FIELD OF THE INVENTION

The present invention discloses and claims ((lS,9S)-l l-{[4-(l,3-benzodioxol-5- ylamino)-8-methyl-2-quinazolinyl]methyl}-7, 1 l-diazatricyclo[7.3.1.0-2.7~| trideca-2,4-dien-6-one) (Formula I) as CRY-binding small compound and stabilizer of CRY s, and a method of using said compound Formula I for treating circadian rhythm associated diseases, including diabetes. Pharmaceutical compositions comprising Formula I and methods for the preparation of Formula I are also disclosed and claimed.

BACKGROUND

The circadian clock generates a 24-hour rhythm through which physiology and behavior are adapted to daily changes in the environment. Many biological processes like hormone secretion, and sleep-wake cycles are controlled by the circadian clock. Therefore, an innate malfunctioning of the circadian clock and the related pathways can cause various pathologies. Sleep disorders, altered metabolism, obesity, diabetes, mood disorders, cancer, and cardiovascular diseases are all linked with abnormal circadian rhythm.

At the molecular level, the clockwork of the cell involves several proteins that participate in positive and negative transcriptional/translational feedback loops (TTFL). BMAL1 and CLOCK are transcription factors that bind E-box elements (CACGTG) in clock-controlled genes including Period and Cryptochrome and thereby exert a positive effect on circadian transcription. The mammalian PERIOD (PER) and CRYPTOCHROME (CRY) proteins form heterodimers that interact with casein kinase Is (CKIs) and then translocate into nucleus where CRY acts as a negative regulator of BMALl/CLOCK-driven transcription. Upon phosphorylation CRYs are ubiquitinated by E3 ubiquitin ligases e.g. FBXL3 and FBXL21 and directed to proteasome for the degradation. FBXL21 and FBXL3 act antagonistically on CRY to regulate its stability differentially in the cytosol and nucleus, respectively. The FBXL3 protein participates in the negative feedback loop by binding to the CRYl and CRY2 proteins to facilitate their polyubiquitination and their subsequent degradation by the proteasome [1, 2]

In addition there is a second feedback loop consists of retinoic acid receptor- related orphan receptors (RORs) and REV-ERBs which control the transcription of the Bmall gene and, in turn, regulate molecular clock [3]

Since the circadian system regulates several aspects of our physiology, it is not surprising that disturbed circadian rhythm can lead into diseases in human. It is, therefore, essential to find small molecules to correct disturbed circadian rhythm. There are several studies have been carried out to find small molecules affect circadian rhythm based on phenotypic changes in the circadian rhythm of reporter cells using high-throughput screening assay. These studies result in identification several molecules affect different features of circadian rhythm [4, 5, 6, 7] For example a molecule (named as GSK4112) was shown to enhance REV-ERB's repressor function toward Bmall transcription and greatly altered circadian clock and metabolism [8] Another example, identification of KL001 molecule, increases stability of the CRYs and suppresses the gluconeogenesis [5]

It has been shown that disturbed circadian rhythm can lead to various diseases including sleep disorders, glucose metabolism disorders, cardiovascular diseases, and cancer. Therefore, it is essential to find small molecules to correct disturbed circadian rhythm. In recent years, many studies focused on the discovery of small molecules interacting with circadian clock proteins for the treatment of these diseases. These studies result in identification several molecules affect different features of circadian rhythm [9, 10, 11, 12] For example, identification of KL001 molecule, increases stability of the CRYs and suppresses the gluconeogenesis.

Alternatively, structure-based drug design approach can be utilized to design small molecules by using the available crystal structure core clock proteins [13, 14] . The crystal structure of the CRY -FBXL3 revealed the critical region (FAD binding pocket) on CRY for the FBXL3 interaction [14]

An effective method to identify small molecules that perturb a biological system is structure-based design. Since the crystal structures of clock proteins are now available, this approach can lead to identification of clock modulating compounds targeting clock proteins.

In the European patent document EP2408906A1, the role of AMPK in circadian rhythms and methods of screening for small molecules that modulate such rhythms are disclosed. The disclosure demonstrates that AMPK phosphorylates the transcription repressor CRY1 and CRY2 and stimulates their proteasomal degradation. According to this invention, the use of an AMP kinase agonist or antagonist is disclosed for the manufacture of a medicament to modulate circadian rhythms in a subject.

Many physiological variables require a robust circadian clock for their proper function. There is a need for circadian rhythm controlling molecules and compositions as potential treatments for clock-related diseases, including diabetes. It has therefore become increasingly interesting to identify small molecules that can specifically modulate regulatory core clock proteins since they have the potential to manage these diseases.

