Masters thesis of science investigation into the modulation of circadian clock proteins by dietary compounds and small molecules

Investigation into the Modulation of Circadian Clock Proteins by Dietary Compounds and Small Molecules A thesis submitted in fulfilment of the requirements for the degree of Master of Science Eleni Pitsillou B.Sc (Hons) Pathology, University of Melbourne School of Science College of Science, Technology, Engineering and Maths RMIT University March 2022 Declaration I certify that except where due acknowledgement has been made, this research is that of the author alone; the content of this research submission is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines have been followed In addition, I certify that this submission contains no material previously submitted for award of any qualification at any other university or institution, unless approved for a joint-award with another institution, and acknowledge that no part of this work will, in the future, be used in a submission in my name, for any other qualification in any university or other tertiary institution without the prior approval of the University, and where applicable, any partner institution responsible for the joint-award of this degree I acknowledge that copyright of any published works contained within this thesis resides with the copyright holder(s) of those works I give permission for the digital version of my research submission to be made available on the web, via the University’s digital research repository, unless permission has been granted by the University to restrict access for a period of time I acknowledge the support I have received for my research through the provision of an Australian Government Research Training Program Scholarship Eleni Pitsillou 03/03/2022 ii Acknowledgements Firstly, I would like to thank Dr Andrew Hung and Dr Tom Karagiannis for their guidance and support over the past few years Their words of encouragement and feedback have been invaluable I appreciate the opportunity to work on this project I would also like to thank Julia Liang for her ongoing support and for all the knowledge that she has provided Thank you to Raymond Beh, Seda Catak, Jaqueline Prestedge, and Vivian Xu for their assistance and I am grateful to be part of such a wonderful team I would like to acknowledge McCord Research (Iowa, USA) for financial support and Reaction Biology (QPatch; Malvern, PA, USA) for performing the patch clamp assays as described in Chapter I would also like to acknowledge the generous allocation of computational resources provided by the National Computing Infrastructure (NCI), Pawsey Supercomputing Centre in Australia (funded by the Australian Government), the Spartan High Performance Computing service (University of Melbourne), and the Partnership for Advanced Computing in Europe (PRACE) for awarding the access to Piz Daint, hosted at the Swiss National Supercomputing Centre (CSCS), Switzerland Last but not least, I would like to say thank you to my family and friends for being there over the past couple of years and for their understanding iii Table of