www.sciencemag.org/cgi/content/full/science.1223710/DC1

Supplementary Materials for

Identification of Small Molecule Activators of Cryptochrome

Tsuyoshi Hirota, Jae Wook Lee, Peter C. St. John, Mariko Sawa, Keiko Iwaisako, Takako Noguchi, Pagkapol Y. Pongsawakul, Tim Sonntag, David K. Welsh,

David A. Brenner, Francis J. Doyle, III, Peter G. Schultz,* Steve A. Kay*

*To whom correspondence should be addressed.

E-mail: skay@ucsd.edu (S.A.K.); schultz@scripps.edu (P.G.S.)

Published 12 July 2012 on Science Express

DOI: 10.1126/science.1223710

This PDF file includes:

Materials and Methods Figs. S1 to S18 Tables S1 to S4 References

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Materials and Methods Compound screening and cell-based circadian assays

Approximately 60,000 uncharacterized compounds corresponding to diverse chemical scaffolds (26, 27) were screened at a final concentration of 7 �M by using a high-throughput circadian assay system as described previously (7). In brief, stable U2OS reporter cells harboring Bmal1-dLuc were suspended in the culture medium [DMEM (11995-073, Gibco) supplemented with 10% fetal bovine serum, 0.29 mg/ml L-glutamine, 100 units/ml penicillin, and 100 �g/ml streptomycin] and plated onto 384-well white solid-bottom plates at 20 �l (2,000 cells) per well. After 2 days, 50 �l of the explant medium [DMEM (12800-017, Gibco) supplemented with 2% B27 (Gibco), 10 mM HEPES, 0.38 mg/ml sodium bicarbonate, 0.29 mg/ml L-glutamine, 100 units/ml penicillin, 100 �g/ml streptomycin, 0.1 mg/ml gentamicin, and 1 mM luciferin, pH 7.2] was dispensed to each well, followed by the application of 500 nl of compounds (dissolved in DMSO; final 0.7% DMSO). The plate was covered with an optically clear film and set to luminescence monitoring system equipped with a CCD imager (ViewLux, Perkin Elmer). The luminescence was recorded every 2 h for 4 days. In follow up studies, the luminescence rhythms of Bmal1-dLuc and Per2-dLuc reporter U2OS cells (22) were recorded every 100 min for 5 days with a microplate reader (Infinite M200, Tecan).

NIH-3T3 cells were plated on 35-mm dishes, cultured for 1 day to reach 80-90% confluent and transfected by PolyFect (Qiagen) with 0.5 �g luciferase reporter plasmid. After 1 day, dexamethasone (final 100 nM) was added to the medium. After 2 h, the medium was replaced with the explant medium containing various concentrations of KL001 (final 0.2% DMSO), and the luminescence was recorded every 10 min for 5 days with a luminometer (LumiCycle, Actimetrics). Reporter plasmids pGL3Basic-E2 and pGL3Basic-mutE2 (18) were from Dr. Joseph S. Takahashi (University of Texas Southwestern Medical Center).

Explants of SCN and lung were dissected from mPer2Luc knockin mice and cultured as described previously (28) with modifications. EML [DMEM (90-013, Mediatech) supplemented with 2% B27, 10 mM HEPES, 0.35 mg/ml sodium bicarbonate, 0.58 mg/ml L-glutamine, 50 units/ml penicillin, 50 �g/ml streptomycin, and 1 mM luciferin, pH 7.4] and the explant medium were used for SCN and lung, respectively. The luminescence was recorded with LumiCycle. The medium was changed every 120 h with increasing concentration of KL001 each time (from 0 to 24 �M, final 0.2% DMSO).

Fibroblasts derived from tails of mPer2Luc knockin mice (28, 29) were plated on 35-mm dishes and cultured for 2 days to reach confluency. Then, the medium was replaced with the explant medium containing various concentrations of KL001 (final 0.2% DMSO), and the luminescence was recorded with LumiCycle. When indicated, the cells were treated with dexamethasone (final 100 nM) for 2 h before the medium replacement.

Knockdown studies using Per2-dLuc U2OS cells were performed as described previously (22) with modifications. The cells were reverse transfected on white solid-bottom 96-well plates by Lipofectamine 2000 (Invitrogen) with total 3 pmol siRNA [control si (D-001810-10, Dharmacon), CRY1 si (CRY1HSS102308, Invitrogen), CRY2 si

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(CRY2HSS102311, Invitrogen) or FBXL3 si (GS26224, Qiagen)]. After overnight incubation, the medium was replaced with the fresh culture medium. After 2 days, the medium was replaced with 180 �l of the explant medium, followed by the addition of 1.3 �l KL001 (final 0.7% DMSO). The luminescence was recorded every 36 min for 5 days with Infinite M200.

The period parameter was obtained from the luminescence rhythm by curve fitting program MultiCycle or LumiCycle (Actimetrics). The luminescence intensity parameter was calculated by averaging the intensity during the experiment. The first day data were excluded from the analysis because of transient luminescence changes upon the medium change. For CRY1 si-treated Per2-dLuc U2OS cells that showed unusual baseline pattern, 24-h moving average instead of polynomial curve was used for baseline subtraction to obtain the period parameter.

In vitro kinase assay

Effects of compounds on CKI�, CKI� and CK2 activities in vitro were analyzed as described previously (9, 10).

Pull-down assay

Compound-interacting proteins were affinity purified as described previously (9) with modifications. Unsynchronized U2OS cells kept in confluence (2 × 108 cells) were homogenized by using Dounce homogenizer in 5 ml of lysis buffer 1 [25 mM MOPS, 15 mM EGTA, 15 mM MgCl2, 1 mM DTT, 60 mM �-glycerophosphate, 15 mM p-nitrophenyl phosphate, 1 mM Na3VO4, 1 mM NaF, 1 mM phenyl phosphate, 10 �g/ml soybean trypsin inhibitor, 100 �M benzamidine, Complete Protease Inhibitor Cocktail (Roche); pH7.2]. After sonication, the homogenate was supplemented with NP-40 (final 0.5%) and incubated on ice for 5 min, followed by centrifugation (16,000 × g) at 4°C for 20 min. The resulting supernatant was split into three, and each portion was incubated with 0, 20 or 50 �M KL001 (final 0.5% DMSO) at 4°C for 30 min with rotation. Then, 120 �l of agarose-conjugated KL001-linker [50% slurry in bead buffer 1 (50 mM Tris, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% NP-40, 5 mM NaF, 10 �g/ml soybean trypsin inhibitor, 100 �M benzamidine, Complete Protease Inhibitor Cocktail; pH7.4)] was added to the mixture and incubated at 4°C for 1 h with rotation. The agarose beads were washed six times with 1 ml of the bead buffer 1 and boiled with SDS sample buffer to elute bound proteins.

For target identification, the proteins were separated by SDS-PAGE (4-12% gradient gel, Invitrogen) and stained with CBB. The gel lane for each condition was cut horizontally into 24 pieces and subjected to protein mass spectrometry analysis as described previously (9).

Lysates of HEK293T cells transiently overexpressing clock proteins (see below) were diluted with lysis buffer 2 [25 mM MOPS, 15 mM EGTA, 15 mM MgCl2, 0.1% NP-40, 1 mM DTT, Complete Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 1 and 3 (Sigma); pH7.2] and subjected to pull-down assay. Bead buffer 2 (50 mM Tris, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% NP-40, Complete Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 1 and 3; pH7.4) was used to wash the agarose beads.

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Transient transfection for protein expression in HEK293T cells HEK293T cells (2.5 × 106 cells) were reverse transfected on 6-well plates with 2 �g

expression vector by Lipofectamine 2000. After 28 h, the cells were collected in ice-cold PBS and resuspended in 100 �l of incubation buffer (50 mM Tris, 50 mM NaCl, 2 mM EDTA, 10% glycerol, 1 mM DTT, Complete Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 1 and 3; pH8.0). The mixture was supplemented with NP-40 (final 1%) and incubated on ice for 15 min, followed by centrifugation (16,000 × g) at 4°C for 10 min. The supernatant was used for assays. Expression vectors for C-terminally 3XFlag-tagged mCRY1 and its mutants, mCRY2, mPER1, mPER2 and mBMAL1 were based on p3XFLAG-CMV-14 (Sigma). For C-terminally 3XFlag-tagged mCLOCK, pcDNA3-intron, which contains the �-globin gene intron [from Dr. Xuan Zhao (The Salk Institute for Biological Studies)], was used because of low expression with p3XFLAG-CMV-14 (9).