Cryptochrome 1 and 2 (Cryl and 2), one of the key circadian clock genes, plays an important role in circadian clock and clock-related diseases. Recent studies suggested that CRYs are associated with diabetes and may act as a novel switch in hepatic fuel metabolism by limiting glucose production [19, 20] CRYs may be a prognostic biomarker and a promising therapeutic target for treatment of diabetes. Despite advances in drug discovery directed to identifying therapeutic small molecules for CRY binding, there is still a scarcity of compounds that are potent, efficacious, and selective stabilizer of cryptochromes. Furthermore, there is a scarcity of compounds effective in the treatment and/or prevention of disorders associated with the circadian rhythm. These needs and other needs are satisfied by the present invention.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a CRY-binding compound of Formula I or pharmaceutically acceptable salts thereof. Accordingly, a broad embodiment of the invention is directed to a CRY -binding compound of Formula I:

The other aspect of the present invention is to provide a CRY -binding compound for treating and/or preventing a circadian rhythm associated diseases or disorders, including diabetes, wherein the compound is characterized by stabilizing of Cryptochromes (CRY s).

In a further aspect, the present invention relates to a CRY-binding compound of the invention for use in increasing the stability of CRY 1/2 in a mammal.

Another aspect of the present invention is to provide a CRY -binding compound capable of controlling circadian rhythm via lengthening the period length. Another embodiment of the present invention relates to a method for identifying a compound for stabilizing the CRY proteins.

A further aspect relates to a process for the synthesis of the CRY-binding compound.

The invention can be used for the preparation of a medicament useful in the treatment and/or prevention of disorders by means of the stabilization of cryptochromes.

Yet another objective of the present invention is to provide a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

This object and other objects of this invention become apparent from the detailed discussion of the invention that follows. Brief Description of Figures

The present invention is illustrated in the accompanying figures wherein;

Figure 1 is an illustration of FORMULA I increases the half-life of CRY1 and 2. FORMULA I increased the degradation rate of Cryl-dLuc and Cry2-dLuc dose dependently. HEK 293T cells were transfected with Cryl-dLucand Cry2-Luc plasmids. 24h after transfection cells were treated with different doses of FORMULA I or solvent (DMSO final 0.5%) as control. 24h after molecule treatment cells were treated with cycloheximide (20pg/ml final) and bioluminescence was recorded. Normalized half-life is shown with ± SEM (n=4 with triplicates). *p<0.05 **p<0.01, all others are non-significant). Figure 2 is an illustration of bioluminescence rhythms of U20S Bmal-dLuc cells treated with various doses of FORMULA I. A) Raw data recorded for 7 days. B) Dose-dependent effect of FORMULA I on period (hr) and amplitude (counts/s) of the oscillation. Data are given as mean ± SEM from at least 3 individual experiments. Statistical analysis performed using one-way ANOVA with Dunnett's multiple comparisons test (p*<0.05, **p<0.01, ***p<0.001, all others are non-significant).

Figure 3 is an illustration of bioluminescence rhythms of NIH3T3 mPerl-dLuc cells treated with various doses of FORMULA I.

Figure 4 is an illustration of western blot analysis of unsynchronized U20S cells treated with FORMULA I. A) CRY1, B) CRY2, and C) PER2 protein level normalized with b-ACTIN. Data are given as mean ± SEM from at least 3 individual experiments. Statistical analysis performed using one-way ANOVA with Dunnetfs multiple comparisons test (*p<0.05, ***p<0.001, all others are non-significant).

Figure 5 is an illustration of RT-qPCR analysis of unsynchronized U20S cells treated with FORMULA I. mRNA levels of Cryl, Cry2, Per2, Dbp, Rev-erba, Rora, Clock, and Bmall relative to RplpO. Data are given as mean ± SEM from at least 3 individual experiments. Statistical analysis performed using one-way ANOVA (*p<0.05, **p<0.01, ns: non-significant).

Figure 6 is an illustration of dose-dependent effects of FORMULA I on CRYl- DLUCand CRY1-W399L-LUC mutant half-life relative to DMSO. Data are given as mean ± SEM from at least 3 individual experiments. Statistical analysis performed using one-way ANOVA (**p<0.01, ns: non-significant).

Figure 7 is an illustration of effect of FORMULA I on gluconeogenesis in HepG2 cells. A) RT-qPCR analysis of gluconeogenic Pckl and G6pc genes when cells were treated with FORMULA I and induced with glucagon. B) Glucose production amount of cells treated with FORMULA I and induced with glucagon. Statistical analysis performed using one-way ANOVA (*p<0.05, ***p<0.001).

Figure 8 is an illustration of synthesis of Formula 1.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, it is aimed to discover a novel CRY-binding small compound using a structure-based approach by targeting the FAD binding pocket which is critical for the FBXL3 interaction. As a result, the interaction of a CRY- binding compound with CRYl and CRY2 has been predicted in silico and then demonstrated experimentally. The CRY-binding compound was identified that stabilizes cryptochromes (CRYs; CRYl/2; CRYl and CRY2) and decrease the degradation of CRYs in vitro. The stability of CRY proteins is important because they can alter the period and amplitude of the circadian rhythm, in turn, affecting metabolism.

Furthermore, it was discovered that, as a result of this interaction, the subsequent degradation of CRYs by proteasome is affected by the CRY-binding compound resulting blocking the proteasome-mediated degradation of CRYl and CRY2 thereof. The CRY-binding compound leads to increase in CRYl or CRY2 half- life. These results also lead to period lengthening and amplitude reduction.

