Circadian rhythms are regulated by endogenous molecular clocks and can be entrained by external stimuli [[1], [2], [3]]. These environmental cues, known as zeitgebers, include the light/dark cycle, temperature, diet, and exercise [1,4]. Biological clocks play an important role in controlling physiological processes and circadian misalignment has been linked to several pathological conditions [1]. Studies have shown that components of the mammalian circadian machinery can be targeted by small drug molecules including the reverse orientation c-erb (REV-ERBs) and retinoic acid-related orphan receptors (RORs), Period (Per) and Cryptochrome (Cry) proteins, and casein kinases [5].

Nuclear receptors are ligand-dependent transcription factors that regulate a number of biological processes including the circadian rhythm, metabolism, immune regulation, and cell development and differentiation [6]. The nuclear receptor superfamily consists of the steroid hormone receptors, non-steroid hormone receptors and the orphan nuclear receptors [6]. The retinoic acid-related orphan receptor (ROR) subfamily is comprised of RORα, RORβ, and RORγ [6]. There are several receptor isoforms and the expression of these transcription factors is tissue-specific [[6], [7], [8], [9]]. The activity of RORs can be modulated by small molecules, such as sterol ligands and synthetic compounds [10]. Retinoic acid-related orphan receptors α and γ have been extensively investigated, while the therapeutic potential of RORβ requires further elucidation [11]. In a study by He et al., the polymethoxylated flavone nobiletin was identified as a clock amplitude-enhancing small molecule and its efficacy in protecting against metabolic syndrome was explored in mouse models [12]. Amplitude refers to the difference between the peak and the trough of the circadian cycle, and a dampened amplitude is closely related with chronic diseases [13]. Using a competitive radioligand binding assay, the RORs were determined to be direct protein targets for nobiletin and a higher affinity was observed for RORγ [12].

In addition to RORα and RORγ, the human circadian locomotor output cycles kaput (CLOCK):aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) (CLOCK:BMAL1) complex was of interest in this study. The CLOCK:BMAL1 heterodimer is a major component of the transcription-translation feedback loops that regulate the circadian molecular oscillator [14]. The RORs bind to retinoic acid-related orphan receptor response elements (ROREs) in the promoter region of Bmal1 and this leads to the expression of Bmal1 being activated [14]. The REV-ERB proteins, which repress the transcription of Clock and Bmal1, compete with the RORs for binding of the ROREs and this leads to the circadian pattern of Bmal1 expression [14]. The REV-ERBs and RORs consequently interconnect the positive limb of the core circadian clock, which includes CLOCK and BMAL1, with the negative limb [14]. The direct modulation of the CLOCK:BMAL1 complex by small molecules is less well-explored however, Doruk et al. found that CLK8 was a CLOCK-binding small molecule using in silico, in vitro, and in vivo methods [15]. They showed that CLK8 could regulate the circadian rhythm amplitude at the cellular level and this compound could be used to gain further insight into the circadian clock regulatory mechanism [15].

The therapeutic efficacy of small molecule modulators of the circadian clock, including dietary compounds, requires further investigation [16]. There is evidence to suggest that adhering to the Mediterranean diet, which is characterised by food products that are rich in polyphenols and polyunsaturated fatty acids, can help to prevent circadian rhythm disorders [17,18]. While the effects of natural compounds, such as caffeine, cinnamic acid, resveratrol, and docosahexaenoic acid, on the circadian rhythm have been previously reported, more research is needed to explore their mechanisms of action [19,20].

Using in silico methods, we aimed to evaluate the binding characteristics of dietary compounds to proteins involved in regulating mammalian circadian rhythms. The OliveNet™ database consists of over 600 compounds identified from Olea europaea and are characterised into 13 main classes [21]. This includes natural sterols and triterpenic acids that may bind to and modulate the activity of RORα and RORγ [21]. The sterols, triterpenic acids, and phenolic compounds were screened against the known binding site of cholesterol sulphate and ursolic acid in the structures of RORα and RORγ, respectively. The structural similarity of the olive sterols and triterpenic acids to the co-crystallised ligands was also evaluated. The binding affinities and protein-ligand interactions were examined. Moreover, a homology model of the human CLOCK:BMAL1 complex was generated and used to investigate the binding properties of CLK8 and a selection of dietary compounds.