erb agonists can potentially inhibit the microglial amyloid clearance and increase amyloid plaque deposition, the signature hallmarks of Alzheimer’s disease [105]. The biological half-life of the drugs should also be taken into consideration. Several Rev-erb agonists and antagonists have a short half-life ranging between 0.17 to 4–5 h and are readily eliminated from the body with low bioavailability [106]. Studies should investigate how chemical modifications of the existing clock modulators can increase their bioavailability while not compromising the functional activity using preclinical models.

Chronotherapy: a novel personalized medicine approach to treat chronic lung diseases

Circadian medicine remains incomplete without the discussion of chronotherapy, which is the timed administration of drugs for better efficacy and reduced toxicity. Zhang et al. reported that 56 of the 100 top-selling drugs in the US target a circadian gene product. The rhythmic expression of these druggable target genes thus allows enormous room to optimize drug delivery [68]. Given the diurnal nature of asthma, chronotherapy undoubtedly is a rational approach to the treatment and management of this disease. It is interesting to note that 75% of patients experience worsening asthmatic symptoms at night or early morning hours [9]. Studies have reported that afternoon or night (compared to morning) administration of several systemic steroids including prednisone and methylprednisolone have been found to significantly increase FEV₁ and decrease inflammatory cells like neutrophils, eosinophils, and macrophages in BAL fluid [69–71]. However, methylprednisolone should be avoided between 00:00 and 4:00 AM as it causes severe adrenal suppression and interferes with endogenous corticosteroid production. Methylprednisolone administration between 08:00 to 16:00 seems to be the safest option which ensures that there is little to no interference with adrenal suppression [71]. Other inhaled corticosteroids including Triamcinolone, Beclomethasone, and Mometasone, long-acting β2-agonists including Salmeterol, and leukotriene receptor antagonists including Montelukast have shown better or similar results with afternoon or evening administration [72]. However, our understanding of how chronic corticosteroid exposure modulates the circadian machinery at the molecular level is still unclear. Koyanagi et al. reported that chronic exposure to corticosteroids (prednisolone) remarkably induced the expression of Per1 while greatly suppressing the serum-induced oscillation of other core clock genes including Per2, Rev-erbs, and Bmal1 in HepG2 cells. Interestingly, this suppressive activity of prednisolone was not observed when a single dose of prednisolone was administered at a time of the day that corresponded to the acrophase of endogenous glucocorticoids [73]. Similar findings were confirmed by another study where dexamethasone, a glucocorticoid, was found to increase human Per1 (hPer1) levels significantly and rapidly in human bronchial epithelial cells and peripheral mononuclear cells [74]. This suggests that time of the day can be exploited to maintain the endogenously operating circadian machinery. Other clinical studies have found that once-daily administration of inhaled corticosteroids is equally effective as twice-daily administration in controlling mild to moderate asthma [75]. Thus, eliminating the need for a second dose and increasing compliance to asthma treatment (Figure 2).

Figure 2. Suggested chronotherapy regimen for Asthma and COPD.

Nighttime worsening of Asthma and COPD symptoms are well recognized. Clinically, asthma exacerbations occur between 12:00 AM and 6:00 AM and account for more than 70% of all asthma-related deaths. These exacerbations are mainly attributed to declining levels of adrenaline, reduced FEV1, PEF, FVC, and cortisol levels, while upregulation of muscarinic receptors with increased cholinergic tone, and vasoconstriction. In COPD patients, around the same time, patients experience increased difficulty breathing, nocturnal oxygen desaturation, and declining pulmonary function. This attributes to sleep disturbances. Corticosteroids including methylprednisolone and prednisolone should be avoided between 00:00 and 4:00 AM as it causes adrenal suppression, thus inhibiting endogenous production of glucocorticoids. However, Methylprednisolone does not cause adrenal suppression when administered between 8:00 AM and 4:00 PM. Other asthma drugs including Triamcinolone, Beclomethasone, Mometasone, Salmeterol and Montelukast are recommended between 3:00 PM to 8:00 PM for best results in alleviating the nocturnal symptoms. Chronotherapy for COPD is not well established due to the heterogeneous nature of the disease. Patients with moderate to severe COPD are recommended a combination of both LABA and LAMA. Tiotropium (LAMA) taken in the morning (8:00 AM) followed by Formoterol (LABA) twice throughout the day (8:00 AM and 8:00 PM) is effective in treating moderate to severe COPD, and for patients with nocturnal symptoms, tiotropium taken in the evening (8:00 PM) with two doses of formoterol (8:00 AM and 8:00 PM) is most effective. Both regimens in COPD chronotherapy greatly minimize the need for rescue medication, Albuterol.