Moreover, the CRY-binding compound caused the repression of gluconeogenic genes Phosphoenolpyruvate carboxykinase 1 ( Pckl ) and Glucose 6 phosphatase (' G6pc ) and lowered the glucose production during glucagon-induced gluconeogenesis. In previous studies, it was discovered that increase in CRY proteins levels inhibit glucagon-induced gluconeogenesis. These results show the therapeutic potential of the compound, a CRY stabilizer, for the treatment of type 2 diabetes mellitus. As used herein, the terms "treat", "treatment" and "treating" refer to therapeutic treatments includes the reduction or amelioration of the progression, severity and/or duration of a disease or disorder.

The present invention relates to a CRY-binding compound (Formula I).

Unless specified otherwise, the term "Formula I " or “compound” or “FORMULA I” refers to compounds of Formula I, prodrugs thereof, salts of the compound and/or prodrug, hydrates or solvates of the compound, stereoisomers, tautomers, isotopically labeled compounds, and polymorphs. It is an object of this invention to provide a CRY -binding small compound having the chemical name ((lS,9S)-l l-{[4-(l,3-benzodioxol-5-ylamino)-8-methyl-2- quinazolinyl]methyl}-7,l l-diazatricyclo[7.3.1.0-2, 7~] trideca-2,4-dien-6-one) IUPAC) as stabilizer of CRY proteins. In accordance with the purpose(s) of the invention, as embodied and broadly described herein, the present invention relates to a CRY-binding compound, capable of stabilizing CRYl and CRY2 proteins. In this regard, the invention relates to a compound having the following Formula I: or a pharmaceutically acceptable salt thereof.

In a general context, the present invention is represented by the compound of Formula I for use as a medicament.

In yet another aspect, the invention relates to said compound of Formula I for use of treating and/or preventing a circadian rhythm associated diseases or disorders, including diabetes, wherein the compound is characterized by stabilizing of Cryptochromes (CRYs; CRY 1 and CRY 2).

Moreover, the invention relates to said compound of Formula I for use as a CRY protein stabilizer, for the treatment of type 2 diabetes mellitus. In a further aspect, the present invention relates to a compound (Formula I) that binds to a CRYs and positively modulates CRY activity.

The present invention relates to a novel CRY-binding small molecule that inhibits the degradation of CRYl and CRY2 proteins in the nucleus through the stabilization of the CRYl and CRY2 and thus increases CRYl/2 protein levels significantly in a dose dependent manner.

When Formula I is present, it interacts with CRYs by binding to FAD binding pocket which is the pocket critical for the FBXL3 interaction and the degradation of CRYs by proteasome. Formula I forms p-p interactions with two residues W399 (W417 in CRY2) and W292 (W310 in CRY2). These residues are found in the FAD binding pocket where FBXLs bind for the ubiquitination of CRY. Docking results suggest that W399 amino acid residue in the CRYl and W417 amino acid residue in the CRY2 contribute to stable binding with energy of -10.1 kcal/mol. Upon binding of Formula I to CRY proteins, this interaction reduces the ubiquitination of CRY by FBXL3 and its proteasomal degradation, hence FORMULA I stabilizes the CRY proteins. Formula I exhibits period-lengthening activities by binding to the FAD-biding pocket of CRYl and CRY2 in competition with FBXL3. The compound directly targets the clock proteins, CRYl and CRY2, and lengthens the period of the circadian rhythm in human cell lines. Although period shortening molecules were previously disclosed as CRY destabilizers, GO044, GO200, and G0211 shortened the period while enhancing the CRY stability [15] .

In the present invention it is found that the compound affects period lengthening and amplitude reduction while enhancing the CRY stability.

In one embodiment of the invention, the disclosed compound exhibits selectivity and high affinity for the CRYl and CRY2 proteins. Thus, the interaction is potent. In the present invention the affinity ranges from -9 to -13.5 kcal/mol, more preferably from -10 to -11 kcal/mol.

As used herein, the term "stabilize", "stabilization" or "stabilizing" refers to inhibition of degradation or increased half-life of CRY1 and CRY2 in the nucleus. The term "a therapeutically effective amount" of a compound of the present invention refers to a non-toxic and sufficient amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of the protein activity or protein: protein interaction, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease or disorder, etc.

All of the various embodiments of the present invention as disclosed herein relate to methods of treating and/or preventing various diseases and disorders as described herein. As stated herein the compound used in the method of this invention is capable of stabilizing CRYl and CRY2 proteins. It is known that cryptochromes also affect human health, including diabetes, sleep disorders, and many physiological disorders.

The invention further provides methods for the treatment or prevention of circadian rhythm related disorders and diseases. Non-limiting examples of circadian rhythm disorders include aging, sleep disorders, altered metabolism (metabolic syndromes), obesity, diabetes, mood disorders, cancer and cardiovascular diseases. Mood disorders including major depressive disorder, bipolar I disorder; sleep disorders including circadian rhythm sleep disorders such as shift work sleep disorder, jet lag syndrome, advanced sleep phase syndrome, non-24-hour sleep-wake syndrome, irregular sleep-wake rhythm and delayed sleep phase syndrome. In a preferred embodiment of the invention, the circadian rhythm related disease is diabetes, more preferably type 2 diabetes mellitus.