Protein immunoblot

Protein immunoblot analyses were performed as described previously (9). Anti-mCRY1 and mCRY2 antibodies (5) were from Dr. Katja A. Lamia (The Scripps Research Institute). Other antibodies are as follows: anti-hPER1 (KAL-KI044, Cosmo Bio), anti-CLOCK (PA1-520, Affinity BioReagents), anti-�-actin (MAB1501, Millipore), anti-lamin A/C (sc-20681, Santa Cruz Biotechnology), anti-Flag (F1804, Sigma), anti-HA (12013819001, Roche), anti-MBP (E8032S, New England Biolabs) and anti-penta-His (Qiagen). A nonspecific band detected by the anti-CLOCK antibody was described previously (30).

Preparation of purified CRY1 expressed in mammalian cells

HEK293T cells (2.0 × 107 cells) were reverse transfected on 15-cm dish with 32 �g of the expression vector for C-terminally 3XFlag-tagged mCRY1. After 48 h, the cell lysate was prepared as described above with 1.5 ml of the incubation buffer. Prior to purification, 100 �g of the anti-Flag antibody was cross-linked with 600 �l of Dynabeads Protein G (100-09D, Invitrogen). The cell lysate (250 �l) was subjected to immunoprecipitation with 45 �l of anti-Flag antibody cross-linked with Dynabeads Protein G for 1 h at 4°C. After wash with wash buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% TritonX-100, 1 mM DTT, 10 mM MG132, Complete Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 1 and 3; pH8.0) for five times, CRY1-Flag protein was eluted with the wash buffer supplemented with 500 �g/ml 3XFlag peptide (F4799, Sigma) twice for 15 min at 4°C and twice for 15 min at 30°C. The 30°C elution fractions were pooled and used for the pull-down assay.

Heterologous CRY expression and protein purification in E. coli

The coding region of mCry1 was amplified from the plasmid mCry1/p3XFLAG-CMV-14 using the primers (ATTAA GCTTG CGGCC GCCAC CATG and ATACT CGAGT TAGTT ACTGC TCTGC CGCTG GACTT TG) and cloned via NcoI and XhoI into pTSKay22 (His6-MBP-TEVsite-MCS) resulting in the expression plasmid pTSKay24 (His6-MBP-TEVsite-mCRY1). To generate pTSKay22, the maltose-binding protein (MBP)

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gene followed by the Tobacco etch virus (TEV) cleavage site was PCR amplified from the plasmid pTS48 (31) with the primers (ATACA TATGG CCCAT CACCA TCACC ATCAC GGATC TAAAA TCGAA GAAGG TAAAC TGG and TATCC ATGGT GCCCT GAAAA TAAAG ATTCT CG) and cloned into pET42b (Novagen) via NdeI and NcoI. For the protein expression, the E. coli B21-Gold (DE3) strain (Stratagene) was used. After transformation by pTSKay24, the cells were grown in LB medium supplemented with 50 mg/ml kanamycin and 150 �g/ml riboflavin at 37°C until reaching an OD600 of 0.6. The cultures were shifted to 22°C and grown for ~30 min before 100 nM isopropyl 1-thio-�-D-galactopyranosid was added. The expression was performed in the dark and lasted overnight (~15-17 h). Subsequently the cells were harvested by centrifugation, resuspended in Ni-NTA buffer A (50 mM Tris, 300 mM NaCl; pH 8.0) and lysed by using a French Press. The lysate was centrifuged at 30,000 × g and the soluble fraction was subjected to affinity chromatography using Ni-NTA Agarose (QIAGEN). The protein was eluted by using Ni-NTA buffer B (50 mM Tris, 300 mM NaCl, 250 mM imidazole; pH 8.0). After dialysis of the elution fractions against buffer 1 (50 mM Tris, 1 mM EDTA, 1mM DTT; pH 8.3), they were subjected to anion exchange chromatography. Proteins were bound by a HiTrap Q HP anion-exchange column (5 ml bed volume, GE Healthcare) and eluted with buffer 2 (50 mM Tris, 1 mM EDTA, 1mM DTT, 500 mM NaCl; pH 8.3). A single fraction that did not contain bacterial chaperone proteins was dialyzed against assay buffer (50 mM Tris, 150 mM NaCl, 1mM DTT; pH 8.0) and further concentrated. For TEV protease digestion, the fraction was incubated with 10-fold excess of purified TEV protease overnight in the assay buffer at room temperature.

RT-qPCR

RT-qPCR analyses were performed as described previously (22). CFX384 Real-Time PCR Detection System (Bio-Rad) was used for qPCR. The primers are listed in (22) and Table S2.

Protein degradation assay

For protein degradation assay in transiently transfected HEK293T cells, 6.0 × 104 cells were reverse transfected on 96-well white solid-bottom plates by Lipofectamine 2000 with 40 ng of expression vector for C-terminally luciferase-fused CRY1 (CRY1-LUC) or its D387N mutant (in p3XFLAG-CMV-14). For luciferase (LUC), 6 ng of expression vector with 34 ng of empty vector (p3XFLAG-CMV-14) was used because of its efficient expression. After 24h, 500 nl of the compound (final 0.5% DMSO) was added to the medium. After 24 h, the medium was supplemented with luciferin (final 1 mM) and HEPES-NaOH (pH7.2; final 10 mM). After 1 h, cycloheximide (final 20 �g/ml) was added, and the luminescence was recorded every 10 min for 18 h with Infinite M200. Half-life was obtained by one phase exponential decay fitting with Prism software (GraphPad Software).

HEK293 cells were transiently transfected by Lipofectamine 2000 with expression vectors for CRY1-LUC, CRY2-LUC or LUC (in p3XFLAG-CMV-14, which contains the neomycin resistance gene) and treated with 600 �g/ml G418 to establish stable cell lines. The stable cell lines (1.0 × 104 cells) were plated onto 384-well white solid-bottom plates at

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50 �l per well. After 24 h, 500 nl of the compound (final 1% DMSO) was applied to the medium. After 24 h, protein half-life was determined (see above).

In vitro ubiquitination assay

HEK293T cells were reverse transfected with expression vector for C-terminally 3XFlag-tagged mCRY1 or HA-ubiquitin (Plasmid 18712, Addgene) (32), and cell lysates were prepared as described above. Cell lysate for CRY1-Flag was incubated with 0 or 50 �M KL001 for 30 min at 4°C with rotation. In vitro ubiquitination was performed at 30°C in a reaction mixture (total 100 �l) containing 50 mM Tris (pH7.6), 5 mM MgCl2, 0.6 mM DTT, 2 mM ATP, 3 �l of HA-ubiquitin lysate and 5 �l of CRY1-Flag lysate. The reaction was terminated by adding 2 mM EDTA (pH8.0) and NP-40 (final 1%) at each time point. All samples were subjected to immunoprecipitation with 10 �l of anti-Flag antibody cross-linked with Dynabeads Protein G for 1 h at 4°C with rotation. After washing with the incubation buffer supplemented with 10 �M MG132 and 1% NP-40 for four times, proteins were eluted with SDS sample buffer and subjected to protein immunoblot analysis.