contents Declaration ii Acknowledgements iii Table of contents iv List of abbreviations vii List of figures x List of tables xvi Abstract Chapter Introduction 1.1 Introduction 1.2 Mammalian transcription-translation feedback loops 1.3 Post-translational modifications and interactions with chromatin-modifying complexes 1.4 Disruption of circadian rhythms 11 1.5 The circadian rhythm, metabolic disorders, and major depressive disorder 12 1.6 Potential modulation of the circadian rhythm by pharmacological and dietary compounds 17 1.7 Conclusion and future directions 25 1.8 In silico molecular docking and molecular dynamics simulations 25 Chapter Identification of novel bioactive compounds from Olea europaea by evaluation of chemical compounds in the OliveNetTM library: in silico bioactivity and molecular modelling, and in vitro validation of hERG activity 30 2.1 Introduction 30 2.2 Materials and Methods 32 2.2.1 Analysis of the OliveNetTM database: SwissADME, QikProp and Discovery Studio 32 2.2.2 Molecular docking to the human ether-à-go-go-related gene channel and P-glycoprotein 33 2.2.3 Steered molecular dynamics simulations 34 2.2.4 Human ether-à-go-go-related gene patch clamp assays 35 2.3 Results 35 2.3.1 Human intestinal absorption and membrane permeability 35 2.3.2 Blood-brain barrier permeability and central nervous system activity 39 2.3.3 Selection of 30 compounds 40 2.3.4 P-glycoprotein substrates and inhibitors 42 2.3.5 Binding to plasma proteins and metabolism 43 iv 2.3.6 Toxicity 45 2.3.7 Bioavailability, synthetic accessibility, and leadlikeness 47 2.4 Discussion 49 2.5 Conclusion 53 Chapter Modulation of circadian core clock proteins by dietary compounds 54 3.1 Introduction 54 3.2 Materials and Methods 56 3.2.1 Homology model of the human CLOCK:BMAL1 complex 56 3.2.2 Protein structures and ligands 56 3.2.3 Molecular docking and blind docking 57 3.2.4 Molecular similarity analysis 57 3.2.5 Ligand binding site prediction 58 3.3 Results and Discussion 58 3.3.1 Homology modelling and docking to the CLOCK protein 58 3.3.2 Molecular docking and blind docking using the homology model of the human CLOCK:BMAL1 complex 59 3.3.3 Molecular similarity of the sterols and triterpenic acids in OliveNetTM to ROR ligands 67 3.3.4 Molecular docking to the ligand-binding domain of ROR and ROR 69 Chapter Molecular mechanisms of action of selected olive phenolics against epigenetic modifiers and the structurally related monoamine oxidase enzymes 73 4.1 Introduction 73 4.2 Materials and Methods 75 4.2.1 Crystal structures and chemical structures of the ligands 75 4.2.2 Molecular docking and blind docking 75 4.2.3 Protein-peptide docking 76 4.2.4 Ligand binding site prediction 76 4.3 Results and Discussion 77 4.3.1 Lysine-specific demethylase (LSD1) and LSD1+8a 77 4.3.2 SET domain containing lysine methyltransferase (SET7/9) 82 4.3.3 Protein-peptide docking 86 4.3.4 Monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) 90 4.4 Conclusion 96 Chapter Conclusions and future directions 97 Chapter References 99 Appendix A 131 Appendix B 132 v Appendix C 159 Appendix D 194 vi List of abbreviations (ATP)-binding cassette (ABC) 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) 5’-AMP-activated protein kinase (AMPK) Absorption, distribution, metabolism, excretion, and toxicity (ADMET) Adenovirus E4 promoter binding protein 4/nuclear factor, interleukin regulated (E4BP4/NFIL3) Albumin D-site binding protein (DBP) Amine-oxidase like (AOL) AMP-activated protein kinase-nicotinamide phosphoribosyl transferase- silent mating type information regulation homolog pathway (AMPK-Nampt-Sirt1) Aryl hydrocarbon receptor nuclear translocator-like protein (ARNTL/BMAL1) Basic helix-loop-helix (bHLH) Blood-brain barrier (BBB) Calcitonin gene-related peptide (CGRP) Casein kinase 1 (CK1/) Casein kinase (CK2) Central nervous system (CNS) Chronic sleep disorders (CSDs) Circadian locomotor output cycles kaput (CLOCK) Clock-controlled genes (CCGs) CREB-binding protein (CBP) Cryptochrome (CRY1 and CRY2) Cyclic AMP response binding element protein (CREB) Cyclic nucleotide-binding homology domain (CNBHD) Cytochrome P450 (CYP) Daytime feeding (DF) Diagnostic and Statistical Manual of Mental Disorders (DSM-5) Dim-light melatonin onset (DLMO) Enhancer of zeste homolog (EZH2) Extra virgin olive oil (EVOO) Familial advanced sleep phase syndrome (FASPS) F-box/leucine-rich repeat protein 21 (FBXL21) vii F-box/leucine-rich repeat protein (FBXL3) Flavin adenine dinucleotide (FAD) Gastrointestinal (GI) Glycogen synthase (GSK3) Hepatic leukemia factor (HLF) High-fat high-sucrose diet (HFHSD) Histone lysine (H3K4) Histone deacetylase (HDAC1) Histone deacetylase (HDAC3) Histone deacetylase enzyme (HDAC) Human ether-à-go-go-related gene (hERG) Hypothalamic-pituitary-adrenal axis (HPA) Inwardly-rectifying potassium channels (Kir3) Lysine-specific demethylase 1+8a (LSD1+8a) Lysine-specific demethylase (LSD1) Lysine-specific demethylase 5A (KDM5A) Lysine-specific demethylase 6A (KDM6A) Lysine-specific methyltransferase 2D (KMT2D) Madin-Darby Canine Kidney (MDCK) Major depressive disorder (MDD) Melatonin receptors (MT1/MT2) Mitogen-activated protein kinases (MAPKs) Mixed lineage leukemia protein-1 (MLL1) Molecular dynamics (MD) Monoamine oxidase enzymes (MAOs) Monoamine oxidase inhibitors (MAOIs) Mouse embryonic fibroblasts (MEFs) Multi-drug resistance and (MDR1 and MDR3) N-ethyl-N-nitrosourea (ENU) Nicotinamide adenine dinucleotide (NAD+) Night-time feeding (NF) N-methyl-D-aspartate receptors (NMDAR) Nuclear receptor corepressor complex (NCoR) viii p300/CBP-associated factor (PCAF) Per Arnt Sim (PAS) Period gene (Per) P-glycoprotein (P-gp) Phosphatase (PP1) Pituitary adenylate cyclase activating peptide (PACAP) Pituitary adenylate cyclase-activating polypeptide type receptor (PAC1) Proline and acidic amino acid-rich basic leucine zipper (PAR-bZIP) Protein kinase C (PKC) RCSB Protein Data Bank (PDB) Retinohypothalamic tract (RHT) Retinoic acid-related orphan receptor (ROR , , ) Retinoic acid-related orphan receptor response elements (ROREs) Reverse orientation c-erb proteins (REV-ERB , ) Root-mean-square deviation (RMSD) S-adenosylmethionine (SAM) S-adenosyl-L-homocysteine (SAH) SET domain containing lysine methyltransferase (SET7/9) Sirtuin (SIRT1) Skp1-Cullin-F-box E3 ligase (SCF) Small interfering RNA (siRNA) Steered molecular dynamics (SMD) Su(var)3-9 and Enhancer of Zeste-Trithorax (SET) Suprachiasmatic nucleus (SCN) Swi3/Rcs8/Moira (SWIRM) Thyrotroph embryonic factor (TEF) Transcription-translation feedback loops (TTFLs) Tumour necrosis factor alpha (TNF-) Voltage-gated calcium channels (VDCC) White adipose tissue (WAT) Wildtype (WT) -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) -transducin repeat-containing protein (-TRCP) ix List of figures Figure 1.1 An overview of the circadian entrainment pathway Natural or artificial light is detected by photoreceptor cells in the retina This stimulates the presynaptic retinal ganglion cell neuron to release neurotransmitters and neuropeptides, such as glutamate and pituitary adenylate cyclase-activating peptide (PACAP), that then bind to receptors on the surface of the postsynaptic neuron This includes N-methyl-D-aspartate receptors (NMDAR), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), voltage-gated calcium channels (VDCC), pituitary adenylate cyclaseactivating polypeptide type receptor (PAC1), melatonin receptors (MT1/MT2) and inwardly-rectifying potassium channels (Kir3) A number of signal transduction cascades are consequently activated, which leads to the phosphorylation of cyclic AMP response binding element protein (CREB) and changes in clock gene expression Figure 1.2 Transcription-translation feedback loops that regulate the circadian rhythm in mammalian cells The aryl hydrocarbon receptor nuclear translocator-like protein (BMAL1 - dark pink) and circadian locomotor output cycles kaput (CLOCK light pink) proteins heterodimerise in the cytoplasm and translocate to the nucleus, where they bind to regulatory sequences in DNA This results in the transcription of clock genes and clock-controlled genes, including Period (Per) and Cryptochrome (Cry) The PER (light brown) and CRY (dark brown) proteins also form heterodimers, translocate to the nucleus and inhibit the activity of CLOCK:BMAL1 The reverse orientation c-erb and retinoic acid-related orphan receptor proteins (REV-ERBs and RORs) are also transcribed and they are able to bind to response elements within DNA to regulate the expression of Bmal1 This process is influenced by signalling pathways, epigenetic modifications, and post-translational modifications, such as phosphorylation and ubiquitination Figure 1.3 Crystal structures of epigenetic regulators from the RCSB Protein Data Bank (PDB) The active site of the lysine-specific demethylase (LSD1) protein is depicted (A) and the flavin adenine dinucleotide (FAD) cofactor is coloured red The residues Y761, E379 and D556 are also shown (B) In addition to LSD1, the crystal structures of histone deacetylase (HDAC3) in complex with its corepressor protein (C) and sirtuin (SIRT1) (D) can be seen Inositol tetraphosphate and nicotinamide adenine dinucleotide are coloured red in HDAC3 and SIRT1, respectively The zinc ions are also depicted in black 10 Figure 1.4 Proteins that are involved in the circadian entrainment pathway and