Previous studies have revealed that glucagon-mediated expression of gluconeogenic genes Pckl and G6pc reduce when CRY 1/2 is either adenovirally overexpressed in mice [16] or stabilized by a small molecule in mouse hepatic cells [5] Moreover, glucose levels were also reduced in these studies. In the present invention qPCR and glucose production assay were performed to test whether the stabilization of CRYl/2 by FORMULA I also represses the glucagon- mediated gluconeogenesis. It is found that the gluconeogenic genes Pckl and G6pc were repressed significantly in a dose dependent manner when Formula I treated cells were induced with glucagon. In line with this observation, treatment of cells with Formula I resulted in significantly decreased glucose production in a dose dependent manner. Overall, Formula I represses glucagon-induced gluconeogenesis in HepG2 cells by stabilizing CRY and hence reducing GPCR (G-protein-coupled receptor)-mediated cAMP levels.

According to the invention, it is found that the compound as CRYs stabilizer is also glucagon-mediated gluconeogenesis repressor. In another embodiment of the invention, the compound is used as novel therapeutic agent for diabetes, especially for the treatment of type 2 diabetes mellitus. The invention can be used for the preparation of a medicament for treatment and/or prevention of diseases or disorders associated with the circadian rhythm by means of the stabilization of cryptochromes. Yet another embodiment of the present invention is a compound defined above for the preparation of a medicament. Moreover, the invention relates to a pharmaceutical composition comprising such compounds, uses and methods of use for such compounds in the treatment and/or prevention of disorders associated with the circadian rhythm. In other embodiment of the present invention, a pharmaceutical composition comprising the compound is useful in the treatment and/or prevention of circadian rhythm related diseases and disorders due the stabilization of CRY1 and CRY2. In a further object, said pharmaceutical composition is for use as a medicament.

The present invention relates to pharmaceutical compositions comprising at least one pharmaceutical carrier and a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

The present invention also relates to pharmaceutical compositions to treat and/or prevent a CRYl/2 -mediated disorders and/or diseases, such as diabetes related to the circadian rhythm.

Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices.

In one aspect, the disclosure relates to a method for the manufacture of a medicament for stabilizing CRY 1/2 in a mammal comprising combining a therapeutically effective amount of a disclosed compound with a pharmaceutically acceptable carrier or diluent.

The present invention provides a method for identifying a compound for stabilizing CRYl/2. The method can be established using systems for pharmaceutical screening that are well known in the art.

In one aspect, the present invention provides a method for identifying a compound that stabilize CRYl/2, wherein the method comprises contacting a compound with CRYl/2 proteins under conditions allowing for the interaction and determining whether the compound leads to increase in CRYl/2 level by using a system that uses a signal and/or a marker generated by the interaction between the compound and CRYl/2 to detect presence or absence or change of the signal and/or the marker. The term "signal" as used herein refers to a substance that can be detected directly by itself based on the physical properties or chemical properties thereof.

These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention. SPECIFIC EMBODIMENTS

In these embodiments, a structure-based design was applied to find small molecules that specifically bind to the CRY proteins. After identifying candidate molecules by virtual screening, experimental studies lead to discover a compound (Formula I, also mentioned as TW68 below) that specifically binds to CRYs. The compound, named hereafter FORMULA I, increases the stability of CRYl/2 and lengthening the period of circadian rhythm in Bmall-dLuc U20S cells. Moreover, Formula I caused the repression of gluconeogenic genes Pckl and G6pc, and lowered the glucose production during glucagon-induced gluconeogenesis in hepatic HepG2 cells. Examples

Example 1 In silico search for compounds that interact with CRY1 and CRY2

Molecular Dynamics Stimulation

The mouse CRY1 (mCRYl) (PDB ID: 4K0R) and mouse CRY2 (mCRY2) (PDB ID: 4I6G) structures were obtained from protein databank. For each structure, using the NAMD (v. 2.6) and VMD (v. 1.9.1) program packages each protein was solvated in a rectangular box with TIP3P water molecules and neutralized with counter ions. Then the system was minimized and heated up to physiological temperature with 10K increments by running lOps simulation at each temperature. CHARMM-PARAM22 force field was used for the molecular dynamics (MD) simulations. After the equilibration of the system, MD simulation was run at 310 ^° K for desired time. Pressure was controlled by Langevin piston method during the simulations. Time step was set to 2fs and the bonded interactions, the van der Waals interactions (12Ά cutoff), long-range electrostatic interactions with particle-mesh Ewald (PME) were included for calculating the total force acting on the system. Last frame of the simulation was used as the “receptor” for the docking simulations.

RMSD calculations were done using VMD RMSD utilities. Backbone atoms (C, CA, and N) of each residue were used for RMSD calculation by excluding their translational motions. The last frame of the simulation was utilized as the receptor for the docking analysis.