Preparation of nuclear fraction

Unsynchronized U2OS cells kept in confluence on a 10-cm dish were treated with the compound for 48 h, collected in ice-cold PBS and resuspended in 300 �l of buffer A (10 mM HEPES-NaOH, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, Complete Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 1 and 3; pH 7.8). The mixture was supplemented with NP-40 (final 0.02%) and incubated on ice for 2 min, followed by centrifugation (700 × g) at 4°C for 5 min. The nuclear pellet was washed with buffer A, resuspended in 60 �l of buffer C [20 mM HEPES-NaOH, 0.4 M NaCl, 5 mM MgCl2, 1 mM EDTA, 2% (v/v) glycerol, 1 mM DTT, Complete Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 1 and 3; pH 7.8], and rotated at 4 °C for 1 h. The mixture was centrifuged at 20,000 × g at 4°C for 30 min, and the supernatant was collected as the nuclear fraction. Protein concentration was determined by the Lowry method.

Primary hepatocyte culture

Primary hepatocytes were prepared at ZT9-11 from fed mice (8 to 12-week old C57BL/6, male) by collagenase perfusion as described previously (33). The cells suspended in the medium [Medium199 (11150-059, Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 100 nM dexamethasone and 2 �g/ml insulin] were plated onto 12-well plates at 2 × 105 cells (in 1 ml) per well. After 3 h, the medium was replaced with 1 ml glucose-free Krebs-Ringer bicarbonate buffer (gfKRB; 118.5 mM NaCl, 4.74 mM KCl, 1.18 mM KH2PO4, 23.4 mM NaHCO3, 2.5 mM CaCl2, 1.18 mM MgSO2, and 25 mM HEPES; pH7.6) supplemented with 0.29 mg/ml L-glutamine, 100 units/ml penicillin, and 100 �g/ml streptomycin. Then 2.4 �l KL001 (final 0.24% DMSO) was applied. After 18 h, glucagon (final 10 nM) was added to the buffer. For RT-qPCR analysis, the cells were collected after 2 h. For glucose production assay, after 3 h, the cells were washed 3 times with warm gfKRB supplemented with 1% BSA and incubated with 0.5 ml gfKRB supplemented with 1% BSA, 20 mM sodium lactate, and 2 mM sodium pyruvate. After 4 h,

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the buffer was collected and subjected to glucose measurement by using Glucose assay kit (BioVision).

Mathematical modeling

Overall design of the mathematical model. We constructed a simple mathematical model of the PER-CRY negative feedback loop, focusing on the roles of CRY1 and CRY2 in determining the period. The model consists of only eight state variables (Fig. 4A): three mRNA species (Per, Cry1 and Cry2), three protein species (PER, CRY1 and CRY2) and two protein complexes (PER-CRY1 and PER-CRY2). The differential equations for each state were formulated by using standard Hill-type repression, Michaelis-Menten and mass action kinetics (Table S3), with the following assumptions: (i) PER and CRY bind and enter the nucleus in a 1:1 stoichiometric ratio through the formation of a PER-CRY complex (34), (ii) PER is present in lower quantities and thus the stoichiometric limiting factor in the formation of the PER-CRY complex (35, 36), (iii) CRY degradation rates play an important role in determining the period length (19, 20, 23), (iv) CRY1 is more potent than CRY2 in repressing CLOCK-BMAL1-dependent transcriptional activation (37) and (v) knockout of Cry1 and Cry2 evokes opposite period changes, i.e., period shortening and lengthening, respectively (38, 39). For the full list of model assumptions, see “Construction of the rate equations” below. The 21 unknown kinetic parameters were found (Table S4) by fitting the stoichiometric data from Lee et al. (35), requiring correct period changes for the Cry1 and Cry2 knockouts (see section below, “Solution of the mathematical model”). The time course plots of the state variables display reasonable phases and amplitudes with a period of 23.7 h (Fig. S15A).

Construction of the rate equations for the mathematical model. Rate equations are shown in Table S3. To derive these equations, some additional assumptions were made about the kinetics of CRY1 and CRY2:

� CLOCK and BMAL1 are considered to be constitutively expressed, and their activity is represented by the vtxn parameters.

� Repression of CLOCK-BMAL1 activity is attained through Hill-type inhibition. The Hill coefficient is fixed at 3, which was found to provide sufficient nonlinearity for oscillations. See below for the derivation of the repressor input function used in Tables S3A and S3B.

� The degradation kinetics of the mRNA species and cytoplasmic proteins are assumed to be standard Michaelis-Menten terms.

� Nuclear protein species are known to exist in both a PER-CRY complex and as free proteins. We assume this equilibrium is fast, and only consider one state variable for the total nuclear CRY1 and nuclear CRY2 states. Nuclear PER is not explicitly considered, but is assumed to be associated with the nuclear CRY states.

� The degradation kinetics of the nuclear complexes are assumed to follow Michaelis-Menten kinetics. However, because both CRY1n and CRY2n are thought to be degraded by the same pathway in the nucleus, kinetic equations using the pseudo steady-state hypothesis were derived for two substrates sharing the same enzyme. As a result, each CRY isoform acts as an inhibitor to the other’s

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degradation. To find the rate equations associated with a shared-enzyme degradation, we derive them from the equilibrium relationships:

[E]� [S1] k1

k�1 � [ES1]

kd,1 [E]

[E]� [S2] k2

k�2 � [ES2]

kd,2 [E]

[E t ]� [E]� [ES1]� [ES2] We are interested in solving for the degradation rates of the two enzyme complexes, namely:

rd ,1 � kd ,1 [ES1]rd ,2 � kd ,2 [ES2]

We invoke the standard pseudo steady-state assumption and set d[ES]dt

� 0 , and obtain the

following production = consumption equalities for [ES1]: k1[E][S]� k-1[ES1]� kd,1[ES1]

Simplifying the equations and defining a combined constant K1 k�1 � kd ,1

k1, we obtain the

following kinetic equation:

rd ,1 �kd ,1[E]t[S1]

K1 � [S1]�K1K2

[S2]

With an equivalent analysis for S2, and setting K1 = K2, we obtain the rate equations used in Table S3A (degradation-based model). To derive the equations for the Hill-type inhibition, allowing for different repressive activities (Table S3B, activation-based model), we start with the standard equation for Hill-type regulation.

vmax[A]n

Kmn � [A]n

Allowing for competitive inhibition with two inhibitors, Km is replaced with the apparent Michaelis-Menten constant

Kmapp � Km 1�

[I1]Ki ,1

�[I2 ]Ki,2

�

���

��

By assuming constitutive activator concentrations and non-dimensionalizing ,

we obtain

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Assuming equal repressive activity (m = 1), we obtain the rate equation used in the degradation-based model (Table S3A).

Solution of the mathematical model. The model equations, specifying the state and parameter dependent time derivatives of each concentration variable, were written in python using the CasADi computer algebra package (40). The model was simulated using the SUNDIALS suite of ODE solvers (41). A parameter-dependent cost function was developed which assigns numerical values reflecting how well a parameter set fits desired features (taken from (35)). The first step in the cost function evaluation is the numerical solution of the limit cycle (42). If a limit cycle was unable to be found, the cost function returns a maximum value. Otherwise, the cost function returns a squared difference from the desired value. A priority weight was also attached to each cost entry, such that more important costs would be prioritized. A description of each entry in the cost function (Weight), along with the value for experimental (Desired, from (22, 35, 38)) and final model (Deg model, degradation-based model; Act model, activation-based model) is shown in the table below.