COPD exacerbations are also observed more frequently in the early morning hours [1]. However, chronotherapy for the treatment of COPD is not established as in the case of asthma mainly due to the heterogeneous nature of the disease. Long-acting beta-agonist (LABA) and long-acting muscarinic antagonists (LAMA) are two classes of long-acting inhaled bronchodilators that are used to treat patients with moderate to severe COPD. While it is recommended that patients are prescribed only one type of bronchodilator (either LABA or LAMA), those with higher symptom burdens have often been prescribed a combination of both bronchodilators [76]. Combination therapy with tiotropium (LAMA) and formoterol (LABA) provides a better effect throughout the 24-h dosing interval [77]. Particularly, tiotropium in the morning (8 AM) followed with formoterol twice throughout the day (8 AM and 8 PM) was found to be most effective in patients with moderate to severe COPD, and in patients with nighttime symptoms, tiotropium administration in the evening (8 PM) with two doses of formoterol (8 AM and 8 PM) was most effective. In both regimens, the use of rescue medication albuterol use was minimized [78] (Figure 2). Similar to the corticosteroids used for asthma treatments, very little is known about LABA and LAMA in terms of the clock genes that directly influence the core circadian machinery. However, interestingly it was identified that beta-agonists also remarkably induced hPer1 expression in the human bronchial epithelium [79]. This implies that the observed benefits of chronotherapy could be linked to the circadian machinery but would need further research to confirm. Chronotherapeutic effects in PF using antifibrotic drugs nintedanib and pirfenidone, or PAH anti-hypertensive drug hydrochlorothiazide remains unclear. A recent report showed the involvement of one of the fundamental clock genes, Rev-erbα, in its ability to inhibit PF and certain PAH phenotypes, indicating the direct or indirect involvement of the clock, and thus should be further explored to optimize the efficacy of the drugs [6,9].

Chronotherapy for chronic lung diseases should be a personalized medicine approach and should not be generalized to the entire population. Chronotherapy is based on controlled exposure to light-dark cycles. Humans have evolved as diurnal species being active during the day and inactive at night. However, in the modern world where light is available 24 h, an individual’s circadian rhythms can manifest as morning and evening chronotypes. Thus, generalizing the timing of the drugs to the entire population defeats the purpose of a possible benefit from chronotherapy. Chronotherapy should assess the individual’s body clock (chronotype) and how it may differ from the external environmental cues. We believe that appropriate characterization of the individual’s chronotype and identification of differential biomarkers if there exists any, may help further maximize the efficacy of the already existing drugs (see Box 2). When considering clinical studies, we, therefore, recommend the use of standardized sleep surveys/questionnaires or wearable technologies that would determine sleep patterns before enrolling subjects to determine the chronotherapy efficacy of a drug. If confounding variables are strictly controlled, we believe that chronotherapy holds enormous potential in the treatment and management of chronic lung diseases.

Concluding remarks and future directions

Research from the past decade helped us to recognize that circadian clock disruption in the lung during chronic pulmonary diseases is among the major contributing factors in the pathobiology of the disease. Thus, a great deal of research initiatives has been recently undertaken to selectively target the circadian clock molecules in chronic lung disease. Herein, we critically discuss how modulation of the circadian clock can be potentially utilized to manage, reverse or ameliorate chronic inflammatory lung disease phenotypes such as inflammatory response, autoimmunity, fibrogenesis, dysregulated apoptosis, and cancer. However, this field of chronotherapeutics in chronic lung diseases is still in its infancy and most of the concepts and approaches are currently being evaluated in basic and clinical translational research (see Outstanding Questions). Thus, it is essential that we equally appreciate the challenges and potential barriers as much as we appreciate the promising results summarized here. A better mechanistic understanding of the complex circadian machinery and its involvement in normal lung function would not only be critical in the treatment and management of chronic lung diseases but can also help us identify the disease in its earliest forms. We strongly believe that the challenges mentioned in this article are addressable with ongoing research using emerging advanced next-generation OMICs technologies (e.g., single-cell RNA sequencing, single nuclear RNA seq, and spatial transcriptomics) and if successful, the circadian clock modulators alone or in combination with already existing drugs can markedly improve efficacy and treatment outcomes in chronic pulmonary diseases.

Outstanding Questions.

• How can we increase the half-life and the bioavailability of synthetic ligands (small molecules) that are used to modulate the circadian clock without compromising its cellular action and molecular functions?

• How do we evaluate the efficacy and safety of novel clock-based drugs for the potential treatment of chronic inflammatory lung diseases?

• What are the other pleiotropic roles of clock-modulating compounds both in in vitro and in vivo models of lung disease?

• How do we conduct studies using translationally relevant models including in vitro (primary cells in culture 2D and 3D models), in vivo (rodent models), and ex vivo (precision-cut lung slices and lung organoids) methods to determine if clock-based therapeutics alone or existing drugs combined with clock modulators can provide better outcomes in the treatment and management of chronic lung diseases?