Docking Setup

More than 8 million small molecules with non-identified functions were used as ligands for the docking. Molecules were filtered according to the following criteria to eliminate non-relevant molecules: molecules should have less than 7 El- bond donor, less than 12 H-bond acceptor, less than 600 Da molecular weight, logP < 7, less than 8 rotatable bonds, at least 3 aromatic rings, and at least 4 rings. Openbabel, Autodock4.2, Autodock Tools4 [17] and Autodock [18] program packages which are free for academic purposes, were utilized to prepare ligands (small molecules) for the docking. Finally, more than 1 million compounds were docked to target pockets by using the Autodock Vina program. Target pocket for FAD and FBXL3 binding site was determined based on the CRY -FBXL3 crystal structure [14] Autodock Tools4 or PyMol (http://pymol.sourceforge.net/) software were used to visualize the docking results and protein structure, respectively.

Small molecules with highest binding energies were identified by molecular docking studies on the CRYl/2 and FBXL3 interaction site. Molecular docking results of TW68 to CRY1 revealed several residues on FBXL3/21 binding site that interact with TW68. TW68 forms p-p interactions with two residues W399 (W417 in CRY2) and W292 (W310 in CRY2). These residues are found in the FAD binding pocket where FBXLs bind for the ubiquitination of CRY [14, 22;]. Docking results suggest that W399 amino acid residue in the CRYl and W417 amino acid residue in the CRY2 contribute to stable binding with energy of -10.1 kcal/mol. Replacing the amino acid residue to another amino acid should eliminate binding of the CRYs to TW68.

Example 3 Cell Culture

HEK293T, U20S, NIH3T3 and HepG2 cell lines were used for different experiments. The cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% heat-inactivated FBS (Gibco), 100 pg/ml streptomycin and 100 pg/ml penicillin (Gibco). The cell cultures were grown in an 85% humidified incubator at 37°C with 5% C02. To subculture the cells when they reach confluency, first the medium was removed, and the cells were washed with 5mL PBS (137 mM NaCl, 2.7 mM KC1, 10 mM Na2HP04, 1.8 mM KH2P04, pH 7.4). Then, cells were detached by adding lmL Trypsin/EDTA (0.25%:0.02%, PAN Biotech) and resuspended with 4 mL fresh medium. Finally, in a new 10cm plate (NEST Biotechnology), 1 mL of the resuspended cells were mixed with 9 mL fresh medium.

Example 4 Real-Time Bioluminescence Monitoring of Circadian Rhythm

This assay was performed with two different equipments: Synergy HI (BioTek) with 96-well plate and LumiCycle luminometer (Actimetrics) with 35mm plates. Initial screening of molecules to determine their effect on the circadian period was performed in 96-well plate via Synergy HI. Clear 35-mm plates are used in LumiCycle luminometer which provides high resolution data describing the circadian oscillation. For 96-plate tests, 50000 U2-OS Bmall-dLuc cells were seeded on an opaque 96-well plate and cultured overnight. Next day cells were reset by adding dexamethasone (DXM) (0.1 mM final) for 2h. Then medium is changed to bioluminescence recording media which contains the following in 1L: DMEM powder (sigma D-2902, 10X 1L), 0.35gr sodium bi-carbonate (tissue culture grade, sigma S5761), 3.5gr D(+) glucose powder (tissue culture grade, sigma G7021), lOmL 1M HEPES buffer (Gibco 15140-122), 2.5 mL Pen/Strep (lOOug/ml), 50mL 5% FBS and up to 1L sterile milliQ water. Luciferin is added freshly with O.lmM final concentration. Molecules were added to the bioluminescence recording media at desired concentration (0.5% final DMSO concentration). Plates were sealed with optically clear film to prevent evaporation and gas exchange thereby to maintain homeostasis of the cells. Luminescence values were recorded at 320C for each 30 minutes with 15 seconds integration time via Synergy HI luminometer for a week.

For LumiCycle, 400x103 U2-OS Bmall-dLuc or NIH3T3 mPer2-dLuc cells were seeded to 35mm plates and then procedure given above was followed with a change in the last step. Plates were sealed with vacuum grease and placed to LumiCycle. Each plate was recorded continuously every 10 minutes for 70 seconds at 370C via photomultiplier tubes. Period and amplitude data were obtained from LumiCycle Analysis software.

To detect the effect of FORMULA I in the absence of CRYs, Cryl-/-/Cry2-/- mouse embryonic fibroblasts (CRYDKO MEFs) transiently transfected with pGL3-Per2-Luc (luciferase reporter) were used. 3x105 CRYDKO cells were seeded in 35mm clear plates. Next day, cells were transfected with 4000ng pGL3- Per2-Luc via Fugene6 transfection reagent according to the manufacturer’s instruction. In short, 3 : 1 ratio of Fugene6 in mΐ against transfected DNA amount in pg was kept in transfections. 72h after transfection cells were synchronized with DXM for two hours. Then, medium was changed with lumicycle medium having DMSO or molecule, sealed with vacuum grease, and placed to LumiCycle.