# Description Weight Desired Deg model Act

model

1 per mRNA peak-trough ratio ymax (Per)ymin (Per)

0.5 >20 large large

2 Cry1 mRNA peak-trough ratio ymax (Cry1)ymin (Cry1)

0.5 2.155 8.942 3.748

3 Cry2 mRNA peak-trough ratio ymax (Cry2)ymin (Cry2)

0.5 2.236 7.813 3.484

4 PER protein peak to trough ratio ymax (PER)ymin (PER)

5 >20 large large

5 CRY1 protein peak-trough ratio ymax (CRY1)ymin (CRY1)

3 3.247 6.385 1.847

6 CRY2 protein peak-trough ratio ymax(CRY2)ymin (CRY2)

3 1.975 8.094 2.347

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7 Fraction PER of total protein

ymax (PER)ymax (PER)� ymax (CRY1)� ymax(CRY2)

3 0.105 0.169 0.073

8 Fraction CRY1 of total protein

ymax (CRY1)ymax (PER)� ymax (CRY1)� ymax(CRY2)

3 0.555 0.473 0.554

9 Fraction CRY2 of total protein

ymax (CRY2)ymax (PER)� ymax (CRY1)� ymax(CRY2)

3 0.341 0.358 0.373

10 Cry1 siRNA sensitivity

�T�vdeg,CRY1

5 0 >0

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shown in Table S3B), possibly because of the simple structure of the model. Instead, we investigated whether the difference in potency between CRY1 and CRY2 could be explained through a difference in nuclear degradation rates. To this end, we allowed the degradation rates of nuclear CRY1 and CRY2 to differ, while holding their inhibition constants equal (degradation-based model, Table S3A). Using this configuration, the structure was able to fit the experimental sensitivities. In the optimized parameter set, the degradation rate of nuclear CRY2 is higher than that of CRY1, such that for a constant total nuclear CRY level, higher fractions of CRY2 cause a faster clearance of the repressive complexes. Since PER is the limiting factor in nuclear entry, the amount of total CRY that enters the nucleus is largely insensitive to perturbations in cytosolic CRY level. In effect, the two isoforms of CRY must compete for the available PER, and thus the nuclear CRY1/CRY2 levels are constrained with a ratio proportional to their relative expression level. The period of our in silico clock model is thus governed by the nuclear CRY1/CRY2 ratio, where higher amounts of CRY1 shift the clock closer to the Cry2 knockout phenotype, and higher amounts of CRY2 shift the clock closer to the Cry1 knockout phenotype.

Generation of testable hypotheses. The finished mathematical model was then used to investigate potential mechanisms of KL001 action. Since KL001 stabilized CRY, equal stabilization of CRY1 and CRY2 was tested on both the cytoplasmic and nuclear state variables by decreasing the relevant degradation parameters. Parameter-dependent period and state profiles (Fig. S15D) were found by solving for the steady state limit cycles at each of 100 points. Only through nuclear stabilization was the model able to reproduce the experimental period lengthening result.

Compound synthesis

General. All chemicals and solvents were obtained from commercial suppliers (Acros, Alfa Aesar and Aldrich) and used without further purification. Unless otherwise indicated, all reactions were run under argon gas. Anhydrous solvents were obtained by passage through an activated alumina column. 1H and 13C NMR spectra were recorded on a Bruker 500 MHz spectrometer. Chemical shifts are reported relative to internal CDCl3 (Me4Si, � 0.0), DMSO-d6 (� 2.50 for 1H) and CD3OD (Me4Si, � 0.0). All compounds were identified by LC-MS from Agilent Technology, using a C18 column (20 � 4.0 mm), with 3.5 minutes elution using a gradient solution of CH3CN-H2O (containing 0.05% trifluoroacetic acid), with UV detector and an electrospray ionization source.

Scheme S1. The synthesis of KL001.

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The synthesis of N-(furan-2-ylmethyl)methanesulfonamide (2). To a solution of furfuryl amine (500 mg, 5.15 mmol) in Pyridine (4 ml) was slowly added methansulfonyl chloride (760 mg, 6.17 mmol, 500 �l) at 0°C. After stirring at room temperature overnight, the reaction mixture was extracted with dichloromethane (200 ml) and water (100 ml). Organic layer was washed with 5% HCl (100 ml, X2) and dried with MgSO4. The organic layer was evaporated under reduced pressure. Dark yellow color oily compound (767.6 mg) was obtained and used without further purification.

The synthesis of N-(furan-2-ylmethyl)-N-(oxiran-2-ylmethyl)methanesulfonamide (3). To a solution of N-(furan-2-ylmethyl)methanesulfonamide (500 mg, 2.85 mmol) in anhydrous DMF (10 ml) was slowly added 95% NaH (85 mg,3.42 mmol) at 0°C. After stirring for 30 min at room temperature, epi-bromohydrin (465 mg, 290.7 �l) was slowly added into the reaction mixture at 0°C. After heating at 50°C overnight, the reaction mixture was extracted with ethyl acetate (200 ml) and water (100 ml). Organic layer was washed with brain solution (100 ml, �2) and dried with MgSO4. The organic layer was evaporated under reduced pressure. Dark yellow color oily compound (767.6 mg) was obtained and used without further purification.

The synthesis of N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)-N-(furan-2-ylmethyl)methanesulfonamide (KL001). To a solution of carbazole (71.8 mg, 0.43 mmol) in anhydrous DMF (10 ml) was slowly added 95% NaH (12.5 mg, 0.5 mmol) at 0°C. After stirring for 30 min at room temperature, N-(furan-2-ylmethyl)-N-(oxiran-2-ylmethyl)methanesulfonamide (3) (100 mg, 0.43 mmol) in DMF (4 ml) was slowly added to the reaction mixture at 0°C. After heating at 50°C overnight, the reaction mixture was extracted with ethyl acetate (100 ml) and water (50 ml). Organic layer was washed with brain solution (100 ml, �2) and dried with MgSO4. The organic layer was evaporated under reduced pressure. Crude compound was purified by column chromatography with ethyl acetate/hexane to afford compound. 1H-NMR (DMSO-d6): 2.90 ppm (3H, CH3, s), 3.20 ppm (1H, m), 3.34 ppm (1H, m), 4.10 ppm (1H, br s), 4.18 ppm (1H, m), 4.33 ppm (1H, m), 4.44 ppm (2H, m), 5.30 ppm (1H, OH, br s), 6.30 ppm (1H, d, J=3 Hz), 6.37 ppm (1H, t, J=3 Hz), 7.18 ppm (2H, t, J=7.5 Hz), 7.44 ppm (2H, t, J=7.5 Hz), 7.51 ppm (2H, d, J=8.5 Hz), 7.57 ppm (1H, m), 8.13 ppm (2H, d, J=8.5 Hz)

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Scheme S2. The synthesis of KL001-linker.

The synthesis of 9-(oxiran-2-ylmethyl)-9H-carbazole (4). To a solution of carbazole (10 g, 0.059 mol) in DMF (400 ml) was slowly added NaH (1.77 g, 0.07 mol, 1.2 equiv.) at 0°C. The reaction mixture was stirred for 30 min at room temperature. epi-chlorohydrin (8.0 g, 0.059 mol, 6.8 ml) was added to reaction mixture at 0°C. After stirring at 4°C overnight, the reaction mixture was extracted with ethyl acetate (500 ml) and water (200 ml). Organic layer was washed with brain solution (200 ml, �3) and dried with MgSO4. The organic layer was evaporated under reduced pressure. Crude compound was purified by column chromatography with ethyl acetate/hexane=1:1 to afford white color solid.

The synthesis of 1-(9H-carbazol-9-yl)-3-((furan-2-ylmethyl)amino)propan-2-ol (5). To a solution of 9-(oxiran-2-ylmethyl)-9H-carbazole (284 mg, 1.26 mol) was added furfuryl amine (1.2 g, 12.6 mol, 1.2 ml, 10 equiv.). After stirring at room temperature overnight, the reaction mixture was extracted with ethyl acetate (500 ml) and water (200 ml). Organic layer was dried with MgSO4. The organic layer was evaporated under reduced pressure. Crude compound was purified by column chromatography with ethyl acetate/hexane/MeOH to afford white color solid. 1H-NMR (CDCl3+CD3OD): 2.95 ppm (2H, m), 4.35 ppm (1H, m), 4.46 ppm (2H, m), 6.30 ppm (1H, m), 6.35 ppm (1H, m), 7.24 ppm (2H, m), 7.3 ppm (1H, m), 7.44-7.46 ppm (4H, m), 8.07 ppm (1H, d, J=7.5 Hz)

The synthesis of N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-N-(furan-2-ylmethyl)acetamide (6). To a solution of 1-(9H-carbazol-9-yl)-3-((furan-2-ylmethyl)amino)propan-2-ol (5) (230 mg, 1.0 mol) in DMF (2 ml) was added DIEA (192 mg, 1.5 mol, 240 �l) and followed HATU (456 mg, 1.2 mmol). After stirring at room temperature for 10 min, the solution of 2-(2-(2-(2-

