A CLOCK-binding small molecule disrupts the interaction between CLOCK and BMAL1 and enhances circadian rhythm amplitude

Abstract

Proper function of many physiological processes requires a robust circadian clock. Disruptions of the circadian clock can result in metabolic diseases, mood disorders, and accelerated aging. Therefore, identifying small molecules that specifically modulate regulatory core clock proteins may potentially enable better management of these disorders. In this study, we applied a structure-based molecular-docking approach to find small molecules that specifically bind to the core circadian regulator, the transcription factor circadian locomotor output cycles kaput (CLOCK). We identified 100 candidate molecules by virtual screening of ∼2 million small molecules for those predicted to bind closely to the interface in CLOCK that interacts with its transcriptional co-regulator, Brain and muscle Arnt-like protein-1 (BMAL1). Using a mammalian two-hybrid system, real-time monitoring of circadian rhythm in U2OS cells, and various biochemical assays, we tested these compounds experimentally and found one, named CLK8, that specifically bound to and interfered with CLOCK activity. We show that CLK8 disrupts the interaction between CLOCK and BMAL1 and interferes with nuclear translocation of CLOCK both in vivo and in vitro. Results from further experiments indicated that CLK8 enhances the amplitude of the cellular circadian rhythm by stabilizing the negative arm of the transcription/translation feedback loop without affecting period length. Our results reveal CLK8 as a tool for further studies of CLOCK's role in circadian rhythm amplitude regulation and as a potential candidate for therapeutic development to manage disorders associated with dampened circadian rhythms.

The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm

“…Site-directed mutagenesis For site-directed mutagenesis, Phusion polymerase based quick-change method was used. Primers were designed using the recommended guidelines (57). The PCR reaction mixtures contained 0.3 mM dNTP, 5 μl of 10X Phusion GC Buffer (Thermo Scientific), 3% DMSO, 1 μM of each primer, 30 ng of the template plasmid (mCry1 in pcDNA4A) and 1 unit of Phusion DNA polymerase in a 50 μl of final volume.…”

“…Pull-down assay 4x10 5 HEK293T cells (per well) were seed to 6-well tissue plate 24h before the transfection. Cells were transfected with Cry1-His-Myc (WT or variants) and Cry2-His-Myc in pcDNA4-A with Flag-CMV-Bmal1, Flag-CMV-CLOCK via PEI transfection reagent for IP with BMAL1, CLOCK and CRY1 as described in (57). Flag-PER2-CMV plasmid was also transfected along with BMAL1, CLOCK and Cry1 plasmids to pull-down four clock proteins.…”

In silico identification of widely used and well-tolerated drugs as potential SARS-CoV-2 3C-like protease and viral RNA-dependent RNA polymerase inhibitors for direct use in clinical trials

“…FDA approved drugs were retrieved from the ZINC database (http://zinc15.docking.org/catalogs/fda) and prepared for docking simulations (Irwin and Shoichet, 2005). Virtual screening was performed as described previously (Armutlu et al, 2009;Cakir et al, 2012;Doruk et al, 2020). AutoDock Vina was used to predict the optimal conformations of the receptor-ligand complex.…”

In silico identification of widely used and well-tolerated drugs as potential SARS-CoV-2 3C-like protease and viral RNA-dependent RNA polymerase inhibitors for direct use in clinical trials

“…The library was prepared by collecting 3948 FDA approved drugs from commercially available ZINC database (http://zinc15.docking.org/catalogs/fda) 34 . Virtual screening was performed as described previously 35,36 . For molecular docking simulations, AutoDock Vina is used to predict the optimal conformations of the receptor-ligand complex and report binding affinity scores by assuming a structure model with a rigid receptor (protein) and a flexible ligand.…”