Example 5 SDS-PAGE and Western Blotting

U20S cells were seeded on 35mm plates with a density of 3.5x105 cells/plate and incubated at 37°C with 5% C02. After 24 hours, small molecules (final 0.4% DMSO) were added at various doses. The cells were incubated for another 24 hours, and then washed and harvested with ice-cold PBS using a cell scraper (Nest Scientific). Harvested cells were centrifuged at 3000 x g, 4°C for 7 minutes and then supernatant was removed. For cell lysis, RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 0.5% SDS) is prepared with freshly added 1 mM phenylmethylsulfonyl fluoride (PMSF) and IX Protease Inhibitor Cocktail (PIC, Thermo Scientific). While keeping the cells on ice, 60 pL RIPA buffer was added to cell pellets and samples with buffer were pipetted up and down every 5 minutes for 20 minutes. Then, the lysed cells were centrifuged at 13,000 x g, 4°C for 15 minutes to eliminate cell debris. The supernatants were collected, and total protein concentration of the samples were measured using Pierce Protein Assay (Thermo Scientific). The protein samples were mixed with 16.7pL 4X Laemmli Buffer (0.1% bromophenol blue, 8% SDS, 400 mM b-mercaptoethanol, 40% glycerol). Then, the samples were incubated in 95°C for 10 minutes and finally put on ice. For quantification of proteins, the proteins were separated according to their molecular weight using SDS-PAGE gel electrophoresis and then detected by Western blotting with corresponding antibodies. The samples were loaded to 10% SDS-PAGE gel. After the gel was run at 100 mV for 90 minutes, the proteins in the gel were transferred to polyvinylidene difluoride membrane (0.45 pm pore size Hydrophobic PVDF Transfer Membrane, Merck-Millipore) by electrophoresis. The membranes were blocked by incubation with 5% fat free milk powder in TBS-0.015% Tween 20 solution for 1 hour with gentle shaking at room temperature. Following this step, the membranes were incubated with primary antibody for 18 hours with gentle shaking at 4°C and consecutively incubated with secondary antibody for 1 hour with gentle shaking at room temperature. The primary antibodies used for detection of proteins are: Anti- CRY1 (Bethyl), Anti-CRY2 (Bethyl), Anti-PER2 (Bethyl), and Ahΐί-b-ACTIN (Cell Signaling). Anti-rabbit-HRP antibody (Cell Signaling) was used for CRY1, CRY2, and PER2 as the secondary antibody. Anti-mouse-HRP antibody (SantaCruz) was used for b-ACTIN as the secondary antibody. Finally, the proteins were visualized with ECL (Enhanced Chemiluminescence) HRP substrate (Advansta) using ChemiDoc Imaging System (Bio-Rad).

Example 6 RNA Isolation and cDNA Preparation

To investigate the effect of small molecules on expression levels of circadian genes, we utilized U20S cell line whereas, for genes involved in gluconeogenesis we used HepG2 cell line. U20S cells were harvested using the methodology described in Section 3.5. HepG2 cells were treated and harvested as described in Section 3.8. Total RNA isolation from the samples was performed with the NucleoSpin RNA kit (Macherey-Nagel) including DNase treatment. RNA is eluted with 50 pL DNase RNase free water. Using NanoDrop 2000 (Thermo Scientific) the approximate concentration and purity of RNA was measured. The integrity of the eluted RNA was confirmed by agarose gel electrophoresis where 1% agarose gel in prepared using DEPC (diethylpyrocarbonate) treated water.

First step of cDNA synthesis is denaturation and annealing of template RNA and oligo(dT)23VN (New England Biolabs). For this purpose, a mixture of 500 ng of RNA, 2 pL of oligo(dT)23, 1 pL of 10 mM dNTP mix was completed to 10 pL with DNase RNase free water. The mixture was incubated at 65 °C for 5 minutes and consecutively placed on ice (+4°C). The second step is the extension of cDNA where 2 pL from 10X M-MuLV buffer (New England Biolabs), 1 pL from M-MuLV Reverse Transcriptase (200 U/pL), 0.2 pL from RNase Inhibitor (40 U/mI) and 6.8 pL DNase RNase free water were added into the RNA-oligo(dT) mixture in the tube. The samples were kept at 42°C for 1 hour for extension reaction and then at 65 °C for 20 minutes for inactivation of the enzyme. Produced cDNA samples were diluted 1:5 with DNase and RNase free water for storage at - 20°C and serially diluted 1:25 to be used in quantitative PCR reactions.

Example 7 Real Time - Polymerase Chain Reaction (RT-PCR)

Real time - quantitative PCR method was implemented for detecting the effect of small molecules on expression levels of core clock genes and gluconeogenic genes. Primer sequences are listed in Appendix B. The reference gene used in this project was ribosomal protein, large, P0 (RplpO) [202] The reaction mixture consisted of 8 pL SYBR Green (Bioline), 8 pL DNase free water, 1 pL primer mixture (forward and reverse primers: lOpmol each), and 3 pL cDNA obtained as described in Section 3.6. After 4 minutes of polymerase activation at 95°C, the cycling protocol is 8 seconds at 95°C (denaturation), 10 seconds at 60°C (annealing), 20 seconds at 72°C (elongation) using CFX Connect Real-Time PCR detection system (Bio-Rad). The product of qPCR was also analyzed by melt curve (Tm) and agarose gel imaging for checking the quality.