14

azidoethoxy)ethoxy)ethoxy)acetic acid (280 mg, 1.2 mmol) in DMF (3 ml) was slowly added into the reaction mixture. After stirring at room temperature overnight, the reaction mixture was extracted with ethyl acetate (500 ml) and water (200 ml). Organic layer was dried with MgSO4. The organic layer was evaporated under reduced pressure. Crude compound was purified by Prep-HPLC with 0.05% trifluoroacetic acid-water/0.05% trifluoroacetic acid- acetonitrile to afford yellow compound. HRMS calcd for C28H33N5O6 (M+H): 536.2503, found: 536.2512

The synthesis of N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)-2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(furan-2-ylmethyl)acetamide (7). To a solution of N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-N-(furan-2-ylmethyl)acetamide (6) (153 mg, mmol) in MeOH (10 ml) was added 10% Pd-C (20 mg) and H2 gas. After stirring at room temperature overnight, the reaction mixture was filtered through frit with celite. Organic solvent was evaporated and purified with prep-HPLC with 0.05% trifluoroacetic acid-water/0.05% trifluoroacetic acid-acetonitrile to afford yellow compound.

The synthesis of KL001-linker-agarose (8). To a solution of compound 7 (4.49 mg, 0.01 mmol) in DMSO was added triethylamine (13.5 �l, 0.1 mmol, 10.0 eq) and agarose bead [Affi-gel 10 Gel (153-6046, Bio-Rad), activated by N-hydroxysuccinimide, (0.01 mmol/ml), (3.3 ml, 3.3 eq)]. After shaking at room temperature overnight, the reaction was analyzed by LC-MS. After 12 h, LC-MS indicated that all of the starting material had been consumed, and the reaction mixture was treated with 2-ethanolamine (6.1 mg, 0.1 mmol, 10.0 eq) and stirred at room temperature overnight. The reaction mixture was filtered through frit. The agarose beads were washed with DMSO (2 ml, �3) and PBS (2 ml, �3). The agarose beads were stored at 4°C in a PBS solution containing 0.05% NaN3.

Scheme S3. The synthesis of KL002-linker.

15

The synthesis of methyl 4-(methylsulfonamido)benzoate (10). To a solution of compound 9 (1.03 g, 6.8 mmol) in Pyridine (10 ml) was added TsCl (3.1 g, 27.2 mmol, 2.1 ml). After stirring at room temperature overnight, 1N NaOH/MeOH was added to the reaction mixture. After stirring at room temperature for 1 h, the reaction mixture was extracted with ethyl acetate and water. Organic layer was dried with MgSO4 and evaporated with reduced pressure. The crude was purified by column chromatography to afford compound.

The synthesis of methyl 4-(N-(oxiran-2-ylmethyl)methylsulfonamido)benzoate (11). To a solution of compound 10 (500 mg, 2.18 mmol) in DMF (10 ml) was added NaH (109 mg, 4.36 mmol). After stirring at room temperature for 30 min, epi-bromohydrin (449 mg, 3.3 mol) was slowly added and the reaction mixture was heated at 40°C overnight. The reaction mixture was purified by column chromatography.

The synthesis of methyl 4-(N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)methylsulfonamido)benzoate (12). To a solution of compound 11 (242 mg, 0.85 mmol) in DMF (5 ml) was added NaH (41 mg, 1.7 mmol). After stirring at room temperature for 30 min, the solution of carbazole (250 mg, 1.5 mmol) in DMF (5 ml) was added. The reaction mixture was stirred at room temperature overnight. The crude compound was purified by column chromatography.

The synthesis of 4-(N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)methylsulfonamido)benzoic acid (13). To solution of compound 12 (60 mg) in MeOH (10 ml) was added 1N of NaOH solution (10 ml). The reaction mixture was stirred at room temperature for 10 h. After reaction complete, the reaction mixture was acidified with 1N HCl and extracted with ethyl acetate (30 ml �3). The crude compound was used for the next reaction.

The synthesis of tert-butyl (2-(2-(2-(4-(N-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)methylsulfonamido)benzamido)ethoxy)ethoxy)ethyl)carbamate (14). To solution of compound 13 (50 mg, 0.114 mmol) in DMF (4 ml) was added DIEA (18 mg, 0.14 mmol) and followed HATU (53.2 mg, 0.14 mmol). After stirring at room temperature for 10 min, the solution of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (35 mg, 1.4 mmol) in DMF (3 ml) was slowly added into the reaction mixture. After stirring at room temperature overnight, the reaction mixture was purified by Prep-HPLC with 0.05% trifluoroacetic acid-water/0.05% trifluoroacetic acid-acetonitrile to afford compound.

The synthesis of KL002-linker. To solution of compound 14 (16. 8 mg, 0.03 mmol) in dichloromethane (10 ml) was added TFA (1 ml). After stirring at room temperature for 1 h, solvent was evaporated. THF (3 ml) was added to the crude compound. DIEA (8 �l) and acetic anhydride (5 �l) was added to the reaction mixture. After stirring at room temperature overnight, the reaction mixture was purified by prep-HPLC with 0.05% trifluoroacetic acid-water/0.05% trifluoroacetic acid-acetonitrile to afford compound.

16

Fig. S1. Representative luminescence traces (n = 2) of Bmal1-dLuc and Per2-dLuc U2OS cells continuously treated with various concentrations of carbazole derivatives or longdaysin.

KL002

Lum

ines

cenc

e

0 24 48 72 96 1200

100

200

300 KL003

0 24 48 72 96 1200

100

200

300

0 24 48 72 96 1200

200

400

600

Time (h)0 24 48 72 96 120

0

200

400

600

Longdaysin

0 24 48 72 96 1200

100

200

300

0 24 48 72 96 1200

200

400

600

Bmal1-dLuc

Per2-dLuc

U2OS cells

0 �M0.03 �M0.1 �M0.3 �M0.9 �M

2.7 �M8 �M24 �M71 �M

17

Fig. S2. Effect of KL001 on cellular ATP levels. CellTiter-Glo assay (Promega) was performed after treatment of Bmal1-dLuc and Per2-dLuc U2OS cells with various concentrations of KL001 for 120 h. The signal intensity relative to DMSO control is shown as mean ± SEM (n = 4).

KL001

-9 -8 -7 -6 -5 -40.0

0.5

1.0

log[conc] (M)

Rel

ativ

e AT

P le

vels

Bmal1-dLucPer2-dLuc

18

Fig. S3. Effect of KL001 on circadian expression of transiently transfected Bmal1-dLuc and Per2-dLuc reporters in mouse NIH-3T3 fibroblasts. Representative traces (n = 2) are shown in left panels. The changes of period and luminescence intensity (an average of 24-120 h) relative to DMSO control are shown in right panels as mean ± SEM (n = 4). When arrhythmic, the period was not plotted.

KL001 (�M)

Bmal1-dLuc

Lum

ines

cenc

e

0 24 48 72 96 1200

1000

2000

3000

Per2-dLuc

Time (h)0 24 48 72 96 120

0

500

1000

1500

20000 �M0.9 �M2.7 �M8 �M

0 0.9 2.7 80.0

0.5

1.0

Rel

ativ

e in

tens

ity

0 0.9 2.7 8

0

2

4

Bmal1-dLuc

Per

iod

chan

ge (h

)

Per2-dLuc

NIH-3T3 cells

19

Fig. S4. Effects of carbazole derivatives on CKI , CKI and CK2 activities in vitro. Data are mean of duplicates. TBB is an inhibitor of CK2.