mammalian clock machinery This includes various core clock components, nuclear x Hydroxytyrosol acetate Safinamide 204 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 -6.2 -6.1 -6.1 -6.1 -6.0 -6.0 -6.0 -6.0 -5.9 -5.9 -5.8 -6.9 -6.9 -6.8 -6.8 -6.8 -6.7 -6.6 -6.6 -6.6 -6.6 -6.6 -6.6 -6.5 -6.5 -6.4 -6.4 -6.4 -6.4 -6.4 -6.3 -9.7 -9.2 -9.1 -9.1 -8.7 -8.6 -8.6 -8.5 -8.1 -8.1 -8.1 -7.9 -7.9 Y Y Y Y Y Y Y Y Y Y N Y Y Y Y Y Y Y N Y N Y Y Y Y Y Y Y N Y N Y Y Y Y Y Y Y Y N N N N N 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 MMHTE Oleacein 205 -7.8 -7.8 -7.8 -7.8 -7.7 -7.7 -7.6 -7.8 -7.7 -7.6 -7.5 -7.4 -7.4 -7.4 -7.2 -7.2 -7.2 -7.1 -7.1 -7.1 -7.1 -7.1 -7.1 -7.0 -7.0 -6.9 -6.9 -8.4 -8.3 -8.2 -8.1 -8.1 -8.0 -8.0 -8.0 -8.0 -8.0 -7.9 -7.9 -7.8 -7.8 -7.8 -7.8 -7.7 N N N N N N N Y Y Y Y N N Y N N Y N N Y N Y N N Y N N Y Y Y Y N Y Y N N N Y Y N Y N Y N 18 19 20 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 19 20 Oleocanthal Tyramine 206 -7.7 -7.6 -7.6 -8.2 -8.2 -7.8 -7.6 -7.5 -7.5 -7.5 -7.4 -7.4 -7.4 -7.4 -7.4 -7.4 -7.3 -7.3 -7.3 -7.3 -7.2 -7.1 -7.1 -6.2 -6.2 -6.2 -6.1 -6.1 -6.0 -5.8 -5.8 -5.7 -5.7 -5.7 -5.6 -5.6 -5.6 -5.6 -5.6 -5.5 -5.5 -5.4 -5.4 N N N Y Y Y Y Y N N Y Y N N N N N Y Y N N N N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Table D4 Blind docking results of control compounds and dietary compounds for SET domain containing lysine methyltransferase (SET7/9) Protein SET7/9 (PDB ID: 4JLG) Ligand (R)-PFI-2 Sinefungin Pose 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 19 207 Binding affinity (kcal/mol) Target binding site -9.5 -9.0 -8.8 -8.7 -8.7 -8.7 -8.6 -8.4 -8.4 -8.4 -8.4 -8.3 -8.3 -8.3 -8.3 -8.2 -8.2 -8.2 -8.0 -8.0 -8.4 -8.3 -8.3 -8.2 -8.2 -8.1 -8.0 -8.0 -8.0 -7.9 -7.9 -7.8 -7.8 -7.8 -7.8 -7.8 -7.7 -7.7 -7.5 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Cyproheptadine Zavegepant Hydroxytyrosol 10 11 12 13 14 15 16 17 18 19 10 11 12 13 14 15 16 17 18 19 208 -8.5 -7.9 -7.6 -7.5 -7.5 -7.4 -7.4 -7.4 -7.3 -7.1 -7.1 -7.0 -6.9 -6.9 -6.8 -6.8 -6.8 -6.8 -6.8 -12.7 -12.6 -12.5 -12.4 -12.3 -12.3 -12.3 -12.2 -11.9 -11.9 -11.8 -11.8 -11.7 -11.6 -11.6 -11.5 -11.3 -11.2 -11.1 -5.8 -5.7 -5.6 -5.6 -5.5 -5.5 Y Y N N Y N Y Y Y N N N N Y Y N N N N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Hydroxytyrosol acetate MMHTE 10 11 12 13 14 15 16 17 -5.4 -5.4 -5.3 -5.2 -5.2 -5.2 -5.2 -5.2 -5.1 -4.9 -4.8 Y Y Y Y Y N Y Y Y N N 10 11 12 13 14 15 16 17 18 19 10 11 12 13 14 209 -6.2 -6.1 -6.1 -6.1 -6.0 -6.0 -6.0 -5.9 -5.8 -5.8 -5.8 -5.8 -5.8 -5.7 -5.7 -5.6 -5.6 -5.6 -5.5 -6.7 -6.6 -6.6 -6.5 -6.5 -6.5 -6.5 -6.4 -6.4 -6.4 -6.4 -6.4 -6.4 -6.4 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Oleacein Oleocanthal 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 210 -6.4 -6.3 -6.3 -6.3 -6.3 -6.3 -7.5 -7.4 -7.3 -7.2 -7.2 -7.2 -7.1 -7.1 -7.1 -7.1 -7.1 -7.1 -7.0 -7.0 -7.0 -7.0 -6.9 -6.9 -6.9 -6.9 -7.5 -7.3 -7.3 -7.3 -7.3 -7.2 -7.2 -7.2 -7.2 -7.2 -7.1 -7.1 -7.0 -7.0 -7.0 -7.0 -6.9 -6.9 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 19 20 211 -6.9 -6.8 Y Y Table D4 The protein-peptide docking results for the 10 top-ranking conformations of the histone H3 peptide