Example 8 Gluconeogenesis Experiments

The effect of small molecules on glucagon-induced gluconeogenesis was determined by comparing the expression of gluconeogenic genes using RT-qPCR and the glucose levels performing glucose production assay. HepG2 cells were seeded on 12-well plates with a density of 2x 105 cells/well in low glucose (1000 mg/L) DMEM with 10% FBS, 100 pg/ml streptomycin and 100 pg/ml penicillin. The medium was supplemented with 2 pg/ml insulin and lOOnM dexamethasone. Cells were incubated for 3 hours, then the medium was exchanged with low glucose DMEM without FBS. Subsequently, the small molecules (final 0.4% DMSO) were added at desired concentrations. After 18 hours, the cells were treated with lOnM glucagon. Cells used in RT-qPCR analysis were harvested after 2 hours with ice-cold PBS using a cell scraper. After 3 hours, the cells used in glucose production assay were washed with warm glucose-free Krebs-Ringer bicarbonate buffer (118.5 mM NaCl, 4.74 mM KC1, 23.4 mM NaHC03, 1.18 mM KH2P04, 1.18 mM MgS02, 2.5 mM CaC12, and 25 mM HEPES - pH 7.6) containing 1% BSA, 100 qg/ml penicillin, 100 qg/ml streptomycin, and 0.29 mg/ml L-glutamine. Then, the cells were incubated in this buffer additionally containing 2 mM sodium pyruvate 20 mM sodium lactate for 4 hours. After the collection of the buffer, the glucose levels were measured using Glucose and Sucrose Assay Kit (Sigma, MAK013).

Example 9 CRY-LUC Degradation Assay

40ng of Cry 1 -Luc, Cry2-Luc, mutant Cryl-dLucplasmids or 5ng Luc plasmid were reverse transfected to 4x104 HEK293T cells on opaque 96-well plate with flat bottom via PEI transfection reagent. 24h after transfection, cells were treated with molecules or solvent (DMSO). 24h of post molecule treatment cells were treated with luciferin (0.4mM final) and HEPES (lOmM final and pH=7.2). After 2h, cycloheximide (20qg/ml final) was added to wells to stop protein synthesis. Plate was sealed with optically clear film and placed to Synergy HE Luminescence readings were recorded every lOmin at 320C for 24 h. Half-life of protein was calculated via one-phase exponential decay fitting function in GraphPad Prism5 software. For each molecule or control at least three replicates were done in each experiment.

Example 10 Statistical Analysis All statistical analysis was performed using GraphPad Prism 5 software. In the comparison of a single dose of the small molecule with control (DMSO), Student’s t-test was employed to calculate significance (p-value). For dose- dependent experiments, as a group all doses were compared, and p-value was calculated by employing one-way ANOVA (analysis of variance) followed by Dunnett's multiple comparisons test. Dunnett's multiple comparisons test compares each dose with the control (DMSO) separately in a pair-wise manner.

Example 11 Synthesis of FORMULA I (Figure 8)

Synthesis of Compound 1 ((lS,5R)-3-((8-methyl-4-oxo-3,4- dihydroquinazolin - 2-yl) methyl)-l,2,3,4, 5, 6-h exahydro-8H- 1, 5-meth anopyrido[ l,2-a][l, 5 ] diazocin-8-one) :

A mixture of 2-(chloromethyl)-8-methylquinazolin-4(3H)-one (0.208 g, lmmol), (-) cytisine (0.380 g, 2mmol) and K2C03 (0.552 g, 4 mmol) in dry DMF (8 mL) was stirred for 24 h at room temperature and then the reaction was terminated by LC-MS control. Reaction mixture was concentrated under pressure. Then it washed with brine solution and dried with Na2S04 and filtrated.

LCMS m/z :[M+H], found 363. C21H22N4O2 requires 362,43 .

Synthesis of Compound 2 (lS,5R)-3-((4-chloro-8-methylquinazolin-2- yl)methyl)-l,2,3,4,5,6-hexahydro-8H-l,5-methanopyrido[l,2-a] [l,5]diazocin-8- one :

Excess amount of POC13 (3 mL) was added dropwise to Compound 1 (0.363 g, lmmol) and the mixture was refluxed for overnight. Reaction mixture was concentrated under pressure. The residue was washed with 10% aqueous NaHC03 solution and dried to obtain compound 2. LCMS m/z :[M+H], found 381. C ₂₁ H ₂₁ C ₁ N ₄ O requires 380,88.

Synthesis of FORMULA I (lS,5R)-3-((4-(benzo[d][l,3]dioxol-5-ylamino)-8- methylquinazolin-2-yl)methyl)-l,2,3,4,5,6-hexahydro-8H-l,5-m ethanopyrido [1,2-a] [l,5]diazocin-8-one:

A mixture of Compund 2 (0.381 g, lmmol), 3,4-(Methylenedioxy)aniline (0.274 g, 2mmol) and K2C03 (0.552 g, 4 mmol) ) in dry DMF (8 mL) was stirred for 24 h at room temperature and then the reaction was terminated by LC-MS control. Reaction mixture was concentrated under pressure. Then it washed with brine solution and dried with Na2S04 and fdtrated. Then the solvent was evaporated in a rotary evaporator. LCMS m/z :[M+H], found ? C28H27N5O3 requires 481,56