0

50

100

-8 -7 -6 -5 -4

TBB

0

50

100

-8 -7 -6 -5 -4

0

50

100

-8 -7 -6 -5 -4

0

50

100

-8 -7 -6 -5 -4

0

50

100

-8 -7 -6 -5 -4

Inhi

bitio

n (%

)

log[conc] (M)

KL001 KL002 KL003 LongdaysinCKI�CKI�CK2

20

Fig. S5. The chemical structure of KL001-linker, KL002-linker and KL004 and their effects on the circadian period in Bmal1-dLuc U2OS cells. Data are mean ± SEM (n = 4, bottom panels). An ethylene glycol linker was attached to KL002 at a position corresponding to furfurylamine position in KL001 (see Fig. 1A). Note that the period effect of KL002-linker was much less than KL001-linker. Data for KL001 are from Fig. 1C.

KL002-linker

A

KL001-linker

B

KL004

-9 -8 -7 -6 -5 -4

0

2

4

6

log[conc] (M)

Per

iod

chan

ge (h

) KL004KL001

-9 -8 -7 -6 -5 -4

0

1

2

log[conc] (M)

Per

iod

chan

ge (h

) KL001-linkerKL002-linker

21

Fig. S6. Reactivity of anti-mCRY1 and anti-mCRY2 antibodies against human CRY proteins. (A) Comparison of amino acid sequences of antigen region (5) between mouse and human CRY proteins. Conserved amino acid is highlighted by gray box. (B) Protein immunoblot analysis with anti-mCRY1 and anti-mCRY2 antibodies. Human U2OS cells were transfected with siRNA against CRY1 (CRY1 si) or CRY2 (CRY2 si). Asterisk indicates a nonspecific band. Note that anti-mCRY1 and anti-mCRY2 antibodies specifically recognize human CRY1 and CRY2, respectively.

CRY1

�-actin

CRY2*

anti-mCRY1

anti-mCRY2

anti-�-actin

Human U2OS cell lysate

mCRY1 583 SGKRPSQEEDAQSVGPKVQRQSSN 606 hCRY1 563 GGKRPSQEEDTQSIGPKVQRQSTN 586

mCRY2 563 EAAEEPPGEELTKRARVTEMPTQEPASKDS 592hCRY2 585 EAAEEPPGEELSKRARVAELPTPELPSKDA 614

A

B

Contr

ol si

CRY1

si

CRY2

si

Contr

ol si

CRY1

si

CRY2

si

22

Fig. S7. Interaction of KL001 with the core clock proteins. Flag-tagged CRY, CLOCK or BMAL1 was transiently overexpressed in HEK293T cells and subjected to pull-down assays with agarose-conjugated KL001-linker compound in the presence or absence of free KL001 (20

M) as a competitor. Bound proteins were subjected to protein imunoblotting with anti-Flag antibody. Note that only CRY1 and CRY2 interacted with the linker compound, and the interaction was competed off by free KL001.

- + - +competitor:

CRY1

CRY1/2-Flag

Input

- +

CRY2CLOCK-BMAL1

Pull-down ppt

CLOCK-Flag

BMAL1-Flag

CRY1

CRY2

CLOC

K-

BMAL

1

23

Fig. S8. Interaction of KL001 with purified CRY1 protein. (A) Interaction of KL001 with purified CRY1-Flag protein overexpressed in HEK293T cells. CRY1-Flag was affinity purified from HEK293T cell lysate (left panel) and subjected to pull-down assay with KL001-agarose conjugate in the presence or absence of free KL001 (50 M) as a competitor (right panel). Pre, pre-purification; Post, post-purification. (B to D) Interaction of KL001 with purified His6-MBP-TEVsite-CRY1 protein expressed in E. coli. (B) CBB stain of His-tag affinity purified proteins. The fraction contained partial degradation products (indicated by an open arrowhead and an asterisk). (C) In vitro TEV protease digestion of the proteins. Calculated molecular weights are 112.9 kDa for His6-MBP-TEVsite-CRY1 and 44.8 kDa for His6-MBP-TEVsite (cleaved). The treatment caused disappearance of two protein species indicated by solid and open arrowheads, showing that they are corresponding to full-length His6-MBP-TEVsite-CRY1 and a C-terminally truncated species, respectively. (D) Pull-down assay with agarose-conjugated KL001-linker compound in the presence or absence of free KL001 (50 M) as a competitor. The degradation product indicated by an open arrowhead was almost absent in pull-down samples, showing that the interaction is specific for full-length CRY1.

A

25015010075

50

(kDa)

37

25

CRY1-Flag

Pre

Post

Silver stain WB: anti-Flag

25015010075

50

(kDa)

37

25

: competitor (50�M)- +Pull-down ppt

Post

Input

25015010075

50

(kDa)

37

2520

15

CBB stain

His6-MBP-TEVsite-CRY1

*

25015010075

50

(kDa)

37

25

20

TEVprotease: - +

WB: anti-penta-His

*

250

150

100

75

50

(kDa)37

- + : competitor (50�M)

Pull-downppt

Input

WB: anti-MBP

*

B C D

24

Fig. S9. Effects of FAD, FMN, riboflavin and NAD on interaction of CRY1 with KL001-agarose conjugate. A lysate of HEK293T cells overexpressing CRY1-Flag was subjected to the pull-down assay in the presence of compounds.

50 500 5000

NAD

Input : competitor (�M)

CRY1-Flag

50 100 500

Riboflavin

50 500 5000

FMN

50 500 5000

FAD

50

KL001

0

Pull-down ppt

25

Fig. S10. Effect of CRY1/2 double knockdown on the action of KL001 against Per2-dLuc reporter in human U2OS cells. Luminescence rhythms of control si or CRY1/2 si transfected cells were monitored in the presence of various concentrations of KL001. Representative traces (n = 2) are shown in left panels. The signal intensity relative to DMSO control is shown in right panel as mean ± SEM (n = 4).

Control si

Time (h)

CRY1/2 siPer2-dLuc U2OS cells

Control siCRY1/2 si

KL001 (�M)

Lum

ines

cenc

e

0 24 48 72 96 1200

200

400

600

0 24 48 72 96 1200

400

800

12000 �M0.9 �M2.7 �M8 �M

0 0.9 2.7 80.0

0.5

1.0

Rel

ativ

e in

tens

ity

26

Fig. S11. Effect of E2 enhancer mutation on the action of KL001 against Per2-Luc reporter in NIH-3T3 cells. The cells were transiently transfected with wild type (pGL3Basic-E2) or E2 enhancer-mutated (pGL3Basic-mutE2) Per2-Luc reporter. Data are mean ± SEM (n = 3).

0 �M0.9 �M2.7 �M8 �M

pGL3Basic-E2

Lum

ines

cenc

e

0 24 48 72 960

10000

20000

30000

24 48 72 960

1000

2000

3000

4000

pGL3Basic-mutE2

Time (h)0 24 48 72 96

0

10000

20000

30000NIH-3T3 cells

pGL3Basic-E2pGL3Basic-mutE2

0 0.9 2.7 8KL001 (�M)

0.0

0.5

1.0

Rel

ativ

e in

tens

ity

27

Fig. S12. Effect of KL002 and KL004 on CRY1 and CRY2 stability in HEK293 stable cell lines expressing CRY1-LUC, CRY2-LUC or LUC. The cells were treated with various concentrations of compound for 24 h and subsequently incubated with cycloheximide for the luminescence recording. Profiles are shown by setting peak luminescence as 1. Data are mean ± SEM (n = 4).

Rel

ativ

e in

tens

ity

Time (h)

CRY1-LUC LUC

KL002

KL004

0 �M 2 �M 8 �M24 �M

0.0

0.5

1.0

0 2 4 6 8

0.0

0.5

1.0

0 2 4 6 8

0.0

0.5

1.0

0 2 4 6 8

0.0

0.5

1.0

0 2 4 6 8

0.0

0.5

1.0

0 2 4 6 8

0.0

0.5

1.0

0 2 4 6 8

CRY2-LUC

28

Fig. S13. Effect of KL001 on ubiquitination of CRY1 and CRY1D387N in vitro. Flag-tagged CRY1 (A) or CRY1D387N (B) was transiently overexpressed in HEK293T cells. Cell lysates were mixed with HA-tagged ubiquitin expressing lysate and incubated in the presence of 2 mM ATP with 0 or 50 M KL001 for in vitro ubiquitination. The proteins were immunoprecipitated with anti-Flag antibody and subjected to protein immunoblotting with anti-HA or Flag antibody.