are provided for lysine-specific demethylase (LSD1) are provided Protein LSD1 (PDB ID: 2V1D) Ligand Apo Hydroxytyrosol Hydroxytyrosol acetate MMHTE 212 Mode l RMSD (Å) Experimentall y determined binding site 10 10 0.0 10.1 7.4 8.5 7.9 9.0 6.8 8.0 7.9 6.9 0.0 11.0 9.5 10.8 8.5 6.6 11.5 7.1 9.3 8.2 Y Y Y N Y Y Y Y Y Y Y Y Y Y N Y Y Y Y N 10 8.5 9.4 9.7 7.4 7.3 8.0 7.3 9.2 9.6 8.3 8.5 7.5 7.1 9.0 6.8 8.3 N Y Y Y Y Y Y Y Y Y N Y Y Y Y Y Oleacein Oleocanthal 4-[2-(4methylphenyl)-5(piperidin-4ylmethoxy)pyridin3yl]benzenecarbonitril e 213 10 10 10 8.0 7.8 8.1 10.9 11.0 10.4 6.8 8.5 7.7 7.0 7.8 7.7 9.3 10.6 6.8 8.5 8.2 7.3 8.2 8.2 6.1 9.2 8.9 10.2 Y Y Y Y Y Y Y N Y Y Y N Y Y Y N Y Y N N N Y Y Y 10 8.5 8.9 7.7 7.2 7.4 8.2 8.5 7.6 6.8 7.3 N Y N Y Y N Y Y Y N Table D4 The protein-peptide docking results for the 10 top-ranking conformations of the histone H3 peptide are provided for lysine-specific demethylase 1+8a (LSD1+8a) are provided Protein Ligand LSD1+8a (PDB ID: 2X0L) Apo Hydroxytyrosol Hydroxytyrosol acetate MMHTE 214 Model RMSD (Å) 10.1 Experimentally determined binding site Y 10 10 8.4 0.0 9.4 6.8 9.1 7.8 10.1 8.0 7.4 0.0 6.0 8.4 6.9 9.5 7.4 7.9 8.4 8.0 6.8 8.4 Y Y Y Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y 10 6.0 7.4 7.1 6.7 7.3 7.2 7.1 7.5 10.0 7.8 8.0 6.9 7.4 9.1 7.6 Y N Y Y Y N Y N Y Y Y Y N Y Y Oleacein Oleocanthal 4-[2-(4methylphenyl)-5(piperidin-4ylmethoxy)pyridin-3yl]benzenecarbonitril e 215 10 10 10 8.4 6.7 7.2 7.5 8.4 6.7 8.0 7.4 9.6 6.8 7.2 7.5 7.2 9.4 8.4 9.1 8.1 9.7 7.4 9.5 6.8 9.5 7.4 6.8 6.3 Y Y N N Y Y Y N Y Y N N Y Y Y Y Y Y N Y Y Y Y Y Y 10 8.5 7.4 7.4 6.3 7.2 7.8 7.4 7.5 7.1 Y N Y Y N Y Y N Y Table D4 The protein-peptide docking results for the 10 top-ranking conformations of the histone H3 peptide are provided for SET domain containing lysine methyltransferase (SET7/9) Protein SET7/9 (PDB ID: 1O9S) Ligand Apo Hydroxytyrosol Hydroxytyrosol acetate MMHTE Model RMSD (Å) Experimentally determined binding site 10 10 0.0 5.4 5.8 6.1 4.6 4.6 5.3 5.8 6.3 5.8 4.6 6.5 5.8 6.3 6.1 5.4 5.6 5.3 5.8 6.3 Y Y Y N Y Y N Y Y Y Y Y Y Y Y Y Y N Y Y 10 4.6 6.1 5.8 5.9 5.3 5.7 6.7 5.7 5.9 5.8 5.6 4.4 6.1 5.0 5.3 5.3 Y Y Y Y N Y Y Y Y Y Y Y N Y N Y 216 Oleacein Oleocanthal (R)-PFI-2 Sinefungin 10 10 10 10 10 217 4.5 6.3 5.4 5.8 5.3 6.1 5.9 5.8 5.5 6.0 6.0 5.1 4.2 5.2 5.3 6.1 5.9 5.8 5.5 6.0 6.0 5.1 4.2 5.2 5.3 5.9 5.5 6.1 5.6 5.5 6.0 4.9 4.2 4.9 5.3 5.9 5.3 5.5 6.0 6.1 5.8 5.8 4.2 4.9 Y Y Y Y N N N N N Y N Y N Y N N N Y N N N N N N N N Y N N N N Y N N N N Y N N N N N N N Zavegepant Cyproheptadine 10 10 218 5.5 5.3 6.3 6.1 5.9 6.1 5.3 5.9 5.5 6.0 5.3 4.9 6.5 5.9 5.5 6.0 5.9 5.3 4.2 4.9 Y N Y N N Y N Y N N N Y Y N N N N N N N ... including diet (323, 324) Therefore, research into the modulation of the circadian clock by dietary compounds and small molecules will allow scientists to gain further insight into how certain diseases... 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... whether the compounds would preferentially bind to the site of zosuquidar in the structure of P-gp The cryo-EM structure of P-gp and the compounds from the OliveNetTM database were imported into