Results

Identification of the Molecule Effect on the Half-Life of the CRYl/2

The stabilization and degradation mechanism of CRY by ubiquitination is an important factor affecting the pace of the mammalian circadian clock. Therefore, we focused on CRY and FBXL3/21 interaction sites for the identification of small molecules stabilizing CRYs. Small molecules with highest binding energies were identified by molecular docking studies on the CRY 1/2 and FBXL3/21 interaction sites. Initially, the mouse CRY1 (mCRYl) structure (PDB ID: 4K0R) was subjected to molecular simulation was performed on CRY1 to bring the structure near their physiological conditions as follow: After structure of CRY1 was solvated in a rectangular box and neutralized with counter ions, the system was minimized, heated up to physiological temperature (310°K) and simulated for 10ns. Root mean square deviation (RMSD) of backbone atoms showed convergence after initial increase upon minimization. The last frame of the simulation was utilized as the receptor for the docking analysis. Commercially available small molecule libraries with non-identified function were used as ligands. AutoDock Vina was used to screen approximately 2 million commercially available small molecules filtered by “Lipinski’s Rule of Five”. A final number of 32 compounds with affinities ranging from -9 to -13.5 kcal/mol were then tested experimentally.

The toxicity of these top 32 molecules were evaluated on cells prior to in vitro screening using U2-OS cells by MTT at the dose of 20 mM. The small molecule doses yielding more than 90% cell viability were selected for further characterization. The 20 molecules were identified to be nontoxic at given concentration and selected for further characterization.

Effect of FORMULA I on Half-life of Crytpochrome 1 and 2

The effect of the TW68 on CRY1 and CRY2 half-life was assessed using CRY1- dLuc and CRY2-dLuc as dose dependent manner. Plasmids, contains Cryl and Cry2 genes fused with destabilized Luciferase genes, were transfected to HEK 293T cells. The cells treated with TW68 and cycloheximide inhibit protein synthesis. The samples were incubated for 16 hours in Synergy to be able to calculate half-life of luciferases based on bioluminance decay. Analysis of the results indicated that TW68 increased the half-life of CRYs in dose dependent manner.

Effect of FORMULA I on Circadian Rhythm

The effect of the TW68 on circadian clock was further investigated using U2-OS and NIH 3T3 cells stably expressing dLuc reporter, under the Bmall and Per2 promoters, respectively. Cells treated with TW68 had longer periods compared to control. TW68 lengthens period about 3 hours in U2-OS and about 2.6 hours in NIH 3T3 cells. Further analysis of amplitude in both cell lines in the presence of the TW68 revealed that the molecules also reduced the amplitude.

Effect of FORMULA I on Protein Levels

To assess the effect of the TW68 at biochemical level U2-OS cells treated with molecules. After preparation of cell lysate from U2-OS cells treated with molecule and DMSO, samples were subjected to the SDS-PAGE followed by Western blot analysis using anti-CRYl, anti-CRY2 and anti-PER2. Analysis of the result indicated that both CRY1 and CRY2 levels were higher in the cells in dose dependent manner compared to cells treated with DMSO control. This result suggested that TW68 stabilized both CRYs, which is in agreement with Cryl- dLuc and Cry2-dLuc assay.

Effect of FORMULA I on ntRNA Levels

The effect TW68 on transcription of clock output genes using U2-OS cells. U20S cells were treated with TW68 and DMSO and levels of clock genes transcripts were determined by RT-qPCR. The mRNA levels measured genes were normalized with respect to RplpO gene expression because its expression level is independent from circadian genes. In agreement with stabilization of CRYs proteins, TW68 treatment resulted in significant decrease of Cryl, Cry2, Per2, Dbp and Rev-erba expression in a dose-dependent manner. Furthermore, there was no significant effect of TW68 treatment on Bmall and Clock expression. These results are consistent with the effects of CRYl and CRY2 stabilization since CRY-PER heterodimer represses the expression of E-box regulated circadian genes.

Effects of FORMULA I on Gluconeogenesis

It has been shown that stabilization of CRY s by stabilizer molecule would reduce glucagon-mediated gluconeogenesis via its interaction with G _s a subunit of heterotrimeric G-protein (16). In order to test the effects of TW68 on glucagon- induced gluconeogenesis, HepG2 cells treated with TW68 and the transcriptional level of the glucagon-induced Pckl and G6pc genes, key genes in gluconeogenesis, were measured. Pckl and G6pc significantly reduced upon exposure TW68. We expected that glucose production in this cell line would reduce upon treatment of TW68. We, therefore, measured glucose level secreted from this cell line and results indicated that glucose levels were decreased dose- dependently. Overall, these results indicate that TW68 acts by stabilizing CRYs and subsequently reducing glucagon-mediated gluconeogenesis.

Effect of FORMULA I in Cryl-/-/Cry2-/~ Mouse Embryonic Fibroblast Cells To eliminate the possibility of TW68 might affect other proteins and, in turn, circadian rhythmicity we tested the effect of these molecule in CryP ^/ Cry2 ^/ mouse embryonic fibroblast cells (DKO-MEF), generated by Udea group (21), transfected with mPer2-dluc plasmid. Results indicated that TW68 had no effect on luminance in dose dependent manner in the absence of the CRY s and suggest that effect of TW68 was through CRYs.

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