• Can we employ OMICS approaches such as phosphoproteomics, metabolomics, and transcriptomics [single cell RNA-sequencing, single nuclei ATAC-sequencing, and spatial transcriptomics] to understand chronotherapeutics in lung development, regeneration, and repair?

• What are the major canonical signaling pathways affected during chronic pulmonary diseases that lead to circadian clock disruption?

• Can pharmacological manipulation of the circadian clock-based therapeutics partly or fully attenuate affected canonical signaling pathways and circadian clock disruption in chronic pulmonary diseases?

Highlights.

• Circadian molecular clock disruption occurs due to dysregulated immune-inflammatory response in the lung which further contributes to the pathobiology of chronic pulmonary diseases.

• Recent evidence from pre-clinical models strongly suggests that selective manipulation of core circadian clock molecules (chronopharmacology) may contribute to the development of novel clock-based therapeutics against chronic lung diseases.

• We provide novel insights into the application of chronopharmacology in chronic pulmonary diseases, and also discuss the clock-based therapeutic challenges in the field.

Glossary

Adaptive immunity

A type of immunity that a person develops following exposure to an antigen or a pathogen mediated by specific immune cells (e.g., dendritic cells, macrophages, and B cells) and antibodies that block the foreign agents and thus prevent disease in the future due to immunological memory

Asthma

Asthma is a chronic inflammatory lung condition characterized by airway narrowing, swelling, and production of increased mucus that leads to breathing difficulty, coughing, wheezing, and shortness of breath

Agonist

A drug or small molecule that binds to the receptor and causes activation of the receptor in as similar ways as its natural ligand (mimics the endogenous ligand)

Antagonist

A drug or small molecule that binds to the receptor and blocks/inhibits the activation of the receptor

Basophils

A certain type of white blood cell that coordinate their function along with the body’s immune system to defend against allergens, pathogens and parasites

Core clock genes (CCGs)

Genes that are primarily responsible for internal circadian timekeeping are referred to as core clock genes

Clock genes

Clock genes (e.g., Clock, Bmal1, Nr1d1, Nr1d2, Cry1, Cry2, Per1, Per2, Rorα, Dbp, Npas2, etc.) mediate circadian oscillation of gene transcription by transcriptional feedback interaction with transcription factor binding to E-box/RORE/D-box elements

Chronotherapy

An emerging treatment modality in circadian medicine that adapts the internal biological time of the individual to have a maximum drug response with minimal side effects

Chronotherapeutics

Chronotherapeutics is the novel approach to optimizing the effects of drugs that can minimize toxicity by timed administration of medications according to the circadian rhythms of disease

Chronotype

The chronotype of an individual refers to the specific entrainment and/or activity-rest preference of the induvial in a 24-hour day

Chronopharmacology

The branch of chronobiology that is used to study the effects of drugs that vary with biological timing and endogenous periodicities

COPD

Chronic obstructive pulmonary disease that is characterized by airflow limitation and decline in pulmonary function associated with breathing difficulty, cough, mucus production, and wheezing mainly caused because of exposure to inhaled irritants (e.g., gases, particulate matter, cigarette smoke, etc.)

Cystic Fibrosis

Cystic fibrosis is an inherited genetic disorder that affects the lungs, digestive system, and other organs such as the pancreas. The defect in the cystic fibrosis transmembrane regulator (CFTR) gene leads to the inability to clear the mucus secretion that is sticky and thick

Circadian gene

Any gene which is expressed in a diurnal fashion with a near 24-hr period. The circadian clock is believed to influences the rhythmic expression of these circadian genes

Dendritic cells

Specialized immune cells also termed as antigen-presenting cells that are found in our body and boosts immune response by presenting the antigens on its surfaces to other cells of the immune system

Histone acetylation

The epigenetic modification that adds the acetyl group to specific lysine residues of the histones that changes the chromatin structure to an open or closed conformation thus regulating gene expression

Homeostasis

The ability of the organism or cell to maintain internal equilibrium by adjusting to its normal physiological processes is termed homeostasis

Inverse agonist

A drug or small molecule that binds to the receptor and antagonizes the effect but exerts the opposite response by suppressing the spontaneous receptor signaling

Mast cells

A type of white blood cell found in the connective tissues which contains basophilic granules (e.g., histamine and heparin) ultimately responsible for allergic responses

Pulmonary Fibrosis

Chronic age-associated lung condition that causes lung tissue damage and scarring that is irreversibly associated with progressive decline in lung function

Pulmonary Arterial Hypertension

A medical condition leading to narrowing of the blood vessels in the lungs, which ultimately leads to slower blood flow through the lungs and increased pulmonary arteriole pressure