25015010075

50

Anti-Flag(CRY1D387N)

(kDa)

0 0.5 1 2 3

Input

- +

Time (h) :

KL001 :

Anti-Flag IP ppt

Anti-HA(Ub)

HA-U

b

CRY1

D387

N-Fla

g

Marke

r

4 0 0.5 1 2 3 4

75

A

250

150

100

75

50

Anti-Flag(CRY1)

(kDa)

0 0.25 1 2 3 0 0.25 1 2 3

Input

- +

Time (h) :

KL001 :

Anti-Flag IP ppt

Anti-HA(Ub)

HA-U

b

CRY1

-Flag

Marke

r

CRY1

(-)

75

B

29

FBXL3 si

0 24 48 72 96 1200

100

200

300Control si

Time (h)

Lum

ines

cenc

e

0 24 48 72 96 1200

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Control si

Time (h)

Lum

ines

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e

0 24 48 72 96 1200

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800 FBXL3 si

0 24 48 72 96 1200

50

100

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0 0.3 0.9 2.7 80.1

1

Rel

ativ

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tens

ity

0 0.3 0.9 2.7 80.1

1

Rel

ativ

e in

tens

ity

0 0.3 0.9 2.7 8

0

2

4

6

Per

iod

chan

ge (h

)

0 0.3 0.9 2.7 80

5

10

15

Per

iod

chan

ge (h

)

Control si

Time (h)

Lum

ines

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e

0 24 48 72 96 1200

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0 24 48 72 96 1200

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Control si

Time (h)

Lum

ines

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e

0 24 48 72 96 1200

200

400

600

800 FBXL3 si

0 24 48 72 96 1200

50

100

150

200

0.3 �M0.9 �M

2.7 �M8 �M

0 �M

Bmal1-dLuc U2OS cells

Per2-dLuc U2OS cells

KL001 (�M)

KL001 (�M)

Longdaysin (�M)

Bmal1-dLuc U2OS cells

Per2-dLuc U2OS cells

Longdaysin (�M)

Control siFBXL3 si

A

B

0.3 �M0.9 �M

2.7 �M8 �M

0 �MControl siFBXL3 si

30

Fig. S14. Effect of FBXL3 knockdown on the action of KL001 and longdaysin in Bmal1-dLuc or Per2-dLuc reporter U2OS cells. Luminescence rhythms of FBXL3 si transfected Bmal1-dLuc or Per2-dLuc reporter cells were monitored in the presence of various concentrations of KL001 (A) or longdaysin (B). Representative traces (n = 2) are shown in left panels. Changes of the period (in Bmal1-dLuc reporter cells) and Per2-dLuc reporter intensity relative to DMSO control are shown in right panels as mean ± SEM (n = 4). Period and intensity plots for KL001 treatment are from Fig. 3F. Knockdown efficiency of the FBXL3 si was shown in our previous study (22).

31

Fig. S15. Construction of a simple mathematical model of the PER-CRY negative feedback loop. (A) Time course plots of the mathematical model. The three polar plots show the time-varying levels of each clock component for optimal parameter set (Table S4, degradation-based model). Rising mRNA levels (left plot) caused by low repressor concentrations (CT12) result in accumulating cytosolic protein (middle plot). Lower levels of PER limit the amount of CRY entering the nucleus. High levels of nuclear repressors halt transcription until both CRY species in the nucleus (CRY1n and CRY2n) have been degraded (right plot). (B and C) Effects of Cry1 or Cry2 knockdown (B) and cytosolic CRY2 stabilization (C) on the period and nuclear CRY levels in silico. Knockdowns were achieved through increasing the degradation parameter of the mRNA state variable, and cytosolic CRY2 (CRY2c) stabilization was similarly achieved by decreasing the degradation parameter of the protein. State variable responses were time-averaged. Cyt, cytosolic. (D) Effect of in silico nuclear CRY stabilization on the period and expression levels of Per and Cry. Period plot is from Fig. 4B.

mRNA

CRY1

CRY2

PER2

4

60

1215 9

18

21 3

6CRY1n

CRY2n0.2

1.0

1.40

1215 9

18

21 3

6

0.60.5

1.01.5

2.00

1215 9

18

21 3

6PerCry2

Cry1

Cytosolic protein CRYn protein

Circadian time (h)

B C

CRY1n

CRY2n

CRY1n

CRY2n

CR

Yn

prot

ein 400

200

0

mR

NA

150

500

100

Per

iod 120

100

80

1 2 3 1 2 3Cry mRNA degradation rate (fold)

Cry1 knockdown Cry2 knockdown

Cry1

Cry2

PerPer

Cry2

Cry1

Rel

ativ

e ch

ange

(%)

CRY1n

CRY2n

1.0 0.9 0.8CR

Yn

prot

ein 140

100

60

Cyt

pro

tein 250

50

150

Per

iod 95

9085

120

80

100CRY2c stabilization

CRY1

CRY2

PER100

200

Rel

ativ

e ch

ange

(%)

CRY2c degradationrate (fold)

A

105110115120

Cyt

pro

tein 100

60

mR

NA

Per

iod

80

100

CR

Yn

prot

ein

110

90

120

100

80

90

100

CRYn degradationrate (fold)

CRY1n

CRY2n

Per

Cry1Cry2

CRY1CRY2

PER

1.0 0.9 0.8 0.7

CRYn stabilization

Rel

ativ

e ch

ange

(%)

D

32

Fig. S16. Effect of KL001 on nuclear CRY1, CRY2 and PER1 levels in unsynchronized U2OS cells. The confluent cells were treated with various concentrations of KL001 for 48 h, and the nuclear fraction was subjected to protein immunoblot analysis.

PER1

CRY1

CRY2

Lamin ALamin C

0 0.9 2.7 8

Nuclear fraction

: KL001 (�M)

Unsynchronized U2OS cells

33

Cry2 KO

Cry1 KO

0 0.9 2.7 8

0

2

4P

erio

d ch

ange

(h)

KL001 (µM)

Arrhythmic

0 24 48 72 96 1200

200

400

600

800

Time (h)

Lum

ines

cenc

e

0 24 48 72 96 1200

100200300400500 0 µM

0.9 µM2.7 µM8 µM

mPer2Luc fibroblasts(without Dex pulse treatment)

0 0.9 2.7 80.0

0.5

1.0

0 0.9 2.7 80.0

0.5

1.0

Rel

ativ

e in

tens

ity

KL001 (µM)

A

CRY2 si

CRY1 si

Time (h)0 24 48 72 96 120

0

200

400

600

800

Lum

ines

cenc

e

0 24 48 72 96 1200

200400600800

1000 0 µM0.9 µM2.7 µM8 µM

Per2-dLuc U2OS cells

0 0.9 2.7 8

0

2

4

6

0 0.9 2.7 8

0

2

4

6

Per

iod

chan

ge (h

)

KL001 (µM)

0 0.9 2.7 80.0

0.5

1.0

0 0.9 2.7 80.0

0.5

1.0Rel

ativ

e in

tens

ity

KL001 (µM)

C

0 0.9 2.7 8 240.0

0.5

1.0

0 0.9 2.7 8 240.0

0.5

1.0Rel

ativ

e in

tens

ity

KL001 (µM)KL001 (µM)

Cry2 KO

Cry1 KO

0 0.9 2.7 8 24

0

2

4

0 0.9 2.7 8 24

0

2

4

Per

iod

chan

ge (h

)

mPer2Luc SCN

Lum

ines

cenc

e

0 µM 0.9 µM 2.7 µM 8 µM 24 µM

0 120 240 360 480 6000

500

1000

1500

Time (h)0 120 240 360 480 600

0

500

1000

1500

KL001DMSO

D

0 0.3 0.9 2.7 80.0

0.5

1.0

0 0.3 0.9 2.7 80.0

0.5

1.0Rel

ativ

e in

tens

ity

KL001 (µM)

mPer2Luc fibroblasts(with Dex pulse treatment)

