Investigation into the modulation of circadian clock proteins by dietary compounds and small molecules
Creators: name: Pitsillou, Eleni
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 340401
subject: Biologically active molecules
classificationCode: 340402
subject: Biomolecular modelling and design

description: A number of physiological processes are regulated by circadian rhythms and when disruptions occur in the circadian machinery, the activities of the proteins and genes implicated in the intricate network of transcription-translation feedback loops (TTFLs) can be altered. Based on the pre-existing literature, it is apparent that disturbances of the circadian rhythm can contribute to the development of several diseases and disorders. This includes depression and its associated comorbidities, including the metabolic syndrome. There is a clinical need to investigate more effective treatment options for patients suffering from depression and related comorbidities. This depends on the shared pathophysiological pathways being elucidated further and key proteins being identified. Research is being performed into the potential protective properties of natural and synthetic compounds and in this project, dietary compounds were of particular interest. In Chapter 1 of this thesis, a literature review on the circadian machinery, its relationship with metabolic disorders and depression, and the modulation of proteins involved in the TTFLs by small molecules is provided. 

The beneficial health effects of the Mediterranean diet continue to be explored and in 2018, our lab published a curated database of over 600 compounds from Olea europaea (OliveNetTM). Oleuropein and hydroxytyrosol are the major constituents that have been extensively investigated for their potential therapeutic efficacy however, the vast majority remain unexplored. To assess the pharmacokinetic properties of the olive compounds and identify potential novel lead compounds that can be synthesised, an in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis was performed in Chapter 2. Molecular docking was used to examine the binding properties of the olive compounds to the human ether-à-go-go-related gene (hERG) ion channel and P-glycoprotein (P-gp), which are important measures of toxicity and absorption. In vitro patch clamp assays were performed to assess hERG inhibition and steered molecular dynamics simulations were used to examine membrane permeability of selected compounds. The work from this chapter resulted in the identification of eight non-commercially available compounds with favourable ADMET properties. 

In Chapter 3, the potential interactions of dietary compounds with the human circadian locomotor output cycles kaput (CLOCK) protein and retinoic acid-related orphan receptors (ROR-alpha and ROR-gamma) were investigated. The CLOCK protein and RORs play a critical role in regulating clock gene expression and circadian rhythms. Molecular docking was used to screen a selection of well-known phenolic compounds from the OliveNetTM database and several dietary compounds that have been reported to modulate the circadian rhythm against the CLOCK protein. Due to the crystal structure of the human CLOCK protein complex being unavailable, a homology model was generated. Potential ligand binding sites were investigated using blind docking and the machine learning tool P2Rank. The binding properties of the phenolic compounds, sterols, and triterpenic acids from the OliveNetTM library were also evaluated for ROR-alpha and ROR-gamma. The MACCS fingerprint analysis revealed that several sterols and triterpenic acids from OliveNetTM were structurally related to the ROR ligands cholesterol and ursolic acid. 

The inhibitory activities of a selection of olive phenolic compounds against lysine-specific demethylase 1 (LSD1) and the structurally related monoamine oxidase enzymes (MAOs) have previously been investigated by our lab in vitro. Lysine-specific demethylase 1 is involved in a number of physiological pathways including the circadian rhythm, while the MAOs are implicated in the pathophysiology of depression. To investigate the molecular mechanisms of action of the olive phenolic compounds further, in silico tools were used to evaluate the predicted ligand binding affinities, their orientation within the target binding site, and potential intermolecular bonds formed with the protein residues. The findings are presented in Chapter 4 of the thesis. The predictions from the current computational docking and simulated studies have helped elucidate the potential mechanisms of action of key dietary compounds on circadian rhythm pathways, and provide a number of molecular leads which may be validated by experimental assays.