Introduction

The circadian clock, driven by a transcriptional feed-back loop coupled with translational and posttranslational regulations, generates the ∼24-h rhythm in physiology and behavior [1, 2]. This evolutionarily conserved time-kee** mechanism imparts pervasive temporal control in diverse biological processes, including metabolic regulation, cell cycle and stem cell behavior [3,4,5,6,7,8]. Disruption of circadian clock regulation, increasingly prevalent in a modern lifestyle, leads to the development of metabolic disorders [9, 10], a myriad of cancers [11], and dysregulation of tissue remodeling [6,7,8]. Therapeutic targeting of circadian clock and its biological output pathways may have applications in metabolic disorders, cancer treatment and prevention, and dystrophic muscle diseases [6, 11,12,13,14,Plasmids and shRNA

pcDNA3-BMAL1-His plasmid was purchased from Addgene (Addgene 31367). pcDNA3.0-6XMyc-Clock and pcDNA3.0-3XFLAG-Cry2 were generated by PCR amplification and sub-cloned into the pCDNA3.0–6XMyc or pCDNA3.0–3XFLAG vector separately. The primers used for PCR amplifications were: CLOCK-s: GCGGCCGCATGGTGTTTACCGTAAGC, CLOCK-as: CTCGAGTTCAGCCCTAACTTCTGCA; Cry2-s: GCGGCCGCATGGCGGCGGCTGCTGTGGT Cry2-as: CTCGAGTCAGGAGTCCTTGCTTGCT. The cysteine 267 mutation of CLOCK gene was generated using site-directed mutagenesis. The primer sequences used to generate this mutation were: CLOCK-C267A-s: TTCATCAAGGAAATGGCTACTGTTGAA, CLOCK-C267A-as: GCCATTTCCTTGATGAACTGAGGTG. The short hairpin RNAs of CLOCK used to generate stable knockdown U2OS cells were purchased from Sigma-Aldrich (TR0000018976) and cloned into the PLKO.1 puro vector.

Generation of stable cell lines

Stable clones of U2OS cells containing control or shCLOCK were generated by lentiviral transduction and stable selection. HEK293A were transfected with lentiviral packaging plasmids (psPAX2 and pMD2.G) and lentivirus vectors PLKO.1 or PLKO.1-shCLOCK using PEI Max. At Forty-eight hours after transfection, lentiviruses were collected through a 0.45-µm filter to remove cell debris. U2OS cells were then infected using the lentiviral medium supplemented with polybrene (Santa Cruz, SC-134220). Stable cell colonies were selected in the presence of 1 μg/ml puromycin at 24 h following lentiviral infection,

Transient transfection luciferase assay

Luciferase assays were performed as described [23, 24, 38, 58, 59]. So far, the identified Cry modulators stabilize the protein and augment its clock repressor function by inhibiting its proteosome-mediated degradation [14, 25, 60]. To date, clock-modulating compounds that directly target CLOCK and Bmal1, the key transcription activators that drive core clock transcription feed-back loop have yet to be identified. This study is the first to report a CLOCK protein-targeting molecule, CHX, with activity in promoting myogenic differentiation. We provided experimental evidence for the target specificity of CHX, with loss of its clock-modulatory activity in clock mutant and its pro-myogenic effect in clock-deficient myoblasts. Mutating CLOCK protein cysteine 267 abrogated CHX-induced CLOCK/Bmal1 association, demonstrating the targeting specificity of the compound. Future experiments are needed to determine the binding affinity and specificity of CHX with CLOCK. Nonetheless, by testing CHX activity using CLOCK mutant protein, Bmal1-deficient myoblasts and CLOCK knockdown U2OS cells, current findings indicate that CHX activity in stimulating clock gene induction, myogenic differentiation and proliferation requires a functional clock.

CHX is a cationic bisbiguanide molecule capable of binding to the bacterial cell wall with broad bacteriostatic or bactericidal activity [61]. It is used as a topical skin antiseptic agent and in mouth wash due to its anti-plaque and anti-gingivitis activity [62, 63]. CHX binds to proteins on skin and mucous membranes with limited systemic absorption. Potentially due to its chemical properties, we found that CHX, at concentrations higher than 1–2 µM, exhibited toxicity, although distinct cell types showed variable sensitivity to CHX concentration and duration of treatment. Dose response analysis of bioluminescence activity using Per2-Luc U2OS cells revealed a broader nontoxic concentration range (0.2–2 μM) than functional assays of pro-myogenic activity in C2C12 myoblasts (0.2–0.5 µM). We confined experiments within the lower concentration range to avoid adverse effects. This contrasts with other studies for clock modulator screening that typically used 0.5–10 μM [50] or investigation of biological functions of clock-targeting compounds [24]. Nonetheless, CHX could have clock-independent off-target effects of CHX, particularly as related to pro-myogenic activities in myoblasts. Despite its significant pro-myogenic action, the observed clock-modulatory effects of CHX on period lengthening were relatively moderate. This could be due to its mechanism of action by promoting CLOCK interaction with Bmal1. As our results revealed that CHX augmented the affinity of these interacting partners, there could be a limit posed by the binding kinetics of these proteins in vivo, and thus, not as efficacious as inhibitors that disrupt endogenous interaction. In line with this, the clock-inhibitory molecules we discovered from this screen were more potent in inhibiting Bmal1/CLOCK interaction and altering period length (unpublished data). These observations highlight the need for optimization of CHX chemical structure in order to develop new efficacious clock-activating molecules with pro-myogenic activities, while minimizing toxicity and off-target effects. Optimization of this lead compound may lead to development of novel agents for dystrophic muscle disease or metabolic disease applications.

Current pharmacological modulators of circadian clock components are directed toward metabolic diseases and cancer [14, 19]. Epidemiological investigations and experimental studies have provided compelling evidence that disruption of clock regulation leads to the development of obesity and insulin resistance [64]. We reported that loss of Bmal1 resulted in obesity [37], and chronic shiftwork induced adipose tissue expansion with inflammation [

Conclusions

In summary, our study is the first report to demonstrate the feasibility of a screening pipeline for discovering circadian clock modulators by targeting the CLOCK protein that has myogenic regulatory properties. The discovery of CHX as a pro-myogenic molecule targeting the clock modulation may have therapeutic applications in muscular dystrophy or related muscle remodeling processes involving muscle stem cells.