Cry2 KO

Cry1 KO

B

34

Fig. S17. Effect of KL001 on the period and Per2 reporter intensity in Cry deficient models. (A) Effect of KL001 in Cry1 or Cry2 knockout mPer2Luc knock-in mouse fibroblasts. Luminescence rhythms were monitored in the presence of various concentrations of KL001. Representative traces (n = 2) are shown in left panels. The changes of period and luminescence intensity relative to DMSO control are shown in middle and right panels, respectively, as mean ± SEM (n = 4). Note that Cry1 KO cells were arrhythmic without a dexamethasone (Dex) pulse treatment as described previously (28). (B) Effect of KL001 in Cry1 or Cry2 knockout mPer2Luc knock-in mouse fibroblasts after 2-h Dex pulse treatment. (C) Effect of KL001 in CRY1 or CRY2 knockdown human U2OS cells. Luminescence rhythms of CRY1 si or CRY2 si transfected Per2-dLuc reporter cells were monitored in the presence of various concentrations of KL001. Data are mean ± SEM (n = 4). (D) Effect of KL001 in SCN explant of Cry1 or Cry2 knockout mPer2Luc knock-in mice. Luminescence rhythms were monitored in the presence of increasing concentration of KL001 (each for 120 h). Data are mean ± SEM (n = 6 for Cry1 KO and 12 for Cry2 KO, right panels).

35

Fig. S18. Effect of KL001 on cellular lactate dehydrogenase (LDH) activity in mouse primary hepatocytes. The hepatocytes were treated with various concentrations of KL001 for 18 h, stimulated with 10 nM glucagon for 3 h, and then incubated with glucose-free buffer containing 20 mM sodium lactate and 2 mM sodium pyruvate. After 4 h, the cells were collected and subjected to LDH activity measurement by Cytotoxicity Detection Kit (Roche). Data are mean ± SEM (n = 3).

0100200300400500

LDH

act

ivity

(uni

t/L)

Control Glucagon

0 �M 2 �M 4 �M8 �M

36

Table S1. Identification of potential KL001 binding proteins. Agarose-conjugated KL001-linker compound was incubated with lysate of unsynchronized U2OS cells in the presence of various concentrations of free KL001 as a competitor. After pull-down, bound proteins were subjected to liquid chromatography-tandem mass spectrometry analysis. Listed

upon competition with 50 M free KL001 in experiment 1 and also identified in experiment 2. Note that only CRY1 showed dose-dependent and strong competition even at 20 M KL001 in both experiments, suggesting that CRY1 is a high-affinity target.

Spectra numberExperiment 1 Experiment 2 MolecularCompetitior (�M) Competitior (�M) Weight

Protein name 0 20 50 0 20 50 (kDa)CRY1 Cryptochrome-1 5 1 1 3 0 0 66

MCCC2 Methylcrotonoyl-CoA carboxylase beta chain, mitochondrial precursor 5 2 0 3 3 3 61

MMS19L MMS19-like protein 7 3 1 8 0 7 113RABAC1 Prenylated Rab acceptor protein 1 8 6 1 6 4 6 21PNPLA6 Neuropathy target esterase 6 5 1 5 3 2 146LDHA L-lactate dehydrogenase A chain 6 5 1 21 20 16 37NUP133 Nuclear pore complex protein Nup133 6 6 0 3 5 6 129

37

Table S2. Primers for qPCR analysis. Gene Forward primer Reverse primerhTbp CCCGAAACGCCGAATATAATCC GACTGTTCTTCACTCTTGGCTC mRplp0 (36B4) GGCCCTGCACTCTCGCTTTC TGCCAGGACGCGCTTGT mPck1 AAGCATTCAACGCCAGGTTC GGGCGAGTCTGTCAGTTCAAT mG6pc TCGGAGACTGGTTCAACCTC AGGTGACAGGGAACTGCTTTAT mTbp ACCTAAAGACCATTGCACTTCG GCTCTCTTATTCTCATGATGACTGC

38

Table S3. Equations for the mathematical model. (A) Model equations for the degradation-based model. Lower case letters (p: Per, c1: Cry1, c2: Cry2) are mRNA state variables. Uppercase letters (P: PER, C1: CRY1, C2: CRY2) are the free (cytosolic) proteins. C1N: CRY1n and C2N: CRY2n are the nuclear proteins. dpdt

�vtxn,p

ktxn,p � C1N �C2N� �3 �

vdeg,p pkdeg,p � p

(1)

dc1dt

�vtxn,c1

ktxn,c � C1N �C2N� �3 �

vdeg,c1 c1kdeg,c � c1

(2)

dc2dt

�vtxn,c2

ktxn,c � C1N �C2N� �3 �

vdeg,c2 c2kdeg,c � c2

(3)

dPdt

� ktln,p p �vdeg,P P

kdeg,P � P� va,CP P C1� vd,CP C1N � va,CP P C2 � vd,CP C2N

(4)

dC1dt

� c1�vdeg,C1 C1kdeg,C �C1

� va,CP P C1� vd,CP C1N (5)

dC2dt

� c2 �vdeg,C2 C2kdeg,C �C2

� va,CP P C2 � vd,CP C2N (6)

dC1Ndt

� �vdeg,CP C1N

kdeg,CP �C1N �C2N� va,CP P C1� vd,CP C1N

(7)

dC2Ndt

� �vdeg,CP mC2N� �C2N

kdeg,CP � C2N �C1N� va,CP P C2 � vd,CP C2N

(8)

(B) Changed equations for the activation-based model. In these equations, the nuclear CRY states C1N and C2N share the same degradation rate, while the repressive potency (KI) is allowed to differ (see Materials and Methods). dpdt

�vtxn,p

ktxn,p � mC2N C1N �C2N� ��

vdeg,p pkdeg,p � p

(1*)

dc1dt

�vtxn,c1

ktxn,c � mC2N C1N �C2N� ��

vdeg,c1 c1kdeg,c � c1

(2*)

dc2dt

�vtxn,c2

ktxn,c � mC2N C1N �C2N� ��

vdeg,c2 c2kdeg,c � c2

(3*)

dC2Ndt

� �vdeg,CP C2N

kdeg,CP �C2N �C1N� va,CP P C2 � vd,CP C2N

(8*)

39

Table S4. Parameter sets for the degradation-based model (Deg model, Table S3A) and activation-based model (Act model, Table S3B). While the period of oscillation was scaled to 23.7 hours, no amplitude scaling of the model was performed. The units of the state variables are therefore arbitrary relative concentrations, and the parameters are also in arbitrary units. Rate parameters, starting with “v”, are in units of arbitrary units/hour. Number Parameter Description Deg model Act model

1 vtxn,p Per transcription rate 0.195 0.2762 vtxn,c1 Cry1 transcription rate 0.131 0.0623 vtxn,c2 Cry2 transcription rate 0.114 0.0534 ktxn,p Per repression constant 0.425 0.4255 ktxn,c Cry1/2 repression constant 0.259 0.2626 vdeg,p Per max degradation rate 0.326 0.4727 vdeg,c1 Cry1 max degradation rate 0.676 0.3228 vdeg,c2 Cry2 max degradation rate 0.608 0.2909 kdeg,p Per degradation constant 0.011 0.02410 kdeg,c Cry1/2 degradation constant 1.149 0.80911 vdeg,P Max PERc degradation rate 2.97 2.04112 kdeg,P PERc degradation constant 0.034 0.03413 vdeg,C1 Max CRY1c degradation rate 1.523 1.04814 vdeg,C2 Max CRY2c degradation rate 1.686 1.13415 kdeg,C CRYc degradation constant 2.017 2.02816 vdeg,CP CRYn degradation rate 0.101 0.07017 mC2P CRY2n degradation multiplier 3.318 3.33418 kdeg,CP CRYn degradation constant 0.053 0.05319 va,CP CRYn association rate 0.041 0.02820 vd,CP CRYn dissociation rate 0.002 0.00121 ktln,P PER translation rate 3.000 1.000

40

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